Irene Esposito

A Revised Classification System and Recommendations From the Baltimore Consensus Meeting for Neoplastic Precursor Lesions in the Pancreas.

International experts met to discuss recent advances and to revise the 2004 recommendations for assessing and reporting precursor lesions to invasive carcinomas of the pancreas, including pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm (IPMN), mucinous cystic neoplasm, and other lesions. Consensus recommendations include the following: (1) To improve concordance and to align with practical consequences, a 2-tiered system (low vs. high grade) is proposed for all precursor lesions, with the provision that the current PanIN-2 and neoplasms with intermediate-grade dysplasia now be categorized as low grade. Thus, “high-grade dysplasia” is to be reserved for only the uppermost end of the spectrum (“carcinoma in situ”–type lesions). (2) Current data indicate that PanIN of any grade at a margin of a resected pancreas with invasive carcinoma does not have prognostic implications; the clinical significance of dysplasia at a margin in a resected pancreas with IPMN lacking invasive carcinoma remains to be determined. (3) Intraductal lesions 0.5 to 1 cm can be either large PanINs or small IPMNs. The term “incipient IPMN” should be reserved for lesions in this size with intestinal or oncocytic papillae or GNAS mutations. (4) Measurement of the distance between an IPMN and invasive carcinoma and sampling of intervening tissue are recommended to assess concomitant versus associated status. Conceptually, concomitant invasive carcinoma (in contrast with the “associated” group) ought to be genetically distinct from an IPMN elsewhere in the gland. (5) “Intraductal spread of invasive carcinoma” (aka, “colonization”) is recommended to describe lesions of invasive carcinoma invading back into and extending along the ductal system, which may morphologically mimic high-grade PanIN or even IPMN. (6) “Simple mucinous cyst” is recommended to describe cysts >1 cm having gastric-type flat mucinous lining at most minimal atypia without ovarian-type stroma to distinguish them from IPMN. (7) Human lesions resembling the acinar to ductal metaplasia and atypical flat lesions of genetically engineered mouse models exist and may reflect an alternate pathway of carcinogenesis; however, their biological significance requires further study. These revised recommendations are expected to improve our management and understanding of precursor lesions in the pancreas.

CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice

Significance Assigning biological relevance and molecular function to large catalogues of mutated genes in cancer is a major challenge. Likewise, pinpointing drivers among thousands of transcriptionally or epigenetically dysregulated genes within a cancer is complex and limited by the lack of tools for high-throughput functional cancer genomic analyses. We show here for the first time, to our knowledge, application of the CRISPR/Cas9 genome engineering system for simultaneous (multiplexed) mutagenesis of large gene sets in adult mice, allowing high-throughput discovery and validation of cancer genes. We characterized applications of CRISPR/Cas9 multiplexing, resulting tumor phenotypes, and limitations of the methodology. By using defined genetic or environmental predisposing conditions, we also developed, to our knowledge, the first mouse models of CRISPR/Cas9-induced hepatocellular carcinoma and show how multiplexed CRISPR/Cas9 can facilitate functional genomic analyses of hepatobiliary cancers. Here, we show CRISPR/Cas9-based targeted somatic multiplex-mutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.

A conditional piggyBac transposition system for genetic screening in mice identifies oncogenic networks in pancreatic cancer

Here we describe a conditional piggyBac transposition system in mice and report the discovery of large sets of new cancer genes through a pancreatic insertional mutagenesis screen. We identify Foxp1 as an oncogenic transcription factor that drives pancreatic cancer invasion and spread in a mouse model and correlates with lymph node metastasis in human patients with pancreatic cancer. The propensity of piggyBac for open chromatin also enabled genome-wide screening for cancer-relevant noncoding DNA, which pinpointed a Cdkn2a cis-regulatory region. Histologically, we observed different tumor subentities and discovered associated genetic events, including Fign insertions in hepatoid pancreatic cancer. Our studies demonstrate the power of genetic screening to discover cancer drivers that are difficult to identify by other approaches to cancer genome analysis, such as downstream targets of commonly mutated human cancer genes. These piggyBac resources are universally applicable in any tissue context and provide unique experimental access to the genetic complexity of cancer.

Resectability After First-Line FOLFIRINOX in Initially Unresectable Locally Advanced Pancreatic Cancer: A Single-Center Experience

BackgroundFOLFIRINOX is an active but relatively toxic chemotherapeutic regimen for patients with metastatic pancreatic ductal adenocarcinoma (PDAC). The increased frequency of responding tumors shift interest to neoadjuvant approaches. We report our institutional experience with FOLFIRINOX for therapy-naïve patients with locally advanced and initially unresectable PDAC.MethodsAll patients with unresectable locally advanced PDAC who underwent treatment with FOLFIRINOX at a single center between 2011 and 2014 were identified and evaluated retrospectively regarding chemotherapy response, toxicity, conversion to resectability, and survival. Resectability, response to chemotherapy, and postoperative complications were reported according to NCCN-guidelines, RECIST-criteria, and Clavien–Dindo-classification, respectively.ResultsOverall, 14 patients received FOLFIRINOX as first-line therapy for locally advanced and unresectable PDAC. Fifty-seven percent of the patients had severe tumor-related comorbidities at the time of diagnosis, and in 86xa0%, dose reduction due to toxicity was necessary during a median of seven cycles. Nevertheless, only one patient had progressive disease during FOLFIRINOX, whereas the others experienced stable disease (nxa0=xa06) or partial remission (nxa0=xa06; no restaging in one patient). Oncological tumor resection was possible in 4 patients (29xa0% of all patients) with no postoperative mortality and only one grade 2 surgical complication. After a median follow-up of 10xa0months, 4 of the 14 patients were still in remission, 5 were alive with stable disease under ongoing systemic chemotherapy, and 5 died tumor-related.ConclusionsFOLFIRINOX is a powerful first-line regimen that leads to resectability in a substantial portion of patients with initially unresectable pancreatic cancer.

Intraductal tubulopapillary neoplasms of the bile ducts: clinicopathologic, immunohistochemical, and molecular analysis of 20 cases

Intraductal tubulopapillary neoplasm is a well-established entity in the pancreas. A similar, if not identical, tumor occurs also in the biliary tract. We conducted a multicenter study of 20 such lesions, focusing on their clinicopathologic characteristics and molecular profile. Biliary intraductal tubulopapillary neoplasms were seen in patients in their 60s (mean 62 years). The tumors were intrahepatic 70%, extrahepatic 10%, and perihilar 20%; mean tumor size was 6.9u2009cm. Histologically, all intraductal tubulopapillary neoplasms showed, in addition to their typical tubular pattern, solid areas (70%) or abortive papillae (50%). Necrosis was common (85%), predominantly focal (40%), and with ‘comedocarcinoma-like pattern’ in 40%. Immunohistochemically, these neoplasms were characterized by the expression of MUC1 (80%) and MUC6 (30%) and by the absence of MUC2 and MUC5AC. Associated invasive carcinomas were present in 16 (80%), mainly conventional tubular adenocarcinoma (50%). The molecular alterations observed included CDKN2A/p16 (intraductal components 44%, invasive 33%) and TP53 (intraductal components 17%, invasive 9%). Mutations in KRAS (intraductal 6%, invasive 0%), PIK3CA (intraductal 6%, invasive 0%), and loss of SMAD4/DPC4 (intraductal 7%, invasive 0%) were rare. No alterations/mutations were identified in IDH1/2, BRAF, GNAS, EGFR, HER2, and β-catenin. Follow-up information was available for 17 patients (85%) with mean follow-up 44 months. Overall combined survival rates showed favorable prognosis: 1 year 100%, 3 years 90%, and 5 years 90%. In conclusion, despite the relatively high incidence of invasive carcinoma (80%), available follow-up suggests that biliary intraductal tubulopapillary neoplasms have an indolent behavior. Molecular analyses highlight the low prevalence of alterations of common oncogenic signaling pathways in intraductal tubulopapillary neoplasm. Further studies using whole-exome sequencing are required to discover yet unknown molecular changes and to understand the carcinogenesis of intraductal tubulopapillary neoplasms.

Structure-Preserving Color Normalization and Sparse Stain Separation for Histological Images

Staining and scanning of tissue samples for microscopic examination is fraught with undesirable color variations arising from differences in raw materials and manufacturing techniques of stain vendors, staining protocols of labs, and color responses of digital scanners. When comparing tissue samples, color normalization and stain separation of the tissue images can be helpful for both pathologists and software. Techniques that are used for natural images fail to utilize structural properties of stained tissue samples and produce undesirable color distortions. The stain concentration cannot be negative. Tissue samples are stained with only a few stains and most tissue regions are characterized by at most one effective stain. We model these physical phenomena that define the tissue structure by first decomposing images in an unsupervised manner into stain density maps that are sparse and non-negative. For a given image, we combine its stain density maps with stain color basis of a pathologist-preferred target image, thus altering only its color while preserving its structure described by the maps. Stain density correlation with ground truth and preference by pathologists were higher for images normalized using our method when compared to other alternatives. We also propose a computationally faster extension of this technique for large whole-slide images that selects an appropriate patch sample instead of using the entire image to compute the stain color basis.

Mechanisms of Insulin Resistance in Primary and Secondary Nonalcoholic Fatty Liver.

Nonalcoholic fatty liver disease is associated with hepatic insulin resistance and may result primarily from increased hepatic de novo lipogenesis (PRIM) or secondarily from adipose tissue lipolysis (SEC). We studied mice with hepatocyte- or adipocyte-specific SREBP-1c overexpression as models of PRIM and SEC. PRIM mice featured increased lipogenic gene expression in the liver and adipose tissue. Their selective, liver-specific insulin resistance was associated with increased C18:1-diacylglycerol content and protein kinase Cε translocation. SEC mice had decreased lipogenesis mediated by hepatic cholesterol responsive element–binding protein and featured portal/lobular inflammation along with total, whole-body insulin resistance. Hepatic mitochondrial respiration transiently increased and declined with aging along with higher muscle reactive oxygen species production. In conclusion, hepatic insulin resistance originates from lipotoxicity but not from lower mitochondrial capacity, which can even transiently adapt to increased peripheral lipolysis. Peripheral insulin resistance is prevented during increased hepatic lipogenesis only if adipose tissue lipid storage capacity is preserved.

Modeling Therapy Response and Spatial Tissue Distribution of Erlotinib in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is likely the most aggressive and therapy-resistant of all cancers. The aim of this study was to investigate the emerging technology of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) as a powerful tool to study drug delivery and spatial tissue distribution in PDAC. We utilized an established genetically engineered mouse model of spontaneous PDAC to examine the distribution of the small-molecule inhibitor erlotinib in healthy pancreas and PDAC. MALDI IMS was utilized on sections of single-dose or long-term–treated mice to measure drug tissue distribution. Histologic and statistical analyses were performed to correlate morphology, drug distribution, and survival. We found that erlotinib levels were significantly lower in PDAC compared with healthy tissue (P = 0.0078). Survival of long-term–treated mice did not correlate with overall levels of erlotinib or with overall histologic tumor grade but did correlate both with the percentage of atypical glands in the cancer (P = 0.021, rs = 0.59) and the level of erlotinib in those atypical glands (P = 0.019, rs = 0.60). The results of this pilot study present MALDI IMS as a reliable technology to study drug delivery and spatial distribution of compounds in a preclinical setting and support drug imaging–based translational approaches. Mol Cancer Ther; 15(5); 1145–52. ©2016 AACR.

Pathology, genetics and precursors of human and experimental pancreatic neoplasms: An update.

Over the past decade, there have been substantial improvements in our knowledge of pancreatic neoplasms and their precursor lesions. Extensive genetic analyses, recently using high-throughput molecular techniques and next-generation sequencing methodologies, and the development of sophisticated genetically engineered mouse models closely recapitulating human disease, have improved our understanding of the genetic basis of pancreatic neoplasms. These advances are paving the way for refined, molecular-based classifications of pancreatic neoplasms with the potential to better predict prognosis and, possibly, response to therapy. Another major development resides in the identification of subsets of pancreatic exocrine and endocrine neoplasms which occur in the context of hereditary syndromes and whose genetic basis and tumor development have been at least partially defined. However, despite all molecular progress, correct and careful morphological characterization of tissue specimens both in the context of experimental and routine diagnostic pathology represents the basis for any further genetic investigation or clinical decision. This review focuses on the current and new concepts of classification and on the current models of tumor development, both in the field of exocrine and endocrine neoplasms, and underscores the importance of applying standardized terminology to allow adequate data interpretation and promote scientific exchange in the field of pancreas research.

New insights into the origin of pancreatic cancer. Role of atypical flat lesions in pancreatic carcinogenesis

The identification and characterization of precursor lesions is fundamental to develop screening programs for early diagnosis and treatment, aiming at reducing cancer-related mortality. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that becomes clinical apparent only in advanced stages. In order to enable screening procedures for early detection of PDAC, an exact characterization of precursor lesions is of utmost importance. Pancreatic intraepithelial neoplasias (PanIN) are the most frequent and best characterized precursors of PDAC and are lesions with a ductal phenotype thus indicating a ductal cell origin of PDAC. However, evidence from genetically engineered mouse models suggests that tubular complexes (TC) originating through a process of acinar-ductal metaplasia (ADM) form atypical flat lesions (AFL) that may represent an alternative pathway of pancreatic carcinogenesis. Based on a thorough morphological and genetic analysis of murine TC, AFL and PanIN and their human counterparts, a new dual model of pancreatic carcinogenesis is proposed taking into account the role of AFL as possible new precursors of PDAC.ZusammenfassungDie Identifizierung und Charakterisierung von Krebsvorläuferläsionen hat bei verschiedenen malignen Neoplasien die Entwicklung von Screeningprogrammen ermöglicht, mit deren Hilfe die Mortalitätsrate reduziert werden konnte. Dies gilt bislang nicht für das pankreatische duktale Adenokarzinom (PDAC), das nach wie vor klinisch erst erkannt wird, wenn es invasiv geworden ist. Um die Entwicklung diagnostischer Verfahren zur Früherkennung des PDAC zu ermöglichen, ist eine genaue Kenntnis seiner Vorläuferläsionen notwendig. Die häufigste bislang bekannte Vorläuferläsion ist die pankreatische intraepitheliale Neoplasie (PanIN), die einen duktalen Phänotyp zeigt und einen duktalen Ursprung des PDAC nahe legt. Genetisch konstruierte Mausmodelle des PDAC zeigen jedoch, dass außer PanIN auch tubuläre Zellkomplexe (TC) zentroazinären Ursprungs durch eine azinär-duktale Metaplasie (ADM) atypische flache Läsionen (AFL) als alternative Vorläufer des PDAC ausbilden können. Die TC, AFL und murinen PanIN wurden von uns im Mausmodell eingehend morphologisch und molekulargenetisch charakterisiert und mit den menschlichen Pankreasläsionen verglichen. Auf der Basis unserer Befunde stellen wir ein duales Modell der Entwicklung des PDAC vor, das die Rolle der AFL als Vorläuferläsion berücksichtigt.AbstractThe identification and characterization of precursor lesions is fundamental to develop screening programs for early diagnosis and treatment, aiming at reducing cancer-related mortality. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that becomes clinical apparent only in advanced stages. In order to enable screening procedures for early detection of PDAC, an exact characterization of precursor lesions is of utmost importance. Pancreatic intraepithelial neoplasias (PanIN) are the most frequent and best characterized precursors of PDAC and are lesions with a ductal phenotype thus indicating a ductal cell origin of PDAC. However, evidence from genetically engineered mouse models suggests that tubular complexes (TC) originating through a process of acinar-ductal metaplasia (ADM) form atypical flat lesions (AFL) that may represent an alternative pathway of pancreatic carcinogenesis. Based on a thorough morphological and genetic analysis of murine TC, AFL and PanIN and their human counterparts, a new dual model of pancreatic carcinogenesis is proposed taking into account the role of AFL as possible new precursors of PDAC.