Evan J. Walker

A Phase I Study of FOLFIRINOX Plus IPI-926, a Hedgehog Pathway Inhibitor, for Advanced Pancreatic Adenocarcinoma.

Objectives In mouse models of pancreatic cancer, IPI-926, an oral Hedgehog inhibitor, increases chemotherapy delivery by depleting tumor-associated stroma. This multicenter phase Ib study evaluated IPI-926 in combination with FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, oxaliplatin) in patients with advanced pancreatic cancer. Methods Patients were treated with once-daily IPI-926 plus FOLFIRINOX. A 3 + 3 dose escalation design was used, with cohort expansion at the maximum tolerated dose. A subset of patients underwent perfusion computed tomography to assess changes in tumor perfusion. Results The maximum tolerated dose was identified 1 dose level below standard FOLFIRINOX. Common treatment-related adverse events included liver function test abnormalities, neuropathy, nausea/vomiting, and diarrhea. Objective response rate was high (67%), and patients receiving IPI-926 maintenance showed further declines in CA19-9 levels even after FOLFIRINOX discontinuation. Treatment did not result in consistent increases in tumor perfusion. The study closed early when a separate phase II trial of IPI-926 plus gemcitabine indicated detrimental effects of this combination. Conclusions This is the first study to demonstrate the feasibility of using FOLFIRINOX as the chemotherapeutic backbone in a clinical trial design. Although robust antitumor activity and acceptable safety were observed with the addition of IPI-926 to this regimen, future development of Hedgehog inhibitors in pancreatic cancer seems unlikely.

Beyond first-line chemotherapy for advanced pancreatic cancer: An expanding array of therapeutic options?

While an increasing number of therapeutic options are now available for the first-line treatment of locally advanced or metastatic pancreatic cancer, the optimal choice for treatment in the second-line setting and beyond is less well defined. A variety of cytotoxic agents, either alone or in combination, have been evaluated, although primarily in the context of small single-arm or retrospective studies. Most regimens have been associated with median progression-free survival rates in the range of 2-4 mo and overall survival rates between 4-8 mo, highlighting the very poor prognosis of patients who are candidates for such treatment. Targeted therapies studied in this chemotherapy-refractory setting, meanwhile, have produced even worse efficacy results. In the current article, we review the clinical evidence for treatment of refractory disease, primarily in patients who have progressed on front-line gemcitabine-based chemotherapy. In the process, we highlight the limitations of the available data to date as well as some of the challenges in designing appropriate clinical trials in this salvage setting, including how to select an appropriate control arm given the absence of a well-established reference standard, and the importance of incorporating predictive biomarkers and quality of life measures whenever possible into study design.

Low density contrast agents for x-ray phase contrast imaging: the use of ambient air for x-ray angiography of excised murine liver tissue

We report a new preparative method for providing contrast through reduction in electron density that is uniquely suited for propagation-based differential x-ray phase contrast imaging. The method, which results in an air or fluid filled vasculature, makes possible visualization of the smallest microvessels, roughly down to 15 microm, in an excised murine liver, while preserving the tissue for subsequent histological workup. We show the utility of spatial frequency filtering for increasing the visibility of minute features characteristic of phase contrast imaging, and the capability of tomographic reconstruction to reveal microvessel structure and three-dimensional visualization of the sample. The effect of water evaporation from livers during x-ray imaging on the visibility of blood vessels is delineated. The deformed vascular tree in a cancerous murine liver is imaged.

Statin use and risk of pancreatic cancer: Results from a large clinic-based case-control study

Statins are cholesterol‐lowering medications with pleiotropic effects, including alterations in growth signaling, as well as immunomodulatory and anti‐inflammatory effects that may alter cancer risk. Evidence from previous epidemiologic studies is inconsistent about whether statin use is associated with a reduced risk of pancreatic cancer (PC).

Metformin use among type 2 diabetics and risk of pancreatic cancer in a clinic-based case–control study

A better understanding of the association between diabetes and pancreatic cancer (PC) may inform prevention and/or early detection strategies. Metformin has been associated with reduced risk of certain cancers, including PC, in some observational clinical studies. We assessed whether metformin use was associated with PC risk among those with type 2 diabetes (DM2), and whether metformin use modulated the association between DM2 and risk of PC. In total, 536 PC cases and 869 frequency‐matched controls were recruited predominantly from University of California San Francisco medical clinics from 2006 to 2011. Eligible participants completed direct interviews using a structured risk factor questionnaire. The association between metformin use and PC risk was assessed using propensity score‐weighted unconditional logistic regression methods in analyses restricted to diabetics and adjusted multivariable logistic models in the total study population. Ever use of metformin was not associated with PC risk in analyses restricted to DM2 (N = 170) participants (adjusted OR: 1.01, 95% CI: 0.61–1.68). In the total study population (N = 1,405) using nondiabetics as the referent group, PC risk was inversely associated with diabetes duration (ptrend < 0.001). Further, when DM2 participants were grouped by ever/never use of metformin and compared with nondiabetics, metformin use did not affect the association between DM2 and PC risk (never users: OR: 1.44, 95% CI: 0.78–2.67; ever users: OR: 1.19, 95% CI: 0.72–1.99). Results from our clinic‐based case–control study suggest that metformin use is not associated with PC risk among those with DM2 and does not alter the association between DM2 and PC risk.

A patient with cholangiocarcinoma demonstrating pathologic complete response to chemotherapy: exploring the role of neoadjuvant therapy in biliary tract cancer

The role of neoadjuvant chemotherapy and/or radiation for localized or potentially resectable cholangiocarcinoma (CCA) has not been well established. We present here the case of a patient with an extrahepatic CCA who achieved a pathologic complete response after undergoing preoperative gemcitabine-based chemotherapy, without sequential or concurrent use of radiation. Further evaluation of neoadjuvant strategies in CCA, including not only combined-modality therapy but also the use of chemotherapy exclusively, is warranted in prospective study design.

Loss of the SxxSS Motif in a Human T-Cell Factor-4 Isoform Confers Hypoxia Resistance to Liver Cancer: An Oncogenic Switch in Wnt Signaling

Purpose Aberrantly activated Wnt/β-catenin signaling is important in hepatocellular carcinoma (HCC) development. Downstream gene expressions involving the Wnt/β-catenin cascade occur through T-cell factor (TCF) proteins. Here, we show the oncogenic potential of human TCF-4 isoforms based on the expression of a single conserved SxxSS motif. Methods We investigated the TCF-4J and K isoform pair characterized by the presence (K) or absence (J) of the SxxSS motif. The mRNA expression profiles were examined in 47 pairs of human HCCs and adjacent non-cancerous liver tissues by RT-PCR. Proliferation, sphere assays and immunoblot analysis were performed under normoxia and hypoxia conditions. The ability of HCC cells overexpressing TCF-4J (J cells) and K (K cells) to grow as solid tumors in nude mice was explored. Results TCF-4J expression was significantly upregulated in HCC tumors compared to corresponding peritumor and normal liver and was preferentially expressed in poorly differentiated HCCs. In contrast, TCF-4K was downregulated in those same HCC tumors. TCF-4J-overexpressing HCC cells (J cells) revealed a survival advantage under hypoxic conditions, high proliferation rate and formation of aggregates/spheres compared to overexpression of TCF-4K (K cells). The hypoxic J cells had high expression levels of HIF-2α and EGFR as possible mechanisms to promote tumorigenesis. Increased stability of HIF-2α under hypoxia in J cells was associated with a decreased level of von Hippel-Lindau (VHL) protein, a known E3 ligase for HIF-αs. In a xenograft model, the J cells rapidly developed tumors compared to K cells. Tumor tissues derived from J cells exhibited high expression levels of HIF-2α and EGFR compared to the slow developing and small K cell derived tumors. Conclusions Our results suggest that the specific TCF-4J isoform, which lacks a regulatory SxxSS motif, has robust tumor-initiating potential under hypoxic conditions.

Referral frequency, attrition rate, and outcomes of germline testing in patients with pancreatic adenocarcinoma

Hereditary predisposition is estimated to account for 10% of all pancreatic cancer cases. However, referral patterns and clinical workflow for germline testing in this disease differ significantly by institution, and many at-risk patients may not undergo appropriate counseling and testing. We undertook an analysis of patients diagnosed with pancreatic cancer (PDAC) who were referred to the Clinical Genetics program of a high-volume academic center over a 3-year period to assess referral frequency, evaluate the yield of germline testing in this selected patient cohort, and elucidate the reasons individuals did not undergo recommended germline testing. Medical records of patients with PDAC referred for genetic counseling between January 2015 and October 2017 were reviewed for demographic, medical/family history, and disease-specific data. If testing did not occur, reasons were documented. Genetic test results were categorized as negative, variants of unknown significance, or established pathogenic mutations. Descriptive statistics included means with standard deviations; associations were analyzed with t test and Fisher’s exact test. 32% (137 of 432) of PDAC patients were referred for genetic counseling, but only 64% attended their appointment and 60% ultimately underwent germline testing. Common reasons for attrition included worsening disease severity, lack of patient follow-up, insurance concerns, and logistic/travel challenges. Pathogenic germline mutations were detected in 20% (16 of 82) of patients tested, distributed across races/ethnicities, and significantly associated with younger age and positive family history of breast cancer. PDAC patients frequently do not undergo genetic counseling/germline testing despite appropriate referrals, highlighting a need to develop streamlined processes to engage more patients in testing, especially those with high-risk features.

Abstract 2160: Metformin use is not associated with pancreatic cancer risk in a clinic-based case-control study conducted in the San Francisco Bay Area, California

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Patients with pancreatic cancer (PC) are commonly diagnosed at advanced stages of the disease, at which point cure is no longer possible. A better understanding of risk or protective factors for PC may inform prevention and/or early detection strategies. Metformin, a commonly used drug for type II diabetes (DM2), has been shown in some observational clinical studies to reduce the risk of several cancer types, including PC. Its antineoplastic effects occur through inhibition of mammalian target of rapamycin (mTOR) and induction of cell cycle arrest and apoptosis, among other mechanisms. The current case-control study addresses two distinct research questions: whether metformin use is associated with PC risk among those with DM2, and whether metformin modulates the risk of PC associated with DM2. We recruited 536 cases and 869 controls, predominantly from University of California, San Francisco (UCSF) medical and surgical oncology clinics (cases) and general medicine clinics (controls), over a six-year period (2006-2011). Controls were frequency-matched to cases by sex and age in 5-year groups. Case diagnoses were confirmed by patient medical record and cancer registry data. Direct interviews were conducted by trained interviewers using an epidemiological risk factor questionnaire. The association between metformin use and PC risk among diabetics was analyzed using propensity score-weighted unconditional logistic regression, whereas the effect of metformin use on the association between DM2 and PC in the total study population was analyzed using standard multivariable logistic models. Metformin was the most common anti-diabetic drug used by DM2 study participants (66.5%). Among DM2 participants (N=170), ever use of metformin was not associated with PC risk in adjusted models (OR: 1.01, 95%CI: 0.61-1.68). Similarly, duration of metformin use was not statistically significantly associated with PC risk when also adjusted for DM2 duration. Among the total study population (N=1405), DM2 was not statistically significantly associated with PC risk (adjusted OR: 1.28, 95%CI: 0.81-2.00). However, risk was inversely associated with DM2 duration; participants whose DM2 was diagnosed 1-5 years prior to PC diagnosis/interview were at markedly increased risk (OR: 2.47, 95%CI: 1.25-4.85). Stratification of participants with DM2 by metformin use revealed no difference in risk between never (OR: 1.44, 95%CI: 0.78-2.67) and ever users (OR: 1.19, 95%CI: 0.72-1.99) relative to non-DM2 participants. PC risk also did not differ across groups of metformin or DM2 duration. In this clinic-based case-control study of PC, metformin use was not associated with PC risk. Specifically, our results suggest that metformin is neither associated with PC risk in those with DM2, nor does it attenuate or exacerbate PC risk associated with DM2. Citation Format: Evan J. Walker, Andrew H. Ko, Elizabeth A. Holly, Paige M. Bracci. Metformin use is not associated with pancreatic cancer risk in a clinic-based case-control study conducted in the San Francisco Bay Area, California. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2160. doi:10.1158/1538-7445.AM2014-2160

Abstract 1933: Preferential ubiquitination of hypoxia-inducible factors depends on the SxxSS motif in human T-cell factor-4 isoforms derived from hepatocellular carcinoma

The T-cell factor (TCF)-4 is a key transcriptional protein activated by Wnt/β-catenin signaling. We previously cloned 14 TCF-4 isoforms derived from human hepatocellular carcinoma (HCC) cell lines. The TCF-4J and K pair were characterized based on the presence (K) or absence (J) of the functional motif SxxSS. Recently, we have demonstrated that loss of the SxxSS motif in the TCF-4 isoforms conferred robust tumorigenic potential to HCC, involving the hypoxia-inducible factor (HIF)-2α (AASLD Oct. 30, 2010; abstract #560). It is known that the cellular amount of HIF proteins is tightly regulated by ubiquitination and such proteins appear to be important in cancer stem cell biology. Thus, the AIM of the current study was to examine how HIF protein expression levels were regulated in the SxxSS motif-dependent manner. Methods: The human HCC cell line HAK-1A was used in this study. Hypoxia was induced with cobalt chloride (CoCl2) or 1% oxygen exposure. TCF-4K mutants were prepared with substitution of serine (S) to alanine (A) by site-directed mutagenesis. Cell clones overexpressing TCF-4J, K, and K mutants such as S269A, S272A, and S273A were established. Ubiquitination of HIF proteins were assessed by immunoprecipitation (IP)/Western blot analysis using the proteasome inhibitor MG-132. Results: Increased expression of both HIF-1α and HIF-2α was preferentially found in the TCF-4J-overexpressing cells (J cells) under severe hypoxia, which was in contrast to the levels in the TCF-4K-overexpressing cells (K cells) and the mock-transfected cells (mock cells). This increase in both HIF expressions in J cells was accompanied by downregulation of Von Hippel Lindau (VHL) protein, a component of E3 ligase for HIFs. When cells were incubated with 10 μM MG-132 for 4 h under severe hypoxia, the expression levels of HIF-1α and HIF-2α were highly increased in K cells, suggesting degradation of HIFs in an ubiquitin-dependent fashion. Indeed, both the robust poly-ubiquitination of HIF-2α and the interaction between VHL and HIF-2α were detected by the IP/Western analysis in K cells. To further elucidate the detailed mechanisms of the augmented HIF-2α ubiquitination through SxxSS, the possible impact derived from the K mutants on the ubiquitination process was investigated. As a result, the S269A mutant demonstrated predominant intracellular accumulation of HIF-2α in the treatment with MG-132 under severe hypoxia, suggesting that de-phosphorylation at serine 269 in SxxSS facilitated the proteasomal degradation of HIF-2α. Conclusion: This is an initial demonstration that the TCF-4 isoforms are involved in the proteolytic processing of HIFs in HCC. The findings from this study may provide insights into the understanding and possible role of the canonical Wnt/β-catenin/TCF-4 signaling pathway in the regulation of HIF-2α, which is a novel cancer stem cell marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1933. doi:10.1158/1538-7445.AM2011-1933