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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 dosedependent antitumour activity Solvent-based taxanes provoke formation of micelles in circulation Large micelle • Micelle formation in the circulation entraps paclitaxel in plasma • Resulting non-linear pharmacokinetics contribute to a lack of dose-dependent antitumour activity Control plasma Plasma + solvent-based paclitaxel Aapro et al. EJC Suppl. 2008;6:3–11 Hamad et al. 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 Paclitaxel (nCi/g) 140 120 AUC Ka (nCi•hr/g) (hr1) nab-paclitaxel 3,632 0.43 Taxol 2,739 0.13 100 80 Tumor AUC nab-paclitaxel = 1.33 x Taxol p < .0001 ANOVA 60 40 0.01 0.1 1 10 100 Hours • 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. 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 Albumin Paclitaxel nab-Paclitaxel particle nab® is a registered trademark of Celgene Corporation. 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. Albumin-Mediated Transcytosis of Paclitaxel Endothelial cells Subendothelial space Tumor cells SPARC, Secreted Protein Acidic and Rich in Cysteine. Investigation of the functional importance of SPARC with respect to nab-paclitaxel is ongoing. SPARC level in heterogeneous tumors affects relative response to nab-paclitaxel Control Abraxane 15 mg/kg q4x3 Saline Abraxane 15 mg/kg q4dx3 Control Abraxane, 10 mg/kg, qdx5, 2 cycles HT29 MDA-MB435 3000 Tumor Volume (mm3) 2500 2000 1500 1000 2500 Tumor volume (mm3 ) Tumor Volume (mm3) MDA-MB-231 3000 1500 2000 1500 1000 500 1000 500 500 0 0 0 0 10 20 30 Days Low SPARC (TGI = 36%) 40 0 20 40 Days Medium SPARC (TGI = 60%) 60 0 20 40 Days High SPARC (TGI = 81%) 60 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 SPARC Expression/Function Hepatocellular Carcinoma 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 Glioblastoma Multiplle Myeloma Meningioma Prostate Carcinoma Reference Lau et al. 2006 Pen et al. 2007 Turk et al. 2005 Remple et al. 1999 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 Bladder Cancer High/Marker of poor prognosis Melanoma High levels correlate with metastasis Esophageal Cancer High/Marker of poor prognosis Breast Cancer High/Marker of poor prognosis; shows + correlation with stage & grade 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 # 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 Preclinical Platform Rubio et al, CCR 2006 Tumors regressed 50% of its initial size (%) Average Response Rate in Xenografts (n = 11) 60 50 40 30 20 10 0 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 17 Effects of nab-paclitaxel on Tumor Stroma Collagen Type I Staining Effects of nab-paclitaxel on Blood Vessels B Concentration ng/g tumor (Mean ± SEM) Effects of nab-paclitaxel on Gemcitabine Delivery 8000 GEM alone 7000 GEM+ABI 6000 5000 4000 3000 2000 1000 0 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 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 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) Fraction (%) of Patients Responding SPARC status SPARC Positive Antibody P (Tumor Cell) (N=32) 8/10 (80%) 2 CR / 6 PR SPARC Negative 8/22 (36.4%) 0 CR / 8 PR P-value Abbreviations: M = antibody M; NS = not significant; P = antibody P 0.027 Antibody M (Tumor Cell) (N=32) Antibody P (Stromal Fibroblasts) (N=27) Antibody M (Stromal Fibroblasts) (N=27) 2/5 (40%) 8/16 (50%) 3/5 (60%) 14/27 (52%) 7/11 (64%) 12/22 (55%) NS NS NS 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 nabpaclitaxel in this trial in terms of overall survival 1. Celgene data on file. 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 25 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 nabpaclitaxel 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. 30 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 33 34