Here, we show CRISPR/Cas9-based targeted somatic multiplexmutagenesis 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.in vivo CRISPR/Cas9 | somatic multiplex-mutagenesis | hepatocellular carcinoma | intrahepatic cholangiocarcinoma | chromosome engineering F or decades, a major bottleneck in cancer research has been our limited ability to identify genetic alterations in cancer. The revolution in array-based and sequencing technologies and the recent development of insertional mutagenesis tools in animal models enable the discovery of cancer-associated genetic alterations on a genome-wide scale in a high-throughput manner. Nextgeneration sequencing (NGS) of cancer genomes and transposonbased genetic screening in mice, for example, are currently creating large catalogs of putative cancer genes for principally all cancer types (1-3). A challenge for the next decades will be to validate the causative cancer relevance of these large gene sets (to distinguish drivers from passengers) and to understand their biological function. Moreover, pinpointing downstream targets of mutated cancer genes or drivers among the thousands of transcriptionally or epigenetically dysregulated genes within individual cancers is complex and limited by the lack of tools for high-throughput functional cancer genomic analyses.The development of technologies for targeted manipulation of the mouse germ line has opened tremendous opportunities to study gene function (4, 5). Mouse models recapitulate the extensive biological complexity of human cancer and have given insights into many fundamental aspects of the disease that can be studied only at an organismal level (6). However, the speed and efficiency of such studies is limited by the long time frames needed to genetically engineer, intercross,...

Somatostatin receptor 2A expression is a feature of well-differentiated neuroendocrine neoplasms and is important for their diagnosis and therapy. Little is known about somatostatin receptor 2A expression in poorly differentiated neuroendocrine neoplasms in relation to TP53 and RB1 status and how these features may contribute to the separation of well from poorly differentiated neuroendocrine neoplasms with a proliferation index above 20%. This study investigates the expression of somatostatin receptors, p53 and Rb1, and TP53 alterations in pancreatic and extrapancreatic well and poorly differentiated neuroendocrine neoplasms (Ki67-index >20%). Thirty-seven poorly differentiated neuroendocrine neoplasms of pancreatic (n=12) and extrapancreatic origin (n=25) as well as 10 well-differentiated neuroendocrine neoplasms of the pancreas (n=9) and rectum (n=1) with a Ki67-index >20% were immunostained for synaptophysin, chromogranin A, Ki67, CD56, p53, Rb1, ATRX, DAXX, progesterone receptor, somatostatin receptor 2A, somatostatin receptor 5, and cytokeratin 20, and sequenced for TP53, exons 5-9. Somatostatin receptor 2A was positive in 6/37 of poorly differentiated and in 8/10 of well-differentiated neuroendocrine neoplasms. One well-differentiated and two poorly differentiated neuroendocrine neoplasms expressed somatostatin receptor 5. Abnormal nuclear p53 and Rb1 staining was found in 29/37 and 22/37 poorly differentiated neuroendocrine neoplasms, respectively, whereas all well-differentiated neuroendocrine neoplasms showed normal p53 and Rb1 expression. TP53 gene alterations were restricted to poorly differentiated neuroendocrine neoplasms (24/34) and correlated well with p53 expression. All cases were progesterone receptor negative. Somatostatin receptor 2A expression is not limited to well-differentiated neuroendocrine neoplasms but also occurs in 16% of poorly differentiated neuroendocrine neoplasms from various sites. Most poorly differentiated neuroendocrine neoplasms are characterized by TP53 alterations and Rb1 loss, usually in the absence of somatostatin receptor 2A expression. In the pancreas, these criteria contribute to separate well-differentiated neuroendocrine neoplasms with a Ki67-index above 20% from poorly differentiated neuroendocrine neoplasms.

Pancreatic ductal adenocarcinoma (PDAC) has generally a poor prognosis, but recent data suggest that there are molecular subtypes differing in clinical outcome. This study examines the association between histopathologic heterogeneity, genetic profile, and survival. Tumor histology from 177 resected PDAC patients with follow-up data was subclassified according to predominant growth pattern, and four key genes were analyzed. PDACs were classified as conventional (51%), combined with a predominant component (41%), variants and special carcinomas (8%). Patients with combined PDACs and a dominant cribriform component survived longer than patients with conventional or other combined PDACs. Genetic alterations in at least two out of four genes were found in 95% of the patients (KRAS 93%, TP53 79%, CDKN2A/p16 75%, SMAD4 37%). Patients with less than four mutations survived significantly longer (p = 0.04) than those with alterations in all four genes. Patients with either wildtype KRAS or CDKN2A/p16 lived significantly longer than those with alterations in these genes (p = 0.018 and p = 0.006, respectively). Our data suggest that the number of altered genes, the mutational status of KRAS and certain morphological subtypes correlate with the outcome of patients with PDAC. Future pathology reporting of PDAC should therefore include the KRAS status and a detailed morphological description.Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor with dismal prognosis. The overall 5-year survival rate is only 6% and after curative surgery less than 25% 1 , making PDAC one of the most lethal tumors among solid malignancies 2 . This poor outcome is related to multiple factors, including resistance to chemotherapy and the relatively late stage of diagnosis due to unspecific symptoms and aggressive tumor biology 1 .Over the last decade major improvements have been made in understanding the mechanisms of molecular carcinogenesis in PDACs 3-7 . The first milestone was the discovery of the molecular fingerprint of PDAC that included the common mutations in KRAS, SMAD4, TP53 and CDKN2A/p16 8 . Recent advances in gene sequencing 9 by introduction of high-throughput molecular methods allowed to further address the genetic complexity of PDAC 10 . The first global analysis of 24 advanced PDACs using comprehensive exome sequencing revealed a high mutation rate with an average of 63 mutations per case connected to 12 core signaling pathways 10 . In 2011, Collisson et al. showed that PDACs and murine PDAC cell lines may be stratified by their transcriptional profiles

Monitoring of blood counts and for evidence of thromboembolic events is essential for patients treated with lenalidomide. Ongoing trials of lenalidomide combination therapy offer a treatment option for patients with advanced cancer and will better define the role of lenalidomide in solid tumors.