Mark Ricigliano

Previous studies have shown that pharmacogenomic modeling of circulating tumor and invasive cells (CTICs) can predict response of pancreatic ductal adenocarcinoma (PDAC) to combination chemotherapy, predominantly 5-fluorouracil-based. We hypothesized that a similar approach could be developed to predict treatment response to standard frontline gemcitabine with nab-paclitaxel (G/nab-P) chemotherapy. Gene expression profiles for responsiveness to G/nab-P were determined in cell lines and a test set of patient samples. A prospective clinical trial was conducted, enrolling 37 patients with advanced PDAC who received G/nab-P. Peripheral blood was collected prior to treatment, after two months of treatment, and at progression. The CTICs were isolated based on a phenotype of collagen invasion. The RNA was isolated, cDNA synthesized, and qPCR gene expression analyzed. Patients were most closely matched to one of three chemotherapy response templates. Circulating tumor and invasive cells’ SMAD4 expression was measured serially. The CTICs were reliably isolated and profiled from peripheral blood prior to and during chemotherapy treatment. Individual patients could be matched to distinct response templates predicting differential responses to G/nab-P treatment. Progression free survival was significantly correlated to response prediction and ΔSMAD4 was significantly associated with disease progression. These findings support phenotypic profiling and ΔSMAD4 of CTICs as promising clinical tools for choosing effective therapy in advanced PDAC, and for anticipating disease progression.

E14018 Background:Standard of care treatment for pancreatic adenocarcinoma (PDAC) consists of FOLFIRINOX or gemcitabine with nab-paclitaxel used in either the front-line or 2^nd line treatment. There is no consensus for 3^rd line treatment. We previously demonstrated that Mitomycin C (M) and Cisplatin (C) as viable 3^rdline treatment options with broad use limited by toxicity. To address the toxicity issues with the prolonged use of M and C, we developed a novel cytotoxic drug conjugate ADR-201 (A), as a treatment option for metastatic PDAC . Methods: (A) is a cytotoxic drug conjugate formed from the APIs of Mitomycin C (M) and Cisplatin (C) conjugated with an amine-to-sulfhydryl crosslinker with a soluble polyethylene glycol (PEG) spacer arm to reduce steric hindrance. In order to conjugate C, the molecule was thiolated prior to crosslinking with a maleimide functional group. In cytotoxic assays, MiaPaca-2 cells were treated with 17uM of A and cell viability was measured at 48hrs, 72hrs and 96hrs. In thin layer chromatography (TLC) A, M and C were spotted at the origin with 0.5uL of 17uM, 10mM and 10mM respectively. Cell cultures comprising circulating tumor and invasive cells (CTICs) obtained from PDAC patients (n = 12) were treated with 17uM of A and cell viability was measured at 72hrs. Results: In TLC, A showed marked solubility in polar neutral solvent EtOAc /hexane (20:80) compared to the polar compounds M and C with Rf values 0.38:1.0:1.0 respectively. In cytotoxic assays with MiaPaca-2 cells, serial dilutions of A at 17uM, 8.5uM and 3.4uM demonstrated a 30%, 11% and 6% reduction in cell viability at 72hrs. In 24 CTIC cultures, A reduced cell viability between treatment v control groups by an average -26%. (range -31% - 0%, paired t-test, p-value = 0.013, ). Conclusions: Improvements in solubility and cytotoxic activity at low dose levels compared to single agents M and C should translate to enhanced permeability and retention (EPR) in tumor tissue with corresponding reduced toxicity.

E15749 Background: Standard of care treatment for pancreatic adenocarcinoma (PDAC) consists of FOLFIRINOX (FFX) or gemcitabine with nab-paclitaxel (G/nab-P) in the front-line setting. The decision to use FFX or G/nab-P is a physician choice as there are no standardized assays to guide treatment selection. Here we report the results of a prospective clinical trial to evaluate a blood-based assay to select patients for G/nab-P in the front line setting. The assay was developed on the premise that if two patients have the same genomic profile, they will have the same response to chemotherapy. Methods: In a prospective study, 37 PDAC patients were pre-selected to receive G/nab-P at Memorial Sloan Kettering as a front-line treatment. Circulating tumor and infiltrating cells (CTICs) were isolated from peripheral blood samples using a cell invasion matrix. RNA was isolated and cDNA synthesized for qPCR gene expression. 83 drug transport genes were used as classifiers to create four treatment templates, one each for G/nab-P, GAX, FFX and FOLFOX. Nearest Template Prediction was used to match the genomic profile of the PDAC patient sample to one of the four a priori defined treatment templates. Results: Front-line PFS was correlated to treatment phenotype with G/nab-P (8.6 mos.), GAX (6.7mos.), FFX (5.6 mos.) and FOLFOX (5.0 mos.). Patients with a positive sensitivity (S+) to G/nab-P demonstrated a median PFS of 7.7 mos. vs. 4.7 mos. compared to those with a negative sensitivity (S-) to G/nab-P (p-value = 0.027). Contingency analysis demonstrated sensitivity (78%), specificity (64%), and likelihood ratio (2.139) with p-value = 0.048. Correlation between G/nab-P sensitivity and PFS was significant as measured by linear regression (R²= 0.90, p-value = 0.013). Conclusions: Phenotype profiling for selecting front-line treatment to G/nab-P is feasible in PDAC. Clinical trial information: NCT01474564.

338 Background: Standard of care treatment for pancreatic adenocarcinoma (PDAC) includes FOLFIRINOX or gemcitabine with nab-paclitaxel used in either the front-line or 2^nd line treatment setting. There is no consensus for 3^rd line treatment for this cohort of poor prognosis patients with clinical trials being the only viable option. We evaluated a cell viability assay using cultures of circulating tumor and invasive cells (CTICs) to identify effective 3^rdline PDAC treatment options. Methods: CTICs were isolated from peripheral blood samples of 17 PDAC patients who previously had received one of the two standard of care regimens using an immunomagnetic separation for EPCAM epitope Ber-EP4 and cultured in enriched DMEM-F12 medium. After 14 days, seven chemotherapeutics (gemcitabine (G), oxaliplatin (O), fluorouracil (F), irinotecan (I), mitomycin C (M), cisplatin (C) and paclitaxel (P)) were added to the cultures and response to treatment was measured 48 hr. after treatment by immunofluorescence using a live-cell resazurin assay. Response to treatment was determined by a reduction of fluorescence in the treated cultures compared to control and the mean florescence (Fm) was calculated for each chemotherapeutic. A one-sample t-test was used to measure the variance between Fm control and treated samples. (p-value rd line PDAC patients.

E15755 Background: We have previously demonstrated that gene expression profiling of CTICs can predict drug response in PDAC. ALDH1A1 has been proposed as a cancer stem cell marker in PDAC and other cancer types. ALDH1A1 controls the oxidation of aldehydes to corresponding acids, and has been proposed to confer drug resistance in ALDH1A1 overexpressing cancer cells. Disulfiram has been shown to inhibit ALDH1A1 activity. Methods: Expression data from 36 PDAC tumor biopsy samples was used to establish baseline ALDH1A1 and GAPDH levels in PDAC patients. A prospective MSKCC study was conducted, 29 treatment naïve patients with metastatic PDAC were accrued. Six mL of heparinized peripheral blood was collected prior to chemotherapy treatment. CTICs were isolated using a previously described cell adhesion assay. Total RNA and cDNA were isolated and qPCR was performed to establish ALDH1A1 levels. ALDH1A1 expression was normalized to levels obtained from tissue biopsy samples. ALDH1A1 levels obtained from CTICs were considered overexpressed at two standard deviations from the mean ALDH1A1 levels of the biopsy samples. ALDH1A1 expression was measured in 5 PDAC cell lines. IC50 to treatment with disulfiram was determined. Cells expressing ADH1A1 were counted by flow cytometry before and after disulfiram treatment. Results: In the 5 cell lines studied a strong negative correlation was observed between ALDH expression and disulfiram IC50 (p = 0.0167). Treatment of cell lines with disulfiram resulted in reduction in ALDH-high expressing cells. 11/29 (38%) patient-derived CTICs overexpressed ALDH1A1. Patients with high ALDH expressing CTICs experienced longer OS compared with low (11.9 v 3.7 mo). Conclusions: ALDH1A1 expression is a candidate biomarker for survival in PDAC. Preclinical studies suggest that disulfiram may be a promising therapy for treatment of ALDH-high expressing PDAC. Clinical trial information: NCT01474564.

142 Background: Pancreatic adenocarcinoma (PDAC) is uniformly lethal; despite this, modern cytotoxics (C) can induce tumor responses and extend life. Xenograft models have shown that pharmacogenomic (PGx) modeling of C can predict efficacy. Chemo-sensitivity and gene expression profiling of tumor progenitor (TP) cells isolated from peripheral blood may predict tumor response, progression and resistance. Methods: A prospective MSKCC study is ongoing with 46 of 60 planned patients accrued. 10 mL of peripheral blood is collected from patients with unresectable PDAC prior to C and at disease progression. Clinical data is prospectively collected. Circulating TP cells are isolated (Vita-Cap, Vitatex Inc.), total RNA is extracted and gene-expression analysis is performed. PGx models for twelve chemotherapy drug combinations used in PDAC were created from the GI50 data obtained from the NCI-60 cell lines (CellPath Therapeutics, Inc., Baltimore, MD). Expression data were normalized through GCRMA then probed to identify expression differences between the 35 patients with continued disease response and the 11 patients with disease progression. Analysis was performed at the pathway and individual gene level. Results: TP cells were isolated and gene expression and chemo-sensitivity patterns were obtained in all 46 evaluable patients prior to initiating C and in all 11 patients at 1st line disease progression. Preliminary analysis demonstrates clinical benefit for patients treated with C predicted to be effective versus C predicted to be ineffective (TTP 4.4 mo versus 2.7 mo, p=0.016). Changes in chemo-sensitivity patterns were evident at disease progression, reflecting treatment resistance. Hedgehog pathway over-expression was associated with resistance to gemcitabine but clinical response to 5-FU based treatment. Pathway analysis revealed that ErbB3, ARE/Nrf2 and Insulin pathways distinguished patients with disease progression. Conclusions: Isolation and gene expression profiling of TP cells can be performed reliably in unresectable PDAC. Preliminary analysis suggests that C profiling can predict response. Repeat PGx profiling identifies key pathways associated with treatment resistance. Clinical trial information: NCT01474564.