SPARC in Pancreatic Cancer

Nab-paclitaxel properties

•

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

Solvent-based taxanes provoke formation of micelles in circulation

• Micelle formation in the circulation entraps paclitaxel in plasma

Large micelle

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

Control plasma Plasma + solvent-based paclitaxel Hamad et al.

Aapro et al.

EJC Suppl

. 2008;6:3 –11

Expert Opin Drug Deliv

. 2008;5: 205 –219

nab -Paclitaxel Results in Higher Tumoral Uptake Compared With paclitaxel Tumor Uptake in Nude Mice Xenografts Following 20 mg/kg Dose of Paclitaxel

140 120 nab-paclitaxel AUC (nCi•hr/g) 3,632 Taxol 2,739 Ka (hr 1 ) 0.43

0.13

100 80 60 40 0.01

0.1

Tumor AUC nab-paclitaxel = 1.33 x Taxol p < .0001 ANOVA 1

Hours

10 100 • The accumulation of paclitaxel in tumors was 33% higher for nab-paclitaxel compared with paclitaxel (P < .0001) 1 AUC, area under the curve; KA, absorption rate.

Desai et al.

Clin Cancer Res

. 2006; 12:1317-1324.

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.

nab

®

The Unique Properties of Albumin Improve the Risk to Benefit Profile of nab-Paclitaxel • 130-nm sized albumin-paclitaxel complexes 1,2 – nab-Paclitaxel is the first nanotechnology-derived agent approved for the treatment of breast cancer – Albumin gives nab-paclitaxel linear pharmacokinetics 3 = predictable drug exposure with dose modification

Albumin Paclitaxel nab-Paclitaxel

2D Conceptualization

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.

Mechanism of Action of nab-Paclitaxel

SPARC, secreted protein acidic and rich in cysteine.

Investigation of the functional importance of SPARC with respect to

nab

-paclitaxel is ongoing.

Endothelial cells Albumin-Mediated Transcytosis of Paclitaxel Subendothelial space Tumor cells

Investigation of the functional importance of SPARC with respect to

nab

-paclitaxel is ongoing.

SPARC, Secreted Protein Acidic and Rich in Cysteine.

SPARC level in heterogeneous tumors affects relative response to nab-paclitaxel 3000 2500 2000 1500 1000 500 0 0

Saline Abraxane 15 mg/kg q4dx3

MDA-MB-231 10 20 Days 30 Low SPARC (TGI = 36%) 40

3000 2500 2000 1500 1000 500 0 0 Control Abraxane 15 mg/kg q4x3

HT29

20 Days 40

Medium SPARC (TGI = 60%)

60

1500 1000 500

Control Abraxane, 10 mg/kg, qdx5, 2 cycles

MDA-MB435 0 0 20 40 Days High SPARC (TGI = 81%) 60 80

Peritumoral Fibroblast SPARC Expression and Patient Outcome With Resectable Pancreatic Adenocarcinoma

•

Stromal SPARC was associated with worse outcome and poor survival

•

Tumoral SPARC did not correlate with survival

Infante 2007

Overexpression of SPARC gene in human gastric carcinoma and its clinic-pathologic significance Diffuse Type Intestinal Type Non-cancerous Mucosa Gastric Cancer Wang, C-S et al - British Journal of Cancer (2004) 91, 1924 – 1930

Summary of SPARC as a marker of poor prognosis

Classification Hepatocellular Carcinoma Glioblastoma Multiplle Myeloma Meningioma Prostate Carcinoma Head & Neck Cancer Tongue Carcinoma Cervical Carcinoma Non-small cell lung cancer Bladder Cancer Melanoma Esophageal Cancer Breast Cancer SPARC Expression/Function Overexpression by stromal myofibroblasts correlates well with angiogenesis & tumor progression Overexpression in juxtratumoral perivascular cells but not non-malignant brain vessels Significant decrease in plasma levels of SPARC has a prognostic value & shows + correlation with Hb levels & platelet counts A diagnostic marker for invasive meningiomas regardless of grade High levels of SPARC mRNA & protein as a marker of CaP metastatic foci High/Marker of poor prognosis High/Marker of poor prognosis High/Marker of poor prognosis High/Marker of poor prognosis High/Marker of poor prognosis High levels correlate with metastasis High/Marker of poor prognosis High/Marker of poor prognosis; shows + correlation with stage & grade Reference Lau et al. 2006 Pen et al. 2007 Turk et al. 2005 Remple et al. 1999 Thomas et al. 2000 Chin et al. 2005 Kato et al. 2005 Sova et al. 2006 Koukourakis et al. 2003 Yamanaka et al. 2001 Massi et al. 1999 Yamashita et al. 2003 Watkins et al. 2005

SPARC Expression in Microarrays Performed on Tumors Taken Directly From Patients Tumor Type Breast Ovary Pancreas Melanoma Adrenal Colon Total † # with SPARC /# studied 6/9 5/15 13/16 15/17 2/5 4/15 76/113 *Increased expression at level of 0.001 versus normal tissue

Slide courtesy Dan Von Hoff, AACR 2006

(%) 67% 33% 81% 88% 40% 27% 67%

Preclinical Platform

Rubio et al, CCR 2006

Average Response Rate in Xenografts (n = 11) 30 20 10 0 60 50 40 GEM ABI GEM+ABI

On a Microscopic level

1. Pancreatic cancers are poorly perfused

• have a poor blood supply

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

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Effects of nab-paclitaxel on Tumor Stroma Collagen Type I Staining

Effects of nab-paclitaxel on Blood Vessels

B

Effects of nab-paclitaxel on Gemcitabine Delivery 8000 7000 6000 5000 4000 3000 2000 1000 0 GEM alone GEM+ABI GEM

Questions in Development

• 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. 21

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 SPARC

1 – SPARC binds albumin 2 – Many tumor types overexpress SPARC compared with normal tissues 3-5 • High SPARC expression has been shown to be a negative prognostic indicator in many cancer types 4,5 SPARC, Secreted Protein Acidic and Rich in Cysteine. 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. 22

SPARC Expression and Clinical Response

D. 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)

SPARC status SPARC Positive SPARC Negative

P

-value Antibody P (Tumor Cell) (N=32) 8/10 (80%) 2 CR / 6 PR 8/22 (36.4%) 0 CR / 8 PR 0.027

Fraction (%) of Patients Responding Antibody M (Tumor Cell) (N=32) Antibody P (Stromal Fibroblasts) (N=27) 2/5 (40%) 14/27 (52%) 8/16 (50%) 7/11 (64%) NS NS Antibody M (Stromal Fibroblasts) (N=27) 3/5 (60%) 12/22 (55%) NS

Abbreviations: M = antibody M; NS = not significant; P = antibody P

P positive pt # 014 P negative pt # 012

SPARC Expression and Clinical Response

D. von Hoff et al. JCO 2011

 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)

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

1.

Celgene data on file.

25

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

Biomarker Development: Requirements

Clinician/Lab education

Commercialisation

Auditing for result consistency Distribution to laboratories

Validation

Regulatory test approval Assay refinement & development IVD test Clinical validation of candidate m. using assay

Discovery

Develop prototype assay Identification of candidate markers Generation of a biomarker hypothesis

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

Clinical use Potentially valuable Early stage assessment 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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