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An inactivating germline mutation in BRCA2 is the most common known genetic basis for familial pancreatic cancer (FPC), accounting for 5-10% of inherited cases. A genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) arising on the backdrop of Brca2 deficiency is likely to elucidate valuable diagnostic and therapeutic insights for FPC. Both Brca2 alleles were conditionally deleted during development within the pancreatic epithelium by generating Pdx1-Cre; Brca2(f/f) (CB) mice; in addition, triple transgenic Pdx1-Cre; Brca2(f/f); LSL-Trp53(R172H) (CBP) mice were generated, in order to determine the impact of p53 deregulation on Brca2-deficient carcinogenesis. Both CB and CBP mice developed non-invasive ductal precursor lesions (murine pancreatic intraepithelial neoplasia or mPanIN), although these were observed at an earlier time point (5 versus 8 months) and with higher prevalence in CBP mice. A minority of CB mice (15%) developed invasive and metastatic PDAC at a latency of 15 months or greater; in contrast, CBP mice of comparable age uniformly developed PDAC with variable histological features. Mortality in the absence of neoplasia in CB and CBP mice was associated with profound loss of pancreatic parenchyma, consistent with progressive elimination of Brca2-deficient cells. Widespread DNA damage, as evidenced by overexpression of the phosphorylated histone H(2)AX(Ser139), was observed in the non-neoplastic exocrine pancreas, as well as in the mPanIN and PDAC lesions of Brca2-deficient mice, independent of p53 status. Loss of Brca2 function predisposes the exocrine pancreas to profound DNA damage, and the frequency of invasive neoplasia is accentuated by the concomitant deregulation of p53.

Background: Specific populations of highly tumorigenic cells are thought to exist in many human tumors, including pancreatic adenocarcinoma. However, the clinical significance of these tumor-initiating (ie, cancer stem) cells remains unclear. Aldehyde dehydrogenase (ALDH) activity can identify tumor-initiating cells and normal stem cells from several human tissues. We examined the prognostic significance and functional features of ALDH expression in pancreatic adenocarcinoma., Methods: ALDH expression was analyzed by immunohistochemistry in 269 primary surgical specimens of pancreatic adenocarcinoma and examined for association with clinical outcomes and in paired primary tumors and metastatic lesions from eight pancreatic cancer patients who had participated in a rapid autopsy program. The clonogenic growth potential of ALDH-positive pancreatic adenocarcinoma cells was assessed in vitro by a colony formation assay and by tumor growth in immunodeficient mice (10-14 mice per group). Mesenchymal features of ALDH-positive pancreatic tumor cells were examined by using quantitative reverse transcription-polymerase chain reaction and an in vitro cell invasion assay. Gene expression levels and the invasive potential of ADLH-positive pancreatic cancer cells relative to the bulk cell population were examined by reverse transcription-polymerase chain reaction and an in vitro invasion assays, respectively. All statistical tests were two-sided., Results: ALDH-positive tumor cells were detected in 90 of the 269 primary surgical specimens, and their presence was associated with worse survival (median survival for patients with ALDH-positive vs ALDH-negative tumors: 14 vs 18 months, hazard ratio of death = 1.28, 95% confidence interval = 1.02 to 1.68, P = .05). Six (75%) of the eight patients with matched primary and metastatic tumor samples had ALDH-negative primary tumors, and in four (67%) of these six patients, the matched metastatic lesions (located in liver and lung) contained ALDH-positive cells. ALDH-positive cells were approximately five- to 11-fold more clonogenic in vitro and in vivo compared with unsorted or ALHD-negative cells, expressed genes consistent with a mesenchymal state, and had in vitro migratory and invasive potentials that were threefold greater than those of unsorted cells., Conclusions: ALDH expression marks pancreatic cancer cells that have stem cell and mesenchymal features. The enhanced clonogenic growth and migratory properties of ALDH-positive pancreatic cancer cells suggest that they play a key role in the development of metastatic disease that negatively affects the overall survival of patients with pancreatic adenocarcinoma.

Pancreatic intraepithelial neoplasia (PanIN) lesions are the most common non-invasive precursors of pancreatic adenocarcinoma. We postulated that accumulating DNA damage within the PanIN epithelium activates checkpoint mechanisms. Tissue microarrays were constructed from 81 surgically resected primary pancreatic adenocarcinomas and an independent set of 58 PanIN lesions (31 PanIN-1, 14 PanIN-2, and 13 PanIN-3). Immunohistochemical labeling was carried out using anti-gammaH2AX(Ser139), anti-phosphoATM(Ser1981), anti-phosphoChk2(Thr68), and anti-p53. A 'histologic score' combining area and intensity of labeling in the nuclear compartment was determined for each lesion. A progressive increase in gammaH2AX(Ser139) labeling, consistent with escalating DNA damage, was observed in the non-invasive precursor lesions (scores of 4.34, 6.21, and 7.50, respectively, for PanIN-1, -2, and -3), compared with the pancreatic ductal epithelium (score 2.36) (ANOVA, P<0.0001). In conjunction, activation of the ataxia telangiectasia mutated (ATM)-Chk2 checkpoint pathway was observed in all histological grades of PanIN lesions. Specifically, pATM(Ser1981) histologic scores for PanIN-1, PanIN-2, and PanIN-3 were 4.83, 5.14, and 7.17, respectively, versus 2.33 for the ductal epithelium (ANOVA, P<0.0001); the corresponding scores for pChk2(Thr68) were 5.43, 7.64, and 5.44 in PanINs-1, -2, and -3, respectively, versus 2.75 in the ductal epithelium (ANOVA, P<0.0001). In contrast, absent to minimal nuclear p53 was observed in the ductal epithelium, and in PanINs-1 and -2 (a histologic score of 0-1.86), with a significant upregulation (corresponding to mutational inactivation) seen only at the stage of PanIN-3 and invasive neoplasia (histologic scores of 4.00 and 4.22). Nuclear p53 accumulation in cancers was associated with attenuation of the ATM-Chk2 checkpoint and a restitution to 'baseline' levels. To conclude, activation of the ATM-Chk2 checkpoint pathway is commonly observed in PanINs, likely in response to the accumulating DNA damage from events such as oncogene mutations and telomere dysfunction. Loss of p53 function appears to be a critical determinant for bypassing this checkpoint and the subsequent progression to invasive adenocarcinoma.

Purpose: Aberrant activation of the Notch signaling pathway is commonly observed in human pancreatic cancer, although the mechanism(s) for this activation has not been elucidated., Experimental Design: A panel of 20 human pancreatic cancer cell lines was profiled for the expression of Notch pathway-related ligands, receptors, and target genes. Disruption of intracellular Notch signaling, either genetically by RNA interference targeting NOTCH1 or pharmacologically by means of the gamma-secretase inhibitor GSI-18, was used for assessing requirement of Notch signaling in pancreatic cancer initiation and maintenance., Results: Striking overexpression of Notch ligand transcripts was detectable in the vast majority of pancreatic cancer cell lines, most prominently JAGGED2 (18 of 20 cases, 90%) and DLL4 (10 of 20 cases, 50%). In two cell lines, genomic amplification of the DLL3 locus was observed, mirrored by overexpression of DLL3 transcripts. In contrast, coding region mutations of NOTCH1 or NOTCH2 were not observed. Genetic and pharmacologic inhibition of Notch signaling mitigated anchorage-independent growth in pancreatic cancer cells, confirming that sustained Notch activation is a requirement for pancreatic cancer maintenance. Further, transient pretreatment of pancreatic cancer cells with GSI-18 resulted in depletion in the proportion of tumor-initiating aldehyde dehydrogenase-expressing subpopulation and was associated with inhibition of colony formation in vitro and xenograft engraftment in vivo, underscoring a requirement for the Notch-dependent aldehyde dehydrogenase-expressing cells in pancreatic cancer initiation., Conclusions: Our studies confirm that Notch activation is almost always ligand dependent in pancreatic cancer, and inhibition of Notch signaling is a promising therapeutic strategy in this malignancy.

Background: Pancreatic cancer is a near uniformly lethal disease and a better understanding of the molecular basis of this malignancy may lead to improved therapeutics. The Axl receptor tyrosine kinase is implicated in cellular transformation and tumor progression, although its role in pancreatic cancer has not been previously documented., Results: Axl labeling was present in 54 of 99 (55%), and was absent in 45 of 99 (45%) cases, respectively. Axl expression in pancreatic cancer was significantly associated with lymph node metastases (p < 0.01), and a shorter median survival (12 versus 18 months, p < 0.01), than in tumors with negative labeling. Stable knockdown of Axl resulted in significant reduction in cell viability (p < 0.001), anchorage independent growth (p = 0.0031), as well as attenuation of migratory (p < 0.001) and invasive properties (p < 0.005), compared to vector-transfected cells. Profound inhibition of p42/p44 MAP kinase and PI-3kinase/Akt effector pathways was observed in MIAPaCa-2 cells with loss of Axl function. The reduction in invasion and migration upon Axl knockdown was mirrored by a decrease in the amounts of activated (GTP-bound) GTPase proteins Rho and Rac, significant downregulation in transcript levels of the epithelial mesenchymal transition (EMT)-associated transcription factors slug, snail and twist, and significant decrease in matrix metalloproteinase MMP-9 mRNA levels., Materials: The immunohistochemical expression of Axl protein was assessed in a panel of 99 archival pancreatic cancers. Endogenous Axl expression was stably downregulated by lentiviral short hairpin shRNA directed against AXL mRNA in MIAPaCa-2 cells, and the effects on cell viability, anchorage independent growth, invasion, migration and intracellular effector pathways was assessed, by comparing to lentiviral vector-transfected cells., Conclusion: Expression of Axl tyrosine kinase in pancreatic cancers confers an adverse prognostic influence, and represents a new therapeutic target in this malignancy.

Background: Intraductal papillary mucinous neoplasms (IPMNs) are non-invasive precursor lesions of pancreatic cancer. Misexpression of microRNAs (miRNAs) is commonly observed in pancreatic adenocarcinoma. In contrast, miRNA abnormalities in pancreatic cancer precursor lesions have not been documented., Experimental Design: Relative expression levels of a panel of twelve miRNAs upregulated in pancreatic cancers were assessed in 15 non-invasive IPMNs, using quantitative reverse transcription PCR (qRT-PCR). Two significantly overexpressed miRNAs-miR-155 and miR-21-were evaluated by locked nucleic acid in situ hybridization (LNA-ISH) in a panel of 64 archival IPMNs. The expression of miR-155 and miR-21 was also evaluated in pancreatic juice samples obtained from ten patients with surgically resected IPMNs and five patients with non-neoplastic pancreato-biliary disorders ("disease controls")., Results: Significant overexpression by qRT-PCR of ten of the twelve miRNAs was observed in the 15 IPMNs versus matched controls (p < 0.05), with miR-155 (mean 11.6-fold) and miR-21 (mean 12.1-fold) demonstrating highest relative fold-changes in the precursor lesions. LNA-ISH confirmed the expression of miR-155 in 53 of 64 (83%) IPMNs compared to 4 of 54 (7%) normal ducts, and of miR-21 in 52 of 64 (81%) IPMNs compared to 1 of 54 (2%) normal ducts, respectively (p < 0.0001). Upregulation of miR-155 transcripts by qRT-PCR was observed in 6 of 10 (60%) IPMN-associated pancreatic juice samples compared to 0 of 5 (0%) disease controls., Conclusions: Aberrant miRNA expression is an early event in the multistage progression of pancreatic cancer, and miR-155 warrants further evaluation as a biomarker for IPMNs in clinical samples.

Nanotechnology has enabled significant advances in the areas of cancer diagnosis and therapy. The field of drug delivery is a sterling example, with nanoparticles being increasingly used for generating therapeutic formulations of poorly water-soluble, yet potent anticancer drugs. Whereas a number of nanoparticle-drug combinations are at various stages of preclinical or clinical assessment, the overwhelming majorities of such systems are injectable formulations and are incapable of being partaken orally. The development of an oral nano-delivery system would have distinct advantages for cancer chemotherapy. We report the synthesis and physicochemical characterization of orally bioavailable polymeric nanoparticles composed of N-isopropylacrylamide, methylmethacrylate, and acrylic acid in the molar ratios of 60:20:20 (designated NMA622). Amphiphilic NMA622 nanoparticles show a size distribution of <100 nm (mean diameter of 80 +/- 34 nm) with low polydispersity and can readily encapsulate a number of poorly water-soluble drugs such as rapamycin within the hydrophobic core. No apparent systemic toxicities are observed in mice receiving as much as 500 mg/kg of the orally administered void NMA622 for 4 weeks. Using NMA622-encapsulated rapamycin ("nanorapamycin") as a prototype for oral nano-drug delivery, we show favorable in vivo pharmacokinetics and therapeutic efficacy in a xenograft model of human pancreatic cancer. Oral nanorapamycin leads to robust inhibition of the mammalian target of rapamycin pathway in pancreatic cancer xenografts, which is accompanied by significant growth inhibition (P < 0.01) compared with control tumors. These data indicate that NMA622 nanoparticles provide a suitable platform for oral delivery of water-insoluble drugs like rapamycin for cancer therapy.

The familial atypical multiple mole melanoma (FAMMM) syndrome is caused by a germline mutation of p16. More than 90% of the sporadic pancreatic carcinomas contain genetic alterations that inactivate p16. Patients with the FAMMM syndrome have an increased risk of developing pancreatic cancer. Ductal adenocarcinoma is the most common cancer of the pancreas and the one encountered in patients with FAMMM syndrome. Undifferentiated carcinoma with osteoclastic giant cells, also referred to as UCOCGC of the pancreas, is a rare variant of pancreatic cancer. An UCOCGC of the pancreas associated with FAMMM syndrome is described in this report. Molecular analysis confirmed a germline p16-Leiden deletion in the UCOCGC, accompanied by somatic loss of heterozygosity of the second p16 allele, and absence of p16 protein expression in the neoplastic cells. It is the first case reported and it provides additional evidence that UCOCGC can be considered as a variant of conventional ductal adenocarcinoma of the pancreas.

Esophageal adenocarcinoma currently has one of the most rapidly increasing tumor incidences in the United States, with the vast majority of cases occurring on the backdrop of metaplastic epithelium (Barrett esophagus). The availability of appropriate cell line models is essential for maintaining the pace of esophageal cancer research and for pre-clinical validation of new therapeutic modalities. The identity of several of the widely utilized esophageal adenocarcinoma cell lines (BIC-1, SEG-1 and TE-7) have recently been called into question. Here we describe the establishment and characterization of a bona fide esophageal cancer cell line, JH-EsoAd1, from a patient with Barrett-associated adenocarcinoma. The rapid dissemination of this cancer cell line to the esophageal cancer research community should help ameliorate the current scarcity of preclinical models in this disease.

Recent evidence suggests that blockade of aberrant Hedgehog signaling can be exploited as a therapeutic strategy for pancreatic cancer. Our previous studies using the prototype Hedgehog small-molecule antagonist cyclopamine had shown the striking inhibition of systemic metastases on Hedgehog blockade in spontaneously metastatic orthotopic xenograft models. Cyclopamine is a natural compound with suboptimal pharmacokinetics, which impedes clinical translation. In the present study, a novel, orally bioavailable small-molecule Hedgehog inhibitor, IPI-269609, was tested using in vitro and in vivo model systems. In vitro treatment of pancreatic cancer cell lines with IPI-269609 resembled effects observed using cyclopamine (i.e., Gli-responsive reporter knockdown, down-regulation of the Hedgehog target genes Gli1 and Ptch, as well as abrogation of cell migration and colony formation in soft agar). Single-agent IPI-269609 profoundly inhibited systemic metastases in orthotopic xenografts established from human pancreatic cancer cell lines, although Hedgehog blockade had minimal effect on primary tumor volume. The only discernible phenotype observed within the treated primary tumor was a significant reduction in the population of aldehyde dehydrogenase-bright cells, which we have previously identified as a clonogenic tumor-initiating population in pancreatic cancer. Selective ex vivo depletion of aldehyde dehydrogenase-bright cells with IPI-269609 was accompanied by significant reduction in tumor engraftment rates in athymic mice. Pharmacologic blockade of aberrant Hedgehog signaling might prove to be an effective therapeutic strategy for inhibition of systemic metastases in pancreatic cancer, likely through targeting subsets of cancer cells with tumor-initiating ("cancer stem cell") properties.

Introduction: Pancreatic ductal adenocarcinoma (i.e., pancreatic cancer) is an almost universally lethal disease. The identification of precursor lesions of pancreatic cancer provides an opportunity for early detection and potential therapeutic intervention before the development of invasive cancer., Discussion: It is now established that pancreatic cancers do not arise de novo but rather exhibit a sequential histological and genetic progression of precursor lesions culminating in frank, invasive neoplasia. Pancreatic intraepithelial neoplasia (PanIN) is the most common non-invasive precursor lesion of pancreatic cancer. The development of a consensus nomenclature scheme for PanINs has facilitated research into pancreatic cancer precursors and enabled standardization of results across institutions., Conclusion: PanINs harbor many of the molecular alterations observed in invasive pancreatic cancer, confirming their status as true non-invasive precursor lesions. Recently developed genetically engineered mouse models of pancreatic cancer also demonstrate the stepwise PanIN progression model, underscoring the commonalities in pancreatic neoplasia between mouse and man.

Pancreatic cancer is an almost universally lethal disease. Research over the last two decades has shown that pancreatic cancer is fundamentally a genetic disease, caused by inherited germline and acquired somatic mutations in cancer-associated genes. Multiple alterations in genes that are important in pancreatic cancer progression have been identified, including tumor suppressor genes, oncogenes, and genome maintenance genes. Furthermore, the identification of noninvasive precursor lesions of pancreatic adenocarcinoma has led to the formulation of a multi-step progression model of pancreatic cancer and the subsequent identification of early and late genetic alterations culminating in invasive cancer. In addition, an increased understanding of the molecular basis of the disease has facilitated the identification of new drug targets enabling rational drug design. The elucidation of genetic alterations in combination with the development of high-throughput sensitive techniques should lead to the discovery of effective biomarkers for early detection of this malignancy. This review focuses mainly on the current knowledge about the molecular insights of the pathogenesis of pancreatic ductal adenocarcinoma., (Copyright 2008 S. Karger AG, Basel.)