Prostate specific antigen (PSA)Measurand characteristics, reference measurement

procedures and reference materials for PSA

JCTLM SymposiumSevres December 14-15, 2004

Ulf-Håkan StenmanDepartment of Clinical Chemistry

Helsinki University Central HospitalFinland

PSA• Why is standardization of PSA assays

important?• Characteristics of PSA• Reference materials• Reference measurement procedures• Conclusions

Prostate cancer, basic factsProstate cancer, basic facts

Most common (nonMost common (non--skin) cancer in menskin) cancer in men•• LifeLife--time risk of acquiring prostate cancer 7 time risk of acquiring prostate cancer 7 -- 17%17%

Second most common cause of cancer deathSecond most common cause of cancer death•• LifeLife--time risk of death from prostate cancer 2 time risk of death from prostate cancer 2 -- 4% 4% •• 5050--70% of cases advanced when giving rise to symptoms70% of cases advanced when giving rise to symptoms

Preclinical stage during which early diagnosis and Preclinical stage during which early diagnosis and curative therapy are possiblecurative therapy are possiblePotential target for screening Potential target for screening

UU--H Stenman 2004H Stenman 2004

Clinical use of PSA• Sensitive marker for prostate cancer

– Expressed by more than 99% of all prostate cancers

– Serum PSA increased by 1 gram tumor

– Prostate-specific rather than cancer specific

• Used for– Monitoring of response to therapy

– Detection of relapse

– Evaluation of prognosis

– Early diagnosis and screening (case finding)

Prostatasyöpäpotilaan seuranta

20001999199819971996.1

1

10

100

1000S-

PSA

(µg/

l)

Castration, androgen suppression

Radiation

PSA during monitoring of a prostate cancer patient

Early detection and screening for prostate cancer with PSA

Pathophysiological basis

Serum PSA at sampling and time to presentationSerum PSA at sampling and time to presentation

0-2-4-6-8-10-120,1

1

10

100

1000

Time before presentation (yr)

PS

A (µ

g/l)

2,5

Stenman et al. Lancet 1994

Prostate cancer incidence and mortality in Finland 1958-2001

20102000199019801970196019500

500

1000

1500

2000

2500

3000

3500

4000

IncidenceMortality

Year

Incidence and Mortality

Use of PSA and prostate cancer incidence in Norway

Prostate cancer incidence and mortality in Finland 1958-2001

20102000199019801970196019500

500

1000

1500

2000

2500

3000

3500

4000

IncidenceMortality

Year

Incidence and Mortality

Survival of cancer patients and serum PSA at samplingSurvival of cancer patients and serum PSA at sampling

2015105000,0

0,2

0,4

0,6

0,8

1,0

<4 µg/l>4 µg/l

Time (years)

PSA cut-off

Stenman et al. Lancet 1994Stenman et al. Lancet 1994

Development of prostate cancer, screening and age

PSA PSA (µg/l)(µg/l)

Lethal stageTumor vol 1 kg

Clinical diseaseTumor vol 32 g

Curable stageTumor vol 1 g

UU--H Stenman 1995H Stenman 1995

1009080706050401

10

100

1000

10000

Age (years)

Are we doing something wrong?• Are we over-diagnosing?• Yes, in 75% of those over 70 years of age• Are we finding the patients that need to be

cured?• Not all, 35% of patients with clinically localized

tumors experience a relapse• We need to improve our diagnostic methods

and procedures– Even earlier diagnosis– More cancer-specific methods– Avoid detection of clinically indolent tumors– Prognostic methods

Characteristics of measurand (PSA)• 30 kDa chymotrypsin-like serine proteinase

• Produced by epithelial cells of the prostate

• Prostate specific, not cancer specific

• Dissolves seminal clot by degrading semenogelin• PSA in plasma is heterogeneous

– Complexes with protease inhibitors– Partially degraded forms and proforms of free

PSA– Crossreacting substances

• hK2

Gel filtration of PSA in prostate cancer serum

7060504030200

1

2

3

4

5

6

7

8

PSA (µg/L)

Fraction no.

PSA

(µg/

L)

30 kDa

90 kDa

Stenman et al. J Nucl Med Allied Sci 1990

Assays for free PSA and PSA-ACT

Eu

PSA PSA

ACT

Sm

Forms of immunoreactive PSA in prostate cancer patients and men with BPH

PCa Controls• PSA-ACT 86% 79%• Free PSA 13% 19%• PSA- API (-AAT) 1% 2%• hK-2 1.5% 1%

• PSA-A2M 2% 4%

67 –43 –

30 –

20 –

14 –

67 –43 –30 –

20 –

14 –

M HIC GF A B C D EM HIC GF A B C D E

kDa kDa

SDS gel electrophoresis of PSA isoenzymes from seminal fluid separated by ion exchange chromatography

Nonreducing conditions Reducing conditions

10080604020200

100

200

300

400 PSA controlACT 24 hACT 240 h

Fraction no.

PSA (ng/mL)

A

B

C

D´ D E

F

PSA-ACT

Amino acid sequence of PSA

M WV PVVFLTL SVT WIGA APLILSR1 7 17 27 37 IVGG WE CEKHSQP W QV LVASRGRAVC GGVLVHPQ WV

LTAAHCIRNK 47 57 67 77 87 SVILLGRHSL FHPEDTGQVF QVSHSFPHPL YDMSLLKNRF LRPGDDSSHD 97 107 117 127 137 LMLLRLSEPA ELTDAVKVMD LPTQEPALGT TCYASGW GSI EPEEFLTPKK 147 157 167 177 187 LQCVDLHVIS NDVCAQVHPQ KVTKFMLCAG RWTGGKSTCS GDSGGPLVCN 197 207 217 227 237

Activation peptidein proPSA

-4 -2Signal peptide

SDS gel electrophoresis of PSA isoenzymes from seminal fluid separated by ion exchange chromatography

67 –43 –

30 –

20 –

14 –

M HIC GF A B C D E

kDaReducing conditions

10080604020200

100

200

300

400 PSA controlACT 24 hACT 240 h

Fraction no.

PSA (ng/mL)

A

B

C

D´ D E

F

PSA-ACT

A B C D E0

20

40

60

80

100Proportion of complex formation (%)

2-dimensional electrophoresis of free PSA in serum and isoelectric focusing of PSA isoenzymes isolated from seminal fluid

Isoelectric point

2-D electrophoresis of serum PSA

Charrier et al. Electrophoresis 20: 1075-81, 1999

Isoelectric focusing of seminal plasma PSA

- 8.15 -

- 7.35 -

- 6.85 -

- 6.55 -

A B C D E pI

Zhang et al. Clin Chem 41:1567-73 1995

Development of PSA standards• Stanford conferences organized by Thomas Stamey in

1992 and 1994

• Preparation of standards for

– Free PSA

– PSA-ACT and free PSA 90/10 mixture

• Establishment of WG on PSA standardization

• Adoption of standards as WHO reference materials

– WHO 96/668 (free PSA)

– WHO 96/670 (PSA-ACT/PSA 90/10)

Rafferty et al. Clinical Chemistry 46: 1310-1317, 2000

Development of PSA standards• Purification of PSA from seminal plasma

• Value assignment by amino acid analysis (mol)

• Determination of MW by mass spectrometry, 28430

• Calculation of mass concentration

• Formation of PSA-ACT in vitro

• A280 of PSA-ACT based on AA compostion = 1.0– Assignment of mass concentration for PSA-ACT

• Preparation of PSA-ACT-free PSA 90/10 mixture

Prestigiacomo AF, Chen Z, Stamey TA. A universal calibrator for prostate specific antigen (PSA). Scand J Clin Lab Invest Suppl 1995;221:57-9.

Figure 4. Potency of human serum sample (97/568) in individual assays calculated using IHR (A) and PSA 90:10 (96/670; B)

Figure 5. Potency of human serum sample (97/566) in individual assays calculated using IHR (A) and PSA 90:10 (96/670; B).

Epitope mapping of PSA

Summary Report of the TD-3 Workshop: Characterization of 83 Antibodies against Prostate-Specific Antigen.Stenman U-H, Paus E, Allard WJ, Andersson I, Andres C, Barnett TR, Becker C, Belenky A, Bellanger L, Pellegrino CM, Börmer OP, Davis G, Dowell B, Grauer LS, Jette DC, Karlsson B, Kreutz FT, van der Kwast T, Lauren L, Leinimaa M, Leinonen J, Lilja H, Linton HJ, Nap M, Nilsson O, Ng PC, Nustad K, Peter A, Pettersson K, Piironen T, Rapp J, Rittenhouse HG, Rye PD, Seguin P, Slota J, Sokoloff RL, Suresh MR, Very DL, Wang TJ, Wigheden I, Wolfert RL, Yeung KK, Zhang W, Zhou Z, Hilgers J. Tumour Biol 1999; 20 (Suppl 1): 1-12.

Epitope mapping of PSA

AA 158-163 Region 3

AA 86-91 Region 1

AA 80-85 Region 2

AA 58-64 Region 5

AA 3-11 Region 6

Catalytically active site

AA 58-64

AA 3-11

1

5a

5b

5c

6a

6b 4b

4a

3b

3a

2a

2c

2b

AA 158-163

AA 86-91

AA 80-85

Stenman et al. Tumour Biol 1999;20 (Suppl 1):1-12.

Correlation HybritechTandem E -Wallac AutoDelfia before 2003

100101.1.1

1

10

100

Access T-PSA

Delfi

a T-

PSA

201510500

5

10

15

20

Access T-PSA

Delfi

a T-

PSA

y = - 6.5112e-2 + 0.88453x R^2 = 0.994

Recalibrated AutoDelfia vs. Access T-PSA (2004)

Intercept : -0.084 [ -0.198 to 0.000 ]Slope : 0.994 [ 0.970 to 1.020 ]

Passing-Bablok agreement test N = 1470 4 8 12 16 20

AutoDelfia0

4

8

12

16

20 Access

Recalibrated AutoDelfia vs. DPC Immulite 2000 T-PSA (2004)

Intercept : -0.050 [ -0.228 to 0.071 ]Slope : 1.352 [ 1.315 to 1.392 ]

Passing-Bablok agreement test N = 1470 5 10 15 20 25 30

AutoDelf ia0

5

10

15

20

25

30 Immulite 2000

Recalibrated AutoDelfia vs. Access F-PSA (2004)

Intercept : 0.033 [ 0.010 to 0.055 ]Slope : 0.963 [ 0.933 to 1.000 ]

Passing-Bablok agreement test N = 1600 2 4 6 8 10 12

AutoDelfia0

2

4

6

8

10

12 Access

Recalibrated AutoDelfia vs. DPC Immulite 2000 F-PSA (2004)

Intercept : -0.022 [ -0.037 to 0.001 ]Slope : 0.787 [ 0.767 to 0.808 ]

Passing-Bablok agreement test N = 1600 2 4 6 8 10

AutoDelfia0

2

4

6

8

10 Immulite 2000

Correlation F/T

0,80,60,40,20,00,0

0,2

0,4

0,6

0,8

AutoDelfia

Beck

man

Acc

ess

y = 8,7969e-3 + 0,95312x R^2 = 1,000

0,80,60,40,20,00,0

0,2

0,4

0,6

0,8

AutoDelifa

DPC

Imm

ulite

y = - 5,8281e-3 + 0,57812x R^2 = 1,000

Probability of prostate cancer in relation to total and free PSAExcel formula avavailable at: www.finne.info

35302520151050

10

20

30

40

50

60

70

80

PSA 4 µg/lPSA 7 µg/lPSA 10 µg/lPSA 20 µg/l

F/T PSA (%)

Prob

abili

ty o

f fin

ding

pro

stat

e ca

ncer

on

biop

sy (%

)

Proportion of free PSA and tumor gradeProportion of free PSA and tumor grade

Bangma J Urol 1997; 157: 544

0.0

0.1

0.2

0.3

0.4

0.5

0.6

BPH Grade 1 Grade 2 Grade 3

Equimolarity of assays for PSA

• Ability to detect free and complexed PSA equally

• Assays based on polyclonal antibodies tend to overestimate free PSA

Effect of proportion of F-PSA on ratio between Access and Delfia total PSA

Tandem-E vs. Delfia Beckman Access vs. Delfia

60504030201000,75

1,00

1,25

1,50

1,75

2,00

F/T (%)

Acc

ess/

Delfi

a

1008060402000,75

1,00

1,25

1,50

1,75

2,00

F/T (%)

Ratio

Acc

ess/

Delfi

a

Recognition of free PSA by Delfia and Access assays

Control containing >90% F-PSA

DelfiaT-PSA

DelfiaF-PSA

Access T-PSA

Sample 1 2.12 2.00 4.1

Sample 2 19.6 19.1 28.0

Recognition of various forms of free PSA by AutoDelfia and Beckman Access assays

AutoDelfia Access RatioF-PSA T-PSA F/T T-PSA Access/AD

PSA-A 96,5 92,2 104,6 114,9 1,25PSA-B 59,5 57,4 103,6 72,7 1,27PSA-C 41,7 40,3 103,6 53,9 1,34PSA-D 55,0 61,6 89,2 77,9 1,26PSA-E 50,6 52,9 95,5 63,8 1,21ProPSA 22,7 23,9 94,9 30,9 1,29

Mean 1,269

Equimolarity of assays for PSA

• Beckman Access uses the same antibodies as Hybritech Tandem E

• What is the difference?

• Assay kinetics– Small molecules react faster

Frequency of aberrant results in 20000 samples analyzed with two methods

Median ratio Access:Delfia = 1.14

Access value >2-fold Delfia 13/2651 0.49%

Access value >1.5-fold Delfia 49/2651 1.8%

Access value <0.67-fold Delfia 1/2651 0.00%

Access value <0.50-fold Delfia 0/2651 -

Comparison of Delfia and Access on samples with aberrant results and benign biopsy

Sample Total PSA Total PSA Free PSA (%)Delfia Access Delfia

A 1,55 18,2 26%B 0,71 5,7 22%C 0,92 6,1 24%D 1,28 5,2 20%E 2,15 4,8 24%F 5,62 12,2 21%G 5,20 30,7 31%

Effect of mouse serum on interference

Beckman AccessT-PSA

Wallac Delfia T-PSA

Wallac Delfia F-PSA

Routine assay 6.33 0.73 0.26

Addition of mouse serum

2.74 1.08 0.26

Conclusions from assay comparisons

• Assay manufacturers compete with speed, capacity and price

• Quality suffers• Who is responsible?• Customer?

– Capable of judging quality?• Manufacturer?• Regulatory bodies?

Reference measurement proceduresAre they necessary?

PSA assays from major manufacturers are unusually well standardized

Room for improvement

Quality of assays from minor companies varies

Stenman U-H. Immunoassay standardization: is it possible,

who is responsible, who is capable? Clin Chem 2001;47(5):815-20.

Is it possible?• Identification of reference antibodies

– Known epitope specificity– Generally available

• Definition of assay format– Sandwich assay– Microtitration well format, sample volume 25 ul– Two-step incubation, 1 + 1 h incubation time– Applicable to any detection method

• ELISA, Delfia, IRMA, luminescence

• Reference laboratories• Calibrated samples• Mass spectrometry?

Who is capable?• I know some• Endagered species• Are they willing?• Who will pay for it?

– Not ”sexy science” that you get grants for

Who is responsible?• WHO?• IFCC?• JCTLM?• We are all responsible• We need to justify the expenses• Calculation of additional health care costs

caused by poor assay quality• Poor quality is expensive• Someone has to take the lead