Sporadic Pancreatic Adenocarcinoma Research Articles

Early TP53 Alterations Shape Gastric and Esophageal Cancer Development.

Simple SummaryRecent evidence establishes that gastric and esophageal (GE) adenocarcinomas are similar cancers at cellular, genomic, and epigenomic levels. Human GE adenocarcinomas develop TP53 mutations at early stages of malignant progression. This contrasts with other gastrointestinal adenocarcinomas, including sporadic colorectal and pancreatic adenocarcinomas, where TP53 alterations occur late. Exposure of the esophagus and stomach to environmental risk factors contributes to the selection of early TP53 mutations and subsequent chromosomal instability, which then lead to activation of mitogen and cell cycle pathways in GE adenocarcinomas by way of focal amplifications rather than mutations. While early TP53 mutations enable GE adenocarcinoma development, they also expose therapeutic vulnerabilities that should be prime for targeted therapy directed against the DNA damage response.Gastric and esophageal (GE) adenocarcinomas are the third and sixth most common causes of cancer-related mortality worldwide, accounting for greater than 1.25 million annual deaths. Despite the advancements in the multi-disciplinary treatment approaches, the prognosis for patients with GE adenocarcinomas remains poor, with a 5-year survival of 32% and 19%, respectively, mainly due to the late-stage diagnosis and aggressive nature of these cancers. Premalignant lesions characterized by atypical glandular proliferation, with neoplastic cells confined to the basement membrane, often precede malignant disease. We now appreciate that premalignant lesions also carry cancer-associated mutations, enabling disease progression in the right environmental context. A better understanding of the premalignant-to-malignant transition can help us diagnose, prevent, and treat GE adenocarcinoma. Here, we discuss the evidence suggesting that alterations in TP53 occur early in GE adenocarcinoma evolution, are selected for under environmental stressors, are responsible for shaping the genomic mechanisms for pathway dysregulation in cancer progression, and lead to potential vulnerabilities that can be exploited by a specific class of targeted therapy.

1252 Isolated Brain Metastasis From Pancreatic Cancer in Lynch Syndrome

INTRODUCTION: Pancreatic cancer is the 4th leading cause of death in US. The incidence of pancreatic cancer metastasis to brain is rare ranging from 0.1 to 0.3% from various studies. Overall from our knowledge, this is the 26th reported case. This is the first reported case of a patient with a history of Lynch syndrome (LS) who was diagnosed with solitary brain metastasis from pancreatic adenocarcinoma and subsequently treated. CASE DESCRIPTION/METHODS: A 69-year-old female with history of LS and sarcoidosis presented complaining of new onset acute expressive aphasia. Patient's oncologic history significant for colon cancer status post (s/p) subtotal colectomy in 2007, pancreatic adenocarcinoma s/p Whipple in 4/2015. The pathology demonstrated isolated tumor cells in two peripancreatic lymph nodes treated with adjuvant gemcitabine. She had surveillance imaging in 9/2017 showing new metastatic disease, PET-avid retroperitoneal lymphadenopathy and cervical lymphadenopathy. Biopsy showed adenocarcinoma of pancreatic origin. She began gemcitabine/abraxane in 10/2017 with complete resolution on interval imaging in 1/2018. She continued chemotherapy with no metastatic disease on surveillance imaging and was well until presenting with aphasia in 2/2019. MRI of the brain revealed 2 cm enhancing left frontal lobe mass with vasogenic edema and 3 mm midline shift. Labwork including Ca 19-9 and CEA were normal. Because of the uncertainty of the origin of the metastatic disease, left frontal craniotomy and tumor resection was performed. Pathology demonstrated IHC positive for CK7, CK19, CA19-9, CDX-2, and negative for TTF-1, CK20, compatible with metastatic pancreatic adenocarcinoma. Subsequently, the patient underwent single fraction 20 Gy of CyberKnife radiosurgery. The patient symptoms improved and surveillance MRI in 6/2019 demonstrated decreased enhancement consistent with response to treatment. DISCUSSION: To our knowledge this is the first documented case of isolated brain metastatic from pancreatic adenocarcinoma in a LS patient. This case suggests awareness and brain imaging are necessary when pancreatic cancer patients present for neurological complaints. LS is due to germline mutations, which has a different pathogenesis from usual sporadic pancreatic adenocarcinoma. This may explain the unusual presentation, excellent response to therapy, and prolonged survival. Treatment with surgical resection and stereotactic radiosurgery can be considered.

Deleterious Germline Mutations in Patients With Apparently Sporadic Pancreatic Adenocarcinoma.

Purpose Deleterious germline mutations contribute to pancreatic cancer susceptibility and are well documented in families in which multiple members have had pancreatic cancer. Methods To define the prevalence of these germline mutations in patients with apparently sporadic pancreatic cancer, we sequenced 32 genes, including known pancreatic cancer susceptibility genes, in DNA prepared from normal tissue obtained from 854 patients with pancreatic ductal adenocarcinoma, 288 patients with other pancreatic and periampullary neoplasms, and 51 patients with non-neoplastic diseases who underwent pancreatic resection at Johns Hopkins Hospital between 2000 and 2015. Results Thirty-three (3.9%; 95% CI, 3.0% to 5.8%) of 854 patients with pancreatic cancer had a deleterious germline mutation, 31 (3.5%) of which affected known familial pancreatic cancer susceptibility genes: BRCA2 (12 patients), ATM (10 patients), BRCA1 (3 patients), PALB2 (2 patients), MLH1 (2 patients), CDKN2A (1 patient), and TP53 (1 patient). Patients with these germline mutations were younger than those without (mean ± SD, 60.8 ± 10.6 v 65.1 ± 10.5 years; P = .03). Deleterious germline mutations were also found in BUB1B (1) and BUB3 (1). Only three of these 33 patients had reported a family history of pancreatic cancer, and most did not have a cancer family history to suggest an inherited cancer syndrome. Five (1.7%) of 288 patients with other periampullary neoplasms also had a deleterious germline mutation. Conclusion Germline mutations in pancreatic cancer susceptibility genes are commonly identified in patients with pancreatic cancer without a significant family history of cancer. These deleterious pancreatic cancer susceptibility gene mutations, some of which are therapeutically targetable, will be missed if current family history guidelines are the main criteria used to determine the appropriateness of gene testing.

Resectable invasive IPMN versus sporadic pancreatic adenocarcinoma of the head of the pancreas: Should these two different diseases receive the same treatment? A matched comparison study of the French Surgical Association (AFC)

PurposeTo compare survival and impact of adjuvant chemotherapy in patients who underwent pancreaticoduodenectomy (PD) for invasive intraductal papillary mucinous neoplasm (IIPMN) and sporadic pancreatic ductal adenocarcinoma (PDAC). MethodsFrom 2005 to 2012, 240 patients underwent pancreatectomy for IIPMN and 1327 for PDAC. Exclusion criteria included neoadjuvant treatment, pancreatic resection other than PD, vascular resection, carcinoma in situ, or <11 examined lymph nodes. Thus, 82 IIPMN and 506 PDAC were eligible for the present study. Finally, The IIPMN group was matched 1:2 to compose the PDAC group according to TNM disease stage, perineural invasion, lymph node ratio, and margin status. ResultsThere was no difference in patient's characteristics, intraoperative parameters, postoperative outcomes, and histologic parameters. Overall survival and disease-free survival times were comparable between the 2 groups. In each group, overall survival time was significantly poorer in patients who did not achieve adjuvant chemotherapy (p = 0.03 for the IIPMN group; p = 0.03 for the PDAC group). In lymph-node negative patients of the IIPMN group, adjuvant chemotherapy did not have any significant impact on overall survival time (OR = 0.57; 95% CI [0.24–1.33]). Considering the whole population (i.e. patients with IIPMN and PDAC; n = 246), patients who did not achieve adjuvant chemotherapy had poorer survival (p < 0.01). ConclusionsThe courses of IIPMN and PDAC were similar after an optimized stage-to-stage comparison. Adjuvant chemotherapy was efficient in both groups. However, in lymph node negative patients, adjuvant chemotherapy seemed not to have a significant impact.

Germline copy number variation in the YTHDC2 gene: does it have a role in finding a novel potential molecular target involved in pancreatic adenocarcinoma susceptibility?

Objective: The vast majority of pancreatic cancers occurs sporadically. The discovery of frequent variations in germline gene copy number can significantly influence the expression levels of genes that predispose to pancreatic adenocarcinoma. We prospectively investigated whether patients with sporadic pancreatic adenocarcinoma share specific gene copy number variations (CNVs) in their germline DNA.Patients and methods: DNA samples were analyzed from peripheral leukocytes from 72 patients with a diagnosis of sporadic pancreatic adenocarcinoma and from 60 controls using Affymetrix 500K array set. Multiplex ligation-dependent probe amplification (MLPA) assay was performed using a set of self-designed MLPA probes specific for seven target sequences.Results: We identified a CNV-containing DNA region associated with pancreatic cancer risk. This region shows a deletion of 1 allele in 36 of the 72 analyzed patients but in none of the controls. This region is of particular interest since it contains the YTHDC2 gene encoding for a putative DNA/RNA helicase, such protein being frequently involved in cancer susceptibility. Interestingly, 82.6% of Sicilian patients showed germline loss of one allele.Conclusions: Our results suggest that the YTHDC2 gene could be a potential candidate for pancreatic cancer susceptibility and a useful marker for early detection as well as for the development of possible new therapeutic strategies.

Analysis of Germline Gene Copy Number Variants of Patients with Sporadic Pancreatic Adenocarcinoma Reveals Specific Variations

Objectives: The rapid fatality of pancreatic cancer is, in large part, the result of diagnosis at an advanced stage in the majority of patients. Identification of individuals at risk of developing pancreatic adenocarcinoma would be useful to improve the prognosis of this disease. There is presently no biological or genetic indicator allowing the detection of patients at risk. Our main goal was to identify copy number variants (CNVs) common to all patients with sporadic pancreatic cancer. Methods: We analyzed gene CNVs in leukocyte DNA from 31 patients with sporadic pancreatic adenocarcinoma and from 93 matched controls. Genotyping was performed with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix). Results: We identified 431 single nucleotide polymorphism (SNP) probes with abnormal hybridization signal present in the DNA of all 31 patients. Of these SNP probes, 284 corresponded to 3 or more copies and 147 corresponded to 1 or 0 copies. Several cancer-associated genes were amplified in all patients. Conversely, several genes supposed to oppose cancer development were present as single copy. Conclusions: These data suggest that a set of 431 CNVs could be associated with the disease. This set could be useful for early diagnosis.

Molecular Analysis of Precursor Lesions in Familial Pancreatic Cancer

BackgroundWith less than a 5% survival rate pancreatic adenocarcinoma (PDAC) is almost uniformly lethal. In order to make a significant impact on survival of patients with this malignancy, it is necessary to diagnose the disease early, when curative surgery is still possible. Detailed knowledge of the natural history of the disease and molecular events leading to its progression is therefore critical.Methods and FindingsWe have analysed the precursor lesions, PanINs, from prophylactic pancreatectomy specimens of patients from four different kindreds with high risk of familial pancreatic cancer who were treated for histologically proven PanIN-2/3. Thus, the material was procured before pancreatic cancer has developed, rather than from PanINs in a tissue field that already contains cancer. Genome-wide transcriptional profiling using such unique specimens was performed. Bulk frozen sections displaying the most extensive but not microdissected PanIN-2/3 lesions were used in order to obtain the holistic view of both the precursor lesions and their microenvironment. A panel of 76 commonly dysregulated genes that underlie neoplastic progression from normal pancreas to PanINs and PDAC were identified. In addition to shared genes some differences between the PanINs of individual families as well as between the PanINs and PDACs were also seen. This was particularly pronounced in the stromal and immune responses.ConclusionsOur comprehensive analysis of precursor lesions without the invasive component provides the definitive molecular proof that PanIN lesions beget cancer from a molecular standpoint. We demonstrate the need for accumulation of transcriptomic changes during the progression of PanIN to PDAC, both in the epithelium and in the surrounding stroma. An identified 76-gene signature of PDAC progression presents a rich candidate pool for the development of early diagnostic and/or surveillance markers as well as potential novel preventive/therapeutic targets for both familial and sporadic pancreatic adenocarcinoma.

Invasive intraductal papillary mucinous neoplasm versus sporadic pancreatic adenocarcinoma

Although invasive intraductal papillary mucinous neoplasm (IPMN) of the pancreas is thought to be more indolent than sporadic pancreatic adenocarcinoma (PAC), the natural history remains poorly defined. The authors compared survival and identify prognostic factors after resection for invasive IPMN versus stage-matched PAC. The Surveillance, Epidemiology, and End Results database (1991-2005) was used to identify 729 patients with invasive IPMN and 8082 patients with PAC who underwent surgical resection. Patients with resected invasive IPMN experienced improved overall survival when compared with resected PAC (median survival, 21 vs 14 months; P<.001). Stratification by nodal status demonstrated no difference in survival among lymph node-positive patients; however, median survival of resected, lymph node-negative, invasive IPMN was significantly improved compared with lymph node-negative PAC (34 vs 18 months; P<.001). On multivariate analysis, PAC histology was an adverse predictor of overall survival (hazard ratio [HR], 1.31; 95% confidence interval [CI], 1.15-1.50) compared with invasive IPMN. For patients with invasive IPMN, positive lymph nodes (HR, 1.98; 95% CI, 1.50-2.60), high tumor grade (HR, 1.74; 95% CI, 1.31-2.31), tumor size>2 cm (HR, 1.50; 95% CI, 1.04-2.19), and age>66 years (HR, 1.33; 95% CI, 1.03-1.73) were adverse predictors of survival. Although lymph node-negative invasive IPMN showed improved survival after resection compared with lymph node-negative PAC, the natural history of lymph node-positive invasive IPMN mimicked that of lymph node-positive PAC. The authors also identified adverse predictors of survival in invasive IPMN to guide discussions regarding use of adjuvant therapies and prognosis after resection of invasive IPMN.

The Inherited Genetic Component of Sporadic Pancreatic Adenocarcinoma

Pancreatic cancer, like many other complex diseases, has genetic and environmental components to its etiology. It is likely that relatively common genetic variants with modest effects on pancreatic cancer risk play an important role in both familial and sporadic forms of the disease, either individually or in interaction with environmental factors. The relatively highfrequencyofsuch variants meansthattheycould potentially explain a substantial portion of disease risk. Here we summarize the findings published to date from genetic association studies1. In general, very few low-penetrance variants have been identified and those that have require replication in independent studies. Possible gene-environment interactions arising from these studies also require replication. More comprehensive approaches are needed to make progress, including global analyses of biologically sound pathways and genome-wide association studies. Large samplesizesare required to do thisappropriatelyand multi-study consortia make this possible. A number of consortia of preexisting studies have already been formed, and these will facilitate the identification of further low-penetrance variants and gene-environment interaction. However, these approaches do not substitute for the design of novel, sufficiently powered studies that apply uniform criteria to case selection, the acquisition of environmental exposure information, and to biological sample collection.

Genetic and Epigenetic Alterations of Familial Pancreatic Cancers

Little is known about the genetic and epigenetic changes that contribute to familial pancreatic cancers. The aim of this study was to compare the prevalence of common genetic and epigenetic alterations in sporadic and familial pancreatic ductal adenocarcinomas. DNA was isolated from the microdissected cancers of 39 patients with familial and 36 patients with sporadic pancreatic adenocarcinoma. KRAS2 mutations were detected by BstN1 digestion and/or cycle sequencing. TP53 and SMAD4 status were determined by immunohistochemistry on tissue microarrays of 23 archival familial pancreatic adenocarcinomas and in selected cases by cycle sequencing to identify TP53 gene mutations. Methylation-specific PCR analysis of seven genes (FoxE1, NPTX2, CLDN5, P16, TFPI-2, SPARC, ppENK) was done on a subset of fresh-frozen familial pancreatic adenocarcinomas. KRAS2 mutations were identified in 31 of 39 (80%) of the familial versus 28 of 36 (78%) of the sporadic pancreatic cancers. Positive immunolabeling for p53 was observed in 57% of the familial pancreatic cancers and loss of SMAD4 labeling was observed in 61% of the familial pancreatic cancers, rates similar to those observed in sporadic pancreatic cancers. The mean prevalence of aberrant methylation in the familial pancreatic cancers was 68.4%, which was not significantly different from that observed in sporadic pancreatic cancers. The prevalence of mutant KRAS2, inactivation of TP53 and SMAD4, and aberrant DNA methylation of a seven-gene panel is similar in familial pancreatic adenocarcinomas as in sporadic pancreatic adenocarcinomas. These findings support the use of markers of sporadic pancreatic adenocarcinomas to detect familial pancreatic adenocarcinomas.

Incidence of Additional Primary Cancers in Patients with Invasive Intraductal Papillary Mucinous Neoplasms and Sporadic Pancreatic Adenocarcinomas

Recent small studies have reported an incidence of 23% to 39% for additional primary cancers in patients with intraductal papillary mucinous neoplasms (IPMN) of the pancreas. There have been no population-based studies evaluating this incidence rate. Using the Surveillance, Epidemiology, and End Results (SEER) database (1983 to 1991), we identified all patients with primary pancreatic cancers (sporadic and adenocarcinomas arising in IPMNs). We determined the incidence of additional primary cancers that developed either before or after the diagnosis of invasive IPMN and compared it to the incidence of additional primary cancers in patients with sporadic pancreatic adenocarcinoma. Nineteen thousand six hundred forty-seven patients were reported with pancreatic cancer. Ninety-five percent of cancers were sporadic and 5.0% were invasive IPMNs. Ten point three percent had one or more extra-pancreatic primary cancers in addition to their pancreatic primary (10.3% in patients with sporadic adenocarcinoma and 10.1% in patients with invasive IPMNs, p = NS). The most common sites of additional primary cancers were colorectal (20.1%), breast (19.9%), prostate (16.6%), urinary system (11.1%), and lung (9.8%). In the 2,017 patients with additional primary cancer, 86% occurred before the diagnosis of pancreatic cancer and 14% occurred after the diagnosis of pancreatic cancer. Our population-based analysis shows that the incidence of additional primary malignancies in patients with invasive IPMNs is 10%. Although not as high as previously reported in smaller studies, the incidence is significant and comparable to the incidence seen in patients with adenocarcinoma. Surveillance for other common malignancies in patients with IPMNs and pancreatic adenocarcinomas should be performed.

LKB1 GENE TISSUE SPECIFIC KNOCKOUT MOUSE MODEL FOR PANCREATIC CANCER

Pancreatic cancer is the fourth leading cause of cancer death in United States. Studies of the molecular genetics of pancreatic cancer as well as molecular biology of pancreatic development may advance our understanding of the mechanisms underlying the pathogenesis of this malignancy. Germline mutations in the LKB1 gene cause Peutz-Jeghers syndrome (PJS), which is characterized by gastrointestinal hamartomatous polyps and abnormal pigmentation. PJS is also a cancer predisposition syndrome with patients exhibiting an increased risk for developing a variety of different cancers including pancreatic cancer. It has been reported that patients with PJS have a 100-fold higher risk for development of pancreatic cancer than the general population and the lifetime-cumulated risk for this disease has been estimated to be 36%. LKB1 is also mutated in a significant proportion of sporadic pancreatic adenocarcinomas. To evaluate the role of LKB1 in pancreatic tumorigenesis, we have created a pancreatic tissue specific mouse model employing Cre-loxp technology. For this purpose, we cloned the mouse LKB1 gene and constructed an LKB1 targeting vector. The LKB13loxp allele with three loxp sites flanking the region of exons 2-8 and an adjacent neomycin resistance gene cassette were integrated into the genome as a result of homologous recombination in mouse ES cells. The chimeric mice derived from recombinant ES clones transmitted the LKB13loxp allele to the offspring. After the mating of LKB13loxp/+ with CMV-cre transgenic mouse, we obtained the LKB1+/− mouse line. Then a series of matings were carried out using LKB1+/− mice, LKB13loxp/+ mice and pdx-cre transgenic mice. The pdx promoter drives expression of Cre primarily in the pancreas, allowing us to generate pancreatic tissue specific null mice for the LKB1 gene (LKB13loxp/−/pdx-cre). Our preliminary data demonstrate that these mice develop intraductal papillary mucinous neoplasm (IPMN) as well as cystadenoma in their pancreata by the age of 4 months. These mice provide a model for studying the potential role of IPMN and cystadenomas in the development of pancreatic adenocarcinoma. IPMN have been implicated as a precursor lesion for human pancreatic adenocarcinoma. Because very little is known about these pancreatic lesions and the role of LKB1 in pancreatic carcinogenesis, the mouse model should be useful in future studies aimed at understanding how mutations in the LKB1 gene can lead to pancreatic cancer.

Increased prevalence of the BRCA2 polymorphic stop codon K3326X among individuals with familial pancreatic cancer

Germline BRCA2 mutations predispose to the development of pancreatic cancer. A polymorphic stop codon in the coding region of BRCA2 (K3326X) has been described, and although an initial epidemiological study suggested it was not disease causing, subsequent studies have been inconclusive. To investigate the biological significance of the K3326X polymorphism, we determined its prevalence in patients with sporadic and familial pancreatic cancer. Using a case-control design, we studied 250 patients with resected sporadic pancreatic adenocarcinomas, 144 patients with familial pancreatic adenocarcinoma, 115 spouses of patients with pancreatic cancer, and a disease control group of 135 patients without a personal history of cancer who had undergone cholecystectomy for non-neoplastic disease. The K3326X polymorphism was detected using heteroduplex analysis and DNA sequencing. The BRCA2 K3326X polymorphism was significantly more prevalent in individuals with familial pancreatic cancer: 8/144 (5.6%) vs 3/250 controls (1.2%) (odds ratio, 4.84; 95% CI, 1.27-18.55, P<0.01). One K3326X carrier with familial pancreatic cancer carried an alteration (IVS 16-2A>G) suspected to be deleterious. Excluding this case did not alter the significance of the association (OR: 4.24, P<0.01). In contrast, there was no difference in prevalence among individuals with sporadic pancreatic cancer - 7/250 (OR: 2.37, 95% CI: 0.61-9.27). The increased prevalence of the BRCA2 K3326X polymorphism in patients with familial pancreatic cancer suggests that this polymorphism is deleterious and contributes to pancreatic cancer risk.

HEDGEHOG AND ITS PATHWAY MEMBERS ARE MISEXPRESSED AND ACTIVE IN THE MAJORITY OF SPORADIC HUMAN PANCREATIC ADENOCARCINOMAS: A BIOLOGICALLY IMPORTANT PATHWAY AND POTENTIAL THERAPEUTIC TARGET.

The Hedgehog (Hh) signaling pathway has been implicated in pancreatic tumorigenesis, where it is believed to play a critical role as both an initiator and maintenance factor. However, the prevalence and magnitude of Hh pathway aberration in sporadic human pancreatic adenocarcinomas remains unknown. Here we present quantitative real-time PCR data from 20 human adenocarcinomas and show increased mRNA expression for 7 representative Hedgehog pathway genes. Methods: Individual samples were obtained from 20 human pancreatic adenocarcinomas freshly resected at our institution. Total RNA was extracted, genomic DNA was removed, and the RNA was subjected to quantitative real-time PCR analysis, using algorithmically generated primers for the genes SHH, IHH, DHH, PTCH, SMO, GLI1, and GLI2. All samples in a given run were normalized to a standardized sample of 18S rRNA, and gene expression levels in tumors were compared to those in an averaged expression level of 10 normal human pancreata. Results: All human tumors showed marked upregulation of the Hedgehog pathway when compared to normal pancreatic controls. All tumors misexpress Hh ligands, principally SHH and IHH (median 168- and 165-fold upregulation, respectively). Furthermore, this pathway is active, as indicated by an impressive upregulation of downstream mediators of the Hh pathway, GLI1 and GLI2 (median 71- and 96-fold). DHH, PTCH, and SMO are identified but demonstrate comparatively less upregulation (median 9-, 2-, and 5-fold). Conclusions: Misexpression of Hh ligands and activation of the Hh pathway is a prevalent finding in human pancreatic adenocarcinoma. The finding that all adenocarcinomas tested to date have an impressive increase in activity suggests that Hh pathway aberration is an important fundamental mechanism in pancreatic tumorigenesis. Thus, inhibition of the Hh pathway may represent a powerful potential treatment modality in pancreatic adenocarcinoma.

Inflammation and Cancer V. Chronic pancreatitis and pancreatic cancer.

Pancreatic inflammation appears to increase the risk of pancreatic cancer. This observation is striking in the hereditary pancreatitis kindreds but also occurs in alcoholic, idiopathic, and tropical chronic pancreatitis and cystic fibrosis. However, the mutations associated with hereditary pancreatitis or cystic fibrosis are not found in sporadic pancreatic adenocarcinomas, suggesting that the effects are indirect by causing recurrent pancreatitis and chronic inflammation. The process of mutation accumulation and clonal expansion that is required for development of invasive pancreatic adenocarcinoma must therefore be accelerated in chronic pancreatitis to account for the high incidence of pancreatic cancer in these patients.

Down-regulation of BRCA1 in chronic pancreatitis and sporadic pancreatic adenocarcinoma.

BRCA1 and BRCA2 are considered to be breast cancer susceptibility genes that may also contribute to pancreatic cancer development because family studies revealed mutation carriers to have an increased risk of developing pancreatic cancer. However, as demonstrated for breast and ovarian cancer, inactivation of BRCA in sporadic diseases is based on alteration in gene expression or functional alteration. To study a potential correlation of BRCA1 and BRCA2 to chronic pancreatitis and development of sporadic pancreatic adenocarcinoma, we have analyzed the expression of these genes by quantitative PCR and performed immunohistochemical analyses in normal pancreatic tissues, chronic pancreatitis, and pancreatic cancer specimens. BRCA1 expression was down-regulated in chronic alcoholic pancreatitis, in particular on the RNA level. Furthermore, our data indicate suppressed BRCA1 expression in pancreatic cancer on both the RNA and protein levels. Quantitative analysis of BRCA1 protein expression demonstrated regular staining in 50% of tumor specimens tested and reduced staining in 50% of tumor specimens tested. Correlation with the clinical outcome revealed a significantly better 1-year overall survival for patients with BRCA1-regular as compared with BRCA1-reduced or BRCA1-absent tumors. In contrast, no substantial differences in BRCA2 expression were found in chronic pancreatitis and pancreatic cancer samples. Our data demonstrate alteration of BRCA1 expression in chronic pancreatitis and sporadic pancreatic adenocarcinoma. We, for the first time, provide evidence for a role of BRCA1 in pancreatic carcinogenesis of noninherited tumors and for clinical outcome.

Correlation between patterns of DNA mismatch repair hmlh1 and hmsh2 protein expression and progression of dysplasia in intraductal papillary mucinous neoplasms of the pancreas.

Defective DNA mismatch repair results from genetic or epigenetic alterations that most frequently inactivate the genes hMLH1 and hMSH2. This is thought to promote tumourigenesis by accumulation of mutations in oncogenes and tumour suppressor genes. This pathway, first reported in colon cancer, has been recently demonstrated in a subgroup of sporadic pancreatic adenocarcinomas. Intraductal papillary-mucinous neoplasms of the pancreas are a special type of pancreatic tumours, characterised by a spectrum of morphological changes from mild to moderate and to non-invasive, and they may associate with adenocarcinoma. An immunohistochemical study of hmlh1 and hmsh2 protein expression was performed on 26 intraductal papillary-mucinous neoplasms. All tumours showed nuclear expression of hmlh1 and hmsh2 proteins. There were two distinctive patterns of protein expression on the basis of the location of cells expressing these markers: the "normal" pattern, observed mainly in adenoma and rarely in intraductal papillary-mucinous neoplasms with moderate dysplasia and the "dysplastic" pattern, frequently encountered in moderate dysplasia neoplasms, non-invasive and invasive carcinomas. These findings suggest that defective DNA mismatch repair, due to inactivation of hMLH1 and hMSH2, does not play a significant role in the pathogenesis of intraductal papillary-mucinous neoplasms of the pancreas. Two patterns of protein expression were observed and were correlated with the progression of dysplasia in intraductal papillary mucinous neoplasms.

Polymorphisms of SPINK1 N34S and CFTR in Patients with Sporadic and Familial Pancreatic Cancer

Long standing chronic pancreatitis is a risk factor for developing pancreatic cancer. Inheritance of polymorphisms in SPINK1 and CFTR are associated with an increased risk of developing pancreatitis. The aim of this study was to determine if patients who carry polymorphisms in SPINK1 and CFTR are at increased risk of developing pancreatic cancer through the development of chronic pancreatitis. DNA from patients with histologically-confirmed surgically-treated chronic pancreatitis, familial and sporadic pancreatic adenocarcinoma and controls were analyzed for the N34S polymorphism of SPINK1 and the 2 commonest polymorphisms of the CFTR gene, the _F508 mutation and the 5T polymorphism. These polymorphisms were determined using restriction fragment length polymorphism, PCR and cycle sequencing methods. The SPINK1 N34S polymorphism was detected in 5 of 172 (2.9%) patients with chronic pancreatitis, in 4 of 200 (2.0%) patients with sporadic pancreatic adenocarcinoma, in 0 of 36 (0%) of patients with familial pancreatic cancer and in 3 of 177 (1.7%) controls of chronic cholecystitis. The CFTR 5T polymorphism was identified in 31 of 334 (9.3%) patients of sporadic pancreatic cancer, in 5 of 43 (11.6%) patients with familial pancreatic cancer and in 10 of 112 (8.9%) controls with colorectal cancer. The CFTR _F508 mutation was recognized in 6 of the 240 (2.5%) patients with pancreatic adenocarcinoma, a prevalence similar to that of control populations. We conclude that the N34S polymorphism of SPINK1 and the 5T and _F508 CFTR polymorphisms do not predispose to the development of pancreatic adenocarcinoma. Furthermore, the N34S polymorphism is rarely found in patients with severe idiopathic chronic pancreatitis.

Loss of Stk11/Lkb1 Expression in Pancreatic and Biliary Neoplasms

We have documented previously somatic mutations of STK11/LKB11, the gene responsible for Peutz-Jeghers syndrome (PJS), in a small proportion of sporadic pancreatic adenocarcinomas, intraductal papillary mucinous neoplasms (IPMNs), and biliary adenocarcinomas. In this report, we characterize the expression of Stk11, the protein product of the STK11 gene, in a larger series of pancreatic and biliary neoplasms. First, the specificity of the Stk11 antibody was established in 23 neoplasms (22 IPMNs and 1 biliary adenocarcinoma) with known STK11 gene status. Complete absence of labeling was seen in the neoplastic cells of 3 of the 3 (100%) cases with previously documented biallelic inactivation of the STK11 gene, whereas 16 of the 20 (80%) IPMNs, presumably with at least one wild-type STK11 gene, retained Stk11 expression in the neoplastic cells. The marked decrease or absence of Stk11 expression in four neoplasms with wild-type STK11 suggests that additional mechanisms may account for the lack of Stk11 expression. Subsequently, to further evaluate Stk11 expression in pancreatic and biliary neoplasms, tissue microarrays (TMAs) were constructed from a series of nearly 100 ductal adenocarcinomas and biliary neoplasms. Stk11 expression was lost in 4 of the 56 (7%) pancreatic adenocarcinomas and 1 of the 38 (2.6%) biliary cancers by immunohistochemistry; the absence of labeling was confirmed by repeated immunohistochemical labeling of complete tissue sections for the same cases. Thus, Stk11 expression is abrogated in a small proportion of pancreatic and biliary neoplasms. The inactivation of Stk11 in 27% (6/22) of IPMNs versus 7% (4/56) of pancreatic adenocarcinomas suggests genetic disparities in the pathogenesis of these closely related neoplasms. Immunohistochemical analysis for Stk11 expression may be a valid surrogate for genetic analysis of STK11 gene mutations in cancers.

Germline and Somatic Mutations of the STK11/LKB1 Peutz-Jeghers Gene in Pancreatic and Biliary Cancers

Peutz-Jeghers syndrome (PJS) is an autosomal-dominant disorder characterized by hamartomatous polyps in the gastrointestinal tract and by pigmented macules of the lips, buccal mucosa, and digits. Less appreciated is the fact that PJS also predisposes patients to an increased risk of gastrointestinal cancer, and pancreatic cancer has been reported in many PJS patients. It was recently shown that germline mutations of the STK11/LKB1 gene are responsible for PJS. We investigated the role of STK11/LKB1 in the development of pancreatic and biliary cancer in patients with and without the PJS. In a PJS patient having a germline splice site mutation in the STK11/LKB1 gene, sequencing analysis of an intestinal polyp and pancreatic cancer from this patient revealed loss of the wild-type allele of the STK11/LKB1 gene in the cancer. Inactivation of STK11/LKB1, by homozygous deletions or somatic sequence mutations coupled with loss of heterozygosity, was also demonstrated in 4-6% of 127 sporadic pancreatic and biliary adenocarcinomas. Our results demonstrate that germline and somatic genetic alterations of the STK11/LKB1 gene may play a causal role in carcinogenesis and that the same gene contributes to the development of both sporadic and familial forms of cancer.