Andrew H. Nguyen

Current Recommendations for Surveillance and Surgery of Intraductal Papillary Mucinous Neoplasms May Overlook Some Patients with Cancer

BackgroundThe 2012 Sendai Criteria recommend that patients with 3xa0cm or larger branch duct intraductal papillary mucinous neoplasms (BD-IPMN) without any additional “worrisome features” or “high-risk stigmata” may undergo close observation. Furthermore, endoscopic ultrasound (EUS) is not recommended for BD-IPMN <2xa0cm. These changes have generated concern among physicians treating patients with pancreatic diseases. The purposes of this study were to (i) apply the new Sendai guidelines to our institution’s surgically resected BD-IPMN and (ii) reevaluate cyst size cutoffs in identifying patients with lesions harboring high-grade dysplasia or invasive cancer.MethodsWe retrospectively reviewed 150 patients at a university medical center with preoperatively diagnosed and pathologically confirmed IPMNs. Sixty-six patients had BD-IPMN. Pathologic grade was dichotomized into low-grade (low or intermediate grade dysplasia) or high-grade/invasive (high-grade dysplasia or invasive cancers). Fisher’s exact test, chi-square test, student’s t test, linear regression, and receiver operating characteristic (ROC) analyses were performed.ResultsThe median BD-IPMN size on imaging was 2.4xa0cm (interquartile range 1.5–3.0). Fifty-one (77xa0%) low-grade and 15 (23xa0%) high-grade/invasive BD-IPMN were identified. ROC analysis demonstrated that cyst size on preoperative imaging is a reasonable predictor of grade with an area under the curve of 0.691. Two-thirds of high-grade/invasive BD-IPMN were <3xa0cm (nu2009=u200910). Compared to a cutoff of 3, 2xa0cm was associated with higher sensitivity (73.3 vs. 33.3xa0%) and negative predictive value (83.3 vs. 80xa0%, NPV) for high-grade/invasive BD-IPMN. Mural nodules on endoscopic ultrasound (EUS) or atypical cells on endoscopic ultrasound-fine needle aspiration (EUS-FNA) were identified in all cysts <2 and only 50xa0% of those <3xa0cm. Forty percent of cysts >3xa0cm were removed based on size alone.Discussion/ConclusionsOur results suggest that “larger” size on noninvasive imaging can indicate high-grade/invasive cysts, and EUS-FNA may help identify “smaller” cysts with high-grade/invasive pathology.

Stromal microRNA-21 levels predict response to 5-fluorouracil in patients with pancreatic cancer.

MicroRNA‐21 (miR‐21) is upregulated and inversely associated with survival in many cancer types, including pancreatic ductal adenocarcinoma (PDAC). We studied the predictive value of miR‐21 levels for gemcitabine or 5‐fluorouracil (5‐FU) response in tumor cells (TCs) or cancer associated fibroblasts (CAFs) in a cohort of PDAC patients from the RTOG 9704 trial.

p85α is a microRNA target and affects chemosensitivity in pancreatic cancer.

BACKGROUNDnWe previously identified a correlation between increased expression of the phosphoinositide 3-kinase (PI3K) regulatory subunit p85α and improved survival in human pancreatic ductal adenocarcinoma (PDAC). The purpose of this study was to investigate the impact of changes in p85α expression on response to chemotherapy and the regulation of p85α by microRNA-21 (miR-21).nnnMATERIALS AND METHODSnPDAC tumor cells overexpressing p85α were generated by viral transduction, and the effect of p85α overexpression on sensitivity to gemcitabine was tested by MTT assay. Primary human PDAC tumors were stained for p85α and miR-21 via immunohistochemistry and in situ hybridization, respectively. Additionally, PDAC cells were treated with miR-21 mimic, and changes in p85α and phospho-AKT were assessed by Western blot. Finally, a luciferase reporter assay system was used to test direct regulation of p85α by miR-21.nnnRESULTSnHigher p85α expression resulted in increased sensitivity to gemcitabine (P < 0.01), which correlated with decreased PI3K-AKT activation. Human tumors demonstrated an inverse correlation between miR-21 and p85α expression levels (r = -0.353, P < 0.001). In vitro, overexpression of miR-21 resulted in decreased levels of p85α and increased phosphorylation of AKT. Luciferase reporter assays confirmed the direct regulation of p85α by miR-21 (P < 0.01).nnnCONCLUSIONSnOur results demonstrate that p85α expression is a determinant of chemosensitivity in PDAC. Additionally, we provide novel evidence that miR-21 can influence PI3K-AKT signaling via its direct regulation of p85α. These data provide insight into potential mechanisms for the known relationship between increased p85α expression and improved survival in PDAC.

Chemotherapy-Induced Inflammatory Gene Signature and Protumorigenic Phenotype in Pancreatic CAFs via Stress-Associated MAPK.

Pancreatic ductal adenocarcinoma (PDAC) has a characteristically dense stroma comprised predominantly of cancer-associated fibroblasts (CAF). CAFs promote tumor growth, metastasis, and treatment resistance. This study aimed to investigate the molecular changes and functional consequences associated with chemotherapy treatment of PDAC CAFs. Chemoresistant immortalized CAFs (R-CAF) were generated by continuous incubation in gemcitabine. Gene expression differences between treatment-naïve CAFs (N-CAF) and R-CAFs were compared by array analysis. Functionally, tumor cells (TC) were exposed to N-CAF– or R-CAF–conditioned media and assayed for migration, invasion, and viability in vitro. Furthermore, a coinjection (TC and CAF) model was used to compare tumor growth in vivo. R-CAFs increased TC viability, migration, and invasion compared with N-CAFs. In vivo, TCs coinjected with R-CAFs grew larger than those accompanied by N-CAFs. Genomic analysis demonstrated that R-CAFs had increased expression of various inflammatory mediators, similar to the previously described senescence-associated secretory phenotype (SASP). In addition, SASP mediators were found to be upregulated in response to short duration treatment with gemcitabine in both immortalized and primary CAFs. Inhibition of stress-associated MAPK signaling (P38 MAPK or JNK) attenuated SASP induction as well as the tumor-supportive functions of chemotherapy-treated CAFs in vitro and in vivo. These results identify a negative consequence of chemotherapy on the PDAC microenvironment that could be targeted to improve the efficacy of current therapeutic regimens. Implications: Chemotherapy treatment of pancreatic cancer–associated fibroblasts results in a proinflammatory response driven by stress-associated MAPK signaling that enhances tumor cell growth and invasiveness. Mol Cancer Res; 14(5); 437–47. ©2016 AACR.

Stiffness of pancreatic cancer cells is associated with increased invasive potential

Metastasis is a fundamentally physical process in which cells are required to deform through narrow gaps as they invade surrounding tissues and transit to distant sites. In many cancers, more invasive cells are more deformable than less invasive cells, but the extent to which mechanical phenotype, or mechanotype, can predict disease aggressiveness in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here we investigate the invasive potential and mechanical properties of immortalized PDAC cell lines derived from primary tumors and a secondary metastatic site, as well as noncancerous pancreatic ductal cells. To investigate how invasive behavior is associated with cell mechanotype, we flow cells through micron-scale pores using parallel microfiltration and microfluidic deformability cytometry; these results show that the ability of PDAC cells to passively transit through pores is only weakly correlated with their invasive potential. We also measure the Youngs modulus of pancreatic ductal cells using atomic force microscopy, which reveals that there is a strong association between cell stiffness and invasive potential in PDAC cells. To determine the molecular origins of the variability in mechanotype across our PDAC cell lines, we analyze RNAseq data for genes that are known to regulate cell mechanotype. Our results show that vimentin, actin, and lamin A are among the most differentially expressed mechanoregulating genes across our panel of PDAC cell lines, as well as a cohort of 38 additional PDAC cell lines. We confirm levels of these proteins across our cell panel using immunoblotting, and find that levels of lamin A increase with both invasive potential and Youngs modulus. Taken together, we find that stiffer PDAC cells are more invasive than more compliant cells, which challenges the paradigm that decreased cell stiffness is a hallmark of metastatic potential.

Low expression of the E3 ubiquitin ligase CBL confers chemoresistance in human pancreatic cancer and is targeted by epidermal growth factor receptor inhibition.

Purpose: Expression of CBL, an ubiquitin ligase, is decreased in 60% of human pancreatic ductal adenocarcinomas (PDAC) and is associated with shorter overall survival. We sought to determine how low CBL directly contributes to clinically more aggressive PDAC. Experimental Design: Human PDACs were stained for CBL, pEGFR, and EGFR. CBL-low was modeled in PDAC cells (Panc-1, L3.6pl, and AsPC-1) via transient transfection (siRNA) or stable knockdown (shRNA). Cell viability and apoptosis were measured by MTT assays and FACS. Immunoblot and a phospho-receptor tyrosine kinase (pRTK) array were used to probe signal transduction. NOD-scid-IL2Rγnull mice were subcutaneously implanted with PDAC or PDACCBL-low cells on opposite flanks and treated with gemcitabine ± erlotinib for ≥4 weeks. Results: There was an inverse correlation between CBL and pEGFR protein expression in 12 of 15 tumors. CBL knockdown increased PDAC resistance to gemcitabine and 5-fluorouracil (5-FU) by upregulating pEGFR (Y1068), pERK, and pAKT. A pRTK array of PDACCBL-low cells revealed additional activated tyrosine kinases but all to a much lower magnitude than EGFR. Increased chemoresistance from low CBL was abrogated by the EGFR inhibitor erlotinib both in vitro and in vivo. Erlotinib+gemcitabine–treated PDACCBL-low cells exhibited greater apoptosis by cleaved PARP, caspase-3, and Annexin V/PI. Conclusions: Low CBL causes chemoresistance in PDAC via stress-induced EGFR activation that can be effectively abrogated by EGFR inhibition. These results suggest that dysregulation of ubiquitination is a key mechanism of EGFR hyperactivation in PDAC and that low CBL may define PDAC tumors likely to respond to erlotinib treatment. Clin Cancer Res; 21(1); 157–65. ©2014 AACR.

Histone deacetylase inhibitors provoke a tumor supportive phenotype in pancreatic cancer associated fibroblasts

Although histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs, thus far, they have been unsuccessful in early phase clinical trials for pancreatic ductal adenocarcinoma (PDAC). One potential reason for their poor efficacy is the tumor stroma, where cancer-associated fibroblasts (CAFs) are a prominent cell type and a source of resistance to cancer therapies. Here, we demonstrate that stromal fibroblasts contribute to the poor efficacy of HDACis in PDAC. HDACi-treated fibroblasts show increased biological aggressiveness and are characterized by increased secretion of pro-inflammatory tumor-supportive cytokines and chemokines. We find that HDAC2 binds to the enhancer and promoter regions of pro-inflammatory genes specifically in CAFs and in silico analysis identified AP-1 to be the most frequently associated transcription factor bound in these regions. Pharmacologic inhibition of pathways upstream of AP-1 suppresses the HDACi-induced inflammatory gene expression and tumor-supportive responses in fibroblasts. Our findings demonstrate that the combination of HDACis with chemical inhibitors of the AP-1 signaling pathway attenuate the inflammatory phenotype of fibroblasts and may improve the efficacy of HDACi in PDAC and, potentially, in other solid tumors rich in stroma.

Assessment of a Revised Management Strategy for Patients With Intraductal Papillary Mucinous Neoplasms Involving the Main Pancreatic Duct

Importance According to the 2012 International Consensus Guidelines, the diagnostic criterion of intraductal papillary mucinous neoplasms (IPMNs) involving the main duct (MD IPMNs) or the main and branch ducts (mixed IPMNs) of the pancreatic system is a main pancreatic duct (MPD) diameter of 5.0 mm or greater on computed tomography (CT) or magnetic resonance imaging (MRI). However, surgical resection is recommended for patients with an MPD diameter of 10.0 mm or greater, which is characterized as a high-risk stigma. An MPD diameter of 5.0 to 9.0 mm is not an indication for immediate resection. Objectives To determine an appropriate cutoff (ie, one with high sensitivity and negative predictive value) of the MPD diameter on CT or MRI as a prognostic factor for malignant disease and to propose a new management algorithm for patients with MD or mixed IPMNs. Design, Setting, and Participants This retrospective cohort study included 103 patients who underwent surgical resection for a preoperative diagnosis of MD or mixed IPMN and in whom IPMN was confirmed by surgical pathologic findings at a single institution from July 1, 1996, to December 31, 2015. Main Outcomes and Measures Malignant disease was defined as high-grade dysplasia or invasive adenocarcinoma on results of surgical pathologic evaluation. An appropriate MPD diameter on preoperative CT or MRI to predict malignant disease was determined using a receiver operating characteristic curve analysis. The prognostic value of the new management algorithm that incorporated the new MPD diameter cutoff was evaluated. Results Among the 103 patients undergoing resection for an MD or mixed IPMN (59 men [57.3%]; 44 women [42.7%]; median [range] age, 71 [48-86] years), 64 (62.1%) had malignant disease. Diagnostic accuracy for malignant neoplasms was highest at an MPD diameter cutoff of 7.2 mm (area under the receiver operating characteristic curve, 0.70; 95% CI, 0.59-0.81). An MPD diameter of 7.2 mm or greater was also an independent prognostic factor for malignant neoplasms (odds ratio, 12.76; 95% CI, 2.43-66.88; Pu2009=u2009.003) on logistic regression analysis after controlling for preoperative variables. The new management algorithm, which included an MPD diameter of 7.2 mm or greater as one of the high-risk stigmata, had a higher sensitivity (100%), negative predictive value (100%), and accuracy (66%) for malignant disease than the 2012 version of the International Consensus Guidelines (95%, 57%, and 63%, respectively). Conclusions and Relevance In this single-center, retrospective analysis, an MPD diameter of 7.2 mm was identified as an optimal cutoff for a prognostic factor for malignant disease in MD or mixed IPMN. These data support lowering the accepted criteria for MPD diameter when selecting patients for resection vs surveillance so as not to overlook cancer in IPMN.

CA19-9 Normalization During Pre-operative Treatment Predicts Longer Survival for Patients with Locally Progressed Pancreatic Cancer.

BackgroundCompared to the widely adopted 2–4xa0months of pre-operative therapy for patients with borderline resectable (BR) or locally advanced (LA) pancreatic ductal adenocarcinoma (PDAC), our institution tends to administer a longer duration before considering surgical resection. Using this unique approach, the aim of this study was to determine pre-operative variables associated with survival.MethodsRecords from patients with BR/LA PDAC who underwent attempt at surgical resection from 1992–2014 were reviewed.ResultsAfter a median duration of 6xa0months of pre-operative treatment, 109 patients with BR/LA PDAC (BR 63, LA 46) were explored; 93 (85.3xa0%) underwent pancreatectomy. Those who received at least 6xa0months of pre-operative treatment had longer median overall survival (OS) than those who received less (52.8 vs. 32.1xa0months, Pu2009=u20090.044). On multivariate analysis, pre-operative treatment duration was the strongest predictor of survival (hazard ratio (HR) 4.79, Pu2009=u20090.043). However, OS was similar in those whose CA19-9 normalized regardless of whether they received more or less than 6xa0months of chemotherapy (71.4 vs. 101.8xa0months, Pu2009=u20090.930).ConclusionsPre-operative CA19-9 decline can guide treatment duration in patients with BR/LA PDAC. We endorse 6xa0months of therapy except in those patients whose values normalize, where surgery can be considered after a shorter course.

Pancreatic cancer patients with lymph node involvement by direct tumor extension have similar survival to those with node-negative disease

Lymph node (LN) involvement is a well‐known poor prognostic factor in patients with pancreatic ductal adenocarcinoma (PDAC). However, there have been conflicting results on the significance of the mechanism of LN involvement, “direct” tumor invasion versus “metastatic,” disease on patient survival.