SPARC in Pancreatic Cancer

• utilises the properties of albumin to reversibly bind paclitaxel, and transport it across the endothelial cell and concentrate it in areas of tumor

• avoids the use of Cremophor EL, which contributes to serious toxicity (e.g. hypersensitivity, axonal degeneration) and requires special infusion tubing, premedication and prolonged infusion

• shows improved bioavailability and linear pharmacokinetics, whereas CrEL forms micelles entrapping the paclitaxel, leading to decreased unbound drug fraction, decreased drug clearance and lack of dose-dependent antitumour activity

Nab-paclitaxel properties

Solvent-based taxanes provoke formation of micelles in circulation

• Micelle formation in the circulation entraps paclitaxel in plasma

• Resulting non-linear pharmacokinetics contribute to a lack of dose-dependent antitumour activity

Aapro et al. EJC Suppl. 2008;6:3–11 Hamad et al. Expert Opin Drug Deliv. 2008;5: 205–219

Largemicelle

Control plasma Plasma + solvent-based paclitaxel

• The accumulation of paclitaxel in tumors was 33% higher for nab-paclitaxel compared with paclitaxel (P < .0001)1

nabnab -Paclitaxel Results in Higher Tumoral Uptake -Paclitaxel Results in Higher Tumoral Uptake Compared With paclitaxelCompared With paclitaxel

Desai et al. Clin Cancer Res. 2006; 12:1317-1324.

Hours

Pac

litax

el (

nC

i/g)

140

80

60

400.1 1 10 1000.01

120

100

Tumor AUC nab-paclitaxel =1.33 x Taxol

p < .0001 ANOVA

nab-paclitaxel

Taxol

AUC(nCi•hr/g)

3,632

2,739

Ka(hr1)

0.43

0.13

AUC, area under the curve; KA, absorption rate.

Tumor Uptake in Nude Mice Xenografts Following 20 mg/kg Dose of Paclitaxel

KEY STEPS in albumin-paclitaxel delivery to tumor

• Albumin initiates the endothelial transcytosis of paclitaxel by binding to a cell surface receptor: 60-kDa glycoprotein (gp60).

• In turn, gp60 associates with caveolin-1 resulting in the invagination of the endothelial cell membrane trapping the complex in vesicular structures called caveolae.

• Clustering of the gp60-albumin complex during vescicle formation reduces receptor affinity for albumin, which permits the release of albumin and any bound ligands to the abluminal side of the cell.

• Albumin accumulates in tumors, possibly due, in part, to the secretion of the albumin-binding protein SPARC (secreted protein, acidic and rich in cysteine, osteonectin or BM-40), which, in turn, may result in preferential intatumoural accumulation of albumin-bound molecules.

The Unique Properties of Albumin Improve the Risk to Benefit Profile of nab-Paclitaxel

• 130-nm sized albumin-paclitaxel complexes1,2

– nab-Paclitaxel is the first nanotechnology-derived agent approved for the treatment of breast cancer

– Albumin gives nab-paclitaxel linear pharmacokinetics3 = predictable drug exposure with dose modification

1. Desai et al. SABCS. 2004 [abstract 1071].2. Kratz et al. J Control Release. 2008;132(3):171-183.

3. Ibrahim et al. Clin Cancer Res. 2002;8(5):1038-1044.nab® is a registered trademark of Celgene Corporation.

Albumin

Paclitaxel

nab-Paclitaxel particle

2D Conceptualization

Mechanism of Action of nab-Paclitaxel

Investigation of the functional importance of SPARC with respect to nab-paclitaxel is ongoing.

SPARC, secreted protein acidic and rich in cysteine.

Albumin-Mediated Transcytosis of Paclitaxel

SPARC, Secreted Protein Acidic and Rich in Cysteine.

Endothelial cells

Tumor cells

Subendothelial space

Investigation of the functional importance of SPARC with respect to nab-paclitaxel is ongoing.

MDA-MB-231

0 10 20 30 400

500

1000

1500

2000

2500

3000

SalineAbraxane 15 mg/kg q4dx3

Days

Tu

mo

r V

olu

me (

mm

3)

Low Medium HighSPARC SPARC SPARC(TGI = 36%) (TGI = 60%) (TGI = 81%)

SPARC level in heterogeneous tumors affects SPARC level in heterogeneous tumors affects relative response to relative response to nabnab-paclitaxel-paclitaxel

• Stromal SPARC was associated with worse outcome and poor survival

• Tumoral SPARC did not correlate with survival

Peritumoral Fibroblast SPARC Expression and Patient Outcome With Resectable Pancreatic Adenocarcinoma

Infante 2007

Overexpression of SPARC gene in human gastric carcinoma and its clinic-pathologic significance

Non-cancerous Mucosa Gastric Cancer

Diffuse Type

Intestinal Type

Wang, C-S et al - British Journal of Cancer (2004) 91, 1924 – 1930

Summary of SPARC as a marker of poor prognosis

Classification SPARC Expression/ Function Reference

Hepatocellular Carcinoma

Overexpression by stromal myofibroblasts correlates well with angiogenesis & tumor

progression Lau et al. 2006

Glioblastoma Overexpression in juxtratumoral perivascular cells but not non-malignant brain vessels

Pen et al. 2007

Multiplle Myeloma Significant decrease in plasma levels of SPARC has a prognostic value & shows + correlation

with Hb levels & platelet counts Turk et al. 2005

Meningioma A diagnostic marker for invasive meningiomas regardless of grade

Remple et al. 1999

Prostate Carcinoma High levels of SPARC mRNA & protein as a marker of CaP metastatic foci

Thomas et al. 2000

Head & Neck Cancer High/ Marker of poor prognosis Chin et al. 2005

Tongue Carcinoma High/ Marker of poor prognosis Kato et al. 2005

Cervical Carcinoma High/ Marker of poor prognosis Sova et al. 2006

Non-small cell lung cancer

High/ Marker of poor prognosis Koukourakis et al. 2003

Bladder Cancer High/ Marker of poor prognosis Yamanaka et al.

2001

Melanoma High levels correlate with metastasis Massi et al.

1999

Esophageal Cancer High/ Marker of poor prognosis Yamashita et al.

2003

Breast Cancer High/ Marker of poor prognosis; shows + correlation with stage & grade

Watkins et al. 2005

SPARC Expression in Microarrays Performed on SPARC Expression in Microarrays Performed on Tumors Taken Directly From Patients Tumors Taken Directly From Patients

SPARC Expression in Microarrays Performed on SPARC Expression in Microarrays Performed on Tumors Taken Directly From Patients Tumors Taken Directly From Patients

Tumor Type # with SPARC /# studied (%)

Breast 6/9 67%

Ovary 5/15 33%

Pancreas 13/16 81%

Melanoma 15/17 88%

Adrenal 2/5 40%

Colon 4/15 27%

Total† 76/113 67%

*Increased expression at level of 0.001 versus normal tissue

Slide courtesy Dan Von Hoff, AACR 2006 Slide courtesy Dan Von Hoff, AACR 2006

Preclinical Platform

Rubio et al, CCR 2006

0

10

20

30

40

50

60

GEM ABI GEM+ABI

Tu

mo

rs r

egre

ss

ed

50

% o

f it

s in

itia

l siz

e (

%)

Average Response Rate in Xenografts (n = 11)Average Response Rate in Xenografts (n = 11)

1. Pancreatic cancers are poorly perfused

2. One factor – an intense fibro inflammatory reaction – stroma – squeezes out blood supply and stops infiltration of immunocytes

3. Need to attack the stroma to improve tumor cells killing

• have a poor blood supply

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On a Microscopic levelOn a Microscopic level

Effects of Effects of nabnab-paclitaxel on Tumor Stroma-paclitaxel on Tumor Stroma

Collagen Type I Staining

B

Effects of Effects of nabnab-paclitaxel on Blood Vessels-paclitaxel on Blood Vessels

0

1000

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5000

6000

7000

8000

GEM

Con

cent

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n ng

/g tu

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(Mea

n ±

SE

M)

GEM alone

GEM+ABI

Effects of Effects of nabnab-paclitaxel on Gemcitabine Delivery-paclitaxel on Gemcitabine Delivery

• Role and regulation of SPARC in Pancreatic Cancer.

• SPARC as • prognostic (which patients need rx) • predictive biomarker (which patients are likely to benefit

from a specific rx).

• Stromal effects.

• Diagnostic test.

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Questions in DevelopmentQuestions in Development

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Hypothesis: Albumin-Binding Proteins May Aid in the Uptake of

nab-paclitaxel Into Tumors

• Accumulation of albumin in tumors may be mediated by the protein SPARC1

– SPARC binds albumin2

– Many tumor types overexpress SPARC compared with normal tissues3-5

• High SPARC expression has been shown to be a negative prognostic indicator in many cancer types4,5

1. Kratz F, et al. J Controlled Release. 2008; 132:171-183.2. Schnitzer JE, et al. J Biol Chem. 1994;269(8):6072-6082.

3. Watkins G, et al. Prostaglandins, Leukotrienes and Essential Fatty Acids. 2005;72:267-272.4. Desai N, et al. Transl Oncol. 2009;2:59-64.

5. Podhajcer OL et al. Cancer Metastasis Rev. 2008;27:691-705. SPARC, Secreted Protein Acidic and Rich in Cysteine.

SPARC Expression and Clinical ResponseD. von Hoff et al. ASCO 2009 Poster # 4525

SPARC status by IHC was available for 32 RECIST evaluable patients (investigator dataset: 2CR, 14PR, 14SD, 2PD)

Staining of tumor cells (and not stromal fibroblasts) by antibody P showed improved response for SPARC+ patients (P = 0.027)

Other epitopes of SPARC showed similar response between SPARC+ve and SPARC-ve groups (P = NS)

Fraction (%) of Patients Responding

SPARC status Antibody P (Tumor Cell)

(N=32)

Antibody M (Tumor Cell)

(N=32)

Antibody P (Stromal Fibroblasts)

(N=27)

Antibody M (Stromal Fibroblasts)

(N=27)

SPARC Positive 8/10 (80%) 2 CR / 6 PR 2/5 (40%) 8/16 (50%) 3/5 (60%)

SPARC Negative 8/22 (36.4%) 0 CR / 8 PR 14/27 (52%) 7/11 (64%) 12/22 (55%)

P-value 0.027 NS NS NS

P positive pt # 014 P negative pt # 012Abbreviations: M = antibody M; NS = not significant; P = antibody P

1. Celgene data on file.

Though high SPARC expression is typically a poor prognostic factor, it actually predicts an improved response to nab-paclitaxel in this trial in terms of overall survival

SPARC status was evaluated in 36 patients:a significant increase in OS was observed for patients in the high-SPARC group versus the low-SPARC group (median OS: 17.8 vs 8.1 months, respectively, P=0.0431)

SPARC Expression and Clinical ResponseD. von Hoff et al. JCO 2011

SPARC Expression and Clinical ResponseD. von Hoff et al. JCO 2011

SPARC Assessment: actual tools• Several antibodies are available and have been

used in publications assessing SPARC protein• Antibodies have different staining patterns, thus

need to optimize an assay:– Compare available antibodies– Standardize antigen retrieval, antibody dilution,

secondary antibody and automated staining systems.– Scoring criteria needs to be simplified and

standardized– Tumor compartments staining with SPARC will vary

based on histology

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Biomarker Development: Requirements

Generation of a biomarker hypothesis

Develop prototype assay

Identification of candidate markers

Discovery

Clinical validation of candidate m. using assay

Assay refinement & development IVD test

Regulatory test approvalValidation

Distribution to laboratories

Auditing for result consistency

CommercialisationClinician/Lab education

Biomarker evaluation in CA046

• Archival tumor tissue for SPARC IHC– Slides and/or blocks for received from 160

patients to date– Further explore role of SPARC in response to nab-

paclitaxel in context of a randomized trial

27

Next steps in developing SPARC prototype assay

• The prototype assay is based on IHC methodologies– a collaboration with EU and USA academic

centers has been established with the goal to define the IHC platform

– M. Hidalgo, Madrid, will lead this collaboration, and the kick-off meeting will take place October 17 in Seville

28

SPARC: where we are today

Potentially valuable

Early stage assessment

Clinical use

Exploratory: current standing of SPARC assessment

In Summary

• Stromal components are strategic targets in Pancreatic Cancer.

• SPARC is a stromal component.

• Gem-Nab-Paclitaxel promising activity.

• Stromal depletion vs stroma modification.

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Back-up

31

SPARC IHC method• Developed at MPI (AZ); transferred to

St.John’s pathology lab (CA)• Two antibodies used; each slide scored

separately– Monoclonal R&D #MAB941(1:250; 30 min)– Monoclonal Haematologica Technologies, Inc,

#A0N5031 (1:250; 30 min)– Polyclonal also evaluated, but not used

• R&D AF941 (1:150; 30 min)

• DAKO Automated stainer, NO Antigen retrieval

32

Scoring criteria

• Tumor compartments assessed separately– Tumor, fibroblasts, blood vessels, inflammatory

cells, background stroma tissue, any normal tissue

• Highest intensity (0 to +4) for a compartment (>10% of cells in that compartment) recorded

• % staining at highest intensity in compartment recorded

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