The use of adjunctive intraoperative photodynamic therapy in Head and Neck cancer

MS THESIS - LONDON UNIVERSITY

THE EFFICACY AND SAFETY OF ADJUNCTIVE

INTRAOPERATIVE PHOTODYNAMIC THERAPY IN REDUCING

THE LOCAL RECURRENCE RATE OF TUMOUR AFTER

RADICAL NECK DISSECTION

M.G.DILKES MB,BS., FRCSEd(GS), FRCS(Otol), FRCS(ORL)

THE EFFICACY AND SAFETY OF ADJUNCTIVE INTRAOPERATIVE PHOTODYNAMIC THERAPY IN REDUCING THE LOCAL

RECURRENCE RATE OF TUMOUR AFTER RADICAL NECK DISSECTION

Acknowledgements

SUPERVISORS: PROFESSOR N.S.WILLIAMS AND MR P.MCKELVIE

FUNDING: THE CANCER RESEARCH CAMPAIGN

Other financial support: Scotia Pharmaceuticals

Physics support, experimental planning: Martin L. DeJode

Animal care, experimental support: Richard Rountree and staff

Histology: Alex Brown and staff

Technical support (vessel work): Steve G. Greenwald

Pharmacological analysis: Chung-Kee Lim and Andy Holroyd

Vessel microvascular anastomosis work: Sandra Simpkin

Fluorescence, general advice: Mike F. Grahn

Photosensitiser: Scotia Pharmaceuticals, Lederle Pharmaceuticals

Drug advice: Brenda Reynolds, Charles Stewart

Ultrasonic Doppler device: Linton Instruments2



Laser devices: QuadraLogicTechnologies

Many thanks to the above - they made the project possible

3



INTRODUCTION PART 1

1:Squamous Cell Carcinoma of the Upper Aerodigestive

tract1a: Incidence 11-14

1b: Aetiology 15

1c: Histology 16

1d: Staging 17-21

1e: Treatment of HNSCC 22-23

1f: Cause of death 24

2: Metastatic Disease2a: Rate of distant metastasis 25-26

2b: Metastatic Neck Disease 27

2c: Incidence 28-29

2d: Prognosis - Neck Metastases 30

2e: Why Lymphatic spread? 31

2f: Treatment of metastatic neck disease. 32

2g: Recurrence after radical neck dissection 33-35

2h: Cause of local recurrence 36

2j: Surgery for metastatic neck disease

2j(i): Radical neck dissection 37-394



2j(ii): Conservative neck dissection 40-41

2k: Treatment of macroscopic residual disease 42

5



3: Radiotherapy3a: Introduction 43

3b: External Beam Radiotherapy 44-45

3c: Brachytherapy 46

3d: Efficacy of Radiotherapy 47

3e: Side Effects of radiotherapy 48-49

3f: Role of radiotherapy in neck malignancy 50

3g: Pre or Post operative radiotherapy? 51

6



INTRODUCTION PART II

4: Photodynamic Therapy4a: Development and Principles 52-54

4b: Photodynamic Therapy for Head and Neck cancer 55-56

4c: History of PDT for Head and Neck cancer 57-59

4d: Why do these advantages occur? 60-61

4e: Light for Photodynamic Therapy 62-65

4f: Potentiation of the Photodynamic effect 66-67

4g: Role of fluorescence in PDT 68-69

5: Chemistry of Photodynamic therapy5a: Drugs for Photodynamic Therapy 70

5b: Examples of first generation drugs 71-73

5c: Second generation Photosensitising Drugs 74-77

5d: Problems 78-79

6: Adjunctive Intraoperative Photodynamic Therapy

(AIOPDT)6a: Preclinical 80

6b: Clinical 80

7: Experimental Rationale 817a: Description 82

7b: First Stage - efficacy 83

7c: Second Stage - Safety Studies 83

7



7d: Third Stage - Clinical Study 83

7e: 6 consecutive preclinical experiments 84

8: Drugs, animals, Laser, Other equipment

8a: Photosensitising Drugs 85

8b: Anaesthetic Drugs 86

8c: Tumour 86-87

8d: Animals 88

8e: Lasers 88

9: Experiment 1 - Preclinical pharmacokinetic studies

of first and second generation photosensitising drugs

9a: Introduction 89-90

9b: Tumour 90

9c: Drug dose and analysis 91

9c(i): mTHPC analysis 92

9c(ii): Photofrin 2 analysis 93

9d: In-vivo validation 94

9e: Results 95-100

8



9f: Discussion 101-106

10: Experiment 2 - The efficacy of Adjunctive

Intraoperative Photodynamic Therapy in a rat

fibrosarcoma model with mTHPC10a: Description 107

10b: Tumour 107

10c: Photodynamic Therapy Parameters 108

10d: Preliminary studies 109

10d(i): Study 1 109

10d(ii): Study 2 110

10d(iii): Study 3 111-115

10e: Results 116-121

10f: Statistical Analysis 122

10g: Discussion 123-125

10h: Conclusion 125

11: Experiment 3 - Preclinical Photodynamic

Safety Studies on Arteries11a: Description 126-127

11b: PDT Details 127

11c: Drug 128

11d: Study Design 128-129

11e: Vessel Analysis 130-133

11f: Statistical analysis 1369



11g: Discussion 137-140

11h: Conclusion 141

12: Experiment 4: Acute phase effects of PDT on

arteries and veins12a: Description 142

12b: Chemical 142

12c: Measurement of flow 143-144

12d: Experimental technique 145-148

12e: Results 148-149

12f: Discussion 150-153

13: Experiment 5 - The effect of high intensity white

and filtered microscope light on the viability of

microvascular anastomoses in photosensitised rats13a: Description 154-156

13b: Plan 156

13c: Methodology

13c(i): Physics assessment 157

13c(ii): Safety study 157

13d: Drug 157

13e: Animals 158

13f: Operative technique 159

13g: Postoperative assessment 159

13h: Results 160-16510



13I: Statistical analysis 166-168

13j: Discussion 169-172

11



14: Experiment 6 - Histological study of large

diameter arteries undergoing photodynamic therapy14a: Description 173

14b: Methodology 173

14c: Results 173

14d: Discussion 174

15: Adjunctive Intraoperative Photodynamic Therapy for

Head and Neck Cancer 175-177

15a: Method 178-183

15b: Results

15b(i): Case #1 184-185

15b(ii): Case #2 186

15b(iii): Case #3 187

15b(iv): Case #4 187

15c: Discussion 188

16: Conclusion 189

17: References 190-225

12



INTRODUCTION

1) Metastatic neck disease in Squamous Cell Carcinoma of the

Head and Neck

2) Photodynamic Therapy for Squamous Cell Carcinoma of the Head

and Neck

13



INTRODUCTION PART 1

Squamous Cell Carcinoma of the Upper Aerodigestive tract

1a: Incidence

These tumours are by far the most common malignant neoplasms of

the Head and Neck, excepting the skin. Those arising from the

upper aerodigestive tract account for 2.4% of all new cancers

presenting each year in men, and 0.7% of all new cancers in

women (see table 1). They tend to present in the 5th and 6th

decades of life, and are more common in men than women, although

the incidence in women is rising.

Head and Neck cancers account for around 4% of all cancer deaths

annually. The figures regarding incidence as a percentage of

total cancer cases, per site, are fairly similar in the Western

World (see tables 1 and 2), although geographical variance does

occur making tumours much more common in some areas than others

(see later).

To clarify descriptions in later tables, regarding the site of

primary tumours, ENT relates to any squamous cell carcinoma of

the Head and Neck (HNSCC) arising from the following described

areas. From the hard/soft palate junction, in a plane extending

down the anterior pillars of the fauces (which is the border of

the oral cavity and the oropharynx), extending caudally to the

end of the subglottic region of the larynx, and the

cricopharyngeal sphincter of the pharynx/oesophagus junction,

14



including the nose and nasopharynx. This is the anatomical

description of the ENT area as taken from Gray’s Anatomy.

Tumours arising anteriorly to the border between oropharynx and

oral cavity are described as oral cavity in origin. This area

extends to the vermilion border of the lip, where it meets the

skin. Other SCC's arising in the Head and Neck area will be from

ear, salivary gland or skin, and are not further discussed,

since the study aims at treating metastatic disease from HNSCC.

For the purposes of this Thesis, that means Oral Cavity and ENT

tumours as described above (although the treatment itself is

potentially applicable to all malignancy of the Head and neck).

There are three main surgical specialities dealing with this

disease, Ear, Nose and Throat (or Otolaryngology),

Oral/Maxillofacial, and Plastic surgeons, in order of number of

new cases seen per year. In the longer term it seems likely that

regional centres combining all three modalities in the same

therapeutic team, along with radiotherapy and support services

will treat the majority of cases, in accordance with Department

of Health Guidelines for cancer treatment (Calman-Hine Report,

1997).

15



Table 1

The incidence of cancers of the Head and Neck as a percentage of

all primary cancers in this country (U.K.), by site of origin

(Powell and Robin 1983).

Site Male

(%)

Female

(%)Skin 8.2 7.0*Oral Cavity 0.6 0.3*Oropharynx 0.4 0.1*Nasopharynx +

Nose

0.1 0.1

* Hypopharynx 0.4 0.1*Larynx 1.3 0.2Thyroid 0.2 0.6Lymphoma 0.3 0.3Sarcoma 0.1 0.1*Total HNSCC 2.8 0.8

16



Table 2

The incidence of HNSCC, as a percentage of all primary cancers

in the USA by site of origin.

Site % of all

cancersLip 0.6Tongue 0.7Other oral 0.9Nasopharynx 0.2Oro/hypo pharynx 0.8Larynx 1.4Nose and paranasal

sinuses

0.2

Unknown Primary 0.2Total HNSCC 5.0

From: Management Guidelines for Head and Neck cancer. Bethesda,

Md., USDHEW NIH Publication number 80-2037, 1987

The figures between the USA and UK are broadly similar, except

that there is a higher incidence of oral cavity cancer in the

USA (2.2% v 0.9%). The reasons for this are unknown, although it

may be to do with the way some cancers, in particular lip, are

classified. Other variations do occur even between similar

societies as the USA and UK. Post-cricoid carcinoma is very rare

in the USA, but relatively more common in the UK, due to the

17



incidence of post-cricoid webs from Patterson Kelly-Brown

Syndrome (Jacobs A. 1962).

18



1b: Aetiology

A number of factors have been implicated in the aetiology of SCC

of the upper aerodigestive tract (HNSCC). The most important of

these is cigarette smoking, with 90% of head and neck cancers

occurring in smokers (Johnston and Ballantyne 1977). This was

first noted in 1918 by Power, when he noted that in tongue

cancer, "tobacco was the greatest irritant". In the state of

Andrha Pradesh in India, the practice of reverse smoking,

whereby the lit end of the cigarette is inserted into the mouth,

causing a high level of smoke exposure to the oral cavity

epithelium, is associated with a very high incidence of oral

cavity SCC (Reddy and Rao 1957). The addition of significant

alcohol intake has a synergistic effect such that the risk of

developing HNSCC rises 3 times (Thompson 1989). Other, less

dominant aetiological associations have been identified (Vaughan

et al 1980), in particular, causes of chronic irrigation such as

poor dental hygiene, syphilis, candidaisis, erosive lichen

planus, iron deficiency anaemia and betel nut chewing. Patients

with the Acquired Immune Deficiency Syndrome (AIDS) also have a

higher incidence of this disease than the normal population.

The disease itself has a fairly uniform incidence rate

throughout the world, although certain areas, such as the Canton

province of Southeast China, have a very high incidence of one

tumour, in this case nasopharyngeal carcinoma, due to a mixture

of causative factors including genetic predisposition, a diet

including significant amounts of salted fish, and endemic

infection with the Epstein Barr virus (Wei et al 1992). The same19



is true of the Normandy region of France, where there is a high

incidence of oral cavity cancer, said to be due to excessive

amounts of alcohol consumed and the chain smoking of high tar

unfiltered cigarettes (Johnston and Ballantyne1977).

20



1c: Histology

The tumours originate from squamous epithelial cells lining the

upper aerodigestive tract. Their appearance may vary, from an

exophytic (verrucous) pattern, to an invasive pattern with

diffuse tissue infiltration and ulceration. The hallmark of

these tumours on pathological examination is the presence of

well formed desmosomal attachments and intracytoplasmic bundles

of keratin. Depending on the degree of expression of these

characteristics, the tumours can be classified into different

levels of differentiation from well differentiated to poorly

differentiated tumours. Those tumours that cannot be classified

in this way by light microscopy should be analysed by electron

microscopy and immunochemistry, since if they are of a squamous

cell origin, intracytoplasmic keratin will be found (Holm et al

1982). Poorly differentiated tumours behave in a more aggressive

manner than well differentiated tumours, although their alleged

higher replication rate that presumably causes their increased

aggression also makes them more sensitive to some forms of

treatment such as radiotherapy (Bauer H.C. 1974).

HNSCC often demonstrates signs of sequential change into overtly

malignant tumour. This is particularly true of the oropharynx

and oral cavity where progression from leukoplakia to

erythroplakia, carcinoma in situ and invasive carcinoma can be

closely monitored in at-risk groups, since it is a relatively

easy area to examine and biopsy. Progression in the degree of

dysplasia can be seen in these samples (Banoczy and Csiba 1976)

until frank malignancy occurs. Efforts have been made to attempt

to stop the progression into overt malignancy occurring, in21



particular with the use of oral retinoid treatment (Shah J.P. et

al 1990).

22



1d: Staging

HNSCC is currently staged using the UICC/AJCC TNM (Tumour, Node,

Metastasis) classification system. This allows more accurate

auditing of results, and also meaningful comparison of results

from clinical trials. This method of staging is somewhat

artificial and rather subjective, but it is nonetheless a

universally accepted yardstick that needs to be retained until a

better method comes along.

See appendix 1 for a full description of current TNM staging.

Appendix 1

TNM staging of Head and Neck cancer.

Oral Cavity tumours:

Stage Description

TX Minimum requirements to assess tumour cannot be met

T0 No evidence of a primary tumour

TiS Carcinoma-in-situ

T1 Tumour diameter 2 cm or less

T2 Tumour diameter 2-4 cm

T3 Tumour diameter more than 4 cm

T4 Tumour with invasion of deeper structures, e.g. through

cortical bone, into deep muscle of tongue, skin.

23



Oropharyngeal tumours:

Stage Description


T1 Tumour diameter 2cm or less

T2 Tumour diameter 2-4 cm

T3 Tumour diameter more than 4 cm

T4 Tumour with deep invasion to bone, soft tissues of the

neck, deep musculature of tongue.

Laryngeal tumours - supraglottic:

Stage Description


T1 Tumour confined to region of origin (one subsite) with

normal cord mobility.

T2 Tumour involving adjacent subsites with normal cord

mobility.

T3 Tumour involving three subsites, or with cord fixation, or

with extension to medial wall of pyriform fossa, postcricoid

or pre-epiglottic space.

24



T4 Tumour extending beyond the larynx or to involve the

laryngeal cartilage.

25



Laryngeal tumours - glottic:

Stage Description

TiS Carcinoma-in-situ.

T1a Tumour confined to one vocal cord with normal cord

mobility.

T1b Tumour confined to both vocal cords with normal cord

mobility.

T2 Supraglottic or subglottic extension with normal cord

mobility.

T3 Tumour limited to the larynx with cord fixation, or

transglottic spread.

T4 Tumour invasion into Thyroid cartilage and/or extension

outside the larynx.

Laryngeal tumours - subglottic

Stage Description

T1 Tumour limited to subglottis

T2 Tumour extends to vocal cord(s) with normal mobility

T3 Tumour limited to larynx with vocal cord fixation

T4 Tumour invades through cartilage and/or extends to other

tissue beyond the larynx

26



Nasal tumours

Stage Description

T1 Tumour confined to mucosa of infrastructure

T2 Tumour confined to mucosa with bone destruction of medial

or inferior walls only

T3 Extensive tumour involving skin, orbit, pterygoid

musculature

T4 Extensive tumour involving cribriform plate and/or sphenoid

sinus and/or nasopharynx and/or skull base

Hypopharyngeal tumours

Stage Description

T1 Tumour confined to one subsite (posterior pharyngeal wall,

pyriform fossa or postcricoid)

T2 Tumour involving 2 subsites

T3 Tumour spreading out of hypopharynx

T4 Tumour involving external structures, e.g. cartilage,

muscle, bone.

Postnasal space tumours

27



Stage Description

T1 Tumour confined to one site of the nasopharynx

T2 Tumour involving 2 sites

T3 Tumour invading nasal cavity or oropharynx

T4 Tumour invading skull or cranial nerves or both

28



Metastatic neck nodes:

Stage Description

NX Minimum requirements to assess the nodes cannot be met.

N0 No clinically positive nodes.

N1 Single node 3 cm or less.

N2A Single ipsilateral node 3-6 cm.

N2B Multiple ipsilateral nodes, none more than 6 cm diameter.

N2C Metastases in bilateral or contralateral nodes, none more

than 6 cm diameter.

N3 Metastases in a lymph node more than 6 cm in greatest

dimension.

From: Spiessl B., Beahrs O.H., Hermanek P. (1989) UICC TNM

Atlas, ed 3, Berlin, Springer-Verlag.

Metastatic spread beyond the neck is staged as simply presence

of disease or not, ie, M+ or M0.

29



1e: Treatment of HNSCC

1ei: History of Treatment of HNSCC

Although laryngeal carcinoma represents only one part of the

spectrum of HNSCC, the history of the surgical treatment of this

condition represents an interesting and useful guide into how

management of all malignant disease of the Head and Neck

progressed. The treatment of laryngeal cancer was initially

developed during the latter half of the 19th century as

pathological diagnosis became possible and it was possible to

make the distinction between benign and malignant disease.

The decade from 1850 to 1860 marked a turning point in the

understanding of pathology of the upper aerodigestive tract.

This was due to the advent of indirect and direct laryngoscopy

which enabled accurate distinction of various laryngeal

abnormalities as described by Virchow in 1858. The technique of

indirect laryngoscopy was first effectively demonstrated by

Babington in 1829 in a presentation to the Hunterian Society of

London. His technique, using a tongue depressor combined with a

mirror and reflected sunlight was never generally accepted,

partly because it was a bulky and cumbersome piece of apparatus,

and partly because Babington never published his findings. It

was not until Manuel Garcia, the so-called "Father of

Laryngology" successfully presented and published work on

indirect laryngoscopy in 1855 that the technique properly caught

on. 30



Following the successful introduction of indirect laryngoscopy,

laryngeal carcinoma was studied in some depth. Initially it was

thought that carcinoma intrinsic to the larynx was rare, but

studies by Semon, Chevalier Jackson, Tucker and Butlin (Thomson

1939) showed that the opposite was in fact true.

31



The surgical treatment of squamous cell carcinoma of the upper

aerodigestive tract was started in the latter part of the 19th

century. Initially, resections were performed either

endoscopically or via a laryngofissure (Buck 1853). However,

this technique involved piecemeal removal of tumour, with no

clear resection margins and consequently the results were poor

(Mackenzie, 1871). Billroth in 1874 performed the first

successful laryngectomy in Vienna (Billroth and Gussenbauer

1874). Although the treatment of Head and Neck cancer was

somewhat unfairly discredited during the infamous episode of

Morrel Mackenzie's treatment of Emperor Frederick the Third of

Germany (Stevenson 1946), the steady advancement of the

treatment of this disease has progressed gradually throughout

the 20th Century (Butlin 1909, Trotter 1913), particularly with

the advent of pedicled, axial and free flaps for the

reconstruction of large defects in the Head and Neck, enabling

increasingly wider resection margins, reducing the risk of local

recurrence. Despite these advances however, little has changed

in terms of survival rate from this disease over the past 20

years. This is due to a number of factors, one of which is the

fact that no successful new modality of treatment has been

introduced during this period - in particular, chemotherapy has

failed to make the expected advances in this area (Amrein 1991).

Other innovative techniques, such as the use of cryotherapy

(Holden and Mckelvie 1972) or isolated segmental perfusion with

chemotherapy, have not found a niche in the treatment of Head

and Neck cancer.32



1eii: Current Treatment

External beam radiotherapy and radical surgery applied singly or

in combination are the current mainstay of treatment (Jesse and

Lindberg 1975, Arrigada et al 1983). The overall outcome remains

almost unchanged over the past 30 years, despite many technical

advances as discussed. Age adjusted 5 year survival rates range

from as little as 10% for hypopharyngeal primary tumours, to 50%

+ for laryngeal tumours (Powell and Robin 1983). Prognosis is

largely determined by site and stage of tumour when discovered

(Vokes et al 1993). The best results have up to 90% 5 year

survival rate, whilst the same tumour, presenting later, has a

similar survival figure of less than 10% (Kumar et al 1987, Hong

et al 1990). Salvage surgery and radiotherapy after initial

“definitive” treatment has a depressingly low survival rate

(Zieske et al 1986).

33



1f: Cause of death

Studies of patients dying from head and neck cancer have shown

that approximately 30% of uncured patients have uncontrolled

disease at the primary site and about 70% have uncontrolled

local lymphatic or soft tissue spread. In these cases, the local

disease was usually directly implicated in the immediate cause

of death (Sloan and Goepfert 1991). About 25% of patients also

have distant metastases , although these tend to be incidental

findings at post-mortem rather than actual cause of death (see

later).

34



2: Metastatic Disease

2a: Rate of distant metastasis

Distant metastasis is defined as spread of tumour beyond its

site of origin and beyond the regional lymphatics for that site.

Crile was the first to look at the frequency of distant

metastases in patients with HNSCC at post mortem. Mentioning the

work of Hutchings (Crile 1923), a distant metastasis rate of 1%

of 4,500 cases was cited. In the same article, Crile said "The

collar of lymphatics about the neck forms an almost impossible

barrier through which tumour rarely penetrates and every portion

of this barrier is readily accessible to the surgeon".

On these rationale, it was difficult not to be enthusiastic

about Criles's operation (block dissection of the neck lymph

nodes - see later), since if local control could be achieved,

the major cause of death was removed, with potential cure being

therefore possible. Studies since then have shown a higher rate

of distant metastasis (see table 3), averaging at around 10% of

cases. As expected, when stage of primary tumour was noted along

with the presence or absence of distant metastases, an increase

in the rate of metastases occurred as the stage of the primary

increased (Merino et al 1977).

35



Table 3

The post-mortem rate of distant metastases in HNSCC:

Author No. %

mets.

Site

Burke (1937) 31 39 ENTPeltier (1951) 200 17 ENTCastigliano and

Reminger (1954)

121 2 ORAL

Price (1934) 87 11 ENTO’Brien (1971) 122 46.7 ENTMerino et al (1977) 5019 10.9 ENTT1 3004 5.2T2 1487 9.6T3 1533 12.7T4 970 16.1Braund and Martin

1941

174 20 ENT

36



2b: Metastatic neck disease

The phrase “metastatic neck disease” implies a malignant tumour

found in the neck that is a secondary deposit from a primary

tumour elsewhere, most commonly HNSCC that has spread into its

regional lymph nodes. Metastatic neck disease is staged

according to the Tumour, Node, Metastasis (TNM) UICC/AJCC system

- see above. Malignant tumours may arise denovo in the neck,

these are usually lymphoma (Jelliffe A.L. 1986), or perhaps

branchiogenic carcinoma from residual neck epithelial tissue

left during embryonic development (McCarthy and Turnbull 1981).

Occasionally malignant neck masses are found that have no

obvious primary site (Strasnick et al 1990). These are usually

undifferentiated or squamous carcinoma, and the most common site

of origin for them when it eventually makes itself known is the

postnasal space or tongue base. In situations where no obvious

primary is found these areas are investigated thoroughly and

treated prophyllactically. Secondary malignant disease in the

neck may also arise in the salivary or thyroid glands. In this

situation the tumour primary is usually distant, and spread

occurs in a blood bourn rather than lymphatic manner. Common

sites for primary tumours in this case include the lungs (Dilkes

and Birchall 1990) and stomach.

Cancer in the neck lymph glands therefore originates from one of

3 sources: as part of primary cancer of the lymph glands

(lymphoma), as part of widespread metastatic disease from

cancers elsewhere in the body, such as stomach or skin, or as

37



locoregional spread from a Head and Neck primary tumour, which

is usually Squamous Cell Carcinoma (SCC). The latter are by far

the most common source of malignant neck glands, and this study

is aimed at this area.

38



2c: Incidence

The incidence of metastatic spread of the primary tumour into

the neck lymph glands varies depending on the site and stage of

the primary tumour: see table 4. Some areas of the Head and Neck

have a much higher rate of metastasis into the neck than others,

this is presumably due to them having a richer network of

lymphatic channels than the lower rate areas. In particular the

supraglottis and tongue are known to have a rich supply of rich

lymphatic vessels. Size of the primary tumour also equates to a

higher incidence of neck metastases, presumably because the

bigger the tumour the longer it has been present, and therefore

if there is a certain “risk per year” of neck metastases, the

longer a tumour has been present, the more likely a lymph node

will be involved. This is generally accepted to be due to two

factors, increased intra-tumour pressure, and increased

likelyhood of lymphatic invasion. Therefore tumours that present

late, such as pyriform fossa or postnasal space carcinomas will

more often have an associated involved lymph node. When treating

the primary tumour, prophylactic treatment of the neck is

usually performed if the primary is at a high risk of neck

metastasis. This would be by either external beam irradiation or

neck dissection, or both.

39



Table 4The percentage incidence of metastatic spread of the primary tumour into the

neck lymph glands varies depending on the site and stage of the primary

tumour:

Site Stage

T1

Stage

T2

Stage

T3

Stage

T4

Alveolar

ridge

16 23 13

Floor of

mouth

26 35 32

Tongue 18 33 60

Supraglott

is

39 34 46 71

Epiglottis 32 50 50 62

Pyriform

fossa

83 93 90

Buccal

pouch

30 63 70 62

Lip 5 52 73

40



Data taken from: Mendelson et al, 1977, DeSanto et al 1977, Razack

et al 1978, Razack et al 1978, Joyce and McQuarrie 1976, Wurman et

al 1975.

41



2c: Prognosis with Neck Metastases

This study is aimed at metastatic squamous cell carcinoma from a

Head and Neck (HNSCC) primary site (definition of Head and Neck

cancer: any malignant tumour arising from the vermilion border

of the lips, down to the upper trachea and cricopharyngeal

sphincter, including the entire nasal area and middle ear,

excluding the orbit and brain). The presence of metastatic neck

disease in a patient with HNSCC significantly reduces the 5 year

survival rate (Snow et al 1982). There is an increased risk of

local recurrence at the primary site when lymphatic metastases

occur. Failure of local control following the treatment of

metastatic neck malignancy is a major cause of death in

patients with HNSCC, 5 year actuarial survival being around 5%

or less (Fletcher G.H 1973, Mendelsohn et al 1977, Pearlman

1979).

Following the successful treatment of the primary and the neck

metastasis, the patient has a reasonable chance of being cured

of his or her cancer (Jesse et al 1970, Shah and Tollefson 1974,

Mendelsohn et al 1976). This is because as mentioned previously

this form of tumour rarely metastasises widely (table 2). This

is unlike many other cancers such as breast cancer, which can be

relatively easily controlled locally by the simple excision of

the cancer mass, or mastectomy, but which often results in the

death of the patient from widespread metastases - this sort of

cancer is not as suitable for extra measures to be taken for

local control.

42



There is therefore a major advantage to patients with HNSCC if a

way of reducing the local recurrence rate after standard methods

of treatment can be devised. Local control of HNSCC is paramount

in improving mortality figures which have remained virtually

unchanged since the 1970’s, the time when the last major

advance, new reconstruction techniques allowing wider local

excision of tumours, was introduced.

43



2d: Why Lymphatic spread?

Spread of the primary tumour to the associated lymphatics is

more common in HNSCC than most other tumours for a number of

reasons: Firstly, it is postulated that the presence of dense

lymphatic tissue in Waldeyer's ring allows easy access of tumour

tissue to the lymphatic system. Secondly, the absence of a dense

subepithelial layer of collagen as compared to normal skin means

that tumours of the Head and Neck mucosa find penetration into

the lymphatic system easier. Thirdly, the frequent forced

movements of chewing, talking and swallowing probably help

propel tumour emboli into and along the lymphatic channels.

These actions can develop pressures of up to 100 mm Hg

(McQuarrie D.G. et al, 1986). These facts do not explain why the

tumours do not disseminate via the bloodstream - this tumour

specific factor is probably due to specific cellular

characteristics that are currently unrecognised.

44



2e: Treatment of metastatic neck disease.

The nineteenth century surgeons initially involved in the

treatment of HNSCC were aware that malignant diseases of the

upper aerodigestive tract metastasized to the cervical lymph

nodes. Initially, the finding of involved neck nodes would be an

indication of inoperability, rather as disseminated liver

involvement in colon cancer is regarded currently. In 1906

however, Crile published his results of treatment in 132 head

and neck cancers, with a reasonable degree of success in

removing the neck lymph glands en bloc, the first properly

described and followed up series that led to the operation of

block neck dissection. This technique of block dissection of

the neck nodes was further clarified and improved upon during

the pre World War 2 era, with increasingly good results being

achieved as added treatments such as antibiotics, blood

transfusion, inhalational anaesthetics through nasotracheal

tubes and improved physical health reduced the perioperative

mortality (Ward and Hendrick 1950). The greatest impetus in the

development of radical surgery for metastatic neck disease with

a primary tumour in the upper aerodigestive tract came in 1951

when Martin et al published an extensive series based on 30

years of experience with the treatment of HNSCC metastatic to

the neck nodes, advocating radical neck dissection in continuity

with resection of the primary tumour, and demonstrating very

impressive results.

45



Following the publication of this paper, there was widespread

acceptance of the operation of radical primary tumour removal

with a block excision of involved nodes taken in continuity with

as wide a pedicle as possible connecting the lymph node

resection with the primary tumour. The role of radiotherapy in

any metastatic HNSCC tumour in the neck over 2cm diameter

appears to be essentially adjunctive to surgery, either given

preoperatively or postoperatively (Wizenburg et al 1972, Jesse

and Lindberg 1975, Leemans et al 1990).

46



2f: Recurrence after radical neck dissection

Despite the advances mentioned above, the operation of radical

neck dissection does have an appreciable local recurrence rate

(see tables 5-7 inc). The rate of local recurrence in the neck

increases with:

1) Nodal stage (see tables)

2) Level of nodes involved (Spiro et al 1974, Kalnins et al

1977).

3) Presence of extra-capsular spread on histological analysis

(Johnson et al 1981).

At first glance, it might seem that the presence of

extracapsular spread would simply be a determinant of node size

and hence stage, and therefore not be an independent variable.

However, work by Annyas et al in 1979 showed that 23% of nodes

of less than 1cm diameter showed some degree of extracapsular

spread.

These figures show that on average there is a local recurrence

rate in the neck after radical neck dissection of around 30%

without pre or postoperative radiotherapy. This rate is

approximately halved by the addition of radiotherapy (table 8).

Since as previously stated, persistent neck disease is a major

cause of death in patients with HNSCC, there is clearly a place

for a successful adjunctive treatment during radical neck

dissection, to reduce the local recurrence rate. Data is not

47



available on the presence or absence of extracapsular spread in

these cases or on the level of the involved nodes.

48



Tables 5,6 and 7

Rates of recurrence following radical neck dissection, by nodal

stage:

Table 5

Author Stag

e

Number Rec % Primary

Site

Kalnins et al

1977

N0 160 18 11.2

5

ENT

Chu et al 1978 N0 57 2 3.5 ENT

DeSanto et al

1982

N0 314 24 7.5 ENT

Weissler et al

1989

N0 49 3 6.0 ENT

Lingeman et al

1977

N0 113 17 14.0 ENT

DeSanto et al

1985

N0 414 29 7.0 ENT

Table 6

Author Stage Number Rec % Primary Site

Faur and Arthur,

1971

N1,N2 42 20 48.

0

Oral and ENT

Strong et al 1969 N1,N2 129 70 54.

3

ENT

49



Kalning et al 1977 N1,N2 93 58 62.

3

ENT

Chu et al 1978 N1,N2 133 41 30.

5

ENT

DeSanto et al 1982 N1,N2 338 100 29.

5

ENT

Jesse and Fletcher

et al 1977

N1,N2 172 33 19.

1

ENT

Lingeman et al 1977 N1,N2 218 42 19.

2

ENT

DeSanto et al 1985 N1,N2 416 106 25 ENT

50



Table 7

Author Stage Number Rec % Primary

Site

Beahrs et al

1961

N0, N1,

N2

615 163 26.

5

ENT

Strong et al

1969

N0, N1,

N2

204 75 36.

3

ENT

Martin et al

1951

N0, N1,

N2

599 203 33.

9

ENT

Table 8 Preoperative radiotherapy given:

Author Stage Numbe

r

Rec % Dose Powe

r

Primary

Site

Jesse and

Lindberg 1975

N2, N3 57 4 7 6,000 MeV ENT

Jesse and

Lindberg 1975

N2, N3 49 2 4 6,000 MeV ENT

Farr and

Arthur 1971

N1, N2 43 12 28 2,000 MeV ENT /

ORAL

Wei et al 1990 N1, N2 50 7 14 7,000 MeV ENT

Strong et al

1969

N0,N1,N

2

144 34 24 2,000 MeV ENT

Weissler et al N0 17 1 6 4,000 - MeV ENT51



1989 6,000

Jesse and

Fletcher 1977

N1,N2,N

3

110 11 10 5,000 MeV ENT

52



2g: Cause of local recurrence

The cause of local recurrence after radical neck dissection is

thought to be due to either the spillage of viable tumour cells

onto the surgical bed during the operation, or the incomplete

excision of all involved lymph nodes (Beahrs and Barber 1962).

An interesting study by Harris and Smith in 1960 demonstrated

viable tumour cells in washings taken from the operative bed

following macroscopically tumour-free head and neck surgery. The

presence of these cells however did not influence the rate of

local recurrence, this may be due to microscopic examination of

the washings missing tumour cells in those cases in which no

cells were seen.

It is interesting to speculate why patients with pathologically

confirmed N0 disease develop any recurrent cancer at all in the

neck as shown in the previous tables. This is most probably due

to the malignant cells being missed during pathological

examination. A pathologist scanning cut sections of a lymph node

would usually be able to detect one abnormal cell from among 100

normal cells, but he/she could not be expected to detect one

abnormal cell in 1000 normal cells. Since 1 gram of tissue

contains 1,000,000,000 cells, if there were one abnormal cell in

1000 missed, there could be 1,000,000 cells in that 1 gram node,

yet no pathologist would call that node positive (Ariyan et al

1977). Therefore, the only definitive report regarding a lymph

node dissection specimen is one reporting a positive finding. A

53



negative report only gives a probability of being free of tumour

(Ariyan 1986).

54



2h Treatment of metastatic neck disease

2h(i) Radical neck dissection

Following initial work by Crile (1906), the technique of block

neck dissection was modified with time by many surgeons, most

notably Brown and McDowell (1944), Ward et al (1959) and Beahrs

et al (1955 and 1977). In time it came to be called a radical

neck dissection, as described by Martin (1951). This was because

he advocated a radical treatment for operable head and neck

cancers, and particularly stressed the importance of removing

the entire block of lymph nodes in the neck, in continuity with

the primary tumour if the operation was simultaneous. Martin

felt that there was no place for partial surgery in the

treatment of neck disease from a primary HNSCC tumour.

Prior to performing an isolated radical neck dissection it is

necessary to fully endoscope the patient, including bronchoscopy

and oesophagoscopy to assess for recurrence at the primary site

and the presence of synchronous primary tumours, both of which

significantly alter the treatment options (Gluckman 1979).

The incision made for the operation depends mainly on whether

the patient has had preoperative radiotherapy, and the surgeon's

own preference. Because the entire lateral neck area needs to be

exposed, an incision will be needed that allows both cranial and

caudal dissection along with anterior and posterior dissection.

This is best achieved using two incisions, an antero-posterior

arm joining a crania-caudal arm. The problem with any of the

incisions based on this premise is that there will be a junction

between the two arms, which will inevitably be a weak point55



that, in the event of poor healing due to radiotherapy, may

result in dehiscence. On its own that would be relatively minor.

However, once a radical neck dissection has been performed, the

carotid artery sits adjacent to the skin - any skin breakdown

could therefore expose the artery and lead to its potential

rupture due to chronic inflammation, with obvious consequences.

The prospects for poor healing and chronic inflammation are

enhanced by the presence of preoperative radiotherapy.

56



Thus if two incisions that join are to be made after

radiotherapy, the junction should be posteriorly based, over the

anterior border of trapezius, minimising the risk of carotid

exposure if dehiscence occurs. Other options include using an s-

shaped incision or two separate anteroom-posterior incisions.

The s-shaped incision has problems with spontaneous dehiscence

of the anterior part of the lower curve due to poor vascularity

of this tip, and the twin incision (Macfee) makes the operation

technically more difficult. Both approaches also compromise a

full view of the neck.

For a non-irradiated neck a caudally curved anteroposterior

incision from mastoid tip to greater cornua of hyoid can be used

in conjunction with a lazy s-shaped caudal incision from the mid

point of the anteroposterior incision, into the supraclavicular

fossa. This gives the best exposure with a good cosmetic result

and little chance of skin flap non-viability. In the irradiated

neck, the previously mentioned posteriorly based incision is

safest, although some restriction of the anterior view is not

uncommon.

Once the skin flaps have been raised and sutured away from the

operative site in a sub-platysmal plane, the dissection can

begin. The key to the operation is to find the floor of the

resection, in the sub-omohyoid plane. There are many ways to do

this, such as by identifying the lower limits of the jugular

vein in the neck, ligating and dividing this, then working

posteriorly along the clavicle along the floor of the posterior

57



triangle sweeping all tissue postero-laterally until the

trapezius muscle is reached. The dissection can then be swept

superiorly along the anterior edge of the trapezius muscle in

the same subomohyoid plane, just superficial to the deep

investing layer of cervical fascia. At the cranial limit, the

dissection is turned anteriorly at the mastoid tip, so

identifying the upper limit of the jugular vein in the neck,

ligating and dividing this and continuing the dissection

anteriorly across the whole neck, including the submandibular

region. In order to reduce venous pressure during the procedure,

the internal jugular vein can be ligated at its cranial end

first, although there is a theoretical risk that this may allow

tumour emboli to pass down the vein during the first part of

surgery.

The common, internal and external carotid arteries and carotid

body are left behind in the bed of the operation, along with the

vagus, hypoglossal and phrenic nerves and the cervico-mandibular

trunk of the facial nerve. The rest of the bed is made up of the

deep investing layer of cervical fascia, and associated

musculature. The midline neck structures such as larynx and

pharynx are usually separated from the operative site by the

strap muscles, other than the omohyoid, unless they are being

removed in continuity. Other important structures in the bed of

the operation include the brachial plexus deep in the floor of

the posterior triangle, between Scalenus Anterior and Scalenus

Medius muscles.. This is usually not seen since it lies too

deep, and for the purposes of this study has not been deemed to

be significant. The parotid gland lies in the superior margin of58



the operative site - this is useful since it protects the upper

3 branches of the facial nerve.

59



2h(ii): Conservative neck dissection

During a radical neck dissection, a number of important

structures such as the accessory nerve, sternocleidomastoid

muscle and internal jugular vein are removed. This is because

all the possible cancer bearing structures are required to be

removed. However, a number of other structures in the same

tissue plane are not removed, e.g. the common carotid artery,

the vagus nerve, the hypoglossal nerve and the phrenic nerve.

The reasons for not removing them are obvious - to do so would

cause an unacceptably high morbidity rate. It does raise the

question however, is it necessary to remove the other structures

mentioned? The aim of the operation is to completely remove the

tissue containing all the lymph nodes. The lateral spaces of the

neck, in which the major vessels and lymph nodes sit, are filled

with loose connective tissue. This tissue surrounds the lymph

nodes and is divided into compartments by a series of fascial

planes, although it is essentially one structure. These

compartments may be removed en bloc from other structures in the

neck such as the muscles and major neurovascular structures by

carefully dissecting them free, whilst keeping the whole block

of lymph nodes unviolated. This is the basis of the modified or

conservative neck dissection. The basic reason for developing

this procedure as an alternative to radical neck dissection was

because of the side effects of removing all the structures named

above (Bocca 1975).

60



The results of this technique have been encouraging (see table

8), although nodal staging data is not available in all the

cases in this table. Increasingly and particularly when treating

the node-negative neck in those patients with primary tumours

and a high likelihood of neck node metastasis, a conservative

neck dissection will be performed in continuity with resection

of the primary site as advocated by Martin. Additional

structures left behind in the bed of this operation above those

left after a standard radical neck dissection include the

sternocleidomastoid and omohyoid muscles, the internal jugular

vein, the accessory nerve and the transverse cervical vessels.

61



Table 9

Comparison of local recurrence rates of conservative and radical neck

dissections

Author Type No. Rec Type No. Rec

Bocca 1975 Con 500 2% Rad 500 2%

Lingeman et al

1977 N0

Con 60 0% Rad 113 17%

N1 Con 30 16% Rad 138 15%

N2 Con 8 25% Rad 80 21%

Chu et al 1978

N0

Con 13 0% Rad 57 3.5%

N+ Con 8 12.5% Rad 133 30.5%

Although the results from Bocca’s series look initially very

good, in fact most of these cases were prophyllactic neck

dissection for N0 disease.

62



2I: Treatment of macroscopic residual disease

Macroscopic disease may be left in the bed of a radical neck

dissection if it involves vital structures, in particular part

of the common or internal carotid arteries (Olcott et al 1981).

Normally such cases would be considered inoperable and radical

surgery not be performed. However, sometimes the extension of

the disease process is greater than initially thought

preoperatively, and such a situation will arise from time to

time. Thus “inoperable” tumours will be operated upon until it

is realised that the tumour is too advanced for the planned

surgery. In the event of macroscopic tumour invading the common

carotid artery, it may still be possible to remove the tumour if

it is only involving the adventitial tissues (Kennedy J.T. et al

1977). However, should it be deeper than this, the risks of

carotid rupture will be too high (Huvos et al 1973). In this

situation, the artery can be ligated and removed, or can be

bypassed and removed. Both of these procedures are associated

with very high morbidity (Biller et al 1988, McReady et al

1989, Moore and Baker 1955). It may be better to simply close up

once advanced disease is found and consider postoperative

chemotherapy or perhaps brachytherapy. In these situations

problems may still occur, since if the tumour is successfully

destroyed, spontaneous carotid rupture might still occur since

the tumour may make up the only viable arterial wall. For this

reason, PDT may cause exactly the same problem and its role in

this situation is unproven. If invasion of the carotid tree is

63



suspected, it is prudent to perform pre-operative digital

subtraction angiography.

Adjunctive intraoperative radiotherapy has been used with some

success, but the logistics of this make it virtually impossible

in the typical clinical situation (Freeman et al 1990).

64



3: Radiotherapy

3a: Introduction

Radiotherapy was first used in the treatment of cancer during

the early part of the twentieth century, radium and its

cytotoxic properties having first been discovered by Marie Curie

in 1898. The first treatments were for superficial lesions,

using orthovoltage (250-400 kilovolt (KV)) radiation, from

sources such as radium. Not only malignant but benign lesions

were treated, although since the discovery of radiotherapy

induced tumours, the practice of treating benign lesions has

been abandoned. The more modern radiotherapy equipment that has

been around for the last 50 years involves the use of radium and

cobalt sources of higher energy, along with linear accelerator X

rays, which can reach energies of up to 25 mega-electron volts

(MeV). Other forms of particle irradiation include neutrons and

protons, although these are still in the evaluatory stage.

The radiation energy used in routine clinical practice tends to

be gamma (particle derived from spontaneous emission from the

atom nucleus) or X (same particle, created artificially)

radiation.

The mode of action of radiotherapy is unsure. Its effect may be

explained by the fact that tumour and normal cells are

distinguished by the former's ability to proliferate

indefinitely. This is associated with a high rate of cell

turnover. Radiotherapy may have its effect because of its

ability to ionize and hence defunction DNA - the more rapidly a65



cell is dividing, the less time there is for DNA repair and

hence eventual loss of DNA function occurs, leading to cessation

of multiplication and cell death. Radiotherapy also causes the

formation of intracellular free radicals, such as singlet

oxygen, which are highly toxic and also cause cell death. The

true mode of action may well be a combination of both forms of

damage.

66



3b: External Beam Radiotherapy

As its name suggests, this form of radiotherapy is given from

outside the body, and is delivered by means of electromagnetic

radiation from one of several different sources. Initially these

sources were from relatively low voltage linear accelerators, or

shielded Cobalt isotopes. Once mega (million) voltage linear

accelerators had been created, the scope for treating larger and

less accessible tumours increased. This is for two reasons:

1) The absorption of electromagnetic radiation is exponential.

Therefore, the more energy a beam of particles has, the deeper

an effect will be seen in tissue.

2) A peculiar characteristic of megavoltage radiotherapy is its

skin sparing effect. Due to physical characteristics of

electromagnetic radiation beyond the scope of this chapter, a

surface sparing effect is seen. The maximum effect of this form

of radiation is therefore seen below the surface, the depth

below the surface being relative to the voltage applied to

produce the electromagnetic radiation. This allows toxic doses

of radiotherapy to structures below the skin, without killing

the skin itself.

67



The sources used for external beam radiotherapy in the Head and

Neck include:

Radium - low energies produced from this source treat

superficial depths only, therefore these machines are useful for

skin and lip cancer.

Cobalt 60 - a very commonly used source for Head and Neck

cancer. Gamma radiation of around 1.2 MeV produced from Cobalt

60 will penetrate most parts of the Head and Neck.

Linear Accelerators - very high energy particle are produced

from these machines, allowing deep penetration to most parts of

the body. Not so useful for Head and Neck cancer, although the

treatment of deep seated nodes or tumours can be effective with

these very high energy particles.

Electrons - electron beam radiotherapy is becoming increasingly

used for Head and Neck cancer. This is because it is associated

with rapid dose build up then equally rapid fall off after a

certain depth, depending on the energy of the electrons

produced. Therefore in those areas where a sharp fall off is

required, such as around the cervical spine or brain, they can

be very useful. However, little skin sparing is seen, so skin

68



necrosis is possible at higher doses. For that reason it is

often used to 'top up' after photon radiation.

69



3c: Brachytherapy

This form of treatment involves the local treatment of tumours

from within, hence it is also called interstitial radiotherapy.

It involves the insertion of a radioactive device into or next

to the tumour to be treated, thus delivering a high dose of

radiation to the tumour, but little to surrounding normal

tissue, since sources producing low penetration beams are often

used, such as Beta radiation sources. This technique is

particularly useful for treating cavities, when a prosthesis

lined with radioactive material can be inserted next to the area

to be treated. The advantages of brachytherapy are maximised

when it is given following external beam radiotherapy, boosting

the usual treatment dose.

The most common form of brachytherapy is with radium needles.

These are inserted into the area to be treated and removed after

the correct dose has been given. However this can cause

excessive radiation exposure to medical and nursing personnel.

Other commonly used techniques include the administration of

radioactive iridium wire or seeds. The safest way to perform

brachytherapy is by the afterloading technique in which a tube

is inserted through the area to be treated followed by the later

mechanical passing of the wire or iridium containing device into

the tumour, with medical personnel observing safely from a

distance.

70



3d: Efficacy of Radiotherapy

Radiotherapy, whether external beam or brachytherapy, is often

effective against early squamous cell carcinoma, but often fails

when tumours present later than T2 stage, which often happens in

the Head and Neck area (Johansen et al 1990). In order to be

able to cure such a tumour, a number of criteria need to be

fulfilled.

1) Small tumour volume, so that a very high concentration of

energy can be achieved.

2) Minimal hypoxia within the tumours. This generally equates to

tumour size, since tumours grater than one centimetre diameter

will often have outgrown their blood supply, leading to central

hypoxia. The presence of oxygen is vital for the cytotoxic

effect of radiotherapy.

3) Easily accessible tumours allowing the use of more than one

field to treat the tumour site, minimizing normal tissue damage.

71



3e: Side Effects of radiotherapy

Radiotherapy, whether external beam or brachytherapy is

associated with not inconsiderable side effects, particularly

when given to the Head and Neck. The standard amount of

radiotherapy that is thought to be safe and tumoricidal is

around 6,000 rads. This has to be divided up into fractions,

usually daily on the premise that normal tissue, being normal,

can repair itself quicker than malignant tissue, being abnormal.

Thus in the 24 hours between doses, the normal tissue has

repaired, the malignant tissue has not. This lack of repair in

malignant tissue ends up with the tumour dying, as previously

mentioned. However, the amount of radiation to the cervical

spine has to be limited to 4000 rads, since any dose greater

than this has a high risk of causing inflammation and

irreversible damage to the spinal cord (transverse myelitis).

Because bone absorbs ionizing radiation strongly, there is a

risk of osteoradionecrosis if too much radiation is given to the

bone, giving great problems with dosimetry in the head and neck,

which has bone within most treatment portals.

The minor and major salivary glands are often irreversibly

damaged by radical radiotherapy with subsequent loss of saliva

secretion leading to xerostomia. Taste buds in the mouth are

often totally destroyed. During radiotherapy, an inflammatory

reaction develops in the mucous membranes of the upper

aerodigestive tract, leading to mucositis, an acutely painful

condition that may cause severe dysphagia, necessitating

nasogastric feeding for several weeks on occasion. 72



Careful attention needs to be paid to the state of the teeth and

gums. Any disease of this area must first be eradicated prior to

radiotherapy, since there is a high risk of osteoradionecrosis

of the mandible and maxilla developing from such lesions during

radiotherapy. Skin care is also important, since the erythema

that almost always develops may be followed by moist

desquamation and breakdown during radiotherapy (Rao and Levitt

1986).

Radiotherapy may induce new malignancies to form in treated

tissue. These usually develop at least 10 years after treatment,

and the majority occur in connective tissue, being sarcomas or

fibrosarcomas on histology (Larson et al 1990).

Radiotherapy may also damage the major vessels of the neck. The

most important of these is the carotid tree. Damage to this set

of vessels includes rupture and stenosis, with an estimated 25%

of vessels being damaged as a direct sequel of treatment

(Elerding et al 1981). Radiotherapy may also cause carotid

artery thrombosis (Call et al 1990) and atherosclerosis (Glick

B., 1972, Hayward R.H., 1972)

Radiotherapy is cumulatively toxic, which means that once the

safe maximum dose of radiation has been given, no more can be

added if treatment fails (Anderson R.E., 1985).

73



3f: Role of radiotherapy in neck malignancy

There is debate as to if and when radiotherapy should be used

for metastatic neck disease from a Head and Neck primary. The

decision to go ahead rests on 3 main factors:

1) No previous radiotherapy to the proposed area of treatment

(most of the lateral neck). This usually excludes those patients

who have had radiotherapy to a primary pharyngeal or laryngeal

lesion.

2) Small node tumour volume - the larger the tumour the more

likely that hypoxic conditions within the tumour will occur,

greatly reducing the efficacy of the treatment, which depends

upon generation of oxygen-based free radicals for its effect on

DNA. The widely touted maximum tumour diameter for curative

radiotherapy is 2cm, although many institutions, particularly in

the U.K. would treat even larger metastases with curative

intent.

3) Disease at the primary site. If there is recurrence at the

primary site or within the upper aerodigestive tract, it is

often better to perform en bloc resection of both sites,

particularly if radiotherapy has been given to the primary site.

New cancers with associated neck nodes are often large in

volume, again despite a small neck node, en bloc resection would

be the treatment of choice.

74



Radiotherapy is very useful in metastatic neck cancer when given

in conjunction with surgery, and can be given pre or post

surgery (Mantravadi et al 1983).

75



3g: Pre or Post operative radiotherapy?

Preoperative radiotherapy: The peripheral tumour that might be

close to surgical margins can be sterilised with preoperative

radiotherapy, preventing burst and spill of viable microscopic

tumour residue into the operative site. Preoperative

radiotherapy might also convert a non-resectable tumour into a

resectable one.

Postoperative radiotherapy: Radiotherapy can be given

postoperatively to kill off any microscopic tumour left behind

at surgery. This has the added advantage that more will be known

about the disease once the majority has been removed, such that

planning of the depth and site of radiotherapy will be more

accurate, and, depending on the specimen, whether radiotherapy

is needed at all. If the metastasis is of a very early, good

prognosis stage, radiotherapy could be held back in case of

further recurrence. Radiotherapy also reduces the rate and

efficacy of wound healing after surgery, and may therefore be

better given after surgery.

76



INTRODUCTION PART II

4: Photodynamic Therapy

4a: Development and Principles

Photodynamic therapy (PDT) as a principle has been around since

the early part of the 20th century. A photodynamic effect was

first described by Raab in 1900, when he reported the death of

Paramecia following the administration of a combination of

acridine and light, though not with either agent solely (Raab,

1900). This effect was first used on tumour cells in 1903, when a

combination of eosin and light was used to treat skin cancer (V

Tappeiner and Jesionek 1903) Further studies throughout the

earlier part of the 20th century began to define some of the

important concepts in this potential treatment, in particular

skin photosensitivity (Meyer-Betz 1913). The history of

Photodynamic Therapy’s development has been summarised by

Daniell and Hill in 1991.

PDT relies upon the fact that some chemically inert compounds

can be activated by light to produce locally toxic effects.

These chemical compounds are called photosensitisers, and the

locally toxic effect is largely due to singlet oxygen production

(Weishaupt et al 1976), a substance that is highly oxidative and

therefore destructive to adjacent biological structures

(Takemura et al 1991). The principle of light activation of

chemical compounds is not new, photosynthesis relies on just 77



such a principle, although the end result of activation with

photosynthesis is constructive rather than destructive as with

photodynamic therapy. Despite this, photosynthesis requires the

presence of orange and yellow pigments to quench spin-off

reactants that arise from photosynthesis.

Drugs that are activated by light tend to have ring structures

similar to that of porphrins, alteration of the configuration

of this ring is what makes one photosenstitising drug different

from another. Ring configurations can be changed to make

photosensitising drugs more efficient, more selective and, by

increasing the activating wavelength, more deeply effective (see

figure 1).

78



The Porphyrin ring structure has the ability to absorb light of

the correct energy, such that when absorption occurs, the drug

molecule is excited to a higher energy level. This can decay to

an intermediate product, then transfer its energy to oxygen –

resulting in the formation of singlet oxygen and at the same

time returning the photosensitiser to it’s ground state ready to

absorb another photon. Singlet oxygen is a highly reactive and

toxic species, which almost instantaneously oxidises any

adjacent structure (Bonnet 1994). If enough singlet oxygen is

created locally within a cell, this leads to cell death due to

overwhelming damage to vital intracellular organelles. Because

those photosensitisers in common use for PDT do not appear to

enter the nucleus, there is little risk of DNA damage and

potential carcinogenicity. This is because the half life of

singlet oxygen and its sheer reactivity would not lend enough

time for it to penetrate into the nucleus from the cytoplasm in

sufficient amounts to cause damage.

Some controversy exists over whether the main site of action is

intracellular as described above (Moan et al 1982, Pass et al

1991), or extracellular – i.e., vascular (Selman et al 1984,

Henderson et al 1984, Henderson et al 1985, Henderson and

Dougherty 1992). The latter relies on the fact that

microcirculatory damage undoubtedly occurs during PDT and that

this may lead to anoxic cell death. More likely, both

mechanisms have a part to play in the overall effect (Nelson et

al 1987, Bown 1990). Autoradiographic and microfluorescence

studies have certainly demonstrated significant levels of

intracellular drug, linked to vital cellular organelles when 79



comparing malignant and normal tissue (Gomer et al 1979,

Bugelski et al 1981), and benign diseased/normal tissue

(Shikowitz et al 1989).

When considering the treatment of malignant disease, it is

important to think about the potentially hazardous effects of

the treatment (Pope and Bown 1991), in particular whether the

treatment itself might extend the stage of the disease. This is

particularly true in the treatment of primary cancers, since

there is always a possibility that in curing the primary, cells

will be shed off into the lymphatics, causing metastases. With

photodynamic therapy being a non-traumatic, non-ablative

biostimulatory procedure, it seems very unlikely that this would

occur. Work by Gomer et al in 1987 helped support this.

Accurate dosimetry of light to the treated area is of obvious

importance, since a supra-threshold amount of light is needed

for tumour destruction. This is an area that may yield great

advantages in selective bleaching of photosensitiser, leading to

a very tumour specific effect (Potter et al 1987).

Photodynamic Therapy has been increasingly used over the past

20 years for the treatment of malignant disease, in particular

Head and Neck cancer (Gluckman J.L. 1991). Treatment has focused

on solid, visible tumours with the best results being achieved

with early disease (Monnier Ph. et al). Other main avenues for

exploration clinically have been skin (Lipson and Baldes 1961),

bladder (Kelly and Snell 1976), brain (Kaye 1987), bronchus

(Edell and Cortese 1987) and gastrointestinal tract (Bown 1990).

80



4b: Photodynamic Therapy for Head and Neck cancer

Photodynamic therapy has a potential to be particularly

beneficial for the treatment of Head and Neck cancer for two

main reasons - its ability to locally and non-thermally destroy

malignant tissue, and its selectivity for tumours above the

surrounding normal tissue. This leads to rapid healing with

retention of form and function, and minimal damage to

surrounding normal tissue. The two other mainstay treatments for

Head and Neck cancer, radical surgery and radiotherapy, do not

share this low morbidity outcome, both being associated with

major degrees of post-treatment morbidity (see Chapter 3). This

is particularly important in the Head and Neck, since loss of

functions such as speech, swallowing, smell, taste are a great

problem to patients, as are the poor cosmetic results of radical

surgery. The other main alternative to surgery or radiotherapy

in the Head and Neck, chemotherapy, has not been found to be

useful so far (Amrein P. 1991). Photodynamic therapy is also of

great value as an alternative treatment because once given, if

local recurrence occurs, the otherwise standard treatments of

radiotherapy or surgery can be given with no loss to the

patient. Indeed, PDT might be one day given to reduce the bulk

of tumours prior to standard treatments, rather like induction

chemotherapy. The rational for this is that both surgery and

radiotherapy work better with lower tumour mass. This is

particularly true of radiotherapy, the success of which is very

much volume-dependant, larger tumours having to some degree

outgrown their blood supply. This leads to an anoxic centre, 81



which will not respond. That is because radiotherapy relies on

the presence of oxygen for its effect, although PDT is also to

some degree limited by this (Henderson and Fingar 1989).

However, the reduction of tumour bulk remains an important

concept in cancer treatment and PDT may eventually find a role

in this.

The main role of PDT in Head and Neck cancer is with respect to

the primary treatment of early cancers, whether initial or

synchronous tumours (Gluckman 1991). These are generally defined

for the purposes of PDT and investigations into its efficacy as

those tumours with a maximum assessed depth of 5 mm, lateral

spread no greater than 3 cm, no regional or distant spread and

no involvement of deep structures.

82



4c: History of PDT for Head and Neck cancer

Following the first published clinical trial using PDT for

cancer (Kelly and Snell 1976), much interest was stirred among

those treating cancer throughout the body. For the reasons

mentioned above, PDT for Head and Neck cancer was particularly

attractive due to the major problems incurred when using

standard methods of treatment. The first mention of PDT being

used therapeutically in the Head and Neck area was in 1978

(Dougherty et al). The first cases treated were usually

palliative, the impressive initial results being independently

confirmed in a paper by Forbes et al in 1980. Dahlman et al in

1983 first reported a large series (20 patients, 26 tumours) in

which there was a complete response (defined as no evidence of

tumour at original site 30 days after treatment) in 5/14 locally

recurrent cancers treated. Keller et al in 1985 were the first

to use Photofrin 2 for the treatment of Head and Neck cancer,

their results in 31 cases again showing promising effect on

early cancers, although it was noted that large tumours and neck

tumours did not respond well. The role of PDT for palliation was

also felt to be encouraging (Schweizer et al 1993). Schuller et

al (1985) also produced a large series of patients, 24, in whom

necrosis of tumour was seen in all cases, with HpD. No mention

is made however of tumour staging or light dose, a problem that

has dogged meta-analysis of published work in PDT. The idea of

PDT being particularly useful for early Head and Neck cancer was

taken up by Gluckman in 1986, treating early tumours of the83



larynx, oral cavity and oropharynx with success. His

recommendations at the end of this article were 5 fold:

1) Because the anatomy of the Head and Neck lends itself to

visualisation down a rigid telescope, and because this often

involves closed-in access down a narrow endoscope, fibre-

delivered light for PDT is ideal in the Head and Neck area

generally.

2) Because of selective necrosis of tumour, large areas of

normal tissue could be safely irradiated, obviating the need for

on-table frozen sections of margins or suspicious areas.

3) Some procedures can be performed in an office setting,

reducing costs.

4) The treatment of field cancerisation or other large area

tumours is particularly appropriate with this treatment, with

optimal preservation of normal tissue.

5) No cumulative toxicity means recurrent lesions can be safely

re-treated, unlike radiotherapy.

A similar drug to Photofrin and HpD called Photosan 3 was also

reported to be effective particularly for skin tumours of the

Head and Neck region, by Feyh in 1990. A very large series was

produced from China in 1991 again showing excellent tumour

necrosis with no residual local tumour seen at 5 years follow up

with 34/72 complete remission cases achieved out of 114

treatments. These studies were backed up by other series during84



the 1980’s and early 1990’s using HpD or Photofrin, including

work by Biel, Schweizer, Grant, Carruth and Monnier (see table).

In all these series, a powerful tumour response to PDT has been

seen with the best results achieved with early disease.

At this stage, new drugs, called “second generation”

photosensitisers began to appear in the clinical field. These

compounds had been manufactured to try to amplify the beneficial

effects of PDT by looking at the chemical properties of

Haematoporphyrin, HpD, Photofrin etc., and picking target areas,

such as the ring structure, or side arm structure, and

continually test every variant as it came out, until more

powerful compounds were produced (Bonnet et al 1989(1)).Of the

second generation drugs, only 2 have currently been used in the

treatment of Head and Neck cancer (see below). These are meta-

Tetra(hydroxyphenyl)chlorin (FoscanR), and delta-Aminolaevulinic

acid (d-ALA). Results with the first drug have been very

encouraging for Head and Neck cancer (Dilkes et al 1994, 1995,

1996, Savary et al 1997). The second drug, d-ALA has been very

useful topically for early skin cancer or solar (actinic)

keratosis, but for more advanced Head and Neck malignancy it is

not powerful enough (Grant et al 1993). Table 9 briefly

summarises those cases available for scrutiny in the literature.

It was not possible to include tumour stage or success rates in

this table because in many cases that data is not adequately

described. This has been one of the failings of the development

of PDT, inadequate description of published data. However, in

all the cases mentioned, tumours treated were found to respond

at least partially to the effects of PDT.85



Table 9 Summary of published cases with PDT treatment for HNSCC

Author Year Number Sensitiser

Dahlman et al

1983 14 HpD

Wile et al 1984 15 HpDMcCaughan 1984 3 HpDCai et al 1985 114 HpDKeller et al

1985 10 Photofrin 2

Carruth et al

1985 3 HpD

Schuller etal

1985 24 HpD

Gluckman etal

1986 3 HpD, Photofrin 2

Buchanan etal

1989 8 HpD

Monnier et al

1990 11 Photofrin 2

Schweizer et al

1990 12 Photofrin 2

Feyh et al 1990 8 Photosan 3Wenig et al 1990 26 PhotofrinZhao et al 1991 114 HpDGrant et al 1993 11 PhotofrinGrant et al 1993 8 ALADilkes et al

1994 17 MTHPC

Biel 1994 11 Photofrin 2Dilkes et 1995 21 Photofrin 2, ALA,

86



al mTHPCDilkes et al

1995 31 MTHPC

Biel 1995 65 Photofrin 2Kulapaditharom

1996 15 Photofrin 2

Ofner et al 1997 5 Photosan 3Savary et al

1997 35 MTHPC

4d: Why do advantages occur with PDT?

PDT relies on non-thermal levels of light energy to activate

photosensitiser (Kinsey et al 1983, Castro et al 1987, Abramson

et al 1990). Cell death occurs by necrosis, though sometimes in

a fashion mimicking apoptosis (Agarwal et al 1991), leaving the

tissue infrastructure, in particular collagen, intact (Barr et

al 1987). This allows rapid healing with retention of function

(Poate et al 1996), since there is a pre-existing normal tissue

matrix for adjacent, undamaged normal cells to grow into. This

selectivity persists when chemically induced tumours are treated

in situ, more tumour damage being seen when compared with normal

tissue (Barr et al 1990). This lead to the principle of

favourable cost:benefit ratio, as mooted by Chevretton (1991).

87



Selectivity with respect to an increase in the amount of damage

sustained by tumour tissue when compared to normal tissue

undoubtedly occurs. It was first described by Policard in 1924

and nicely demonstrated by Figge and Weiland in 1948 and

Rasmussen-Taxdal et al in 1955. Selectivity appears to be true

of virtually all the photosensitising drugs tested since then

(Dougherty et al 1978, Peng et al 1991, Pass et al 1993). There

are at least 6 possible reasons why selectivity occurs (Lin C.W.

1990):

1) Because tumours have an increased affinity for

photochemicals such as Photofrin and HPD over normal tissue.

This may be due to enhanced uptake due to altered tumour

metabolism and leaking, abnormal vasculature (Fingar 1990) or

the upregulation of low density lipoprotein (LDL) receptors in

cancer cells (Kessel D., 1986). These effects may well be

mediated by prostaglandins or other local hormonal mediators

(Ben-Hur et al 1988, Henderson and Donovan 1989).

Photosensitisers tend to be lipophilic and therefore carried on

LDL.

2) The preferential tumour localisation of photosensitizers may

also be caused by special properties of tumour tissue, such as a

low pH (Wike-Hooley L. et al 1984.), the presence of large

numbers of macrophages (Eccles S.A. and Alexander P. 1974), a

high density of lipoprotein receptors , and a relatively large

amount of newly synthesised collagen (El-Far M.A. and Pimstone

N.R., 1985).

3) Tumour selectivity can also be achieved by applying a

treatment to which tumour tissue is more sensitive then88



surrounding normal tissue. Tumour cells generally seem to suffer

from a relative lack of nutrients and oxygen, due to their less

controlled and organised growth. This makes them more sensitive

to external or internal damage than normal tissue (West et al

1990). Since PDT may exert all or part of its effect due to

damage to the microcirculation supplying tissue, the relative

deficiency in tumour cells of vital nutrients and oxygen may

well cause them to be much more sensitive to this effect than

normal cells (West et al 1990, Ben-Hur and Orenstein 1991).

4) The larger than usual volume of interstitial space in tumours

may act as a reservoir for photosensitisers (Jain R.K. 1987).

5) Due to their altered metabolism, cancer cells may not clear

photosensitising drug as quickly as normally functioning cells,

again leading to a degree of selectivity with time.

6) Simply by aiming the light at tumours, hence sparing the

majority of surrounding normal tissue, a form of selectivity

will occur.

89



4e: Light for Photodynamic Therapy

The first light sources for clinical PDT were adapted arc lamps,

often slide projectors, passing through narrow band-width

filters (Lipson and Baldes 1960) to achieve relatively high

(hundreds of milliwatts per cm2) power outputs. However,

although useful for surface applications and diagnostic purposes

(Lipson et al 1961), filtered arc lamps were superseded as the

preferred light source for PDT by the advent of lasers of the

appropriate wavelength. The monochromatic nature of laser

radiation allowed maximal photosensitiser excitation, whilst the

directional properties of the laser emission permitted efficient

coupling of the light into single optical fibres, facilitating

treatment of relatively inaccessible sites, in particular the

Head and Neck region.

Light towards the infra red end of the visible spectrum is

beneficial for most indications of PDT. This is because maximal

penetration of tissue occurs at this wavelength as described by

Wan et al in 1981 on cadaveric tissue, then Anderson and Parrish

in 1982 on skin, and Wilson et al in 1985 on a selection of in vivo

and ex vivo tissue. This is summarised in figure 1. Work by Bown et

al (1986) confirmed these basic facts in an in-vivo model, using

two different photosensitising drugs. However because the energy

of each photon is inversely proportional to its wavelength,

eventually with increase in wavelength to the infra-red this

energy drops to such a level that they will not adequately

excite the photosenstitising drug to the triplet state - this

occurs at around 850 nm laser light wavelength (Moan 1990).90



Therefore photosensitising drugs that act in this manner are

limited to activating wavelengths below 850 nm. Furthermore, as

the 850 nm level is reached, the generation of singlet oxygen

becomes an increasingly inefficient process, increasing

potential treatment times and removing one of the advantages of

the 2nd generation photosensitisers. Photosensitisers that

operate via type 1 mechanisms are not limited to this wavelength

region, and may be efficient up to 1270 nm wavelength, offering

maximal penetration through tissue. These compounds are not

currently available (Moan 1990). Light quanta above this

wavelength do not have the necessary energy to excite the

photosensitising drug up to its triplet state, the level that is

required in order to release the correct quantum of energy to

change the oxygen molecule on decay of the photosensitiser to

the ground state. So far lasers have been used that deliver

light in the 630 - 690 nanometre wavelength, although as new

photosensitising drugs are developed, these wavelengths will

move towards the 800nm range.

91



Figure 1: The central curved line shows the transmittance of

light through tissue, peak transmission occurring at around 800

nm. The separate peaks are the absorption peaks of the different

groups of drugs. Of interest is the porphyrin peak in the blue

spectrum (400 nm), which is mimicked by most other

photosensitisers, especially mTHPC. This can be seen to be a

wavelength which is clinically not very useful due to low

penetration through tissue, but is responsible for a lot of the

sun-damage that occurs in exposed skin following systemic

delivery of photosensitising drugs.

92



Currently used lasers have tended to be the gold vapour laser,

at 628 nm, used to activate Haematoporphyrin Derivative (HpD),

Photofrin 2, and amino-laevulinic acid (ALA), and the Copper

vapour or Argon Ion pumped dye lasers, able to cover the whole

range when used with a variety of dyes. The fact that these

systems produce either pulsed or continuous wave forms of laser

light does not seem to matter (Okunaka et al 1992). More

recently newer light sources have either made much more power

available (KTP-pumped dye laser) or been much cheaper and

portable than previous systems (Diode lasers and LED arrays:

Dilkes and DeJode 1995, DeJode and Dilkes 1995).

This all adds up to the need for a red light laser. The fact that

a variety of wavelengths are often required due to different drugs

being activated at different red peaks means that a tuneable laser

is far more advantageous for research purposes than a fixed

wavelength laser. Thus although the ruby and gold vapour lasers

produce red light of the correct physical characteristics for some

sensitisers, most PDT work is done with tuneable dye lasers,

giving the necessary degree of flexibility. A tuneable dye laser

relies on a higher energy pump laser driving a red light laser,

such as the Rhodamine dye laser.

The red light laser can be tuned to vary the wavelength of light

produced over most of the red spectrum, by means of a variable

light graticule. The pumping laser needs to produce photons of

higher relative energy than the eventual red light (one never gets

energy for nothing). This means that it has to be of a lower

93



wavelength. Thus green light lasers such as the Copper Vapour,

Argon Ion or KTP are used to pump most dye lasers.

The copper vapour or Argon Ion pumped dye lasers have therefore

been the mainstay of light production for PDT research. These are

bulky lasers, with high purchase and running costs. A full time

laser physicist is usually employed to run the lasers and perform

the necessary dosimetry prior to treatment. Because the tube

inside the copper vapour laser needs to heat up to 1500 degrees

Celsius to vaporise the copper prior to lasing, warm up times for

this laser are anything between 1 and 2 hours.

The Laserscope KTP pumped dye laser system is a major advance on

these earlier systems. As previously described it is a frequency

doubled Nd-YAG laser (KTP), which then pumps a Rhodamine Dye

laser. It is therefore a solid state system, with a setting up

time of perhaps 10 minutes. The control panel and light dosimetry

is computerised and with a little training, the operating

clinician can use it without technical support. Purchase costs

remain high however, although running costs are reduced over the

Copper Vapour/ Argon Ion lasers.

Recently, diode lasers of specific PDT wavelengths have been

produced by several different laser manufacturers worldwide. Only

a few have been used clinically, although it seems likely that by

the time this article is published, PDT Diode lasers will be

involved in full scale clinical treatments. The great advantage of

diode lasers for PDT is that they are so small (size of a small

suitcase) that they can be easily transported from centre to94



centre, enabling several different hospitals to use the same

system. They are also relatively cheap to purchase, and have

negligible running costs. The problem with them is that they are

of a fixed wavelength, so they will need to be replaced when

different sensitisers are used, and that in order to attain the

correct wavelength, the diodes have to be cooled – which can cause

problems in warm operating theatres / treatment areas.

The emergence of skin and oral cavity cancer as a target for PDT

has led to the re-consideration of non-laser light sources. This

is because fibre delivery is not necessary for treating these

areas. Development of narrow band filtered arc lap sources and

Light Emitting Diode arrays at a variety of wavelengths for the

treatment of malignancy in these areas has been possible (Dilkes

and DeJode 1995). Although the light produced is not

monochromatic, it is possible to almost exactly mimic the

absorption spectra of drugs like mTHPC and d-ALA in the red

spectrum. This is particularly true with the LED array, meaning

that although not all the light will maximally stimulate the drug,

the next best thing will happen – little of the light is wasted,

leading one assumes to a similar clinical effect although total

light dose may need to be increased (see figure superimposition of

LED output over red mTHPC peak). Early studies have borne this out

(DeJode and Dilkes 1995). The great advantages of these light

sources is that they are not only easily portable (with

development they might become powered by rechargeable batteries in

a torch-like package) but the costs are dramatically reduced. The

dosimetry and delivery of light with these sources should also be

95



easy for a clinician to learn, again without the need for

technical support.

96



4f: Potentiation of the Photodynamic effect

As with any form of clinical treatment, efforts have been made

to increase the effect of PDT by either combining it with other

treatments or increasing the beneficial effects of its

environment. This has taken its shape in several different

forms:

Since PDT exerts its effect by the photoproduction of singlet

oxygen (Weishaupt et al 1976) and the production of this

substance depends on the concentration of oxygen in the tissue

treated (Moan J., and Sommer S., 1985) and the fact that PDT may

also cause a reduction in tissue oxygen concentration because of

its effect on the microcirculation, reducing vascularity,

particularly in tumours (Henderson B.W. 1990), it is possible

that by pulsing or chopping the delivered light and therefore

allowing tumour re-oxygenation between episodes of light

delivery, the effect of PDT may be enhanced (Foster et al 1991).

This principle may be taken one stage further by treating the

patient in a hyperbaric oxygen atmosphere (Jirsa et al 1991).

This is similar to ionising radiation with one major difference

- whereas the effect of ionising radiation is reduced when

oxygen is absent, there is no photodynamic effect at all in this

situation (Henderson and Dougherty 1992). Potentiation of the

photodynamic effect has also been tried with laser and other non

laser thermal devices, with limited success (Waldow et al 1987).

Photodynamic therapy has also been used in combination with

chemotherapy, a study by Nahabedian et al in 1988 combined

Cisplatin and Doxorubicin with PDT in a mouse tumour model, to97



reasonably good effect, particularly with Doxorubicin - a

finding that has been described elsewhere (Cowled et al 1987).

The role of PDT in combination with bioreductive drugs has also

been investigated - this was thought to be useful since these

compounds are essentially inactive unless present in conditions

of reduced oxygen supply. With PDT, the effect on the

microvasculature as described leads to hypoxia within the

treatment site (Reed et al 1989), with subsequent activation of

the bioreductive agent (Bremner et al 1994).

98



Other drugs such as metoclopramide have also been shown to

potentiate the photodynamic effect in preclinical studies,

although no confirmation of this effect has been shown

clinically (Werning J.W. et al 1995). Linking photodynamic drugs

with carriers such as liposomes has been a way of both

increasing the stability and solubility of compounds in

physiological solution and also increasing their affinity for

tumours (Davis R.K. et al 1990), although despite considerable

investment being made in this area, there remains no firm

clinical evidence that this will prove to be advantageous.

The “magic bullet” scenario has been postulated by Donald et al

in 1991, linking photodynamic drugs with monoclonal antibodies,

sadly again no clinical work has yet been undertaken to confirm

or deny this postulate, although cell work has shown some very

impressive efficacy (Mew et al 1985).

99



4g: Role of fluorescence in PDT

Fluorescence was the first avenue down which PDT was explored in

the clinical field (Lipson and Baldes 1961). Its potential in

malignant disease was later confirmed by Gregorie et al in 1968.

The principal of this phenomenon is that certain compounds, such

as photosensitising agents, when stimulated by light of the

correct wavelength, will then re-emit light at a longer

wavelength, allowing this light to be detected independently of

the stimulating light. The property that most photosensitising

drugs seem to have, selectivity for malignant/altered tissue,

then allows that tissue to stand out from surrounding normal

tissue by means of its greater emittance of fluoresced light.

Therefore fluorescent imaging can be used to differentiate

malignant/premalignant tissue from normal tissue (Leonard and

Beck 1970). When applied to the skin, this is fairly easy, since

this area is easily accessible and background light is easy to

eliminate. However, when trying to perform fluorescent diagnosis

within the upper aerodigestive tract, a number of problems

occur:

- Stimulation and emission need to occur down a light carrying

fibre, rather than directly, since an endoscope needs to be

passed to access the area to be measured. This then means that

very low levels of light are obtained on emission, and often a

laser has to be used to stimulate the area. High technology is

needed to gather and amplify the emitted signal.

- Direct visualisation of the suspicious area is required, since

the area cannot be marked and treated later. This requires100



complex and expensive charge-coupled device (CCD) cameras to be

linked up to the fluorescence emittance pathway. This will allow

the suspicious areas to be visualised on a screen by the

surgeon, before immediate PDT is carried out (Mang et al 1993,

Monnier Ph. et al 1995).

If these expensive and time consuming problems are overcome,

fluorescence imaging can play an important role in both

diagnosis and monitoring of treatment (Braichotte et al 1996),

but currently due to the expense these facilities are available

in only a few centres worldwide. The drugs currently being

investigated for fluorescence diagnosis include d-ALA (Campbell

et al 1996), Photofrin 2 (Braichotte et al 1995) and mTHPC

(Braichotte et al 1995).

Our own efforts in this area have been related to using

fluorescence to determine the optimum drug-light interval for

best tumour response, unfortunately the tumour peak with

fluorescence may occur after peak photosensitiser concentration

in the tumour, when compared with the drug assayed tumour peak.

There is a variable gap between the two, giving us little added

information of use (Ansell J et al 1995, Grahn et al 1996).

PDT-induced fluorescence can also be used when studying the

cellular or tissue distribution of sensitising drug, which may

give vital information regarding the site of the PDT effect, in

particular whether there is any penetration of drug into the

nucleus, in which case the potential for mutagenesis arises.

Work so far suggests that this is not the case, although the

actual definite site of action remains unknown, it seems likely

to be cytoplasmic (Barr et al 1988).101



5: Chemistry of Photodynamic therapy

5a: Drugs for Photodynamic Therapy

In order to be useful as a photosensitiser for PDT, a drug must

have at least some and preferably all of the following

characteristics, as described by Moan (1990):

a) Selective uptake of sensitiser by tumour tissue.

b) Efficient photoactivation.

c) A maximum absorption peak well into the red spectrum of

light, allowing maximal tissue penetration.

d) Non-toxic in the dark.

e) Minimal/no skin sensitisation.

f) Have a constant composition and preferably be a pure single

substance.

Since the first description of the photodynamic effect by Raab

(1900), drugs used in PDT have evolved from impure compounds

with many disadvantages, to the stage whereby clinical PDT can

be performed scientifically and with a low risk of complications

(Gomer C.J. 1991). The majority of drugs developed have been

derived from the earlier, crude mixtures, all based on the basic

porphyrin ring structure (Bonnet et al 1989(2)).

Photosensitising drugs tend to be administered parenterally,

preferably intravenously for reliability of distribution (Perry

et al 1991). Much work continues on basic photochemistry to

achieve these goals (Lin C.W. 1991(2)). Virtually no resistance

to the Photodynamic effect has been seen clinically, although

preclinical work by Singh et al in 1991 did show some

resistance, particularly with multi-drug resistant cells.102



5b: Examples of first generation drugs

Haematoporphyrin

This compound is an impure derivative of haemoglobin, it was

first used clinically for PDT in the early part of the 20th

century. It was discovered in the 1940's to be preferentially

retained in malignant tissues, a major step for photodynamic

therapy (Auler and Banzer 1942, Figge et al 1948). It was well

recognised however that Haematoporphyrin was a crude, variable

mixture of numerous porphyrins (Schwartz et al 1955), and the

drug was quickly dropped in favour of more purified compounds as

they became available.

Haematoporphyrin Derivative (HpD), or Photofrin 1

This compound has been used extensively as a sensitiser in

clinical practice, with some promising results. Many early

principles of PDT have been established with this drug, although

its use has now been superseded by drugs derived from it. The

compound itself is a mixture of porphyrins with a peak of

activation in the red spectrum at 628 nm. It is prepared by the

alkaline hydrolysis of haematoporphyrin acetate(Lipson et al

1960). It was the first photosensitising drug to be used with

malignancy (Lipson et al 1961), although in this situation only

for photodetection. A further study in 1972 confirmed the

selectivity of HpD and restated its role in fluorescent

diagnosis (Sanderson et al 1972). Its cytotoxic potential was

initially described by Figge and Weiland in 1949, preclinical103



studies demonstrating its efficacy on tumour models were

convincing (Dougherty et al 1974), although it was not until

1976 (Kelly and Snell) that it was first shown to be effective

for potential cure against primary, early human cancer. Several

thousand patients, with malignancies from different parts of the

body have since been treated with the drug with generally

encouraging results. In particular, several of these studies

have looked at squamous cell carcinoma in the Head and Neck area

(Wenig at al 1990, Patrice et al 1990, Monnier et al 1990, Edell

and Cortese 1992, Ono et al 1992, Weiman and Fingar 1992). There

have been problems with the compound however, preventing its

widespread use. In particular, it remains a complex, variable

mixture of porphyrins that are derived from bovine blood and it

is largely uncharacterised (Dougherty et al 1984). It may have

a variable degree of activity depending on its batch and source.

Its peak absorption in the red spectrum being at 628 nm, gives

it only around 5mm depth of penetration in living tissue. It

also has a variable half life of around 19 days, leading to

prolonged skin sensitivity to light (Brown et al 1992).

104



Photofrin 2

This is a compound derived from HPD (see above), and is the

first photosensitising drug to be licensed for clinical use,

this occurring in Canada in 1993, for in-situ bladder carcinoma.

Despite this, much preclinical work continues, attempting to

further determine the optimum parameters for treatment, still

unclear (Mukhtar et al 1991). Photofrin 2 is extracted from HpD

by gel exclusion chromatography, and is a purified fraction of

the photosensitising elements of HpD. It is also known as DHE

(Dihaemotoporphyrin Ether), or Polyporphyrin. It has been

characterised as an aggregated mixture of haematoporphyrin

molecules, linked together by ether and/or ester bonds. It has

been shown to selectively concentrate in malignant tissue, and

is maximally activated in the red spectrum by light at 630 nm.

There has been a considerable amount of work published regarding

the use of this drug in the treatment of Head and Neck cancer,

with generally encouraging results - see table 9. The dose of

light delivered is variable, most tumour work being carried out

at between 50 and 100 Joules/cm2 (Dougherty and Marcus 1992).

Being a slightly purified version of HpD, this compound suffers

with the same problems of impurity etc. (Byrne C.J. 1990) and

high unit drug dose linked with a long half life again lead to

problems with skin phototoxicity with over exposure from ambient

light (Dougherty 1990).

105



5c: Second generation Photosensitising Drugs

The limitations of HpD and Photofrin (Dougherty T.J. 1989) led

to the search for photosensitising drugs closer to the desired

qualities mentioned above for the ideal photosensitiser, and led

to the development of a new 2nd generation of synthetically

created drugs, based upon the original porphyrin ring and active

components of HpD, but markedly altered so that in some cases

there is little to recognise from the original (Berenbaum et al

1982, Berenbaum M.C. 1989).Such is the interest in these second

generation compounds that scientists have discovered potentially

non-photodynamic properties of compounds such as tin -

etiopurpurin and benzoporphyrin derivative, in which the drug

inhibits neointimal hyperplasia after balloon angioplasty

without light activation (Coates et al 1996).

Examples of second generation photosensitising drugs:

Chlorins

These compounds, (of which meta-TetraHydroxyPhenylChlorin

(mTHPC) is currently being clinically evaluated, Stewart J.C.M.

1994) were developed in order to increase the wavelength at

which a reasonable peak of sensitisation occurs. It was

synthesised from porphyrin by modifying the substitution pattern

of a basic form of the porphyrin molecule (Berenbaum et al

1986). Reduction of porphyrins with diimide gives the

corresponding Chlorin, hence reduction of

tetra(hydroxyphenyl)porphyrin, which had been shown in bioassay106



to have good tumour photosensitising properties, gives

tetra(hydroxyphenyl)chlorin (Bonnet et al 1989(1)). The meta-

and para- isomers showed good selectivity for tumours above

muscle and skin. When this large array of compounds was tested

for activity on a tumour model, one of the compounds with the

best all-round characteristics produced was meta-

TetraHydroxyPhenylChlorin, or m-THPC. This is a chemically and

isomerically pure compound at least 100 times more active mole

for mole than HpD, as assessed using tumour necrosis models

(Chevretton 1993). The drug appears to be far more tumour

selective than HpD, allowing equal clinical effect with reduced

normal tissue reaction (Ris H.B et al 1993(2)). Its activation

deeper into the red spectrum allows more depth of clinical

effect than Photofrin 2, with tumour necrosis up to 1cm deep

being seen in animal and human tumours (Ris H.B et al 1993 (1)).

The light dose given tends to be in the range 2.5-20 joules/cm2,

at 100-300mW/cm2 density (Dilkes et al 1996), leading to

treatment times of under 3 minutes for relatively small tumours,

and, depending on the light source used, also for adjunctive

work. This if in the order of 10 times less than with

Photofrin2. The accumulated evidence so far suggests that this

drug has the most potential for the treatment of Head and Neck

cancer, although the very fact that it is so powerful can lead

to dangerous skin toxicity and other side effects, such as

fistula formation (Bradley P.F. personal communication, Dilkes

et al 1996, Savary et al 1997)

107



Phthalocyanines

These compounds are synthetic porphyrins, with high absorption

peaks in the red spectrum at around 680 nm wavelength (Ben Hur

and Rosenthal 1985, Chan et al 1986). They are chelated for

clinical use with a variety of metals, usually zinc or aluminium

(Selman et al 1986). They are thought to have less side effects

when compared to other photosensitizers, and also have a

powerful photodynamic effect with good tumour localisation

(Tralau et al 1987, Barr et al 1990). The activation deep into

the red spectrum made these drugs good candidates for PDT, by

increasing the potential depth of effect (Tralau 1987). There

have been problems however with stability of the drugs in

solution, with quantification of effect being difficult, and in

the fact that they dissolve poorly. Recently, combination with

liposomes has increased their solubility. Problems remain

however, particularly since high light doses (300 j/cm2) are

needed for effective treatment, leading to prolonged laser-on

time (Chan et al 1991).

Benzoporphyrin Derivative (BPD)

This compound is prepared from Protoporphyrin 9, an endogenously

produced photosensitising agent (see later under ALA). It has

similar properties to the Chlorin family of drugs, also

containing a reduced tetrapyrrhole ring. Its absorption peak is

at 692 nm, and it is very rapidly cleared from the body, leading

to reduced problems with skin photosensitivity (Wolford et al

1995). Little further is currently known about the drug, which108



is in preclinical (Jamieson et al 1993) and early clinical

evaluation in the USA (Nelson et al 1988), although currently

work seems to be concentrating on a role in the treatment of

ocular malignancy and other retinal diseases (Kim et al 1996).

109



ALA

delta - Aminolaevulinic acid (ALA) is a different form of

compound to the more classical photosensitizers. It acts by

being rapidly metabolised into an active photosensitising agent,

Protoporphyrin 9 (PP9) which is then slowly metabolised into

Haem (a rate limiting step) (Kennedy and Pottier 1992). It can

be delivered either systemically (orally) or topically, since

the ALA itself is a small, uncharged lipophilic molecule that

passes through cell membranes with relative ease, down a

concentration gradient. Active photosensitisation if given

systemically only occurs during a fairly short period of time

when PP9 levels are high and this dramatically reduces problems

with skin sensitisation since over a matter of a few hours the

PP9 is metabolised into Haem.. However due to its relative

inefficiency both in terms of depth of penetration and

efficiency compared to other drugs such as mTHPC it can only be

used to treat very superficial lesions such as early basal cell

carcinomas or premalignant conditions (Warloe et al 1992, Grant

et al 1993). The amount of light needed for a good clinical

effect appears to be circa 150 J/cm2, which increases the

treatment time compared to mTHPC, the fact that it is effective

topically is enough to make this a very exciting compound for

full scale clinical use, particularly on the skin (Svanberg K.

et al 1994).

110



5d: Problems

The major problem holding back PDT from more widespread use is

the fact that in almost all cases it involves the systemic

(usually intravenous to ensure good drug delivery) administration

of a photosensitising compound. Since the drugs are only ever

partially tumour specific (Dougherty et al 1990., Wooten et al

1988, Zalar et al 1977, Tralau et al 1986), drug will therefore

accumulate in normal tissue before being metabolised and removed

over a variable period of time. This causes two problems:

1) Photosensitizer in the skin and eye tissue will be activated by

light, including sunlight. Patients therefore need a prolonged

period (up to 2 months with the older sensitizers) of bright light

avoidance, necessitating the wearing of a hat, gloves and sun

glasses on top of normal wear when venturing outside, and making

sure that windows at home are shielded from direct sunlight (eg

with curtains) and that there are no light bulbs of greater than

60 watts output. Even at these light levels, patients are advised

that if they feel their skin tingling at light exposed areas, they

should reduce the ambient light levels. This is of particular

importance when considering that PDT was initially used for

palliation of advanced cancer in most cases, where being kept

indoors for the last few weeks of life was a major burden, and not

acceptable long term. The mechanism of skin phototoxicity is the

same as tumour destruction, namely the release of singlet oxygen

upon photoactivation of the drug (McLear and Hayden 1989). Manyak111



et al have also identified a role for a type 1 hypersensitivity

response, with mast cell degranulation and histamine release being

seen. Much work has been done trying to limit the photosensitive

effect on the skin whilst preserving it in the tumour, by the use

of singlet oxygen quenchers such as beta-carotenes (Mathews-Roth

1982) or N-acetylcysteine (Holdiness 1991), without much benefit.

To minimise the unwanted effects of skin photosensitivity, newer

drugs are being designed to have improved selectivity and a

shorter half life over the earlier compounds.

112



2) Photosensitizer in normal tissue around areas to be treated

will be activated by scattered light used for treatment. This will

cause local damage and pain over a period of around two weeks as

healing progresses. This can be minimised in areas where such

collateral damage may cause unwanted side effects, such as the

larynx or skin, by using adherent black gel that is liquid at room

temperature but adherent and gel-like at body temperature. This

means it can be painted onto the area to be masked, where it will

rapidly reach body temperature, and stick there until wiped away.

Other problems such as depth of penetration etc. are surmountable

by choosing cases to be treated correctly such that the depth of

the tumour to be treated is within the effective range of the

drug-light combination. No photosensitising drug has been reported

as being clinically toxic without photoactivation at normal

therapeutic doses. The second generation photosensitising drugs

have overcome most of the criticisms of earlier agents, in

particular they are now virtually 100% pure compounds that are

easily quantifiable, whereas before the haematoporphyrin

derivatives were an essentially unquantified mixture of active and

inactive compounds.

113



6: Adjunctive Intraoperative Photodynamic Therapy (AIOPDT)

6a: Preclinical

Since it has become quite clear that PDT is effective in

necrosing squamous cell carcinoma of the Head and Neck, the next

obvious role is in potentially mopping up the tumour residue at

radical resection that causes local recurrence, which is, as

previously described, a major cause of morbidity and mortality

following major Head and Neck cancer surgery. Several

preclinical studies have shown that adjunctive intraoperative

PDT (AIOPDT) significantly reduces the local recurrence of

tumour following macroscopically clear tumour removal (Davis et

al 1990, Abulafi et al 1995, van Hillsberg et al 1995). It is

postulated in these papers that AIOPDT destroys microscopic

tumour residue without causing significant damage to structures

in the operative bed, due to tumour selectivity of the drug, and

the rapid healing of damaged normal tissue, with the potential

for full retention of function. Thus following radical

(attempted curative) resection of cancer, prior to closing the

wound, treatment with PDT can be given to the operative bed to

reduce the chance of local recurrence of disease. PDT is known

to exert at least some, if not all its effect at the cellular

level (Gomer et al 1988), which is needed when considering

AIOPDT, since any effect relies on microscopic tumour

destruction, possibly that tumour which may be inadvertently

spilt at surgery, which is unlikely to have its own tumour

microvasculature as a PDT target (Henderson B.W. 1990). 114



6b: Clinical

Clinical studies are in progress looking at the efficacy of

AIOPDT in other parts of the body without the Head and Neck,

such as the colorectal (Allardice et al 1994, Abulafi et al

1993, Herrera-Ornelas et al 1986), neurosurgical (Muller and

Wilson 1995), thoracic (Ris et al 1991) and retroperitoneal

(Nambisan et al 1988) areas . No definitive data is currently

available, although recently published clinical studies in the

Head and Neck area suggest that this form of treatment has

definite potential (Dilkes et al 1995, Biel 1996).

In the face of evidence suggesting that the treatment is

potentially effective, the main question that needs to be

answered is the one regarding safety to other structures,

particularly vital ones, in the potential treatment bed.

115



EXPERIMENTAL RATIONALE

To explore the premise:-

Adjunctive Intraoperative Photodynamic Therapy using a 2nd

generation photosensitising drug is safe and effective for use

in Head and Neck Cancer

116



7: Experimental Rationale

7a: Description

When setting out to investigate the premise that adjunctive

intraoperative photodynamic therapy (AIOPDT) using the second

generation drug mTHPC was safe and effective, there were several

questions that needed answering. The decision was made to use

the new, virtually uncharacterised second generation Chlorin

photosensitiser meta-TetraHydroxyPhenylChlorin (mTHPC), because

having looked at all the drugs available, it was the one that

was closest to Moan’s ideal photosensitiser, being a pure

substance, readily soluble and stable in physiological solution,

with a high co-efficient of extinction deep into the red

spectrum (Berenbaum et al 1986). Given what was known about the

most widely explored photosensitising drug, Photofrin 2, it

seemed likely that this new drug would be more useful

clinically, with lower unit drug doses expected - reducing the

period of skin sensitivity to bright light, lowering light doses

needed for tumour necrosis (both of these due to the greater

efficiency (order of 200x) of mTHPC compared to Photofrin)

leading to shorter treatment times, deeper effective necrosis of

tumours since its activation peak in the red is significantly

further towards the infra-red, and the value of knowing exactly

what the active ingredient is, its purity and stability.

Not only were the basic tumour:normal tissue pharmacodyamics of

the drug not known, but the efficacy in the adjunctive

intraoperative situation of PDT also needed to be determined, 117



with respect to head and neck cancer since this had not been

tested before in any of the 3 previous AIOPDT preclinical

studies.

118



3 Stages to the experimental series were envisaged:

7b: First Stage - efficacy

Clearly there is no point in have a safe procedure that is

ineffective, so the first question to be answered was: does it

work, and at which PDT parameters?

Since the model clinical operation for this treatment is the

radical neck dissection for metastatic squamous cell carcinoma

of the Head and Neck (HNSCC), an animal tumour model was needed

that mimicked this tumour, and the pharmacodynamics of the new

photosensitiser evaluated on this model, compared to a first

generation photosensitiser to demonstrate the advantages of the

new drug (Experiment 1). The efficacy of AIOPDT would then be

tested in this model with the treatment optimised using data

from Experiment 1 (Experiment 2).

At this point, if the treatment was found to be ineffective, the

experimental series would be abandoned. If a statistically

significant positive result was achieved, the next stage of

experimentation would be performed.

7c: Second Stage - Safety Studies

The response to mTHPC PDT of those structures in the operative

bed deemed to be vital to the survival of the patient needed to

be determined . For the purposes of this experiment, this meant

the carotid arterial system, with delayed (histological

assessment, Experiment 3) and acute phase (doppler flow,

Experiment 4) studies. Further to this, the survival of 119



revascularised free flaps needed to be determined, since they

are a vascularly dependant structure often used during major

head and neck surgery, when a radical neck dissection might also

be performed (Experiment 5). Finally, a short study was

performed to confirm the results of the safety data in

experiment 3, on human sized vessels, namely the pig (Experiment

6), should the earlier study show a positive (no significant

damage) result.

7d: Third Stage - Clinical Study

Following the accumulation of satisfactory data (safe, effective

treatment) preliminary clinical studies were planned on patients

undergoing a radical neck dissection, initially in those thought

to have a high risk of local recur (Experiment 7).

120



7e: 6 consecutive preclinical experiments were therefore

performed:

Experiment 1: Determination of the correct PDT parameters for

mTHPC, in particular the drug-light interval. Comparison of

relative drug levels with Photofrin 2.

Title: Preclinical pharmacokinetic studies of first and second generation

photosensitising drugs

Experiment 2: Assessment of the efficacy of mTHPC PDT as an

adjunctive intraoperative therapy.

Title: The efficacy of Adjunctive Intraoperative Photodynamic Therapy with mTHPC in a

rat fibrosarcoma model

Experiment 3: Histological analysis of damage to arteries after

clinically appropriate PDT had been administered as in the

proposed clinical situation.

Title: Preclinical Photodynamic Safety Studies on Arteries

Experiment 4: Doppler flow studies of arteries and veins during

clinically appropriate PDT, as in the proposed clinical

situation.

Title: Acute phase effects of PDT on arteries and veins

Experiment 5: Analysis of survival of microvascular anastomises

in sensitised animals.

121



Title: The effect of high intensity white and filtered microscope light on the viability

of microvascular anastomoses in photosensitised rats

Experiment 6: Confirmatory study on human sized arteries prior

to clinical study

Title: Histological study of large diameter arteries undergoing photodynamic

therapy

On finding positive results a 7th clinical experiment was performed

Experiment 7: Human studies.

Title: Adjunctive Intraoperative Photodynamic Therapy for Head and Neck Cancer

122



8: Drugs, animals, Laser, Other equipment

8a: Photosensitising Drugs: Photofrin 2 was obtained from

Lederle Pharmaceuticals, Gosport, Hants, U.K., as a gift and

stored as sterile dry powder in a fridge, protected from light.

The diluent used was 5% dextrose, to create solution of 5 mg/ml,

which was prepared fresh (within one hour of injection) in all

cases.

mTHPC (Temoporphyrin, Foscan) was obtained from Scotia

Pharmaceuticals, Guildford, U.K., as a gift. It was stored as a

sterile dry powder in a fridge, protected from light.

Solution for injection was made up within 1 hour of delivery, by

weighing the powder on a 7-place balance and reconstituting it

with its solvent (1g ethanol and 1g polyethylene glycol 400 made

up to 5 ml with sterile water for injection for mTHPC, 5%

dextrose for Photofrin 2) to achieve a final concentration of

0.5 mg/ml for mTHPC and 5 mg/ml for Photofrin 2. With both

compounds, meticulous attention was taken to ensure that the

drug had gone into solution. Invariably this meant 2 or 3

minutes of vigorous shaking, ensuring no residue was visible on

the bottom of the tube used for reconstitution. Care was taken

with both drugs to avoid exposure of the photosensitiser to

light during preparation and injection (see picture) to avoid

photobleaching active drug.

123



8b: Anaesthetic Drugs

Rat: Hypnorm (Janssen) - Fentanyl 0.315mg, Fluanisone 10mg, per

ml

Hypnovel (Roche) - Midazolam 5mg/ml

Sagatal (Rhone Merieux) - 60mg/ml pentobarbitone sodium

Atrocare (Animalcare Ltd) - Atropine Sulphate 0.6mg/kg

Pig: Halothane, Nitrous Oxide, Oxygen on an open circuit

(inhalational)

Euthesate (Willows Francis) - 20% Phenobarbitone

Drug Doses

Sedation rats - Hypnorm 0.2ml/kg i/p

General anaesthetic rats - premedicate Atropine sulphate

0.05mg/kg, then Hypnorm/Hypnovel mixture 1:1, 5mg/ml plus same

volume of water for injection, 2.7 ml/kg i/p. To extend the

period of anaesthesia additional doses of Hypnorm are given

(0.1ml/kg every 30 to 40 minutes).

Lethal injection rats - Sagatal 40mg/kg of freshly prepared 10%

solution of Sagatal and

n-saline i/p.

General anaesthetic pigs - Inhalational anaesthetic as above

Sedation pigs - Inhalational anaesthetic as above

124



Lethal injection pigs - Inhalational anaesthetic as above

followed by Euthesate i/v 3.5ml/kg

8c: Tumour: The tumour model HSN fibrosarcoma was chosen because

it displays similar characteristics to Head and Neck Squamous

Cell Carcinoma (HNSCC), the eventual main clinical target. In

particular it is locally invasive see picture 1, non-

encapsulating, non-metastasising, hardy strain of tumour that

has been used before in PDT (Tralau C.J.,et al 1987). It is

available from the Chester-Beatty laboratories at The Royal

Marsden Hospital, Sutton, Surrey.

125



Picture 1: Tumour can be seen invading into surrounding muscle,

NOT encapsulating.

126



8d: Animals:

Chester-Beatty HSN adult rats of both sexes were obtained from

the National Institute for Medical Research, South Mimms,

Hertfordshire, U.K. These are a relatively cheap and hardy

strain of hooded rat, which bear the HSN Fibrosarcoma. The rats

were grown in free single sex cages with open access to food and

water, 5 animals per cage..

For other experiments not involving tumour, adult Wistar rats

were obtained from Charles River Ltd, and kept in free single

sex cages, 5 animals per cage with open access to food and

water.

Suckling pigs for the 6th experiment were obtained from Charles

River and kept in standard pens, food and drink given as per

home office regulations.

In all cases where photosensitising drug had been administered the animals were

kept in conditions of subdued lighting to prevent possible skin or retinal

phototoxicity.

8e: Laser:

Preclinical work - in all cases a copper vapour pumped (model Cu

15) tuneable Rhodamine dye laser was used. This was obtained

from and serviced by Oxford Lasers, Abingdon Rd., Oxford, U.K.

This produced adequate power for the preclinical work, when the

127



maximum light delivery rate necessitated less than 1.5 watts

from the fibre tip in all cases.

In the clinical work, a more powerful Cu20 pumped Rhodamine dye

laser was used (same supplier and maintenance), since demands

for power in adjunctive cases are higher. Some cases used the

KTP pumped Rhodamine dye laser (Laserscope, Raglan House,

Cwmbran, Wales, U.K.), which is the most powerful system

available for purchase, producing up to 7 watts of red light

from the fibre tip.

128



9: Experiment 1 - Preclinical pharmacokinetic studies of first

and second generation photosensitising drugs

The aim of this experiment was to compare the tissue

accumulation qualities of two photosensitising drugs in an

animal tumour model, to determine the optimum drug - light

interval for selectivity and efficacy with a view to future

preclinical and clinical studies. The pharmacological results

would then be corroborated by assessing depth of PDT induced

necrosis in the same tumour model.

9a: Introduction

There is evidence that malignant tumours preferentially retain

or take up photosensitising drugs when compared to normal

tissue. This was initially described by Policard in 1936, and

has been confirmed subsequently in virtually every study of

photosensitising drugs (Gomer 1991). For reasons outlined above,

it was decided to use the relatively unknown photosensitising

drug meta-(tetrahydroxyphenyl)chlorin (mTHPC) in this study

looking at the potential role of PDT in an adjunctive setting

for treatment after radical neck dissection and potentially

other major Head and Neck cancer procedures. The new second

generation photosensitising drugs are claimed to have this

property of selectivity enhanced over 1st generation compounds.

This experiment was therefore designed to compare the basic

pharmacokinetic properties of these typical first and second 129



generation photosensitising drugs using a suitable animal

tumour model (rat fibrosarcoma), skin, muscle and aorta as the

sampled sites.

130



Meta-TetraHydroxyPhenylChlorin (mTHPC) is a good example of the

Chlorin group of second generation photosensitising drugs. which

are known to offer particular advantages over the most widely

used drug for PDT, Photofrin 2, which is a poorly characterised

mixture of esters and ethers of unknown relative molecular

weight and purity. The initial evaluatory work with mTHPC has

shown it to be particularly promising when compared with

Photofrin 2, both in terms of a high quantum yield of free

radicals, leading to lower drug and light doses, and also a high

absorption peak deep into the red spectrum at 652 nm (Berenbaum

et al 1986), giving more efficient tissue penetration than

Photofrin 2 at 630 nm. Preliminary studies had also shown that

tumour selectivity should be increased with this compound

( Bonnet et al 1989). This drug has been designed to fit in as

near as possible with the characteristics of an ideal

photosensitizer (Moan 1990) and although it is not ideal in

these respects, it does represent a considerable improvement on

the originally used clinical photosensitisers HpD and Photofrin

2.

9b: Tumour

Previous work had also shown the HSN Fibrosarcoma tumour to be

useful for measuring the clinical photodynamic effect, in terms

of depth of PDT - induced tumour necrosis (Tralau et al 1987).

Tumour was initially grown in cell culture, using cell culture

flasks containing 10% Foetal Calf serum in Dulbecco’s modified 131



egg medium, enhanced with 10% glutamine, Streptomycin,

Penicillin and Amphotericin B. Once confluence had been reached

as assessed on daily microscopy, the cells were split with

Trypsin, and divided into 10 new flasks prepared as before. Once

these had reached confluence, some cells were frozen in

dimethylsulphoxide and stored in liquid nitrogen, others were

injected into the flank areas of the HSN rats, approximately 2

x107 cells/ml of solution per injected area as measured using a

graticule, under Hypnorm sedation. 1 ml of solution was injected

into each area. Once a solid tumour had grown, live tumour was

maintained by passaging 1mm3 of viable (peripheral) tumour into a

fresh animal. This was then used as the method of tumour

preparation.

132



9c: Drug dose and analysis

In the clinical situation the dose of Photofrin used has

generally ranged from 1 to 5 mg/kg body weight. The dose used

in this experiment for adult rats was 20 mg/kg. The greater dose

in the rat as compared to the human is an extrapolation of the

smaller animal’s increased surface area to volume ratio which

equates to a higher metabolic rate and therefore increased

metabolism / excretion of drugs (Paxton J.W. 1995).

The dose of mTHPC, 1.0 mg/kg body weight was calculated on the

basis of some preliminary toxicology work, the levels used by

another investigator (Ris et al 1993) , and the initially

proposed and used human dose of 0.3 mg/kg body weight (Ris et al

1991), again extrapolated to take into account the higher

relative metabolic rate of small animals when compared to large

ones. Once tumour had reached approximately 1cm largest

diameter (after 7 to 10 days), the animal was weighed and an

intravenous injection of either mTHPC or Photofrin 2 was given,

at a dose of 1mg/kg and 20 mg/kg respectively. Based upon the

concentration of stock solution as described above, the volume

injected was between 0.7 and 1.0 ml, as measured using an

insulin syringe with 0.1 ml gradations. This could be performed

without the need for anaesthesia, the animal being held in a

restraining device, injecting into the tail vein (see picture

2). After injection, the animals were kept in conditions of

reduced lighting, no direct contact with light was allowed. 4

rats were sacrificed at each time interval, these were from 6

133



hours to 14 days in both the mTHPC and Photofrin groups. One

piece of viable tumour of approximately 0.5 cm3 was taken from

each animal and stored fresh at -80 degrees centigrade in a

separate container. At the same time, approximately 1cm3 samples

of shaved abdominal skin, gluteal muscle and thoracoabdominal

aorta (flushed with normal saline) were taken and stored in a

similar fashion (separately). Following the collection of all

the samples, they were sent for analysis.

134



9c(i): mTHPC analysis - performed by Dr C.K. Lim and associates

at the MRC Toxicology Unit, Carshalton, Surrey

Each specimen was thawed on reception and divided into 4

separate pieces, which were analysed separately. The final

figure for each piece of tissue is an average of the 4 levels.

The extraction of mTHPC was performed by High Performance Liquid

Chromatography (HPLC). A Hypersil (5 micron) column was used

with acetonitrile- 0.1% TFA as the mobile phase. The flow rate

was 1 ml/minute. A range of calibration curves was obtained with

standard concentrations of mTHPC before each set of assays. The

tissue to be assayed was homogenized in a medium containing 8

parts of methanol-Dimethylsulphoxide (4:1 v/v) containing para-

TetraHydroxyPhenylChlorin and 1 part water. 200-300mg of the

tissue was homogenized in 2ml of this medium in a Dounce

homogenizer. This was then centrifuged at 2,600g for 10 minutes.

400 microlitres of the supernatant was mixed with 200

microlitres of water and 200 microlitres of this solution was

injected into the HPLC column for measurement of relative

fluorescence (Wang et al 1993).

135



9c(ii): Photofrin 2 analysis - performed by Professor

S.B.Brown’s team at the Biochemistry Department of Leeds

University

Each specimen was thawed on reception and divided into 4

separate pieces, which were analysed separately. The final

figure for each piece of tissue is an average of the 4 levels.

The analysis of Photofrin 2 was performed by spectrophotometry.

Tumour and muscle were homogenised in HEPES/CTAB at pH 7.4,

using a Polytron homogeniser. Skin was digested overnight at 40

degrees Centigrade in 1.0 M Sodium Hydroxide solution. This was

then neutralised with phosphoric acid. Following this, 1 ml

samples of homogenate and blank buffer were taken in duplicate

and added to 5 ml of (4:1 ethyl acetate to glacial acetic acid).

After vigorous mixing the samples were centrifuged at 2,000g for

5 minutes to remove precipitated proteins. The supernatant was

removed to a clean tube and 4 ml of 1.0 M Hydrochloric acid

added and mixed. The upper organic phase was carefully removed

and discarded. The remaining aqueous phase was then incubated

for 30 minutes at 100 degrees Centigrade, in a water bath. This

was to convert ether and ester linked oligomers to monomeric

porphyrin. The fluorescence of these samples was recorded on a

Kontron SFM-25 spectrofluorometer exciting at a wavelength of

400 nm, emission being recorded at 596 nm . The fluorescence of

a standard solution of haematoporphyrin (50 ng/ml) was recorded

at the same time. This allows an apparent concentration of drug

to be calculated. The true concentration is greater because not

136



all the drug is hydrolysable in these conditions- hence the need

for a calibration, which was performed using 14C- labelled drug

injected into animals. Following a delay of a few hours, tissue

was then removed after killing the animal and the amount of drug

within the tissue was measured using a scintillation counter.

This then gave a “real” level, which was used to calibrate the

system (Vernon D.I. et al 1995).

137



9d: In-vivo validation

One the optimum DLI had been determined, a series of experiments

were planned to shown that peak levels of drug in tumour equated

to peak clinical effect. Tumours were grown to approximately 15

mm largest diameter as assessed by palpation and measured using

a steel ruler with 1 mm gradations. Photosensitising drug was

injected at times around the optimum DLI as determined from the

previous experiment - the tumour was exposed after the correct

time interval had passed, under a general anaesthetic of Hypnorm

and Midazolam. A standard light dose of 20 joules/square

centimetre (j/cm2) was given to a 1cm diameter spot on the

superficial surface of the tumour, at 100 milliwatts/cm2

(mW/cm2) using a microlens (Quadra Logic Technologies,

Vancouver, Canada). Laser light at 630 nm (Photofrin 2) or 652

nm (mTHPC) was used, being obtained from a Copper Vapour Laser

(Cu 15, Oxford Lasers, Abingdon Rd., Oxford, U.K.) pumping a

Rhodamine dye laser (Oxford Lasers). Following this the animal

was recovered. After a period of 72 hours had passed for tumour

necrosis to become established, the animals were re-

anaesthetised and given an intravenous injection via the tail

vein of Evans Blue dye (1ml 0.5% Evans Blue in 0.9% saline) to

facilitate identification of the necrotic border (necrotic

tissue is not perfused and therefore will not take up the dye).

The animals were killed 2 minutes later, and the tumours

removed. The tumours were then immediately serially sectioned

perpendicular to the surface of light delivery using a size 20

138



scalpel blade, and the depth of induced necrosis measured from

the surface into the tumour with a travelling microscope, the

average of the 3 largest measurements taken as the final

measurement.

139



9e: Results

Table 10: ug/g mTHPC in extracted muscle preparation

Day Sample

1

Sample

2

Sample

3

Averag

e

1 .03 .03 .07 .04

2 .06 .07 .08 .07

3 .17 .22 .36 .26

4 .12 .13 .15 .13

5 .02 .07 .09 .06

6 .06 .06 .06 .06

7 .1 .1 .1 .1

14 .02 .06 .11 .06

Table 11: ug/g extracted mTHPC from skin preparation

Day Sample

1

Sample

2

Sample

3

Sample

4

Sample

5

Sample

6

Averag

e

1 .01 .04 .03 .028

2 .04 .05 .1 .07

3 .14 .19 .14 .16

4 .16 .26 .14 .14 .19 .12 .17

5 .23 .13 .05 .02 .04 .03 .1

6 .03 .06 .05 .12 .11 .14 .08

140



7 .13 .09 .08 .1 .04 .12 .09

14 .06 .04 .14 .08

Table 12: ug/g extracted mTHPC from tumour preparation

Day Sample

1

Sample

2

Sample

3

Averag

e

1 .01 .04 .14 .06

2 .03 .13 .05 .07

3 .11 .18 .14 .14

4 .13 .31 .28 .24

5 .31 .27 .28 .29

6 .25 .14 .35 .24

7 .18 .02 .14 .15

14 .13 .15 .14 .14

Aorta preparations: No measurable level of drug was found in any of these samples

Table 13: ug/g extracted photofrin from muscle preparation

Day Sample

1

Sample

2

Sample

3

Averag

e

1 .84 .79 .75 .79

2 .48 .65 .63 .58

141



3 .40 .48 .51 .46

4 .42 .54 .57 .51

5 .61 .74 .52 .62

6 .35 .61 .36 .44

7 .36 .39 .39 .38

Contr

ol

.18

Table 14: ug/g extracted Photofrin from tumour preparation

Day Sample

1

Sample

2

Sample

3

Averag

e

1 7.17 10.11 5.96 7.5

2 4.7 5.8 10.00 6.85

3 3.3 2.61 4.05 3.31

4 4.69 3.28 3.24 3.73

5 4.96 5.23 3.87 4.69

6 2.69 4.40 3.52 3.53

7 2.45 3.56 3.99 3.34

Contr

ol

.205

Table 15: ug/g extracted Photofrin from aorta preparation142



Day Sample

1

Sample

2

Sample

3

Averag

e

1 4.01 5.8 4.56 4.79

2 8.42 2.19 11.32 7.3

3 2.31 1.73 2.14 2.05

4 2.17 2.81 1.75 2.24

5 4.18 4.57 3.11 3.96

6 1.69 2.28 1.45 1.80

7 1.97 2.59 3.11 2.56

Contr

ol

.569

Table 16: ug/g extracted Photofrin 2 from skin preparation

Day Sample

1

Sample

2

Sample

3

Averag

e

1 6.53 6.90 7.10 6.85

2 4.50 5.30 5.80 5.20

3 1.70 1.39 2.22 1.77

4 2.53 2.45 3.20 2.72

5 14.06 4.27 4.82 7.71

143



6 2.15 3.68 2.11 2.65

7 2.17 2.83 2.84 2.61

Contr

ol

.366

144



Graph 1: Photofrin results summarised. Insignificant levels of

drug were found in control samples (diluent but no sensitiser

injected)

145



Graph 2: mTHPC results summarised. No recordable drug was found

in the control samples (diluent but no sensitiser injected).

146



9f: Discussion

The main method of assay, namely an extraction procedure

followed by the measurement of resultant supernatant

fluorescence and comparing this with a standard solution is a

widely practised method of analysis of photosensitising drugs.

It is however an indirect process, and until better methods of

quantification and validation, such as measurement of emitted

radiation from isotope doped photosensitisers becomes widely

available (Whelpton R. et al 1996), it is essentially an

unproven process. It is possible that drugs are somehow changed

in the extraction process, or that fluorescence is not a

reliable indicator of amount of active photosensitising drug.

The best method of assay without doubt is a bioactivity assay,

in which the physiological effect, measured for example by depth

of necrosis in tissue, is measured and compared. In this case,

depth of necrosis with a standard light dose and intensity was

to be measured on a daily basis, comparing the two drugs with

their depth of effect at each time interval. This technique was

tried using our fibrosarcoma model. It was not successful mainly

because the predicted depth of necrosis using a clinically

relevant light and drug dose was at least 7 mm in tumour,

generally not optically very dense. Therefore, in order to treat

one surface and not get complete tumour necrosis, so that an

edge between live and necrosed tumour is visible (down to which

the measurement is taken from the incident beam surface), a

tumour at least 10 mm depth is needed, realistically 15 mm. At

this size, in our model HSN fibrosarcoma, central necrosis of

147



the tumour occurred, making any possible interpretation of PDT-

induced depth of necrosis meaningless, even when using Evans

Blue dye to optimise the distinction between live and dead

tumour. This problem can be circumvented by growing very small

tumours and treating with low levels of light and drug, but this

has little relation to the clinical doses used, and we have no

evidence that linear extrapolation of the obtained results would

be valid. Also, the percentage error of the measurements taken (

several, including drug to be injected, light dose and

intensity, and depth of necrosis) all increase as the size of

measurement decreases. Other methods of bioactivity assessment

include the treatment of tumours of known depth to induce

complete necrosis, ie, cure, or measure the reduction in tumour

volume (Orenstein et al 1996, Van Geel et al 1995). Thus if a 6

mm tumour is cured at a set parameter of light and drug doses,

but not cured at 7 mm, this gives a depth of effect of between 6

and 7 mm. On preceding and following days, only 5 mm of necrosis

may be obtained, eventually giving a full description of the PDT

kinetics with this model. However, the model is flawed by the

fact that it is very difficult to accurately measure the depth

of tumours prior to treatment, even if they are grown in the

skin and can be inverted - the thickness of the skin is a

variable that is difficult to predict, and since the tumours

need to be fairly small - around 7 mm diameter, this increases

the percentage error. Bioactivity measurement with tumours is

also flawed by the fact that the tumours are inevitably

transplanted from their original primary site and are therefore

unnatural. Other methods of assessment include surface blue 148



light fluorescence of tumours (Ansell et al 1995). We feel this

is potentially useful although not yet validated, and may be too

difficult to use clinically, particularly in the less accessible

regions of the head and neck.

The levels of Photofrin 2 in muscle show an initial peak at 24

H, followed by a gradual drop over 7 days, at which stage they

are 50% of their level at 24 hours. There is a small peak at 5

days which is mimicked in all the samples. This has not been

reported in other series, and may be real or artefactual. The

skin results show a very similar picture, although the peak at 5

days is more pronounced. In the fibrosarcoma results, there is

again a very similar drop off in levels, with peak tumour levels

occurring early, and being high only 6 hours after injection.

The peak at 5 days is much less pronounced. One possible reason

for this peak is that all 4 animals on day 5 were given an

incorrectly high amount of drug, although this seems unlikely

since all animals were weighed and injected with drug on the

same day, each cage of 4 animals being sacrificed on a different

day. When the average levels are compared (graph 1), it can be

seen that the best tumour to normal tissue ratio occurs within

the first 3 days. Muscle levels are very low when compared to

skin and tumour levels. Eventually at day 5, skin levels exceed

that of tumour, although favourable selectivity returns by day 6

and. 7. As far as treatment times go, at virtually any time

period between 1 and 7 days there is a highly favourable tumour

to muscle ratio, at its peak this reaches 8:1 (day 1). The peak

tumour to skin ratio occurs at day 3, and is 1.6:1, although at

day 2 it is not much lower, at 1.4:1, and drug levels in tumour 149



are that much higher. Thus if one was treating a fairly deep

tumour at the limits of adequate light penetration a drug-light

delay of 2 days would seem sensible, allowing for a fairly large

amount of normal tissue reaction. If a superficial tumour was

being treated, day 3 might be more suitable, because better

selectivity with less normal tissue reaction should occur there.

Other studies have shown these results to be broadly as

expected, and in most cases the clinical treatment times with

Photofrin 2 are between 1 and 2 days. The levels of mTHPC in

tissue show a marked difference to Photofrin 2. More analysis is

available on this drug because it was previously unknown, and

since given the advantages we knew it had over Photofrin 2, it

was likely to be used by us as a clinical agent. In all the

tissues where drug was assayable (muscle, skin and tumour)

initial levels were low. This went on for the first 48 hours.

After this however, levels rose rapidly in all tissue, with

muscle peaking at 3 days, skin between 3 and 4 days and tumour

at 5 days. Peak tumour to normal tissue ratios were 4.5:1 tumour

to muscle and 3:1 tumour to skin, both occurring at day 5, and

coinciding with the highest tumour levels. Fairly high tumour

levels and good selectivity also occurred at days 4 and 6. These

results can be seen clearly when the average levels are compared

(graph 2). Optimum treatment times for the tissues mentioned in

terms of selectivity and amount of drug in tumour therefore

occur at between 4 and 6 days.

150



These findings are in keeping with a previous study looking at

clinical effect with human mesothelioma grown in nude mice,

although in this study no difference was found in the levels of

assayed drug between tumour and normal tissue (Ris et al 1993),

a finding in distinct contrast with this study, and perhaps due

to the different tumour model used, the one in this study being

a natural rat tumour occurring in its tissue of origin, against

a human tumour grown in a mouse away from its site of origin.

Safety studies by another group looking at pancreas and

gastrointestinal tract also found normal tissue levels peak at

around 2 days, although in this case relatively high levels

remained until day 4 (Mlkvy et al 1996). The difference in the

pharmacokinetics of the two drugs raise interesting

possibilities. Clearly with Photofrin, the time immediately

after injection and up to 48 hours after that is when levels in

measured tissue are their highest, suggesting that the drug is

perhaps held in the vascular compartment, since immediate (6 H)

levels are very similar to 48 H levels. The lack of data on

plasma levels makes this difficult to prove however, and this

phenomenon may be simply a factor of drug entering tissue at the

same rate that it is cleared from the vascular compartment. With

mTHPC, there is little drug in tissue for the first 48 hours,

following which a rapid rise occurs. This suggests an active

uptake process, perhaps the long delay being due to enzyme

induction. The small amounts initially present may represent the

vascular component - the rest of the drug may be in another

compartment, such as fat. From here it will gradually be

depleted as active uptake processes occur in other tissue, with 151



plasma being the exchange medium. We presently have no data on

levels of drug in fat. Other studies have lent evidence that

gives weight to this theory, since initially as expected after

intravenous administration, plasma levels are very high. A rapid

fall off is seen over 6-12 hours, which is the stage we would

expect fat accumulation to occur.

The value of the undoubted pharmacological selectivity of these

drugs, in particular mTHPC has not been properly established.

Indeed, it is unclear as to whether assayed level of drug has

any bearing on resultant photodynamic effect, although common

sense suggests the two are directly related. Clearly if it were

to be true, then one could expect to find minimal normal tissue

damage around treated malignancy when using mTHPC, of great

benefit in areas such as the Head and Neck, skin and brain.

Studies have been performed that seem to validate this principal

(Berenbaum et al 1993), but not with this model or in a human

situation. The value of selectivity of effect can be taken one

step further. Theoretically it should be possible to bleach out

active drug from normal tissue at the same rate as tumour within

the same treatment, at a light intensity below the threshold of

PDT-induced damage.

152



Given that there was more drug in tumour than normal tissue,

once all the drug in normal tissue has been photobleached, there

should be significant amounts of drug left in tumour (depending

on the degree of selectivity). At this stage the delivery rate

can be raised to above the putative threshold, and the tumour

necrosed with no damage to surrounding normal tissue, since no

drug is now present in that tissue. The levels of drug in the

tissue to be treated can be monitored by blue-,light

fluorescence, although this only gives a value for the

superficial tissue, the amount of bleaching deeper to this can

be calculated by extrapolation using a Monte-Carlo model (DeJode

M.L. personal communication). Once levels in normal tissue reach

minimal amounts, then is the time to raise the activating light

intensity to above threshold. This theory depends on there being

a measurable threshold of light intensity for PDT-induced damage

to occur. This must happen, since patients’ skin appears to be

undamaged by low levels of ambient light even when at the height

of photosensitisation. However, work in our Unit has not

currently been able to identify such a threshold, and it may be

at such a low level that is clinically non-useful.

The fact that no sensitiser was found in the arterial specimens

with either drug is due to either the amount of tissue being too

small to analyse, which the pharmacologists think is unlikely,

or because the total amount of drug present was too small to

analyse. This is encouraging when considering the effects of PDT

on vessels, although work by Grant et al (1994) shows that with

some drugs the endothelium is the site of photosensitiser

153



accumulation, and very small amount in such a delicate tissue

could still cause severe damage.

Another study has also compared mTHPC with Photofrin 2 and had

more success in assessing bioactivity when using a different

tumour model, RIF 1 (Van Geel I.P.J., et al 1995). They found

better efficacy and selectivity with mTHPC. Ma et al (1994) also

showed greater efficiency of light conversion into singlet

oxygen when comparing mTHPC with mTHPP and Photofrin 2. More

recently, Orenstein et al (1996) found that another Chlorin,

Chlorin E6 was more selective and more powerful than Photofrin 2

when using the Colo-26 tumour model.

154



In conclusion, this study showed that when measuring depth of

PDT induced necrosis from the surface with mTHPC as the

photosensitising drug, the HSN rat fibrosarcoma is not a

suitable model even when optimising the distinction between

normal and necrotic tissue with methylene blue administration.

Pharmacological selectivity was demonstrated with both drugs,

with unit drug doses of mTHPC being much lower due to its higher

efficiency. The optimum drug-light interval with mTHPC is

thought to be between 4 and 6 days post injection and between 1

and 2 days with Photofrin 2, for greatest selectivity and

effect. This study corroborates other evidence suggesting that

for mTHPC the optimum drug-light interval is 96 hours.

155



10: Experiment 2 - The efficacy of Adjunctive Intraoperative

Photodynamic Therapy in a rat fibrosarcoma model with mTHPC

10a: Description

Since PDT works at least partially at the cellular level, (Moan

et al 1982) it may also help to reduce the local recurrence rate

of tumours after surgical excision, by destroying microscopic

tumour residue. This experiment was designed to test this

hypothesis in an animal model. The aim of this study was to

determine whether the tumour model HSN fibrosarcoma, which bears

a close resemblance to our target clinical tumour, squamous cell

carcinoma of the Head and Neck , shows a statistically

significant reduction in local recurrence with the treatment

groups when compared with the control group. The experiment was

performed in such a manner that the operating and evaluating

surgeon (MGD) was blind to the treatment group each animal was

in. This had not been done in the previous studies, to our

knowledge. The other studies also used tumours that bore less

resemblance to the aggressively locally invasive fibrosarcoma

used here, which is similar in character to squamous cell

carcinoma of the Head and Neck.

10b: Tumour

The fibrosarcoma tumour HSN in Chester-Beatty hooded (CBH) rats

was used, as before.

Using a freshly killed donor animal with a tumour previously

inoculated, the tumour periphery (containing viable, non-156



necrotic cells) was excised. Small pieces of tumour

approximately 1mm x 1mm x 1mm were taken and implanted under

direct vision into the flank musculature of the adult CBH rats

under sedation with Midazolam. Either one or both flank areas

were inoculated. Following this the animals were recovered and

daily records made of the largest tumour dimension.

157



10c: Photodynamic Therapy Parameters

The drug dose of mTHPC used was 1.0 mg/kg body weight delivered

intravenously via the tail vein. mTHPC was obtained from Scotia

Pharmaceuticals, Guildford, U.K., as a gift. It was stored as a

sterile powder in a fridge, and reconstituted fresh with 1g

ethanol and 1g polyethylene glycol 400 made up to 5 ml with

sterile water for injection, to make up a solution of 0.5 mg/ml.

When reconstituting the drug for injection, meticulous attention

was taken to ensure that the drug had gone into solution.

Invariably this meant 2 or 3 minutes of vigorous shaking,

ensuring no residue was visible on the bottom of the tube used

for reconstitution.

The drug light interval was 96 hours, as Experiment 1 and other

studies have found this period of time to offer advantages in

terms of potential selectivity within tumour tissue when

compared to surrounding normal tissue whilst retaining excellent

anti-tumour activity (Ris et al 1993, Van Geel et al 1993). The

light dose given for photoactivation was 20 joules/square

centimetre (j/cm2) at an intensity of 100 milliwatts/cm2

(mW/cm2), wavelength 652 nm. These were parameters used in the

first studies with the mTHPC, with good necrosis of malignant

tissue seen (Ris et al 1991).

158



10d: Preliminary studies

The aims of the 2 preliminary studies were to determine:

1) What the feasible maximum size of the tumours was with

respect to maximising the recurrence rate of tumour after

surgery, without an unacceptably high mortality rate.

2) What the growth characteristics of the tumour were, in order

to predict the day at which tumours would reach the previously

determined optimum size.

10d(i): Study 1

Initial studies were performed to determine the optimum size of

tumour in terms of local recurrence rate after macroscopic

excision, and survival of the animal. This was necessary because

clearly the larger the tumour, the more likely it is to recur

locally due to inadequacy of excision, even if the margins are

macroscopically clear. However, resection of very large tumours

will result in a critical loss of fluid, and death of the

animal. Therefore we wanted to know what the safe maximum size

of the tumours was. Tumours were grown bilaterally until they

reached a size of 1cm, 2cm or 3cm largest diameter as measured

externally with the animal awake. At this stage a general

anaesthetic was administered, and both tumours removed locally,

but with full macroscopic clearance (ie no visible tumour left).

The skin was then closed and the animals recovered. Animals were

closely monitored postoperatively for signs of distress. If any

were shown the animals were immediately killed by a schedule 1

technique. Other animals succumbed shortly after surgery. All 159



non-survivors were recorded. The survivors were killed 2 weeks

after surgery and the surgical sites examined for signs of local

recurrence, which was recorded.

160



10d(ii): Study 2

This was performed to determine the predictability of tumour

growth prior to photosensitisation. Once the optimum tumour size

had been determined, it was important to have an idea regarding

the growth rate of the tumour, since the decision had been taken

to sensitise 4 days prior to treatment. Clearly, a tumour

measured at the safe maximum size at injection would be

significantly above that size 96 hours later, given a doubling

time of x hours, where x is any number less than around 300. Ten

tumours were grown in 10 animals, the same way as above, but

unilaterally. Tumours were implanted on day zero. Alternate

daily measurements of the maximum tumour diameter were taken.

Measurements were taken with the animal awake using a steel

ruler with 1mm gradations. Once the tumour reached around 20mm

diameter (the chosen size for the 3rd experiment on the basis of

the first preliminary study), the animal was killed.

Once this data had been gathered, a blind, prospective

controlled study (Study 3) was performed to test the hypothesis

that AIOPDT with mTHPC reduces significantly local recurrence

after macroscopic surgical excision in the HSN rat fibrosarcoma

model:

161



10d(iii): Study 3

4 groups of 30 animals were included in the experiment. These

were:

1) Surgery and AIOPDT.

2) Surgery, mTHPC diluent and light only at the above parameters

(no drug).

3) Surgery and drug only

4) Surgery only.

Two independent tumours were grown in each animal, on the back,

to the left and right side of the midline, well separated

(picture 3), using the method described above. After 10 days,

the animals were admitted into one of the 4 treatment groups..

They were kept in cages of up to 5 animals each, cages were

coded according to the group each animal was in. Each cage was

coded following randomn selection by biological services staff.

Those animals due for drug injection (groups 1 and 2) were

weighed and injected intravenously via the tail vein with 1mg/kg

mTHPC that had been freshly prepared from powder. 4 days later a

general anaesthetic was administered to all animals and the

tumours exposed. The largest diameter measured. Any tumour of

20mm +/- 2mm largest diameter was included in the study and 162



resected so that no macroscopic tumour was left (picture 4).

Tumours beyond these criteria were not used. At that stage

animals due for light administration (groups 1 and 2) were

identified and 20 joules/cm2 of 652 nm laser light of 100

milliwatts/cm2 intensity was given. The light was produced by a

copper vapour laser pumping a rhodamine dye laser (Cu 15, Oxford

Lasers, Abingdon Rd., Oxford, U.K.). The light intensity was

measured using a photodetector linked to an integrating sphere,

and was delivered via a microlens (Quadra Logic Technologies,

Vancouver, Canada).

163



Picture 3: The animal is under general anaesthesia. The skin

over the dorsum has been shaved with an electric shaver. To the

left and right of the midline around a healing incision, 2 small

tumours can be seen - see arrows. The incision is from the

previous surgery to inoculate the tumours. Betadine has been

used to prepare the skin.

164



Picture 4: At the end of the each day’s experimentation, the

tumours are collected and incinerated. The uniform nature of the

tumour growth can be seen from this picture.

165



In all cases a 2.5 cm diameter spot was used with the original

tumour site in the middle, giving a margin of normal tissue

treatment, which was 12.5 % of the original tumour size. The

light spot size was measured using a steel ruler with 1 mm

gradations. The microlens was held steady over the treatment

site by holding it with a clamp fixed to a retort stand.

Following this the skin was closed and the animals were

recovered. At this stage the rats were numbered by puncturing

and marking the ears with Evans Blue dye (Picture 5).

Postoperatively the animals were observed closely. Any animal in

distress was killed immediately and recorded. Any animal

succumbing to the effects of surgery was recorded.

Once obvious macroscopic and potentially distressing tumour

recurrence had occurred in one animal (the experimental end

point), all animals in the groups treated in that session were

killed. The flank areas were opened and examined for tumour

recurrence. Any suspicious areas that were not obviously tumour

were biopsied and sent for histological confirmation of the

result, otherwise the diagnosis of tumour recurrence was made by

sight alone. At this stage and during recording of the results

the examining surgeon was blind to the group the animals were

in, only the experiment had been completed were the codes broken

and the results recorded.

166



Picture 5: Ears being marked. The animals are recovered on a

heated mat to prevent hypothermia.

167



10e: Results

Table 17: Experiment 1

Groups N=10 Recurrence

(%)

Death

(%)1 cm diameter

tumours

5 (50) 0

2 cm diameter

tumours

7 (70) 0

3cm diameter

tumours

6 (100) 4 (40)

Table 18: Experiment 2

Day 2 4 6 8 10 12 14 16Tumour1 1mm 2mm 4mm 12mm 14mm 16mm 18mm 20mm2 1mm 3mm 8mm 14mm 18mm 20mm 22mm 24mm3 1mm 2mm 3mm 6mm 14mm 18mm 20mm 23mm4 1mm 2mm 3mm 7mm 14mm 17mm 19mm 21mm5 1mm 2mm 4mm 9mm 15mm 20mm 23mm 27mm6 2mm 3mm 7mm 12mm 15mm 17mm 19mm 20mm7 1mm 2mm 4mm 11mm 15mm 17mm 20mm 22mm8 1mm 1mm 4mm 9mm 15mm 18mm 20mm 23mm9 1mm 2mm 7mm 12mm 16mm 19mm 21mm 23mm10 1mm 2mm 4mm 10mm 14mm 17mm 19mm 21mm

168



Figure 2: Representation of tumour growth rate, obeying

Gompertzian dynamics

169



Experiment 3:

There were 4 possible results: 0 recurrence, 1 recurrence, 2

recurrences, death prior to experimental end point. The full set

of results for each treatment session and overall are shown in

tables 19-26.

The individual experiment days are tables 19-25 inclusive, the

summated data is in table 26.

Table 19

Treatmen

t

Number Deat

h

Recurren

ceSurgery 3 1,1,2Light

alone

4 0,0,1,2

Drug

alone

3 0,1,2

AIOPDT 3 1 0,1

Table 20

Treatmen

t

Number Deat

h

Recurrence

Surgery 9 1 2,0,0,1,0,

1,1,1Light

alone

5 2,1,2,0,0

Drug

alone

3 2,1,2

170



AIOPDT 9 1 1,0,0,0,1,

0,0,1

Table 21

Treatmen

t

Number Deat

h

Recurrence

Surgery 4 2,1,0,0Light

alone

4 2,2,0,1

Drug

alone

9 1,2,0,1,0,0

,2,1,2AIOPDT 4 1,0,2,1

171



Table 22

Treatmen

t

Number Deat

h

Recurren

ceSurgery 2 2,1Light

alone

5 1 2,2,1,1

Drug

alone

4 1,1,2,2

AIOPDT 4 0,2,1,0

Table 23

Treatmen

t

Number Deat

h

Recurren

ceSurgery 3 1 1,1Light

alone

4 2,1,1,0

Drug

alone

3 1,2,2

AIOPDT 4 1,2,0,0

172



Table 24

Treatmen

t

Number Deat

h

Recurren

ceSurgery 3 1 2,2Light

alone

5 1,0,0,2,

1Drug

alone

5 2,2,0,2,

2AIOPDT 6 1 1,0,0,0,

2

Table 25

Treatmen

t

Number Deat

h

Recurren

ceSurgery 6 2,2,0,1,

1,1Light

alone

6 2,2,0,2,

2,0Drug

alone

6 1,1,1,2,

0,1AIOPDT 6 1,0,0,0,

0,2

173



Table 26: Overall results

Number of recurrences (%)

0 1 2 Death Total

Surgery

(%)

6

(21)

13

(44)

8

(28)

2

(6)

29

Light

alone (%)

10

(30)

9

(27)

13

(39)

1

(3)

33

Drug alone

(%)

6

(18)

12

(36)

15

(45)

33

AIOPDT (%) 18

(50)

10

(28)

5

(14)

3

(8)

36

Total (%) 40

(31)

44

(34)

41

(31)

6

(4)

131

174



10f: Statistical Analysis

A Kruskal-Wallis one way analysis of variance of ranks was used

to compare the 4 treatment groups (1st = Surgery alone, 2nd =

Light and surgery, 3rd = Drug and surgery, 4th = surgery and

AIOPDT). There were significant differences between the 4 groups

(p=0.01). Examination of the distribution of outcome in the 4

groups suggested that the first 3 groups had similar outcomes. A

Kruskal-Wallace anova test was used to compare these first 3

groups and found no significant difference (p=0.6). The 4th

group (surgery and AIOPDT) was then compared with these 3 groups

combined. The Kruskal-Wallace anova demonstrated a significant

difference (p=0.002). A rank transformation was also used on the

data, and a two-way analysis of variance on the ranks with the

factors “treatment group” and “day” was performed. There were no

significant differences between the 7 days on which the study

was performed (p=0.7).

175



10g: Discussion

Table 17 shows that the size of tumour removed is proportional

to the death rate following its removal. This is in keeping with

general mortality figures in surgery, in which size and stage at

surgery have a negative effect on outcome and perioperative

mortality. Size of tumour also influences the local recurrence

rate in a positive manner. Thus a compromise of perioperative

mortality against local recurrence was needed for this study.

The 2cm diameter tumours were chosen on that basis.

Table 18 and figure 2 show that tumours grow in a fairly uniform

manner after intramuscular inoculation of a standard sized

tumour piece. This it important because it allows the prediction

of when the tumour will be 2cm in largest diameter for the

experiment. This is particularly important in PDT

experimentation because of the 96 hour drug-light interval that

is needed before the experiment can begin.

Tables 19 – 25 show the individual experiment days. The

variation of numbers within the groups represents excluded

tumours that were outside the treatment parameters. As discussed

in the statistics section, there were no significant differences

between the groups.

The results (summarised in table 26) show a statistically

significant reduction in the local tumour recurrence rate with

the treatment group as compared to the control groups. Although

there were more deaths postoperatively in this group, this was

not a significant finding, and may just represent the relatively

small numbers tested. The control groups tested the hypothesis 176



that the drug and diluent is toxic to tumours without light

(drug only group), although even in this group there will have

been a mild photodynamic effect due to background theatre

illumination and theatre lights during the excision. However,

the total light dose given during the relatively short

(approximately 5 minutes) time that it took for excision of both

tumours is so low that this effect is unlikely to have been

significant. The control groups also tested the hypothesis that

the drug diluent is an active photosensitising agent (light and

diluent only group), although the hypothesis that 652 nm laser

light at non-thermal intensity could be cytotoxic was therefore

not independently tested. This is extremely unlikely however

since there are many publicly available lasers at around this

wavelength (laser light pointers) that have been shown to be

totally safe except when shone directly into the eyes. The fact

that there was no significant difference between any of the

control groups including the surgery alone suggests that these

hypotheses are invalid.

A study such as this is only valid if the principle that local

tumour recurrence is due to residual disease is agreed. There

are other theories regarding why tumours recur in the excision

bed, such as further metastases from the primary area, or new

primary disease growing in the tumour bed. Studies have shown

that after a macroscopically complete excision, if the tumour

bed is washed with cell growth medium and incubated in the

correct conditions for growth, viable tumour cells can be grown

(Harris and Smith 1960). These cells almost certainly come from

the excised tumour area, either due to "burst and spill" or 177



unwittingly cutting through tumour during the excision, and

contaminating the surgical instruments (Beahrs and Barber 1962).

What is not known is whether these are viable or not in the

clinical situation. Disease recurrence due to residual tumour

can also occur because of involvement of vital structures

rendering the tumour inoperable, or because tumour margins or

not clear due to the disease growing to the defined limits of

the operation (Olcott et al 1981). Other adjunctive

intraoperative methods have been tried in order to reduce the

local recurrence rate of tumour after Head and Neck surgery.

178



Other intraoperative therapeutic methods include:

1) Intraoperative external beam radiotherapy, which was

logistically very difficult, time consuming and damaging to

the carotid artery (Freeman et al 1990).

2) Washing the tumour bed with cytotoxic agents, including

distilled, sterile water which has not been shown to be

effective, presumably because there is little penetration of

effect into the microscopic cracks and crevices on the tumour

bed where viable tumour clumps may lodge.

This tumour model bears a reasonable relationship to the

proposed clinical treatment, namely metastatic neck disease

treated by radical neck dissection. Neither are primary tumour

sites, the animal model being an implanted tumour graft, the

clinical model being a proliferating clump of tumour cells shed

from the primary site into lymphatic channels and caught in the

draining lymph glands. The main difference apart from site of

growth is in the fact that the clinical model lies in a lymph

gland, the animal model in normal surrounding muscle, although

this is important since the tumour is growing in its natural

environment, being a connective tissue tumour (Bown S.G. pers.

comm.). It thus is a more natural model than others used for

AIOPDT studies, such as neuroblastoma in muscle (Davis et al

1990) or adenocarcinoma in subcutaneous tissue (Abulafi et al

1994). However, by the time most secondary tumours in the neck

have been detected, they have reached a size of at least 1cm

diameter, and have usually destroyed the lymph node they lie in.179



The adjacent tissue to most of the lymph nodes in the neck is

mainly muscle, particularly in the lymph node bed, and fat,

which tends to lie superficially. Thus even in the human

situation, the tumour lies in close proximity to the muscle.

This is even more so following radical neck dissection, since

the superficial tissue (mainly fat, internal jugular vein and

the sternocleidomastoid muscle) and tumour is removed, the deep

musculature is left untouched. Thus the treatment site for

AIOPDT in both cases would be a muscle bed. The tumour type

used, HSN fibrosarcoma was also similar to the human model,

squamous cell carcinoma of the upper aerodigestive tract. This

is because it is a locally invasive, non-encapsulating tumour.

Clearly an encapsulating tumour would be easy to shell out with

little chance of local recurrence, whereas a tumour

microscopically invading into the surrounding tissue will recur

unless a wide block of apparently normal tissue is removed with

the specimen, which was not done in this case. The tumour was

removed very close to the main tumour lump, although macroscopic

clearance was complete in all cases. Therefore it is more likely

that microscopic tumour residue occurs, leading to a local

recurrence rate in the control groups of around 70%. The initial

studies on survival after excision also showed this - it would

have been useful to have had an even higher local recurrence

rate as occurred with the 3 cm diameter tumours, but the

postoperative mortality rate made this unacceptable.

The scope of procedures suitable for AIOPDT in surgery is much

greater than just radical neck dissection . It may also have a 180



role in general surgery (Herrera-Ornelas et al 1986, Nambisan

R.L. et al 1988, Abulafi et al 1993), neurosurgery (Muller and

Wilson 1995) and thoracic surgery (Ris et al 1993). Conservation

surgery in the neck, in vogue since it seems to reduce

postoperative morbidity (Bocca 1975) could be made safer by

treating those retained structures such as the accessory nerve

with PDT to mop up any residual tumour around such structures.

The fact that red light penetrates tissue significantly means

that structures such as the accessory nerve can be completely

treated by illuminating onto and through the nerve. The same may

be true of other vital structures in the neck such as the

carotid tree, although the fact that this is of quite large

diameter and filled with blood means that complete penetration

and treatment of the medial surface is unlikely to be effective.

Prior to clinical studies, safety work is therefore needed,

particularly regarding the effect of PDT on arterial structures.

10h: Conclusion

The scope of adjunctive intraoperative photodynamic therapy is

large, in the Head and Neck as well as other areas. This and

other studies have successfully demonstrated, to the

satisfaction of statisticians, the principle that treatment

reduces the local recurrence rate of tumour following

macroscopically clear excision to be true on a preclinical

basis.

181



11: Experiment 3 - Preclinical Photodynamic Safety Studies on

Arteries

11a: Description

The previous study has shown that adjunctive intraoperative

photodynamic therapy (AIOPDT) can significantly reduce the local

recurrence rate of malignant tumours after macroscopically clear

tumour removal. This has implications for most forms of cancer

surgery, but particularly those in which the local recurrence of

disease has a major impact on survival. One such operation is a

radical neck dissection for secondary spread of squamous cell

carcinoma of the upper aerodigestive tract. AIOPDT may be useful

to reduce the local recurrence rate after this operation. Before

AIOPDT could be safely transferred into the clinical sphere,

safety studies were necessary on vital structures that would be

in the photodynamic treatment area. For the operation of radical

neck dissection, this means the carotid tree, a structure whose

damage leads to grave consequences.

The operation of radical neck dissection, as described in the

introduction is a good example of a Head and Neck operation

that has a significant mortality if local recurrence occurs. It

involves the removal of a block of tissue from the side of the

neck, incorporating all of the surgically available lymph nodes 182



on that side. It is classically performed to treat secondary

spread of squamous cell carcinoma of the upper aerodigestive

tract into the lymph nodes on one side of the neck. All the

nodes are removed because it is not possible to know the extent

of spread prior to surgery, although retrospective studies have

suggested that this can usually be predicted. There is a very

high mortality rate due to local recurrence in this area when it

occurs (Pearlman 1979, DeSanto et al 1982). Therefore any

treatment that might reduce this figure is welcome.

Safety work was performed to assess the effect of AIOPDT on

vital structures that would be exposed to PDT in the tumour bed

of this operation, the most important of which is the carotid

tree, consisting of the common carotid artery and its terminal

branches, the internal and external carotid arteries.

183



Other structures in the surgical bed include the vagus and

hypoglossal nerves and the carotid body. Since nervous tissue

has so far been found to be largely resistant to PDT (anecdotal,

unpublished observations in general), and since these structures

are not integral to the survival of the patient, they were not

studied in this experiment. The carotid vessels are vital

however, not least because if the carotid artery wall is

weakened with AIOPDT and it ruptures postoperatively, death due

to exsanguination is likely, as has happened following PDT

treatments to advanced Head and Neck cancers (Schuller et al

1985, Gluckman 1991), although there is a very definite

incidence of carotid artery rupture with this disease, so the

significance of this is not known. Also, the internal carotid

artery is often an "end artery", having little or no collateral

supply. Therefore if it clots off during PDT or stenoses at a

later date due to partial damage, the entire volume of tissue

supplied by the vessel might die. Since this vessel is the major

blood supply to the ipsilateral cerebrum, at the very least a

major cerebrovascular accident (CVA, stroke) can be expected.

11b: PDT Details

Previous studies had shown a suitable drug level for animal

experimentation to be 1.0 mg/kg body weight. The drug light

interval chosen was 96 hours, since again this seemed likely to

give a good tumour necrosis response with minimal normal tissue

damage, and was the DLI used in early and all subsequent

clinical investigation on Head and Neck cancer (Dilkes et al

1994). The light dose given ranged from 10 to 100 joules/cm2 184



(J/cm2), in order to potentially obtain a dose/damage curve and

predict safe levels of light, potentially useful if severe

damage was seen, 500 J/cm2 was given to the non-sensitised

control groups. The light delivery rate was kept at 300

milliwatts (mW)/cm2 and given via a microlens (Quadra Logic

Technologies, Vancouver, Canada) in all cases. The delivery rate

was higher than in previous studies because of the excessive

amount of time it would take to deliver the maximum light dose,

500 J/cm2 at the previously used parameter of 100 mW/cm2. The

light source used was an Oxford Lasers CU15 copper vapour laser

pumping a Rhodamine dye laser tuned to 652 nm. Light intensity

was continually checked using an integrating sphere and

radiometer.

185



11c: Drug

The drug mTHPC was obtained form Scotia Pharmaceuticals,

Guildford, Surrey, U.K. as a gift. It was supplied as dry,

sterile powder of 99.7% purity. When administered it was

dissolved immediately before use (within 1 hour) in its diluent.

Adult female ex-breeder Wistar rats were obtained from Charles

River Ltd. Drug was administered intravenously via the tail vein

using a standard restraining device. Once the animals had been

photosensitised, they were kept out of direct contact with

light, in darkened conditions.

11d: Study Design

The rat superficial femoral artery was chosen as the study

vessel. This structure is a continuation of the external Iliac

artery, arising where the common femoral artery gives off its

profunda vessels. It is easy to expose on the surface of the

adductor muscles in the floor of the femoral triangle, and lies

in a fibrous sheath also containing the femoral vein and nerve

(sensory only). Both (right and left) femoral vessels were

dissected free of surrounding structures under a general

anaesthetic of Hypnorm and Midazolam, using microsurgical

instruments and magnifying loupes where necessary. Prior to

light administration, a small piece of black blotting paper,

soaked in normal saline was gently manoeuvred under the vessel

to shield surrounding structures, making eventual postoperative

dissection easy, and avoiding serious damage to underlying 186



muscle and vein (picture 6). Light was delivered to a 1cm

diameter spot centred over the vessel, measured using a steel

ruler with 1 mm gradations. The light fibre was held in a steel

fibre holder which was supported in a clamp to avoid unnecessary

movement of the spot during light delivery.

Following delivery of light to one side only (the other side was

left exposed with blotting paper beneath it to act as a direct

matched control), the part of the vessel treated was marked with

small 4/0 silk sutures a few millimetres away from the vessel,

in the adductor muscle, in the centre of the treated area, so

that the area treated on the vessel could be identified

postoperatively. Following this the animal was recovered.

187



Picture 6: Freshly dissected femoral artery. The nerve and vein

are left untouched below the black blotting paper, which

prevents any other collateral damage since the 1cm diameter spot

is entirely focused onto this area. The control side is treated

in an identical way, but no laser light is delivered.

188



11e: Vessel Analysis

After a time period of either 3 days, 14 days or 3 months from

treatment, the animals were given a further general anaesthetic.

The chest was opened via a thoraco-abdominal incision, and the

left ventricle of the heart identified. This was punctured using

a 23 gauge needle, and a standard sized cannula with a bulbous

tip was inserted into the ascending aorta. This was secured with

a silk tie placed below the bulbous tip (picture 7). Normal

saline at normal adult rat physiological pressure was then

perfused into the animal, drainage being obtained by an incision

into the right atrium of the heart. The reason for this was to

wash blood out of the system prior to perfusion, again at

physiological pressures, overnight with formol-saline to fix the

femoral arteries in their normal state. This allowed realistic

measurement of vessel lumen diameter and wall thickness.

Following overnight perfusion the femoral areas were dissected

and the femoral arteries removed, on the treated side a 2cm

length of vessel 1cm above and below the marking sutures was

taken (Picture 8). These were then mounted in paraffin blocks,

and cut using a microtome. The resultant slides were stained

with Haematoxylin and Eosin. Using a microscope fitted with a

CCD camera, images of the vessels were captured and measured on

a monitor using a Magiscanner. Three outside circumferences were

measured: lumen, media and adventitia (Picture 9). On each

animal analysed, there was control data from the contralateral

superficial femoral artery, with the ipsilateral side providing

data on PDT effects.189



Picture 7: A cannula is secured in place in the root of the aorta, via the left ventricle. The cannula is connected to a reservoir of saline initially, then formalin, both at physiological pressures, so the arteries are preserved in their usual state.

190



Picture 8: Haematoxylin and Eosin slide of sacrificed vessel, showing preservation of the arterial lumen, and the identifying silk suture in the surrounding muscle bed.

191



Picture 9: Lines drawn around the areas to be measured with the magiscanner. From the calculated area (A- lumen, B- media, C - adventitia), the radii can be determined, and by subtraction, the average width of the three areas determined.

192



Table 27Magiscanner results - relative arterial sizes TREAT DOSE NUMBER TIME LUMEN MEDIA ADVENT1 10 1 1 210 65.9 342 10 1 1 176.1 86.7 24.71 10 2 1 183.1 34.7 44.12 10 2 1 150.9 55.1 28.11 10 4 1 185.8 50.1 312 10 4 1 182.3 78.3 19.91 10 1 2 132.3 48.7 24.52 10 1 2 137.4 55.2 18.91 10 2 2 161.9 57.1 30.22 10 2 2 184.5 47.2 23.51 10 1 3 164.3 45.3 19.72 10 1 3 167 27.8 16.81 10 2 3 229.9 41.5 20.82 10 2 3 103 54.7 20.21 20 4 1 259 18 49.42 20 4 1 214.8 18.6 10.21 20 2 1 261.5 20.4 322 20 2 1 207 26.2 19.41 20 3 1 199.6 32.8 81.42 20 3 1 212.7 33.6 11.81 20 1 1 241.1 53.7 46.92 20 1 1 206.2 22.8 15.11 20 1 2 88.1 79 43.62 20 1 2 216 42.5 22.41 20 2 2 219.7 42.6 46.62 20 2 2 284.2 35.8 24.31 20 3 2 162.7 62 37.62 20 3 2 264 35.8 321 20 1 3 79.6 83.8 70.12 20 1 3 67.7 99.2 691 20 2 3 197 32.6 40.92 20 2 3 210 30.5 25.41 20 3 3 87.2 86.4 462 20 3 3 250.3 23.7 32.1

193



1 50 1 1 229.4 36.4 401 50 2 1 227.2 54.1 56.51 50 3 1 155.4 44.2 73.21 50 4 1 150.6 38.5 72.11 50 1 2 267.3 36.6 27.51 50 2 2 270.5 42.3 29.31 50 2 3 232.9 33.1 18.71 50 3 3 331.7 39 18.61 50 4 3 292.3 25.4 11.62 50 1 1 195.5 35.9 14.82 50 2 1 159 28.8 30.52 50 3 1 158.1 49.5 17.92 50 4 1 105.8 27.3 10.52 50 1 2 229.9 26.7 102 50 2 2 289.6 29.7 132 50 2 3 138.5 32 15.72 50 3 3 253.5 21.1 10.22 50 4 3 205.1 32.1 12.51 100 1 1 241.4 41.5 43.41 100 2 1 110.4 68 167.61 100 3 1 161.4 30.7 781 100 1 2 263 38.5 26.51 100 2 2 320.7 52.3 18.11 100 3 2 267.5 46.8 16.21 100 4 2 273.7 35.9 1.11 100 1 3 369.1 28.3 53.81 100 2 3 105.9 57.9 11.31 100 3 3 259.2 38 10.81 100 4 3 196.8 55.1 16.12 100 1 1 108.1 70 10.52 100 2 1 357.3 32.6 172 100 3 1 96.9 75.7 73.72 100 1 2 279.6 45.8 7.82 100 2 2 354.4 35.3 15.62 100 3 2 192.6 38 8.42 100 1 3 334 36.4 17.82 100 2 3 248.7 26.2 12.72 100 3 3 240.7 30.4 12.52 100 4 3 199 41.8 18.4Treat: 1=Control (no laser light), 2 = PDT,Dose: laser light dose at 652 nm, 300 mW/cm2, given to PDT sideNumber: animal within each group Time: Delay before overnight perfusion, 1=3days, 2=2weeks, 3=3 monthsOther figures are measurements of vessel sizes, in microns, lumen = radius of lumen

194



Other non-sensitised controls (mTHPC diluent only) compared laser light at 500 J/cm 2 , 300 mW/cm 2 on one side, with no laser on the other. No significant differences were seen on Magiscanner analysis.

195



11f: Statistical analysis

An analysis of variance was performed on the differences between

the control and treated side of each animal. One animal with a

very high measurement for adventitial thickness with PDT was

excluded. The p values for this analysis are shown in table 27:

Table 27

Variabl

e

Dose Time Dose x

timeLumen 0.01 0.02 0.07Media 0.05 0.01 0.4Adventi

tia

0.15 0.00

4

0.6

Dose = laser light delivered, time = interval between PDT to

the vessels, and sacrifice of the animal

These results show that the measured results on the PDT (non-

control) side are significantly and directly affected by light

dose except the adventia, which can be seen in graph 1. All

three measurements are significantly affected by time, see graph

2. The earlier the interval from moment of PDT delivery, the

greater the effect seen.

Table 28

This shows the mean and standard deviations for light dose.

Light dose (J/cm2)

Variabl 10 20 50 100 196



e

(width

/radius

)

(n=7) (n=10) (n=9) (n=9)

Mean/

sd

Mean/sd Mean/

sd

Mean/sd

Lumen 24/50 -34/77 47/39 15/78Media -9/17 14/24 7/11 -1/24Adventi

tia

8/5 23/20 24/22 11/15

There is no significant pattern of difference in these results

when performing contrasts to compare each set of means.

197



11g: Discussion

The main significant finding is that of substantial adventitial

oedema occurring in all the treatment groups 3 days after PDT,

when compared to the contralateral (control) side (Picture 10),

but no further significant damage to the media or intima. By the

time 2 weeks have passed this oedema is no longer present. Long

term stricture formation or dilatation of the treated vessel was

not noted 3 months post treatment, at which stage it would be

noticeable if present. This indicates that 96 hours post injection

of mTHPC, there is an insignificant amount of drug in the vessel

wall or endothelium, but still enough in the adventitia to cause

an effect, or that the endothelium and media are resistant to the

effects of PDT, a finding that has not been shown by those looking

specifically at this area (Sobeh M., pers. comm.). The fact that

no decrease in the vessel lumen size occurred in conjunction with

this is encouraging, since flow through the vessel should

therefore be maintained. The lack of endothelial damage is in

contrast to a similar study using different photosensitising

drugs, 5-Aminolaevulinic acid and aluminium phthalocyanine (Grant

et al 1994) . In this case almost total loss of endothelium was

seen in the early phase of the study, although full regeneration

eventually occurred, with no apparent adverse effects of this

damage, in particular no evidence of intravascular coagulation.

Total loss of all cells in the vessel wall also occurred in a

separate experiment (Dilkes M.G., unpublished data, see Picture

11) using Photofrin 2 20mg/kg, DLI 24 hours, light dose 100

J/cm2@100mW/cm2, with the control sides being unaffected, a major

factor in deciding which photosensitising drug to use in this198



series of experiments. Other studies have also looked at PDT for

the treatment of intravascular lesions, in particular atheroma

(Hsiang et al 1994) and neointimal hyperplasia (Nyamekye et al

1996). In both of these studies, light was delivered via the

endoluminal route in order to treat lesions of the vessel wall.

The results achieved were not particularly promising, which is

surprising since it is well known that vessel endothelium is the

site of at least part of the photodynamic effect, at least in

tumours (Henderson et al 1985). It may be that in these situations

the timing of the drug-light interval (DLI) is critical,

explaining the lack of damage seen in our study, although raising

questions therefore as to the potential efficacy of the treatment

if the microvascular effect has been lost. However, in over 100

clinical cases treated with mTHPC at a DLI of 96 hours as in this

experiment, excellent results have been seen in terms of tumour

response (Stewart J.C.M. 1996). It may be that with mTHPC the

vascular effect is of little consequence, tumour necrosis relying

much more on direct cell toxicity. Further studies looking at the

mechanical integrity of vessels following PDT showed that despite

full-thickness cell death (Orteu et al 1992), resistance to

bursting pressures was unaffected when compared to the control

side (Grant et al 1995).

Picture 10: Obvious damage is seen in the adventitial layer, but

no inflammation in the vessel wall, no endothelial damage, and no

relative compression of the lumen.

199



Picture 11: Femoral artery 3 days after PDT with Photorfrin 2 100

J/cm2, DLI 24H, dose 20mg/kg. Total loss of cellularity in the

vessel wall is seen. Established complete thrombosis is present in

the vessel lumen.

200



This is presumably due to maintenance of the strength and function

of collagen in the vessel wall, a finding that has also been seen

in colonic mucosa (Barr et al 1987), and it is probably this

property that is the main cause of the much enhanced healing and

retention of function seen in clinical cases treated with PDT

(Poate et al 1996), an intact collagen matrix allowing the rapid

ingrowth and seeding of normal cells into the space vacated by

the tumour cells, which degenerate in a fashion somewhat

mimicking apoptosis (Agarwal et al 1991).

Studies into the clinical effect of PDT on coronary arteries using

another photosensitiser, Dihaematoporphyrin Ether showed that at

high light doses (>200J/cm2 ) 4/7 treated animals died within 48

hours, and again significant medial damage occurred. This study

also reported adventitial injury, although the exact nature of

this was unclear (Mackie et al 1991).

One reason for the lack of clinically significant damage in our

experiment could be explained by the fact that the drug was

inactive, or the laser incorrectly set-up. However, the fact that

obvious adventitial damage occurred (a definite photodynamic

effect as compared to the other side) means this cannot be so,

true selectivity has occurred between intima/media, and adventia.

The finding of significant adventitial oedema 3 days post

treatment is also interesting because the amount of damage shows a

direct relationship with the amount of light in J/cm2 delivered.

Although on the face of it this is not surprising, recent data

(M.L.DeJode et al 1996) looking at depth of necrosis in a liver

model, showed a plateau-ing of effect at around 40J/cm2, all

other parameters being the same as in this study. In this case201



there is a big jump in effect between 50J/cm2 and 100 J/cm2,

although if one very large result in both the treatment and

control groups in one animal is excluded, the trend is much less

pronounced, and in keeping with this other data that suggests the

plateau for effect is around 20 – 40 J/cm2. The difference in

level of plateau may also be due to different optical properties

of the tissues treated (vessels in this study, liver in the deJode

study).

The lack of damage in animals treated with light only, no drug,

demonstrates 2 points. One is that red light at 652 nm does not

have any directly negative effect on the vessels at this

intensity, the other is that the intensity of light used,

300mW/cm2, does not have any damaging thermal effect when surface

irradiance occurs even up to 500J/cm2 total light dose. This has

been confirmed using a microthermocouple by us (Dilkes et al

unpublished data) and others (Abramson et al 1990). Although some

photosensitising drugs have been shown to affect the intimal layer

of vessels without light activation (Coates et al 1996), no

significant change in the intima was seen in any part of the

study.

The use of ex-breeding adult rats was favourable because these

animals were fully mature - therefore any change in vessel size

over 3 months would not be due to growth of the animal.

202



11h: Conclusion

No significant damage has been seen in this experiment that might

be deleterious to the patient in a clinical situation. This is

relevant because the photodynamic therapy parameters in this

situation are the same as the parameters currently used for the

treatment of clinical tumours (Dilkes and DeJode 1994). Before

clinical trials on AIOPDT with mTHPC however, more data was needed

regarding the effects of PDT on vessels during PDT and

immediately after. This is because the first time point studied in

this case is 3 days, at which stage severe spasm may have

corrected, and thrombus in the vessel may have dispersed - if that

happens however, the short term effect of this on the carotid

artery for example would be disastrous. Also, data is needed

regarding the effects of PDT on larger vessels, although the

larger the vessel the less likely severe damage will be, since the

opposite side of the vessel to the incident surface will be that

much further away from the light, so intensity will be reduced.

203



12: Experiment 4: Acute phase effects of PDT on arteries and

veins

12a: Description

Adjunctive intraoperative photodynamic therapy is being

investigated for use in Head and Neck cancer. Although this and

previous studies have looked at the effects of PDT on vessels

that might be in the AIOPDT site, no study has looked at the

immediate effects of PDT on vessel flow, which has real clinical

significance. We have used an ultrasonic doppler probe to

investigate this problem in rats during and immediately after

PDT, and find no significant changes in arterial flow, but

significant reductions in venous flow rate, particularly in the

external jugular vein which goes into long term spasm.

Our main aim was to look at the role of AIOPDT on reducing the

local recurrence of tumours following radical neck dissection

for secondary spread of Head and Neck cancer. Structures in the

bed of this operation include the carotid arterial tree, and the

jugular vein if a conservative approach is used (Bocca et al

1975). There is currently no good data on the effect of PDT in

the short term, during and immediately after treatment. This is

of great importance clinically, since vessels may go into spasm

or thrombose during PDT, only to have returned to normal or

recanalised 3 days hence. During that period if major vessels

are occluded, serious and irreversible damage may have occurred

204



to the area supplied by that vessel. In the case of the carotid

tree, this could mean infarction of a large part of the brain.

12b: Chemical

The drug mTHPC was obtained form Scotia Pharmaceuticals,

Guildford, Surrey, U.K. as a gift. It was supplied as dry,

sterile powder of 99.7% purity. When administered it was

dissolved fresh (within 1 hour) in its diluent, to make up a

concentration of 0.5 mg/ml. Adult female ex-breeder Wistar rats

were obtained from Charles River Ltd. Drug was administered

intravenously via the tail vein using a standard restraining

device by experienced staff. 1.0 mg/kg body weight was given in

all cases except the light-only controls. Once the animals had

been photosensitised, they were kept out of direct contact with

light, in darkened conditions.

12c: Measurement of flow

The method of blood flow measurement chosen was with a

Transonic flowprobe (Linton Instruments, Stowmarket, Suffolk,

U.K.). This device uses two ultrasonic transducers pointing at a

fixed acoustic reflector (see figure 3), The time taken for

ultrasonic waves to pass between the two transducers is

measured, giving an accurate measure of transit time - which is

affected by the rate of flow in the vessel. By a subtraction

technique of integrated transit times the final measurement is

in volume of flow rather than velocity (Drost C.J. 1978). Using

a selection of flow probes it is possible to measure flow rate 205



in a number of different vessels. The Transonic Flowprobe and

meter was obtained on free long term loan from Linton

Instruments.

206



Figure 3

207



12d: Experimental technique

General anaesthesia was induced using the standard

intraperitoneal injection of a mixture of Hypnorm and Midazolam.

Because it was felt that depth of anaesthesia would directly

influence flow through vessels independent of any other

variables such as PDT, a steady state of anaesthesia was

desirable. This was achieved using an intravenous cannula placed

in the tail vein, attached to a syringe pump delivering a

continuous flow of Hypnorm and Midazolam.

The vessels to be examined were exposed using standard surgical

approaches (Picture 12). Magnifying surgical loupes and

microsurgical instruments were used to delicately free the

vessels from surrounding structures. Using a series of retort

stands and clamps the flowprobe was held in position around the

vessel, aqueous gel being used as the transmission medium (see

picture 13). Laser light at 652 nm wavelength was obtained from

a copper vapour laser pumping a Rhodamine dye laser (Cu15,

Oxford Lasers, Abingdon Rd., Oxford, U.K.), 100 mW/cm2

intensity, using a 1 cm diameter spot delivered via a

microlens. The vessel being in the middle of the spot in all

cases, the light fibre being held in a metal fibre-holder,

gripped again by clamps from a retort stand. With veins the

laser light was delivered distal to the probe, with arteries the

laser light was delivered proximally (with respect to the

heart).

Flow of blood through the vessels was recorded using a

continuous XY recorder (see picture 14) for a period of 5 208



minutes pre light delivery (baseline) and 15 minutes post light

delivery. Although the clinical dose of light was only 20 joules

per square centimetre (J/cm2), a total dose up to 250 J/cm2 was

given, in a continuous manner, to thoroughly evaluate any

changes induced at higher light doses.

209



Picture 12: Exposure of the jugular vein and common carotid

artery. The skin of the front of the neck is completely removed

to facilitate access.

210



Picture 13: Typical set-up during doppler flow measurements

211



Only one vessel was examined per animal. When cases of vessel

spasm occurred, the vessel was bathed in Praxiline 10mg (0.5 ml

of 20mg/ml solution) to try to reverse this.

The following vessels were assessed: Jugular vein, common

carotid artery, abdominal aorta, common iliac artery and vein,

femoral artery and vein. Two of each vessel were measured with

PDT except in the case of the external jugular vein, in which 4

vessels were tested. One of each vessel was a control, except

with the external jugular vein in which there were 2 controls.

12e: Results

All animals tolerated the procedure successfully. The experiment

was sometimes dogged by air bubbles appearing in the gel around

the vessel, and satisfactory readings were not always possible,

despite the best efforts (Picture 14). The results obtained are

shown in graphs 3 and 4. They show that arteries do not appear

to be significantly affected by PDT when delivered in this

manner, but veins, and in particular the internal jugular vein,

do have a significant reduction in blood flow that is sustained

throughout the period of observation. Acute and irreversible

vessel spasm as occurred in the jugular veins was not helped by

Praxiline administration topically. It was not possible to get

accurate readings of aortic flow rate, probably because our flow

probe was too small to fit around the vessel. No significant

changes in flow occurred in any of the control animals.

212



Picture 14: Typical reading during PDT and doppler measurements

of the jugular vein.

213



12e: Discussion

The most striking result obtained was virtually complete

cessation of flow in the jugular veins tested in the PDT groups

(Graph 4). At the time of the procedure the vein was seen to go

into spasm which did not reverse during the entire procedure or

for 15 minutes after. The jugular vein is a structure noted for

its propensity to spasm, even with simple instrumentation, but

this was not seen in the control groups tested. More of these

vessels were studied because no major change in flow was seen in

any of the other vessels treated, although all the veins treated

did show some moderate reduction in flow rate. The use of

Praxilene, a powerful vasodilator which works topically did not

help. This finding has potentially major implications regarding

the role of AIOPDT in Head and Neck surgery, although not with

respect to the operation of radical neck dissection, where

temporary loss of a preserved internal jugular vein is of little

importance, unless perhaps the contralateral vein has already

been taken, or bilateral AIOPDT is planned. In this case there

may be a rise in intracranial pressure with potentially serious

side effects (Jones R.K. 1951, Marks et al 1990). However, the

size of the internal jugular vein in its normal state in the

adult human is much larger than that of a rat, and it is

possible that because of this only one half of the vessel wall,

on the irradiated surface side, will be stimulated by the PDT,

since significantly less light will pass through to the other

side, which is often separated from the lateral side by a lumen

of 1.75cm diameter filled with deoxygenated blood (Doktor et al 214



1996). However, in this case only a 1cm length of vessel was

treated, whereas in the clinical situation the entire length of

the internal jugular vein would be irradiated, some 15-20 cm.

The planned clinical light dose for AIOPDT would be the same as

that used in experiment 2, and in the treatment of early Head

and Neck cancer (Dilkes and DeJode, 1994), because since this is

effective in necrosing solid, visible tumours, it is assumed it

will also work on microscopic tumour residue. The light dose in

this situation is 20 J/cm2, at which stage little of the effect

had set in regarding spasm. From an initial average flow rate of

3.4 ml/minute, by the time 20 J/cm2 had been given, the average

flow had dropped to 2.7 ml/min, a 19% fall, which would not be

significant clinically. We did not wait however, after giving

the 20J/cm2 to see if further spasm occurred, although since

spasm occurs because of an irritative stimulus, we assume that

this is a linear, accumulative relationship, rather than due to

any possibly delayed effect.

215



Graph 3: CIV = Common iliac vein, CFA = Common femoral artery,

CIA = Common iliac artery, CCA = Common carotid artery.

216



Graph 4: IJV = Internal jugular vein.

217



The effect of PDT on veins might however be important in AIOPDT

for other Head and Neck operations. These would be operations

where vascularised free flaps of tissue are re-anastomosed with

local Head and Neck vessels to fill the holes created by radical

excision of tumours, as occurs increasingly commonly. In this

situation fairly small blood vessels are joined together, some

of only around 2 mm diameter. These would not be protected on

the non-irradiated side by blood, since when the vessels are

anastomosed they are empty, and besides which red light at 652

nm will adequately penetrate 2 mm of blood. Although the veins

and arteries to be anastomosed could be shielded from any laser

light at 652 nm used for the AIOPDT, when they are anastomosed a

microscope with very bright white light is needed to be able to

accurately appose and suture the edges of the vessels. This will

contain significant amounts of light that will activate

photosensitisers, with mTHPC this means blue and middle-far red.

Thus spasm could be induced in the veins of the anastomosis

(usually at least one artery and 2 veins are needed to ensure

survival of the graft), even though the internal jugular vein

would usually not be used for such a procedure. If persistent

venous spasm is induced, the graft will fail, with potentially

serious consequences for the patient.

More encouragingly, the arteries tested were all resistant to

all but minor variations in flow with PDT (Graph 3). In

particular the rat common carotid arteries tested showed no

significant variation in flow at all, up to 240 J/cm2 of light.

The control sides demonstrated that even at a relatively high

fluence rate of 300 mW/cm2, no damage due to heating occurred, 218



and it might be beneficial in the clinical situation to use

higher fluence rates to reduce treatment time once more powerful

laser systems are available, although there is some data, as yet

unpublished, to suggest that increased fluence rate decreases

the efficacy of the treatment (DeJode et al, in preparation

1997), presumably due to exhaustion of the oxygen supply for

PDT.

In summary, the ultrasonic flowprobe was a useful way of

measuring acute flow changes in vessels undergoing PDT. The

findings do not preclude clinical investigation of the role of

AIOPDT for the operation of radical neck dissection in which the

internal jugular vein is removed, but do raise questions about

the potential viability of free flaps if AIOPDT is used in other

cancer procedures.

219



13: Experiment 5 - The effect of high intensity white and

filtered microscope light on the viability of microvascular

anastomoses in photosensitised rats

13a: Description

The previous experiment has shown potentially serious damage to

small veins undergoing photodynamic therapy. This has

implications when PDT is planned in patients having a

simultaneous microvascular re-anastomosis of vessels during a

vascularised free flap reconstruction in surgery,. This is

because the high intensity white microscope light that is

necessary to illuminate the small vessels so that the very

accurate microscope controlled anastomosis can be created may

contain sufficient amounts of activating light to a damaging

photodynamic effect within the walls of the vessels being

anastomosed (Picture 15).

This is potentially a major problem, particularly in Head and

Neck cancer surgery where the use of microvascular anastomosis

for vascularised free flaps is increasing rapidly. Since the

late 1940’s small vessels anastomoses have been reliably

achieved (Johns T.W.P. 1947), and the procedure of free

microvascular transfer and re-anastomosis of tissue into local

blood supply is now a mainline method of reconstruction for

defects all over the body. This is particularly true in the Head

and Neck area where, following the radical resection of cancer, 220



these flaps offer better structure and versatility than

previously used flaps (Soutar et al 1983).

Following the previous experiment it seems that veins in

general and in particular the external jugular veins of the rat

were very sensitive to the effects of photodynamic therapy when

stimulated by red laser light at 652 nm and sensitised with

mTHPC, going into sustained spasm under PDT. The fact that veins

used in microvascular reconstruction are often of fairly small

diameter (like those in the rat) may mean that the brilliant

Xenon light source of operating microscopes might contain

enough activating light to damage venous anastomoses and cause

failure of the blood supply to free flaps, with their resultant

necrosis and high subsequent morbidity and mortality, should a

salivary fistula occur. Immediate excision of the failed graft

and its replacement is standard practice should necrosis occur.

221



Picture 15: Rat femoral artery held in an Acland clamp, under

conditions of magnification and brilliant, focused illumination

222



This aspect is particularly important when considering the

second generation photosensitising drugs such as mTHPC, which

have very high absorption peaks in the blue spectrum, which the

Xenon arc lamp sources such as the ones used in operating

microscopes tend to produce in significant amounts (see

diagram). They are also very efficient converters of light

energy into chemical energy (Bonnet 1995), so relatively small

amounts of activating light will cause significant amounts of

damage. Time interval between drug injection and light

administration might also be critical, since there is a well

known vascular effect in the first 6-12 hours sfter intravenous

administration of the drug, due to circulating photosensitiser,

and drug being taken up through the vessel wall into the

interstitial spaces. At 96 hours post injection however, minimal

levels of mTHPC are seen in vessels, as shown in experiment 1.

This might mean that anastomoses performed at the early stage

might fail, but those at a later stage might survive.

13b: Plan

The specific output of the Xenon lamp sources of standard

operating microscopes needed to be equated with the absorption

spectrum of the chosen photosensitising drug mTHPC. If

significant levels were found (>5j/cm2 in the red or blue

band)then it would be necessary to assess the viability of

microvascular anastomoses in sensitised and non-sensitised

animals, the non-sensitised group being a control group. All

other parameters would be kept exactly the same.223



The work was carried out at the Northwick Park Institute for

Medical Research, Surgical Research laboratories, Watford Road,

Harrow, Middlesex HA1 3UJ, with the assistance of Sandra

Simpkin, Senior Technician.

224



13c: Methodology

13c(i): Physics assessment

The output from a Zeiss operating microscope (OPMI 2) was

measured under standard operating conditions for rat femoral

artery and vein microvascular anastomosis, using a thermopile.

Output using incorporated red and blue filters was also

measured. This microscope is the standard instrument used

clinically for this procedure. Information regarding the

spectrum of the lamp emission was obtained from Osram, Forty

lane, Wembley, Middlesex. The absorption spectrum of mTHPC was

available from previous studies (M.F.Grahn, Surgical Unit, The

Royal London Hospital, Whitechapel, London E1 1BB).

13c(ii): Safety study

The adult Wistar rat was used. In order to determine whether the

wavelength of light delivered or the drug light delay was

affecting results, twelve different groups were planned:

Table 29

Drug-light interval (H) Light Group(N=5) Group(N=5)6 white PDT Control96 white PDT Control6 red PDT Control96 red PDT Control6 blue PDT Control96 blue PDT Control

13d: Drug

mTHPC was obtained from Scotia Pharmaceuticals, Guildford, U.K.,

as a gift. It was stored as a sterile powder in a fridge, and 225



reconstituted fresh with 1g ethanol and 1g polyethylene glycol

400 made up to 5 ml with sterile water for injection, to make up

a solution of 1.0 mg/ml. When reconstituting the drug for

injection, as usual meticulous attention was taken to ensure

that the drug had gone into solution. Invariably this meant 2 or

3 minutes of vigorous shaking, ensuring no residue was visible

on the bottom of the tube used for reconstitution.

13e: Animals

Male Sprague-Dawley rats in the weight range 450-600 grammes

were obtained from Charles River Ltd. (U.K.) and used

throughout. The experimental groups were given 1 mg/kg mTHPC and

0.5 ml of N-Saline via the lingual vein under Enflurane sedation

either 6H or 96H prior to microvascular anastomosis. The control

groups were given 1.0 ml N-Saline at the same time points. The

operation was performed under Hypnorm (intramuscular) and

Diazepam (intraperitoneal) anaesthesia.

The femoral triangle of the rat was exposed on one side. The

femoral artery and vein was dissected free from underlying

fascia and the profunda vessels were tied and divided.

A piece of plastic backing material was inserted under the

vessels (Picture 15) and a large damp swab was placed over the

surrounding tissue to protect the area from the potential

effects of the microscope light. This was in order to prevent

226



unnecessary collateral damage that might mask PDT induced damage

to the microvascular anastomosis, such as oedema, pain etc.

An ultrasonic doppler probe (Linton Instruments, Stowmarket,

Suffolk, U.K.) was used to objectively measure flow through the

vessels at 3 time points: On exposure, immediately post

anastomosis and after 80 minutes of microscope light exposure.

227



13f: Operative technique

The vessels were severed and then anastomosed using an ABB-1

Acland approximating clamp and 10/0 Ethilon (Ethicon Ltd)

sutures. Microscope light exposure was for 80 minutes in all

cases. The “white” light used was normal microscope light, “red”

and “blue” light was achieved by using filters set into

microscope filter holders. These were slotted into the

microscope so that the operating field was bathed in the

required light. The filters were RG-630 28mm x 3mm (red) and BG-

25 28mm x 3 mm (blue), both supplied by Schott. After 80 minutes

of light exposure the vessels were assessed for spasm and

patency, and their flow measured using the ultrasonic doppler.

13g: Postoperative assessment

The skin was closed and the animals recovered. Following this

they were closely observed for signs of anastomotic failure, in

particular leg swelling, footpad darkening, limping and general

condition. Close observation of the legs was carried out, in

order to detect any impending vascular insufficiency. Any animal

thought to be in difficulty was killed immediately and the

femoral area exposed to assess the state of the arterial and

venous anastomoses.

One week after the operation, the vessels were exposed and re-

assessed for patency and spasm in the surviving animals. The

flow rate was re-measured. The animals were killed and the

vessels were harvested and preserved in formol saline.,

228



13h: Results

Light source: Using the manufacturers data, the integral was

evaluated numerically between 350-720 nm, contributions beyond

these limits being negligible. The wavelength assessed was set

as 652 nm, the normal wavelength for mTHPC excitation. The

calculated value for the activation efficiency was 0.27.

The total focused output of the microscope was measured with the

thermopile as approximately 800 mW. Using a hot mirror to

eliminate infra red emission, the visible output 350-720 nm was

measured as approximately 500 mW.

The spot diameter was 6.8 cm, an area of 36cm2. This gave an

irradiance in the visible spectrum of 14 mW/cm2 of white light.

Typical operating time was set at 80 minutes, giving a total

light dose of 62J/cm2. In terms of photons absorbed (and

therefore photosensitiser activation) this is equivalent to

approximately 17J/cm2 of 652 nm light.

Through the RG630 longpass filter (Schott), the relative

activation of the lamp emission compared to monochromatic light

at 652 nm is 0.23. The power output using this filter was 170

mW, giving a red light dose of 23 J/cm2, equivalent to 5J/cm2 of

monochromatic 652 nm light for the 80 minute exposure.

The blue filtered light using the RG25 bandpass filter (Schott)

gave a relative activation figure of 0.55. The power output in

this spectrum was 30mW/cm2, giving a blue light dose of 4J/cm2

for the 80 minute exposure, equivalent to 2J/cm2 of 652nm red

light in terms of photon absorption. The results are displayed

in Graph 5.229



Graph 5

230



Table 30: Doppler flow results pre- and immediately post-

microvascular anastomosis

Group Pre -

Artery

Pre -

Vein

Imm -

Artery

Imm-

Vein

Change

-

Artery

(%)

Change

- Vein

6 C W 9.5/1.8 5.7/1.3 4.1/0.6 2.3/0.5 -46 -596RXW 7.4/0.8 5.3/0.8 2.7/0.3 1.6/0.4 -62 -706CB 8.5/1.0 5.0/1.8 6.9/1.7 4.3/1.5 -19 -146RXB 10.2/1.2 5.7/0.8 5.1/0.6 2.6/0.7 -50 -546CR 8.2/1.5 4.0/0.2 3.5/0.6 2.3/0.5 -66 -426RXR 6.5/0.8 4.8/0.9 4.6/0.8 3.0/0.9 -29 -3896CW n/a n/a n/a n/a96RXW 5.3 2.6 4.2 2/0 -21 -2396CB 8.4/0.8 5.8/1.1 6.2/1.5 3.5/0.9 -26 -3996RXB 9.4/1.0 6.7/1.2 5.7/1.3 2.5/1.4 -40 -6396CR 8.7/1 5.1/.4 7.7/1 4.8/0.5 -11 -696RXR 9.1/1.2 5.1/0.7 9.1/1 5.2/0.8 0 +2

C = control animal

RX = photosensitised animal

W = Unfiltered white microscope light

B = Blue only (filtered) microscope light

R = Red only (filtered) microscope light

The figures relate to the mean flow rate (1st) at each assessed

point and the standard deviation of this figure (2nd).

231



Table 31 Observations at experimental end (80 minutes illumination) and comments at

death (where possible all animals were kept alive until 7 days postop)

Group Obs. 80min Obs. 24H Outcome Sur

v

Comments at harvest

6 C W A+VP FD All well End expt 7 All normal6RXW 5/5 V.Thromb, AP 3/5 dead,

others

moribund,

swollen

All died 9H Thrombosis,oedema,

haemorrhage

6CB A+VP,FD All well End expt 7D All normal6RXB A+VP5/6, 1 V

thromb

All well End expt 7D Scar tissue++, 1xV

thromb6CR A+VP, FD All well End expt 7D All normal6RXR A+VP, FD 2/5 dead,

others

moribund,

swollen

All died 9H AP, 1/5V thromb,

oedema

96CW A+VP, FD All well End expt 7D All normal96RXW 1/5 V spasm,

A+VP, FD

1/5 dead, 4/5

Swollen groin

3/4 died, 1/5

end expt

7D AP, V thromb, oedema,

muscle necrosis.96CB A+VP, FD All well End expt 7D All normal96RXB 4/5 V spasm,

AP,FD

All well End expt 7D AP, 2/4 V.Thromb, scar

tissue++96CR A+VP, FD 1 dead, others

well

End expt. 7D All normal

96RXR A+VP, FD 5/5 swollen

groin

End expt 7D 1/5 v thrombosis, all

swollen

C = control animal D = Days post op

232



RX = photosensitised animal H = Hours post op

W = Unfiltered white microscope light Thromb = Thrombosis

B = Blue only (filtered) microscope light V = vein

P = patent as assessed R = Red only (filtered)

microscope light

A = Artery FD = Fully dilated

233



Table 32

Doppler flow results pre- and 80 minutes post- microvascular

anastomosis

Group Pre

-A

Pre

- V

80min

A

80min

V

Change -

artery (%)

Change -

vein (%)6 C W 5.7/

1.3

10.3/

0.8

5.4/1

.1

+8 -5

6RXW 7.4/0

.8

5.3/

0.8

5.6/0

.4

2.0/0

.3

-24 -62

6CB 8.5/1

.0

5.0/

1.8

9.8/0

.6

7.4/1

.9

+15 +48

6RXB 10.2/

1.2

5.7/

0.8

10.1/

1.3

5.6/1

.9

+1 -1

6CR 8.2/1

.5

4.0/

0.2

11.9/

1.8

6.5/1

.6

+45 +63

6RXR 6.5/0

.8

4.8/

0.9

7.8/1

.9

4.6/0

.9

+20 -4

96CW96RXW 5.3 2.6 6.7 0.5 +26 -8096CB 8.4/0

.8

5.8/

1.1

11.4/

2.5

7.2/1

.9

+35 +24

96RXB 9.4/1

.0

6.7/

1.2

8.7/0

.7

3.1/1

.5

-7 -53

96CR 8.7/1 5.1/

.4

11/1.

1

8/1.8 +26 +57

96RXR 9.1/1

.2

5.1/

0.7

11/0.

6

6.9/0

.6

+21 +35

234



C = control animal





Numbers are (mean flow)/(standard deviation of results)

235



Table 33

Doppler flow results pre- and 7 days post- microvascular

anastomosis

Group Pre

-A

Pre -

V

7 day-

Artery

7 day-

Vein

Change -

artery (%)

Change -

vein (%)6 C W 9.5/1

.8

5.7/1

.3

2.3/0.2 1.5/0.3 -76 -74

6RXW 7.4/0

.8

5.3/0

.8

All died All

died6CB 8.5/1

.0

5.0/1

.8

1.8/ 1.4/ -79 -72

6RXB 10.2/

1.2

5.7/0

.8

4.1/0.8 2.3/0.8 -59 -59

6CR 8.2/1

.5

4.0/0

.2

5.1/1.5 3.2/0.8 -38 -20

6RXR 6.5/0

.8

4.8/0

.9

All died All

died96CW n/a n/a 3.3/ 2.5/96RXW 5.3 2.6 *3.6/ *0.6/96CB 8.4/0

.8

5.8/1

.1

5.1/1.8 3.0/0.4 -31 -48

96RXB 9.4/1

.0

6.7/1

.2

3.4/ 1.2/ -64 -82

96CR 8.7/1 5.1/.

4

3.4/ 1.5/ -49 -70

96RXR 9.1/1 5.1/0 2.3/ 1.1/ -75 -78

236



.2 .7

* only one surviving animal




C = control animal


Numbers are (mean flow)/(standard deviation of results)

237



13I: Statistical Analysis

The entire range of results were compared within PDT groups and

against control groups. The significant findings were as

follows:

1) Survival to 7 days following the experiment

Alive 7 days Dead 7 days TotalControl 29 1 30PDT 18 12 30Total 47 13Chi2=11.88, p=0.00057

PDT significantly reduces the survival to 7 days following the

experiment. Within the PDT groups there was no significant

difference between colour of light, although by combining red

and white colours and comparing with blue, a significantly worse

prognosis was indicated (Fisher’s exact test, p=0.001). Low

numbers of animals in each group meant that it was not possible

to find a significant difference between individual colours,

although it was found that colour made a significant difference

to the death rate (Chi2 test, p=0.004). In order to gain a

statistically significant result, it can be calculated that if

alpha = 0.05, and beta = 0.2, predicting a red/blue 95% survival

and a white 60% survival, 21 rats would be needed in each group,

instead of the 10 here.

2) Effect of drug-light interval

6 hours 96 hoursAlive 7 days 5 13 18Dead 7 days 10 2 12Total 15 15 30

238



Using a Fisher’s exact test the probability of the results being

randomly distributed is 0.008, ie the result is significant that

survival is decreased in the 6H group.

239



3) Effect of PDT on flow rate immediately post anastomosisnb. In all cases no significant changes were seen in flow rate with any of

the control groups.

Colour Mean Std. Dev. FrequencyWhite 2.94 0.82 6Red 6.87 3.01 10Blue 5.36 2.33 10Total 5.38 2.77 26

Performing an analysis of variance on these results, Between the

groups there was a statistically significant difference in flow

immediately after anastomosis, p=0.01, with white lower flow

than blue, blue lower than red.

4) Prediction of survival

The flow rate in arteries immediately after anastomosis in the

PDT group significantly predicted survival, ie, in those

surviving, this value was significantly higher at measurement:

Mean Std. Dev. FrequencyAlive 7 days 6.01 2.89 40Dead 7 days 3.80 1.56 11Total 5.53 2.80 51

Performing an analysis of variance, p=0.019 that these results

are significantly different

The flow rate in veins was not significant for this, however,

the flow rate in veins 80 minutes after the start of the

experiment did show a significant prediction for non-survival to

7 days:240



Alive 7 days 6.03 3.75 40Dead 7 days 3.35 2.73 11Total 5.45 3.70 51

Performing an analysis of variance the probability of this

result being significant is 0.032

241



The same is true for the arterial flow at 80 minutes:

Mean Std. Dev. FrequencyAlive 7 days 10.46 3.04 40Dead 7 days 6.91 3.11 11Total 9.69 3.36 51Performing an analysis of variance the probability of this

result being significant is 0.001

5) Effect of colour and drug-light interval on arterial flow

immediately after the anastomosis:

Anova test-

Partial

ss

df ms f p

Colour 34.89 2 17.44 3.88 0.036Time 35.26 1 35.26 7.85 0.01

Thus both colour and drug light interval have a significant

effect on this measurement, although using the same test, trying

to determine whether colour was important at both time

intervals, no significant difference was seen (Anova test,

p=0.06 for 6 hours, p=0.12 for 96 hours).

6) Effect of colour on flow immediately after anastomosis:

Oneway test- analysis of variance

ss df ms f pBetween

gps

58.92 2 29.46 4.21 0.02

242



Thus colour affects the flow through the anastomosis

significantly, white worse than red worse than blue

243



13J: Discussion

Little work has been done on the effect of clinic PDT on the

healing of reconstructed tissue following cancer surgery,

although there is evidence that abdominal bowel anastomoses are

not significantly damaged by PDT (Allardice 1992, Ansell 1996).

Work by Kubler et al in 1996 demonstrated damage to skin suture

lines in a pedicled myocutaneous flap reconstruction model. All

of these studies related to activating red or green light,

rather than the effects of ambient light. The previous study

(Experiment 4) has demonstrated the high sensitivity of small

veins to the effects of laser induced photodynamic therapy.

Usually they would be relatively unimportant, except in the

situation where they provide the sole drainage for a free flap

of skin with or without muscle and bone, as commonly used in the

reconstruction of Head and Neck defects following the excision

of malignancy. As previously stated, damage in this situation

could be catastrophic. Only one other study has mentioned the

use of free flaps such as these in sensitised patients (Biel

1996). This was using the drug Photofrin 2, no damage was

reported. No preclinical assessment such as in this study has

been carried out with any photosensitising drug, to our

knowledge.

Our own experiences with Photofrin on small vessels has however

shown significant damage 3 days postoperatively, at a light dose

of 100 J/cm2, 100 mW/cm2 irradiance (see previous experiments).

244



This was one of the reasons this entire series of experiments

has been mostly devoted to mTHPC.

However, the toxic effects of photofrin PDT on microvascular

anastomoses is mitigated by the fact that the net amount of

light given using a standard operating microscope is less than

20 J/cm2 in the red spectrum, as demonstrated in the first part

of this experiment. This is a small amount of light for an

inefficient photosensitiser such as Photofrin. We have not

however explored the effect of green light on microvascular

anastomoses, a part of the spectrum where there is a high

absorption peak for Photofrin, and where significant amounts of

activation may occur.

This study has instead concentrated on mTHPC, a powerful second

generation agent, for which 20 J/cm2 of activating red light is

a very significant dose, indeed, the therapeutic dose for Head

and Neck cancer treatments (Dilkes et al 1995).

245



In performing the assessment of amount of light in different

parts of the emitted spectrum of the microscope lamp, three

different levels were measured. The first, and most significant,

was the total amount of activating energy contained in the

unfiltered (except for infra-red) white microscope light. This

is the light that would be used in the clinical situation. On

linking the spectrum of the bulb with the action spectrum of

mTHPC, a relative activation efficiency can be calculated, in

the white light measurements this was the equivalent of 17 J/cm2

of monochromatic 652 nm red light for an 80 minute exposure, the

peak activating wavelength of mTHPC in the red spectrum. The 80

minute exposure relates to an approximate illumination time of

vessels undergoing microvascular anastomosis, and is the total

figure for vein and arterial anastomosis (40 minutes each). The

figure does not take into account other ambient operating light

that may also cause a damaging effect, particularly when raising

the flap prior to microvascular anastomosis.

The measurements were repeated using blue and red filters. This

was in order to assess the wavelength that was most critical in

causing damage to microvascular anastomoses in the next

experiment. As would be expected when light is being filtered

out, the relative activation figures were reduced in these

cases, down to 5J/cm2 for the red filter, 2 J/cm2 for the blue

filter.

The next part of the experiment was performed to assess the

sensitivity of the vessels in the microvascular anastomosis to

the effects of this light. Levels of mTHPC were undetectable in246



the abdominal aorta vessel of rats in the pharmacokimetic study

(experiment 1). No assessment was made of venous levels however,

where subsequent experimentation (Exp 4) showed significant

damage at higher light doses, although little effect at around

20 J/cm2. In these situation the drug-light interval was set at

96 hours, since this and other studies had shown this period of

time to be beneficial in terms of effect and selectivity (Exp.

1, Ris et al 1993). However, there is evidence to suggest that

high vascular levels occur in the first stages of

biodistribution following intravenous injection. This is

unsurprising since it is the vascular compartment of the

extracellular fluid that the drug is delivered to. This effect

falls off rapidly after the first 6 hours post-injection (Ronn

et al 1996). Two time points for the experiment were therefore

used, an early phase at 6 hours post injection, and a late stage

96 hours post injection. Control animals were selected at

randomn, and given an equivalent volume of drug diluent with no

sensitiser to prevent possible vascular overloading being an

independent variable. The 96 hour DLI was in keeping with

clinical studies, and the most critical time point, since this

is the stage at which vessels would be illuminated in the

clinical situation. The Linton Instruments doppler probe had

been used by us previously, and found to be a reliable method of

assessment of small vessel flow (exp 4). In this study it was

used for all cases, except early controls (96 hour DLI, white

light) when it was not available. The results comparing the pre-

and immediately post microvascular anastomoses show a fall in

measured blood flow in all groups. This is probably due to mild 247



stricturing of the vessels due to the anastomotic sutures. No

significant differences occurred between PDT with any of the

variables, and the controls. Once 80 minutes of total

illumination had occurred, an increase in blood flow was seen

when compared to the pre-anastomosis figures, for the control

(all groups) and arterial PDT groups. This is presumed to be

due to local and systemic inflammatory mediators causing vessel

dilatation, with some stretching of the anastomosis. The venous

PDT groups show a persistent fall in flow when compared to the

venous controls, a finding that is statistically WHAT??. The

effect is most pronounced in the white light groups, less in the

red and least in the blue filtered light groups. The range of

some of the results means statistical significance is not

reached for this effect, and this is due to the difficulty

maintaining a correct Doppler reading during the whole

experiment. The results comparing pre- and 7 days post

anastomosis are largely unimportant, since by this stage those

animals in whom a significant effect had been seen were dead.

The most clinically significant results were seen in the table

relating to observed findings, and death of animals. All of the

control animals survived to the experimental end point at 7 days

post anastomosis, when they were killed and the vessels

examined. In all these animals the vessels, anastomoses and

surrounding tissue were normal.

In the 6 hour DLI PDT group, damaging effects were seen in all

groups, the majority in the white light group, less in the red

light group and the least in the blue light group. In these

cases, observation at 80 minutes showed significant venous 248



thrombosis, with no arterial damage. 24 hours post anastomosis

the animals were re-assessed, further observation revealed

significant groin swelling particularly in the white light

group, 6/15 animals had already died. Post mortem examination

showed severe thrombosis, haemorrhage and oedema of the veins.

The arteries were undamaged. These findings were confirmed in

subsequent post mortems of those that survived beyond 24 hours.

249



In the 96 hour DLI PDT group, these findings were mirrored,

although the severity was less. Despite this only one of the

white light PDT group survived to 7 days postop, at post mortem

the venous anastomosis was thrombosed. Again in all cases the

arterial anastomosis was undamaged at post mortem, venous

thrombosis was present in 1/5 in the red light group and 2/5 in

the blue light group. In the situation where post mortem

examination at 7 days revealed venous thrombosis, collateral

circulation may have saved the animal.

Some attempt can be made to mask vessels from the effect of the

microscope light whilst they are not being anastomosed, for

example by hiding them behind moist black blotting paper. In

practice both vessels tend to be very close during surgery, and

this would be difficult to achieve. Although using filtered

light undoubtedly reduced the failure and damage rate of the

anastomoses, in practice performing surgery in these conditions

was difficult even in experienced hands, and was not felt to be

advisable in the clinical situation

250



14: Experiment 6 - Histological study of large diameter arteries

undergoing photodynamic therapy

14a: Description

This experiment was performed because the next experimental step

was to be human studies. Although the results of the arterial

studies had been convincing enough in rats, it was felt that

before human studies could be safely started, a human carotid

artery size vessel should be examined at the proposed human

drug, drug light interval, light dose and intensity for

confirmation of the safety seen in rats. The pig carotid artery

was chosen as the experimental target, since the pig has a

similar cardiac output to the adult human, around 5

litres/minute.

14b: Methodology

Suckling pigs of around 30 kg weight were used for the

experiment. They were injected with mTHPC 0.3 mg/kg

intravenously via an ear vein under sedation 96 hours prior to

PDT. After this they were kept in conditions of reduced

lighting. 96 hours later they were given a general anaesthetic

using halothane. The neck was opened on both sides. The carotid

artery was exposed after ligation of the external jugular vein,

division of the sternomastoid muscle and mobilisation of the

internal jugular vein. Black moist blotting paper was placed

medially to the vessel on both sides. One side was irradiated

with 20 j/cm2 red light at 652 nm wavelength, 100mW/cm2

intensity in 2 animals, the other side acted as a control. In 2 251



further animals the carotid vessels were emptied of blood by

holding the vessel between 2 rubber slings and milking the blood

out of the distal end using a vascular clamp. Light at the same

parameters as with the previous 2 animals was given unilaterally

on each animal. Following completion of the photodynamic

therapy, the area treated was marked with black silk sutures in

the muscle directly beneath the areas treated/exposed on both

sides. The skin was then closed and the animals recovered. 3

days later the animals were killed and the carotid vessels

removed for histological analysis.

14c: Results

No animal suffered unnecessarily as a consequence of the PDT to

its carotid artery. In particular there was no evidence of

cerebrovascular insufficiency. All animals were well prior to

the moment of death. The histological analysis revealed no

abnormal findings in PDT or control groups, in particular no

thrombosis or adventitial oedema was seen.

14d: Discussion

This study was performed to finalise safety work preclinically

prior to human studies. The 3 day time scale was chosen because

that is the point at which maximum damage had been seen in

experiment 3, where significant adventitial oedema had been

noted, although seemingly no compromise to blood flow occurred.

In this case there was no damage seen at all, on histological

analysis at this time point. This might be expected since the

vessel is 10x wider in diameter in the pig as compared to the 252



rat, thus it would be difficult for light to penetrate through

to the vessel wall opposite the light - incident surface.

Therefore any damage seen could be expected to be on the light

incident side only. In fact no damage was seen. This is in

contrast to a study performed in Switzerland on the same model,

which anecdotally (Altermatt H.B., pers. Comm. 1995) showed

internal carotid artery thrombosis at these PDT parameters,

particularly when the vessel was emptied of blood, as in this

study. Emptying the vessel of blood removes a significant part

of the light absorbing effect of the larger vessels, since

oxygenated haemoglobin is an important chromophore in the

absorption of light at this wavelength. Therefore it might be

expected that more damage occurs. The fact that no thrombosis

occurred in our experiment suggests that either we were not

adequately removing blood from the irradiated segment, or that

Altermatt and co. were over zealous in doing this, damaging the

intima so leading to thrombosis. The drug dose given, 0.3 mg/kg

was also significantly smaller than the 1.0 mg/kg given to the

rats. However, the rules of allometry suggest that this was a

fair relative dose (Paxton, 1995). The drug-light interval was

the same as in the rat study (96 hours). The light dose was 20

J/cm2, which equals the lowest level used in experiment 3,

although significant damage did still occur at that level in the

rats. Irradiance was the same in both studies (100 mW/cm2).

Other studies have looked at the effect of PDT using other

photosensitisers on larger vessels than rats. In particular,

work by Grant et al (1993) showed that the bursting pressure of

rabbit aorta’s was unaffected by PDT at time intervals up to 12 253



weeks post irradiation. Other safety studies, particularly

looking at PDT irradiation of the pig pelvis, showed no serious

damage to the common iliac vessels, although this was not

examined in depth (Allardice et al 1992).

254



15: Adjunctive Intraoperative Photodynamic Therapy for Head and

Neck Cancer

Having gained experience with the second generation

photosensitiser mTHPC on palliative and early cases of Head and

Neck cancer with very promising results, particularly with the

treatment of early malignancy (Pictures 16 and 17), we report

here our experience when using this treatment adjunctively, in 4

cases, further to the results of the previous 5 experiments. The

rationale and safety aspects are covered in the preceding

chapters.

255



Picture 16: Pre photodynamic therapy picture of an early

squamous cell carcinoma of the soft palate, with surrounding

leukoplakia.

256



Picture 17: 4 weeks post photodynamic therapy, no residual

disease was found on biopsy. 4 years on, no local recurrence has

occurred.

257



15a: Method

mTHPC (Temoporphyrin, Foscan) was obtained from Scotia

Pharmaceuticals, Guildford, U.K. It was stored as a sterile dry

powder in a fridge, protected from light. Solution for injection

was made up within 1 hour of delivery, by reconstituting the

powder with its solvent (1g ethanol and 1g polyethylene glycol

400 made up to 5 ml with sterile water for injection) to achieve

a final concentration of 4.0 mg/ml. The solution was filter-

sterilised as it was injected slowly over 5 minutes, a dose of

0.15 mg/kg body weight being given in all cases..

The drug-light interval was 96 hours in all cases, injection

therefore occurring 96 hours before the day of surgery. Patients

were fully consented regarding this additional treatment, and

particular care was taken to warn them regarding the risks of

exposure of the skin or eyes to direct sunlight for the first 4

weeks following drug injection, since to do this would risk a

serious photochemical skin burn. A full surgical resection of

the proposed site was performed, taking meticulous care to

ensure none of the patient’s skin was exposed to the operating

or theatre lights for a significant period of time. The skin

flaps were raised first, using ambient theatre light instead of

operating lights for illumination, and then folded back on

themselves, sutured in this position with 3/0 silk sutures, and

then covered by moist green cotton operating drapes, clipped to

the exposed edge. Betadine skin preparation was used prior to

surgery, its dark brown colour was felt to be helpful in further

absorbing ambient light during the initial phases of surgery 258



when skin would be exposed to operating theatre lights. The

pulse oximeter as routinely used by the anaesthetic staff was

not used for continuous monitoring, only on an “as required”

basis. This is because the red light emitted by these devices is

at around 630nm, and does activate the photosensitising drug

used here, mTHPC, albeit to a lesser extent than more commonly

used drugs such as Photofrin 2. Once the surgical resection was

complete, the patients were wheeled around to the laser room

still under general anaesthetic, if the Copper Vapour pumped dye

laser was used (Oxford Lasers, Abingdon Rd., Oxford, U.K.), or

treated in the operating theatre if the KTP pumped dye laser was

used (Laserscope, Raglan House, Cwmbran, Wales, U.K.). In order

to treat a uniform area, circular spots produced by the

microlens used (QLT, Vancouver, Canada) were converted to a

square of known area using a black anodised template (see

picture 18). Treated areas were marked with small dots of Evans

Blue dye to prevent missing out areas, and to make sure no over-

treatment occurred. Other methods of shielding treated and

untreated tissue included modifying a steridrape by cutting the

paper backing into uniform squares (Picture 19) and removing one

square at a time in a sequential manner, replacing each square

once the area underneath had been treated (Picture 20).

259



Picture 18: In order to prevent overlapping or under treatment,

the circular spot from the microlens is turned into a square

treatment area.

260



Picture 19: A steredrape is modified by sticking a grid of

squares over the irradiation site

261



Picture 20: Light is then delivered to the square with one area

removed, this is then re-applied and the next square treated

262



Light at 652nm was delivered at 100 milliwatts/cm2 to a total

light dose of 20 joules/cm2 in all cases, over the entire

operative bed. We had previously found this to be an effective

level of light preclinically for AIOPDT and clinically for the

successful destruction of early malignant tumours of the Head

and Neck (Dilkes and DeJode 1994). The treatment time for each

spot was therefore 200 seconds. Using a 2cm x 2cm square (4cm2

area), the spot diameter was 2.8 cm. To treat the entire

operative bed of a radical neck dissection in this manner with

an area of approximately 200 cm2 would therefore take 10,000

seconds, just under 3 hours. In practice the more powerful laser

systems such as the KTP-pumped dye laser can treat up to 70 cm2

in one 200 second period, so the entire area can be treated in

600 seconds, 10 minutes. Thus the spot size is varied depending

on the output of the laser at 652 nm, and the area required to

treat. During PDT any structure deemed unsuitable for laser

light exposure can be masked using black absorbent gel (Black

stuff, Scotia Pharmaceuticals). For practical reasons in this

study this meant any exposed skin edge. Following completion of

photodynamic therapy, the usual drains were inserted, and skin

closure was completed with clips to save exposed skin from

ambient light damage, clips being quicker to insert than

sutures. During the in-patient period, standard precautions were

taken against sunlight exposure, the patient was not allowed

outside between 8 a.m. and 8 p.m., all light bulbs were replaced

with 60 watt bulbs, the windows were covered with black paper,

sellotaped to the glass. Baths and normal use of the toilet was

allowed, for up to 30 minutes per day. The same instructions 263



were given on discharge for 4 weeks post injection. At this

stage the patients were advised to place their hand in bright

light for several minutes. If tingling occurred, this meant

significant levels of sensitiser were still present, and the

patient was asked to wait 48 hours before trying this again.

264



14b: Results

14b(i): Case #1

A 55 year old man presented initially to another Hospital with

throat pain and difficulty swallowing. Full ENT examination

revealed a 3 cm x3 cm infiltrating mass in the tongue base,

subsequent pan-endoscopy showing this to be an isolated

squamous cell carcinoma, moderately differentiated, stage T3 N0

M0. Radical external beam radiotherapy was used to treat the

tumour, a dose of 2880 centigray (cGy) being given to both sides

of the neck and tumour, followed by a further 3000 cGy to the

tumour area alone. Routine follow up showed no symptoms or signs

of recurrent disease until, 12 months later, he developed a

painful mass deep in front of the right ear, with an associated

facial nerve palsy. At this stage he was transferred to our

Unit, where further pan-endoscopy showed no recurrence of tumour

at the primary site, but fine needle aspiration cytology of the

mass showed cells of squamous cell carcinoma. A radical

parotidectomy and neck dissection in continuity was performed,

AIOPDT was given to the upper half of the operative bed,

including the carotid bifurcation and tree. Facial reanimation

with temporalis fascia slings was performed by the plastic

surgeons. Postoperative progress was uneventful, he was

discharged home 7 days after surgery with no skin reaction from

PDT, drains having been removed at 48 and 72 hours, as per

usual. Six weeks after surgery, he died suddenly at home. A post

mortem revealed a massive pulmonary embolus from a pelvic deep

vein thrombosis that had been asymptomatic, a 3 cm x 3 cm

parotid gland node was found to be infiltrated with squamous 265



cell carcinoma. All other nodes in the specimen were clear. With

consent from the next of kin, both treated and untreated (right

and left respectively) carotid arteries were removed and

examined histologically with Haematoxylin + Eosin (routine), Van

Geesen (Collagen) and MSB (Fibrin) stains. No changes

attributable to PDT were found (see picture 21), although some

intimal hyperplasia and atheroma were noted on both sides,

thought to be due to age and previous radiotherapy.

266



Picture 21: Carotid arteries from post-mortem specimen, the

arrow points to the side that had photodynamic therapy. Intimal

hyperplasia can be seen on both vessels, there are no changes

specifically attributable to PDT.

267



14b(ii): Case #2

A 67 year old man presented with a 3 month history of a hoarse

voice. Indirect laryngoscopy revealed a keratinising lesion

confined to the mid-vocal cord on the left side. This was shown

at panendoscopy and biopsy to be a well differentiated squamous

cell carcinoma. Clinical and radiological staging was T1 N0 M0.

A course of radical radiotherapy was given, 5,500 cGy in 20

fractions over 4 weeks. Rapid regression of the tumour was noted

at monthly follow up. 10 months later he presented routinely

with an 8 x 6 cm hard mass arising from the mid-jugular area,

that had come up over the previous month. Needle aspiration and

panendoscopy revealed this to be a metastatic node from the

laryngeal primary, no further tumours being found. A radical

neck dissection was performed with AIOPDT given to the operative

bed, since clinical examination was suspicious of the tumour

involving the carotid tree. At operation this suspicion was

confirmed, tumour had to be peeled off the carotid artery below

the adventitial plane, with a high possibility of residual

tumour in the operative bed. Postoperatively no complications

specific to PDT were encountered. Less than 4 weeks after

surgery the skin on the treated side of the neck became

thickened and tender, biopsy showed carcinoma en cuirass.

Despite additional radiotherapy and chemotherapy the patient

died 2 months after this, from the effects of advanced disease.

268



14b(iii): Case #3

A 69 year old lady presented with a 2 year history of throat

discomfort. Indirect laryngoscopy revealed a 5 x 5 cm ulcerating

mass in the tongue base and vallecula. Panendoscopy and biopsy

showed this to be a moderately differentiated squamous cell

carcinoma, stage T3 N0 M0. This was treated with radical

radiotherapy, 2,500 cGy to neck and an extra 3,000 cGy to the

primary site. Check biopsy 2 months later showed persistent

tumour at the primary site, but no further spread. She turned

down a total laryngopharyngoglossectomy, but accepted a partial

laryngopharyngectomy with excision of the tongue base. AIOPDT

was given to the residual tongue resection margin. A radial

forearm free flap was revascularised locally and used to fill an

extensive anterior mucosal defect. Postoperatively a

pharyngocutaneous fistula developed, it was difficult to assess

the viability of the flap. No further adverse sequelae occurred

that might have been attributable to PDT. 6 weeks after surgery

a fatal carotid artery rupture occurred.

`

15b(iv): Case #4

A 39 year old man presented with a 4 week history of facial

pain, discharge into the roof of his mouth (antro-oral fistula),

and discharge onto the cheek skin (antro-cutaneous fistula).

Naso-endoscopy and biopsy revealed extensive polypoid disease

arising from the lateral nasal wall, shown on histology to be

poorly differentiated squamous cell carcinoma. Computerised

tomography scanning showed this tumour to have invaded the orbit269



and the pterygoid musculature. The clinical stage was T4 N0 M0.

Radical radiotherapy was given, 6,500 cGy to the whole area.

Biopsy 4 weeks after radiotherapy revealed persistent disease.

The was still no sign of metastasis. Radical surgery involving a

total maxillectomy, orbital exenteration and ethmoidectomy was

performed. 6 months after surgery tumour reappeared in the skin

near the nasomaxillary part of the incision. This was debulked

using the Nd-YAG and CO2 combination laser (Broomhead et al

1995), and the whole operative bed and lateral nose was treated

with AIOPDT. This area healed well, no complication due to PDT

occurred. Future tumour recurrence arose in the skull base which

was treated by additional radiotherapy, and chemotherapy. He

eventually died 6 months later of intracranial extension of the

disease.

15d: Discussion

No significant damage occurred that was attributable to PDT in

any case except number 3, when the microvascular anastomosis may

have been affected by the effects of white microscope light,

which we have shown to be possible in an animal model,

experiment 5 above. At this stage we do not recommend that

AIOPDT with mTHPC is attempted when a free flap is to undergo a

microvascular anastomosis. The results otherwise do not show

that tumour recurrence was reduced with AIOPDT, although in

cases 2,3 and 4 very advanced tumours were treated with a low 270



chance of long term survival (Sloan and Goepfert 1991). The

first case had no residual tumour in the site of treatment,

although only one node in the entire specimen was involved with

tumour, and local recurrence 6 weeks after treatment would be

unlikely in this situation anyway. The histological analysis of

the carotid arteries showed changes in keeping with age and

radiotherapy on both sides, no additional changes were seen on

the AIOPDT side. This was in keeping with the previous study

(experiment 3) in which short term (3 days) adventitial oedema

was seen in the femoral arteries, but no significant damage

occurred (Dilkes et al 1996b).

Local tumour recurrence after major head and neck surgery

depends on several factors, such as age and general condition of

the patient, site and extent of disease, pre or post operative

treatment with radiotherapy (Gilbert and Kagan 1974). When

considering the operation of radical neck dissection, the

incidence of local recurrence is further affected by the

presence of extracapsular spread of disease, and/or multiply

involved nodes (Snow et al 1982). The rate of local recurrence

in an N1 or N2 neck after radical neck dissection but without

radiotherapy is around 30% (DeSanto et al 1982). If local

recurrence occurs in a previously treated N+ neck, the salvage

rate is as low as 5% (Mendelsohn et al 1976). Therefore any

procedure that carries a reasonable expectation of increasing

this figure in a safe manner, is welcome. A prospective study is

needed with control (no AIOPDT) to show whether or not AIOPDT

is effective in the human situation.

271



16: Conclusion

This series of experiments has demonstrated the following facts:

mTHPC has a greater degree of tumour selectivity than Photofrin

2 when tested on the rat HSN Fibrosarcoma model, as measured

pharmacologically. The tumour necrosis validation was

unsuccessful. The peak drug-light interval for effect and

selectivity occurred between 96 and 144 hours post intravenous

injection.

The tumour model HSN Fibrosarcoma is a reliable and relatively

uniformly growing tumour with a doubling time of approximately

18 hours. PDT with mTHPC gives a statistically significant

reduction when compared to controls, in the local recurrence

rate after tumour excision and adjunctive intraoperative

delivery to the tumour bed, at an end point limited by

overwhelming tumour recurrence in each experimental series.

Safety studies at therapeutic levels used for adjunctive PDT,

and light doses above and below that showed no significant

damage to the femoral artery of rats, when compared to controls

(directly matched groups). Inconsequential adventitial oedema

occurred in the PDT groups only, 3 days post irradiation.

Ultrasonic Doppler blood flow measurements during and

immediately after PDT showed a significant reduction in flow

through veins, particularly the internal jugular vein, with no

effect on arteries or controls.

Measurement of the light output from operating microscopes used

for microvascular anastomosis showed significant relative

activation levels of light, particularly in unfiltered white272



light. This was found to cause severe compromise to the venous

anastomoses of sensitised rats both at 6 and 96 hours post drug

injection. No damage was seen in arteries, controls were

unaffected.

The pig common carotid artery was not affected by clinical doses

of PDT, even when emptied of blood.

Initial clinical studies have shown that adjunctive

intraoperative photodynamic therapy (AIOPDT) is safe for use in

the Head and Neck surgery performed without formal

reconstruction using tissue from elsewhere.

A prospective, controlled study is needed to assess the effect

of AIOPDT on a simple Head and Neck surgical model, where the

negative effects should be minimal. It is suggested that such a

model is the operation of radical neck dissection.

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