Charmi Patel

Gemcitabine resistant pancreatic cancer cell lines acquire an invasive phenotype with collateral hypersensitivity to histone deacetylase inhibitors

Gemcitabine based treatment is currently a standard first line treatment for patients with advanced pancreatic cancer, however overall survival remains poor, and few options are available for patients that fail gemcitabine based therapy. To identify potential molecular targets in gemcitabine refractory pancreatic cancer, we developed a series of gemcitabine resistant (GR) cell lines. Initial drug exposure selected for an early resistant phenotype that was independent of drug metabolic pathways. Prolonged drug selection pressure after 16 weeks, led to an induction of cytidine deaminase (CDA) and enhanced drug detoxification. Cross resistance profiles demonstrate approximately 100-fold cross resistance to the pyrimidine nucleoside cytarabine, but no resistance to the same in class agents, azacytidine and decitabine. GR cell lines demonstrated a dose dependent collateral hypersensitivity to class I and II histone deacetylase (HDAC) inhibitors and decreased expression of 3 different global heterochromatin marks, as detected by H4K20me3, H3K9me3 and H3K27me3. Cell morphology of the drug resistant cell lines demonstrated a fibroblastic type appearance with loss of cell-cell junctions and an altered microarray expression pattern, using Gene Ontology (GO) annotation, consistent with progression to an invasive phenotype. Of particular note, the gemcitabine resistant cell lines displayed up to a 15 fold increase in invasive potential that directly correlates with the level of gemcitabine resistance. These findings suggest a mechanistic relationship between chemoresistance and metastatic potential in pancreatic carcinoma and provide evidence for molecular pathways that may be exploited to develop therapeutic strategies for refractory pancreatic cancer.

Enteric Duplication Cyst Containing Squamous and Respiratory Epithelium: An Interesting Case of a Typically Pediatric Entity Presenting in an Adult Patient

Enteric duplication cysts are rare congenital malformations that can occur at any point along the digestive tract, most commonly the small bowel. They are characterized by the presence of an outer layer of smooth muscle and an inner lining of mucosa that may resemble any portion of the digestive tract. Less commonly, cases have been reported that also contain mucosal components of nonintestinal origin. This entity is typically diagnosed in young children, but occasionally presents in adolescence and young adulthood. We present a rare case of a 21-year-old male who presented with nonspecific symptoms of abdominal discomfort and weight loss and was later found to have a 9 cm nonenhancing mass in the distal ileum on CT imaging. Laparoscopic dissection of the mass revealed a cystic lesion lined mainly by pseudostratified ciliated columnar respiratory-type epithelium, with patchy areas of squamous epithelium as well as villous columnar epithelium resembling small bowel. The unique histology and advanced patient age make this case a unique presentation of what is already a rare pathological entity.

Label-free multi-photon imaging of dysplasia in Barrett's esophagus.

Barretts esophagus (BE) is a metaplastic disorder where dysplastic and early cancerous changes are invisible to the naked eye and where the practice of blind biopsy is hampered by large sampling errors. Multi-photon microscopy (MPM) has emerged as an alternative solution for fast and label-free diagnostic capability for identifying the histological features with sub-micron accuracy. We developed a compact, inexpensive MPM system by using a handheld mode-locked fiber laser operating at 1560nm to study mucosal biopsies of BE. The combination of back-scattered THG, back-reflected forward THG and SHG signals generate images of cell nuclei and collagen, leading to label-free diagnosis in Barretts.

Intrabiliary Hepatic Metastasis of Colorectal Carcinoma Mimicking Primary Cholangiocarcinoma: A Case Report and Review of the Literature

Intrabiliary metastasis from colorectal carcinoma (CRC) growing within or invading bile ducts is not a very common pattern. However, accurate diagnosis of metastatic lesions is very important for selection of adjuvant therapy and prognosis. We report a case of 71-year-old male who developed painless jaundice due to hepatobiliary obstruction. MRI demonstrated 1.4 cm intraductal mass at hepatic hilum with severe intrahepatic ductal dilation, consistent with cholangiocarcinoma. ERCP (endoscopic retrograde cholangiopancreatography) showed intraductal segmental biliary stricture. Biopsy from the lesion showed adenocarcinoma favoring primary cholangiocarcinoma due to the papillary morphology and location of the mass. His past history was significant for rectosigmoid carcinoma (pT1N0) ten years ago and liver resection for metastatic CRC four years ago. He subsequently underwent central hepatectomy with resection of common bile duct. Grossly, there was a 1.2 cm intraductal mass at the bifurcation of bile ducts with multiple nodules in liver parenchyma. Microscopic examination revealed intraductal carcinoma with papillary architecture colonizing bile duct epithelium with resultant dilation and tortuosity. Occasional liver parenchymal nodules show classical metastatic pattern resembling CRC. Because of two distinct morphologic patterns and patients past history, immunostains were performed. CK7 stained uninvolved bile duct epithelium with no staining in intrabiliary metastatic growth. CK20 and CDX2 were positive, thus confirming intrabiliary growth as metastatic growth from CRC. In summary, findings from our case indicate that intrabiliary growth of metastatic CRC can easily be overlooked with major duct involvement. Pathologic evaluation with use of immunohistochemical stains is very important to achieve correct diagnosis.

Characterization of TCP-1 probes for molecular imaging of colon cancer.

Molecular probes capable of detecting colorectal cancer (CRC) are needed for early CRC diagnosis. The objective of this study was to characterize c[CTPSPFSHC]OH (TCP-1), a small peptide derived from phage display selection, for targeting human CRC xenografts using technetium-99m ((99m)Tc)-labeled TCP-1 and fluorescent cyanine-7 (Cy7)-labeled form of the peptide (Cy7-TCP-1). (99m)Tc-TCP-1 was generated by modifying TCP-1 with succinimidyl-6-hydrazino-nicotinamide (S-HYNIC) followed by radiolabeling. In vitro saturation binding experiments were performed for (99m)Tc-TCP-1 in human HCT116 colon cancer cells. SCID mice with human HCT116 cancer xenografts were imaged with (99m)Tc-TCP-1 or control peptide using a small-animal SPECT imager: Group I (n=5) received no blockade; Group II (n=5) received a blocking dose of non-radiolabeled TCP-1. Group III (n=5) were imaged with (99m)Tc-labeled control peptide (inactive peptide). SCID mice with human PC3 prostate cancer xenografts (Group IV, n=5) were also imaged with (99m)Tc-TCP-1. Eight additional SCID mice bearing HCT116 xenografts in dorsal skinfold window chambers (DSWC) were imaged by direct positron imaging of (18)F-fluorodeoxyglucose ((18)F-FDG) and fluorescence microscopy of Cy7-TCP-1. In vitro(99m)Tc-HYNIC-TCP-1 binding assays on HCT 116 cells indicated a mean Kd of 3.04±0.52nM. In cancer xenografts, (99m)Tc-TCP-1 radioactivity (%ID/g) was 1.01±0.15 in the absence of blockade and was reduced to 0.26±0.04 (P<0.01) with blockade. No radioactive uptake was observed in the PC3 tumors with (99m)Tc-TCP-1 or HCT116 tumors with inactive peptide. Cy7-TCP-1 activity localized not only in metabolically active tumors, as defined by (18)F-FDG imaging, but also in peritumoral microvasculature. In conclusion, TCP-1 probes may have a distinct targeting mechanism with high selectivity for CRC and tumor-associated vasculature. Molecular imaging with TCP-1 probes appears promising to detect malignant colorectal lesions.

An Unusual Case of Gastritis and Duodenitis After Yttrium 90–Microsphere Selective Internal Radiation

he patient was a 55-year-old man with gastroinTtestinal stromal tumor that was metastatic to bilateral liver lobes. After multiple trials of chemotherapy without significant response, he received 2 courses of radioembolization with yttrium 90–labeled microspheres through the right and left branches of the hepatic artery. After radioembolization of the right lobe, embolization of the gastroduodenal artery was performed to prevent extrahepatic delivery of spheres. An arteriogram confirmed no flow to bowel previously supplied by the artery before embolization of the left lobe. In both instances, imaging after radioembolization confirmed delivery to targeted regions only. Seven months after the first infusion, the patient presented with abdominal pain and nausea and was admitted for management and endoscopy. One gastric ulcer and one duodenal ulcer were identified and sampled for histologic evaluation and cytomegalovirus testing (Figure A). Duodenal biopsies showed mucosal ulceration, mucin depletion, and reactive epithelial and stromal changes. Gastric biopsies showed similar, albeit milder, changes, along with plasmacytic infiltrate in the lamina propria. No dysplasia, carcinoma, or metastases were noted in either biopsy. Immunohistochemistry was negative for Helicobacter pylori organisms or cytomegalovirus inclusions. Both biopsies showed foreign round violet particles surrounded by lighter peripheral halos in the lamina propria. These findings were suggestive of radiotherapyinduced changes (Figures B and C). The liver is a common site of metastases from gastrointestinal malignancies, including gastrointestinal stromal tumor. Although traditional treatments include resection, radiotherapy, and chemotherapy, for those patients with metastases, alternatives (radiofrequency ablation, portal vein embolization, and intra-arterial chemotherapy and/or radiotherapy) may reduce morbidity, if not mortality. A subset of radiotherapy, selective internal radiation therapy, involves the infusion of microspheres labeled with the radioisotope yttrium 90 (Y) directly into the hepatic artery. This method allows for direct delivery of radiation to tumor cells, minimizing toxicity to surrounding normal liver parenchyma, and has shown promise in patients meeting specific selection criteria. Despite this significant reduction in morbidity, several recent reports indicate adverse effects as a result of dissemination of microbeads into the extrahepatic circulation. Histopathologic evidence of gastritis and duodenitis has been observed days to months after radioembolization and can be identified microscopically by such changes as epithelial attenuation, foveolar hyperplasia, and apoptosis in the context of yttrium microspheres in the lamina propria. Awareness of yttrium-induced gastroduodenitis and identification of yttrium 90–related changes are necessary as selective internal radiation therapy treatment becomes a more common therapy for primary and secondary liver malignancies and may guide the early institution of care, which is mainly supportive.

Evaluating IPMN and pancreatic carcinoma utilizing quantitative histopathology

Intraductal papillary mucinous neoplasms (IPMN) are pancreatic lesions with uncertain biologic behavior. This study sought objective, accurate prediction tools, through the use of quantitative histopathological signatures of nuclear images, for classifying lesions as chronic pancreatitis (CP), IPMN, or pancreatic carcinoma (PC). Forty‐four pancreatic resection patients were retrospectively identified for this study (12 CP; 16 IPMN; 16 PC). Regularized multinomial regression quantitatively classified each specimen as CP, IPMN, or PC in an automated, blinded fashion. Classification certainty was determined by subtracting the smallest classification probability from the largest probability (of the three groups). The certainty function varied from 1.0 (perfectly classified) to 0.0 (random). From each lesion, 180 ± 22 nuclei were imaged. Overall classification accuracy was 89.6% with six unique nuclear features. No CP cases were misclassified, 1/16 IPMN cases were misclassified, and 4/16 PC cases were misclassified. Certainty function was 0.75 ± 0.16 for correctly classified lesions and 0.47 ± 0.10 for incorrectly classified lesions (P = 0.0005). Uncertainty was identified in four of the five misclassified lesions. Quantitative histopathology provides a robust, novel method to distinguish among CP, IPMN, and PC with a quantitative measure of uncertainty. This may be useful when there is uncertainty in diagnosis.

Abstract A83: Nuclear morphometry differentiates chronic pancreatitis, IPMN, and pancreatic carcinoma

Background: It can be difficult to distinguish between chronic pancreatitis (CP), IPMN, and pancreatic carcinoma (PC) on tissue biopsy. Nuclear morphometry can measure up to 93 unique nuclear features based on standard histopathology. The goal of this work is to build novel, objective, and accurate prediction tools, based on nuclear morphometric signatures in high resolution images of nuclei of histologic sections, for classifying pancreatic tissues into three distinct groups. Materials & Methods: 44 patients who underwent pancreatic resections were identified. 12 cases of CP, 16 cases of IPMN, and 16 cases of PC were utilized in this pilot study. 180 ± 22 nuclei from each lesion were imaged with high resolution microscopy. Clincodemographic data was obtained retrospectively from the medical record. Statistically significant nuclear features were determined by a fully automated penalized multinomial regression algorithm in order to determine a multi-class classifier and simultaneously identify important nuclear features. The LASSO penalty function, and associated regularization parameter, is adaptively chosen by cross validation to prevent over-fitting. In order to test the veracity of the automated algorithm, we randomly removed 25% of the cases as a training set and utilized the remaining cases as a test set; this was repeated 10 times. Results: The average age was 64 ± 15 years, with patients in the CP being slightly younger; 63% were male. Median follow-up time was 3 years in the CP group, 3 years in the IPMN group, and 5 years in PC group. The method described automatically identified 6 unique and statistically significant nuclear features (corrected overall P Conclusions: Nuclear morphometry classifies pancreatic lesions into CP, IPMN, and PC with 84.5% accuracy using a fully automated algorithm to determine statistically significant and unique nuclear features. Since the incorrectly classified lesions had a larger proportion of mixed nuclei, diagnostic uncertainty may be determined in a quantitative manner allowing for a confidence probability estimation of whether a given lesions should be classified as a CP, IPMN, or PC. Further studies will validate these results in a resected cohort as well as a cohort based on biopsied specimens alone. Citation Format: Evan S. Glazer, Hao Zhang, Kimberly A. Hill, Charmi Patel, Stephanie T. Kha, Peter H. Bartels, Michael L. Yozwiak, Hubert G. Bartels, Joseph C. Watkins, David S. Alberts, Robert S. Krouse. Nuclear morphometry differentiates chronic pancreatitis, IPMN, and pancreatic carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A83.