Tumor Suppressor Genes CDKN2A Research Articles

Human genes differ by their UV sensitivity estimated through analysis of UV-induced silent mutations in melanoma.

We hypothesized that human genes differ by their sensitivity to ultraviolet (UV) exposure. We used somatic mutations detected by genome-wide screens in melanoma and reported in the Catalog Of Somatic Mutations In Cancer. As a measure of UV sensitivity, we used the number of silent mutations generated by C>T transitions in pyrimidine dimers of a given transcript divided by the number of potential sites for this type of mutations in the transcript. We found that human genes varied by UV sensitivity by two orders of magnitude. We noted that the melanoma-associated tumor suppressor gene CDKN2A was among the top five most UV-sensitive genes in the human genome. Melanoma driver genes have a higher UV-sensitivity compared with other genes in the human genome. The difference was more prominent for tumor suppressors compared with oncogene. The results of this study suggest that differential sensitivity of human transcripts to UV light may explain melanoma specificity of some driver genes. Practical significance of the study relates to the fact that differences in UV sensitivity among human genes need to be taken into consideration whereas predicting melanoma-associated genes by the number of somatic mutations detected in a given gene.

An acquired stable variant of a dicentric dic(9;20) and complex karyotype in a Syrian childhood B-acute lymphoblastic leukemia case

BackgroundAbout 25 years ago, the acquired chromosome abnormality dicentric dic(9;20)(p11 ~ 13;q11) was seen described as a non-random aberration in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet, about 200 cases were reported. However, dicentric dic(9;20) is a subtle abnormality which easily may be mixed up with monosomy 20 and/or del(9p). The dicentric dic(9;20) can be found as a sole chromosomal abnormality or can be masked within complex rearrangements; also, a dicentric dic(9;20) is often associated with mono- or biallelic loss of CDKN2A gene.Case presentationHere we report a case of 16-year-old male diagnosed with a de novo pre-B-ALL. Molecular approaches (array-based multicolor banding (aMCB) and array comparative genomic hybridization (aCGH)) were applied, and a unique complex karyotype involving six chromosomes was identified. It included three previously unreported chromosomal aberrations: dicentric dic(9;20;X), deletion del(7)(p22.2p15.2) and dicentric dic(7;13). The dicentric dic(9;20;X) also led to monoallelic loss of tumor suppressor gene CDKN2A. After successful chemotherapeutic treatment the patient experienced a relapse with a secondary ALL without complex karyotype but a deletion del(19)(p13). Unfortunately, the patient died after 17 months of the initial diagnosis.ConclusionsTo the best of our knowledge, a comparable childhood ALL associated with such complex karyotype and deletion del(19)(p13) in secondary ALL was not previously reported. Thus, the complex karyotype with dicentrc dic(9;20;X) seems to indicate for a poor prognosis.

272 Targeted reactivation of a dormant tumor suppressor gene CDKN2A inhibits proliferation of skin cancer cells

Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer in the U.S. cSCC frequently manifests with inactivation of the tumor suppressor gene CDKN2A, which encodes p16INK4A, due to not only genetic but also epigenetic alterations. The loss of CDKN2A expression increases malignant phenotypes in cancer. Thus, we investigated whether p16INK4A transcriptional suppression in cSCC cells can be reversed and whether targeted transcriptional reactivation of p16INK4A inhibits cell proliferation.

GSTs polymorphisms are associated with epigenetic silencing of CDKN2A gene in esophageal squamous cell carcinoma.

Esophageal cancer is the eighth most common cancer and the sixth most frequent cause of cancer mortality worldwide. Exposure to polycyclic aromatic hydrocarbons formed by incomplete combustion of organic matter is an important risk factor. Genetic polymorphisms in genes encoding PAH-metabolizing enzymes like glutathione S-transferases (GSTM1, GSTP1, GSTT1) which conjugate glutathione to PAHs for reduction of oxidative stress may affect an individual's response to PAH exposure. Genomic DNA from 50 esophageal squamous cell carcinoma patients extracted from peripheral blood. PCR-RFLP technique was employed to determine GSTM1, GSTT1, and GSTP1 polymorphisms. Aberrant promoter methylation of CDKN2A was applied by methylation-specific PCR technique. Concentration of urinary 1-hydroxypyrene was determined using a HPLC system. About 38.7% showed the null GSTM1 genotype (54% cases and 13% controls), 23.7% showed GSTT1 null genotype (30% cases and 13% controls), and 62.5% were GSTP1 A/A genotype (66% cases and 56% controls). Polymorphic variants of GSTM1 and GSTT1 were significantly associated with aberrant methylation of CDKN2A gene. The null state of GSTT1 was significantly associated with high concentrations of 1-OHP in urea (p < 0.01). There was significant association between methylated states of CDKN2A and high concentrations of 1-OHP in urine (p < 0.01). We identified significant association between polymorphism of GSTs genes and epigenetic silencing of tumor suppressor gene CDKN2A in esophageal squamous cell carcinoma.

Frequency and Role of CDKN2A Deletion in High-Risk Pituitary Neuroendocrine Tumors.

The underlying mechanisms of aggressive pituitary neuroendocrine tumors (pitNETs) are still unclear. The p16 protein, encoded by the CDKN2A tumor suppressor gene on chromosome 9p21, is commonly reported to be lost in numerous types of cancer. For this reason, this study examined to examine the status of homozygous deletion of CDKN2A in high-risk pitNETs. Thirty-eight high-risk pitNETs (30 male, 8 female) were analyzed for CDKN2A deletion by fluorescent in situ hybridization (FISH). Demographic characteristics such as sex, patient age at operation, and sellar magnetic resonance imaging findings including tumor size and invasion status were recorded. The frequency of CDKN2A homozygous deletion by FISH was 3/38 (7.89%) in the high-risk pitNET group. All of these three cases with CDKN2A homozygous deletion were invasive densely granulated lactotroph tumors (p= 0.000). CDKN2A deletion was not correlated with patient age, sex, cavernous sinus invasion (CSI), and tumor size (p> 0.05). The Ki-67 proliferation index was significantly correlated with CDKN2A homozygous deletion (p= 0.003). The mean Ki-67 proliferation index was 10.7% in pitNETs with CDKN2A homozygous deletion and the Ki-67 proliferation index in the whole study group was 4.1%. CSI was significantly correlated with the morphofunctional tumor types including lactotroph tumor, invasive null cell tumor, and invasive gonadotroph tumor (p= 0.021). These findings suggest a close correlation between inactivation of p16 gene and invasive lactotroph tumors. Further investigations are needed to expand on the mechanism of p16 (CDKN2A) gene deletion in high-risk pitNETs.

Inside the USCAP Journals

Inside the USCAP Journals

Inside the USCAP Journals

Inside the USCAP Journals

Frequent Homozygous Deletions of Type I Interferon Genes in Pleural Mesothelioma Confer Sensitivity to Oncolytic Measles Virus.

Oncolytic immunotherapy is based on the use of nonpathogenic replicative oncolytic viruses that infect and kill tumor cells exclusively. Recently, we found that the spontaneous oncolytic activity of the Schwarz strain of measles virus (MV) against human malignant pleural mesothelioma (MPM) depends on defects in the antiviral type I interferon (IFN-I) response in tumor cells. In this study, we studied three independent human MPM bio-collections to identify the defects in the IFN-I responses in tumor cells. We show that the most frequent defect is the homozygous deletions (HDs) of all the 14 IFN-I genes (IFN-α and IFN-β) that we found in more than half of MV-sensitive MPM cell lines. These HDs occur together with the HDs of the tumor suppressor gene CDKN2A also located in the 9p21.3 chromosome region. Therefore, the IFN-I-/- MPM cell lines develop a partial and weak IFN-I response when they are exposed to the virus compared with that of normal cells and MV-resistant MPM cell lines. This response consists of the expression of a restricted number of IFN-stimulated genes that do not depend on the presence of IFN-I. In addition, the IFN-I-/- MPM cell lines infected by MV also develop a pro-inflammatory response associated with stress of the endoplasmic reticulum. Our study emphasizes the link between HDs of IFN-I encoding genes and the CDKN2A gene in MPM and sensitivity to MV oncolytic immunotherapy.

Association of SNPs in CDKN2A (P14ARF) Tumour Suppressor Gene With Endometrial Cancer in Postmenopausal Women

This research was aimed to evaluate the association between three selected single nucleotide polymorphisms (SNPs) within the CDKN2A (P14ARF) tumour suppressor gene and the incidence of endometrial cancer (EC) in postmenopausal women. The study included 194 postmenopausal women; 144 with EC and 50 non-cancer controls. Genotypes in P14ARF rs3088440, rs3731217 and rs3731245 polymorphisms were assayed using PCR-RFLP and confirmed by sequencing. Regarding the rs3088440 polymorphism, CT, and CT-TT genotypes, were more prevalent among EC patients than in controls (OR=5.55, p=0.023, OR=5.29, p=0.027; and OR=2.92, p=0.023, respectively). The T allele within rs3088440 was more prevalent in EC females than in controls (χ2=4.7, p=0.030). Considering rs3731217, TG and TG-GG genotypes were less prevalent among EC (OR=0.34, p=0.024 or p=0.023; and OR=0.38, p=0.035, respectively). Polymorphisms in the CDKN2A gene are associated with EC in postmenopausal women.

Manifestation of Pathological States of Numerous Diseases in the Largest Organ of the Human Body: (II) From Pancreatitis to Pancreatic Cancer Invasion, Formation of Stroma around the Primary Tumor in the Fascia, to Early Detection of Non-Coding microRNAs in Body Fluids and Development of Drugs to Treat Different Stages of Pancreatic Cancer

Patients suffering from pancreatic ductal adenocarcinoma (PDAC) have an average survival time of 4 - 6 months after confirmed diagnosis. The primary tumor is surrounded by a thick interstitial fluid with high pressure and dense distribution of collagen, forming a huge stroma, rendering the tumor resistant to chemo- and radiotherapy. From the genetic point of view, pancreatic carcinogenesis is driven by mutations, resulting in common activation of the oncogene KRAS, and/or inactivation of one or more of the tumor suppressor genes CDKN2A, TP53, SMAD4 [1]. The pancreas is a mixed exocrine and autocrine organ, with different cell types building up the organ. The pathogenesis involves more than 13 signaling pathways at different stages. Off-balance of the function of the proteins in these pathways due to the stated 4 plus other mutations could readily lead to carcinogenesis. We first present the basic mechanism of these 13 relevant pathways. We then provide a detailed analysis of the progression of this disease, from pancreatitis to tumor formation and metastasis, with special attention on the roles played by the newly discover calcium channel Piezo, stellate cells, stem-cell-like cells, and the concept invadopodium. Thirty potential drugs, based on in vitro and xenograft experiments from different groups, are discussed, including vitamins A, Tocotrienols-E, and D, chemical compounds, non-coding micro RNAs, circular RNA, piwi-interacting RNAs. The recent detection of exosomes enclosing many of these RNAs in body fluids gives us hope of developing early detection methodology because these RNAs carry messages for cell-cell communication at a distance. Delivery of potent drugs by nanoparticles gives us chance to send drugs through the stroma to target the tumor. Since body fluids form a circulating system, together with the connective tissues (where the tumor is associated) form the largest organ—the fascia, we conclude that manifestation of successive pathological states of pancreatic carcinogenesis can be found in compartments of the fascia. We present 17 figures, hoping to ease off the complexity of the pathogenesis of this most lethal cancer disease.

Abstract C40: Dissecting key biologic processes in pancreatic cancer metastasis using a genetically defined 3D organoid model

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers, with a dismal 5-year survival rate of 6-8% due to its late detection and limited response to treatment (1,2). Multiple combinations of genetic alterations are frequently found in human PDAC, with KRAS activating mutation representing the main driver, followed by loss of tumor-suppressor genes CDKN2A, TP53, and SMAD4 as the disease progresses (3). 90% of PDAC patients die of metastatic disease (1); however, the cellular and molecular mechanisms involved in establishing the metastatic disease are understudied compared to those involved in tumor initiation. Owing to this, improved biologic understanding of PDAC metastasis remains warranted in order to develop effective therapeutic regimens for this disease. 3D organoid cultures derived from human and mouse PDAC tumors have recently become a model of choice to study PDAC tumorigenesis, gradually replacing more commonly used 2D cancer cell lines. Organoids maintain similar gene expression profiles as the in vivo situation when cultured in a 3D matrix, and genetic manipulation is feasible. We generated a panel of isogenic murine pancreatic intraepithelial neoplasia (PanIN) and PDAC organoids containing genetic mutations commonly found in human PDAC (KRAS, TP53, SMAD4, CDKN2A), either alone or in combination, using CRISPR-Cas9 technology. Since large-scale in vitro production of these PDAC organoids is achievable, the platform can be used for a multitude of downstream analyses in vitro and in vivo, such as drug screening and metastasis-related studies. The parental of these organoids is isolated from C57/Bl6 mice, which can be orthotopically transplanted into immunocompetent mice, and the resulting tumor recapitulates human and GEMM PDAC development. In the process of systematic characterization of these organoid lines, we found that organoids with more mutations exhibit higher proliferation rates in vitro and tumor formation rates in vivo. These organoids also showed genotype-dependent organotropism during metastatic colonization. We further used this unique tool to systematically address if each combination of tumor suppressor loss utilizes distinct molecular mechanisms to drive metastasis initiation and invasion. This will allow us to identify druggable pathways that could inhibit metastasis initiation/invasion and progression. Thus, these organoids provide a powerful platform for disease modeling and drug discovery both in vitro and in vivo. Citation Format: Fong Cheng Pan, Angie Ai-Nok Chong, Steven D. Leach, Shuibing Chen, Todd Evans. Dissecting key biologic processes in pancreatic cancer metastasis using a genetically defined 3D organoid model [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C40.

ES17.01 Mesothelioma Evolution

Malignant pleural mesothelioma (MPM) is mostly caused by prior exposure to asbestos fibres. It has a long but variable latency period following exposure, with a median of around 40 years but a range between 20-70 years, possibly reflecting the length and level of asbestos exposure as well as other environmental and genetic causes. Upon diagnosis, there are limited treatment options, and median survival time is a year, although, again, this is highly variable. Understanding the genetic events in the mesothelium between asbestos exposure and diagnosis of MPM is important for two reasons. Firstly, it will inform the biology of MPM tumour growth and potentially highlight different environmental and genetic factors that cause the variation in latency. Secondly, it will help identify key early driver mutations in MPM informing biology and candidate changes for developing approaches for early detection of the cancer. Evolutionary genetics is based on inferring past events from current genetic/genomic sequences. It has long been recognised that the development of cancer is an evolutionary process, and the availability of large amounts of DNA sequence data have facilitated an understanding of evolution of tumours using methods mostly borrowed from evolutionary genetics. One powerful approach compares a matched tumour and normal genome, infers the somatic mutations in the tumour, and uses the ratio of mutations that change an amino acid to mutations that don’t change an amino acid (dN/dS ratio) across all genes to identify particular genes that have been positively selected during tumour evolution. This approach can also shed light on overall evolutionary processes that have occurred in the tumour. Genomic sequences from multiple regions of the same tumour not only emphasise the molecular heterogeneity of tumours but allow an explicit phylogenetic tree of the evolution of the tumour for each patient, distinguishing somatic mutations that happened early in the tumour's evolution (and are therefore present throughout the tumour) from those that happened late in the tumour evolution. Here, I report preliminary findings from a British Lung Foundation/Mesothelioma UK-funded project entitled MEDUSA – Mesothelioma Evolution: Deciphering drUgable Somatic Alterations as potential targets for synthetic lethal therapy. This project uses multiregional sampling of MPMs, together with matched whole blood, to infer a phylogenetic tree of MPMs. The preliminary data presented focuses on the first 20 patients, with between 4 and 5 regions of the tumour analysed per patient. Using whole exome sequencing, the project aims to identify truncal changes, that is, mutations that happened early in the tumour and are present throughout the tumour that can be potential targets for drugs, with the aim of developing personalised, effective tumour treatment for each patient. We focus on copy number changes (deletions and duplications of genes) and confirm that MPM is highly heterogeneous with extensive copy number changes in the genome. We focus on truncal copy number changes in ∼20-25% of patients affecting the known mesothelioma tumour suppressor genes BAP1, MTOR, CDKN2A and SETD1. Distinguishing patients that have truncal copy number changes in these genes, in contrast to those patients with copy number changes in the terminal branches of the evolution of the tumour, helps to tailor individualised drug therapies. Our approach emphasises the importance of multiregional sampling of tumours to account for MPM heterogeneity. For example, by sampling the posterior costophenic angle of these MPM from these 20 patients, we would find 10 deletions of CDKN2A, of which only 5 are truncal, with the other five localised to only part of the tumour. Multiregional genomic analysis and evolutionary genetics approaches can illuminate the history of a tumour and have the potential to guide therapy. They also provide the framework for follow-on studies in a patient, such as analysing the origin of metastases and identifying the effects on the tumour of treatment. The extra information provided by multiregional sampling supports the idea that this approach should become routine in tailoring the treatment to the tumour in MPM. evolution, Mesothelioma, Genomics

Fluorescence in situ hybridization (FISH) provides estimates of minute and interstitial BAP1, CDKN2A, and NF2 gene deletions in peritoneal mesothelioma.

The aim of this study was to assess the performance of fluorescence in situ hybridization (FISH) in identifying the copy number profiles of the three key peritoneal mesothelioma tumor suppressor genes BAP1, CDKN2A, and NF2, with particular emphasis on minute homozygous deletions, a copy number abnormality recently unveiled at the 3p21 (BAP1) chromosomal region using high-throughput methods. FISH was performed on 75 formalin-fixed-paraffin-embedded peritoneal mesotheliomas and recognized two types of monoallelic loss (monosomy, and hemizygous deletion) and two types of biallelic loss (canonical homozygous deletion with a complete loss of FISH signal and homozygous deletion with diminished signal). Diminished FISH signals revealed deletions occurring within the genomic region covered by the gene-specific probe and affected all three tumor suppressors. BAP1 homozygous deletions with diminished signal outnumbered canonical homozygous deletions (13 vs 3): conversely, canonical homozygous deletions were prevalent for CDKN2A (2 vs 14). Diminished signal homozygous deletion was the only pattern of biallelic loss observed for NF2 (2 cases). Hemizygous deletion mainly affected BAP1 (21 vs 6), while monosomy was prevalent for CDKN2A (14 vs 7) and particularly for NF2 where it accounts for all monoallelic losses. FISH/immunohistochemistry (BAP1, CDKN2A, and MTAP) correlation showed that all homozygous deletions, including those with diminished signals, resulted in a null BAP1 and CDKN2A immunophenotype but only canonical CDKN2A homozygous deletions resulted in MTAP loss of expression. BAP1 hemizygous deletion, but not monosomy, was also invariably associated with loss of protein expression whereas neither type of CDKN2A monoallelic loss correlated with p16 or MTAPimmunohistochemistry. Array comparative genomic hybridization performed on a spontaneously emerging peritoneal mesothelioma cell line provided support for the interpretation of the FISH patterns and allowed us to extend the number of chromatin remodeling factors involved in mesothelioma to SETD7 and PCGF5, two previously unreported genes.

G-tract RNA removes Polycomb repressive complex 2 from genes.

Polycomb Repressive Complex 2 (PRC2) maintains repression of cell type-specific genes but also associates with genes ectopically in cancer. While it is currently unknown how PRC2 is removed from genes, such knowledge would be useful for the targeted reversal of deleterious PRC2 recruitment events. Here, we show that G-tract RNA specifically removes PRC2 from genes in human and mouse cells. PRC2 preferentially binds G-tracts within nascent pre-mRNAs, especially within predicted G-quadruplex structures. G-quadruplex RNA evicts the PRC2 catalytic core from the substrate nucleosome. PRC2 transfers from chromatin to RNA upon gene activation and chromatin-associated G-tract RNA removes PRC2, leading to H3K27me3 depletion from genes. Targeting G-tract RNA to the tumor suppressor gene CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces senescence. These data support a model in which pre-mRNA evicts PRC2 during gene activation and provides the means to selectively remove PRC2 from specific genes.

Clinical and prognostic effects of CDKN2A, CDKN2B and CDH13 promoter methylation in ovarian cancer: a study using meta-analysis and TCGA data

Background: Promoter methylation of tumour suppressor genes (TSGs) CDKN2A, CDKN2B and CDH13 has been reported in ovarian cancer. However, the clinicopathological characteristics and prognostic role of CDKN2A, CDKN2B and CDH13 promoter methylation in ovarian carcinoma remained unclear.Methods: The pooled odds ratio (OR) or hazard ratios (HRs) with their 95% confidence intervals (95% CIs) were calculated in this meta-analysis. The Cancer Genome Atlas data were obtained to confirm the role of CDKN2A, CDKN2B and CDH13 methylation in ovarian cancer.Results: CDKN2A, CDKN2B and CDH13 promoter methylation was higher in ovarian cancer than in normal ovarian tissues. CDH13 promoter methylation was correlated with tumour histology (serous vs. non-serous type: OR = 0.33, p = 0.031). CDKN2A promoter methylation was not linked to overall survival (OS), but it was correlated with a poor prognosis in progression-free survival (HR = 1.55, p = 0.004). TCGA data showed no correlation between CDKN2A, CDKN2B and CDH13 methylation and OS as well as disease-free survival (DFS).Conclusions: CDKN2A, CDKN2B and CDH13 promoter methylation may correlate with the increased risk of ovarian cancer. CDKN2A promoter methylation may be an independent prognostic biomarker for predicting progression-free survival.

Bicistronic transfer of CDKN2A and p53 culminates in collaborative killing of human lung cancer cells in vitro and in vivo.

Cancer therapies that target a single protein or pathway may be limited by their specificity, thus missing key players that control cellular proliferation and contributing to the failure of the treatment. We propose that approaches to cancer therapy that hit multiple targets would limit the chances of escape. To this end, we have developed a bicistronic adenoviral vector encoding both the CDKN2A and p53 tumor suppressor genes. The bicistronic vector, AdCDKN2A-I-p53, supports the translation of both gene products from a single transcript, assuring that all transduced cells will express both proteins. We show that combined, but not single, gene transfer results in markedly reduced proliferation and increased cell death correlated with reduced levels of phosphorylated pRB, induction of CDKN1A and caspase 3 activity, yet avoiding the induction of senescence. Using isogenic cell lines, we show that these effects were not impeded by the presence of mutant p53. In a mouse model of in situ gene therapy, a single intratumoral treatment with the bicistronic vector conferred markedly inhibited tumor progression while the treatment with either CDKN2A or p53 alone only partially controlled tumor growth. Histologic analysis revealed widespread transduction, yet reduced proliferation and increased cell death was associated only with the simultaneous transfer of CDKN2A and p53. We propose that restoration of two of the most frequently altered genes in human cancer, mediated by AdCDKN2A-I-p53, is beneficial since multiple targets are reached, thus increasing the efficacy of the treatment.

The RS4;11 cell line as a model for leukaemia with t(4;11)(q21;q23): Revised characterisation of cytogenetic features.

BackgroundHaematological malignancies harbouring rearrangements of the KMT2A gene represent a unique subtype of leukaemia, with biphenotypic clinical manifestations, a rapid and aggressive onset, and a generally poor prognosis. Chromosomal translocations involving KMT2A often cause the formation of oncogenic fusion genes, such as the most common translocation t(4;11)(q21;q23) producing the KMT2A‐AFF1 chimera.AimThe aim of this study was to confirm and review the cytogenetic and molecular features of the KMT2A‐rearranged RS4;11 cell line and put those in context with other reports of cell lines also harbouring a t(4;11) rearrangement.Methods and ResultsThe main chromosomal rearrangements t(4;11)(q21;q23) and i(7q), described when the cell line was first established, were confirmed by fluorescence in situ hybridisation (FISH) and 24‐colour karyotyping by M‐FISH. Additional cytogenetic abnormalities were investigated by further FISH experiments, including the presence of trisomy 18 as a clonal abnormality and the discovery of one chromosome 8 being an i(8q), which indicates a duplication of the oncogene MYC. A homozygous deletion of 9p21 containing the tumour‐suppressor genes CDKN2A and CDKN2B was also revealed by FISH. The production of the fusion transcript KMT2A‐AFF1 arising from the der(11)t(4;11) was confirmed by RT‐PCR, but sequencing of the amplified fragment revealed the presence of multiple isoforms. Two transcript variants, resulting from alternative splicing, were identified differing in one glutamine residue in the translated protein.ConclusionAs karyotype evolution is a common issue in cell lines, we highlight the need to monitor cell lines in order to re‐confirm their characteristics over time. We also reviewed the literature to provide a comparison of key features of several cell lines harbouring a t(4;11). This would guide scientists in selecting the most suitable research model for this particular type of KMT2A‐leukaemia.

Identification of diverse activating mutations of the RAS-MAPK pathway in histiocytic sarcoma

Recent studies have demonstrated recurrent activating mutations involving the classical MAPK and PI3K signaling pathways in a large proportion of histiocytic neoplasms, such as Langerhans cell histiocytosis. However, very little is known about the molecular genetics of histiocytic sarcoma, a rare aggressive malignant neoplasm that shows pathologic characteristics of mature macrophages. Here we report the genomic characteristics of a large cohort of histiocytic sarcomas (n = 28) using a targeted next-generation sequencing approach to identify driver alterations. We identified recurrent mutations involving the RAS-MAPK signaling pathway (MAP2K1, KRAS, NRAS, BRAF, PTPN11, NF1, CBL) in a majority (57%) of histiocytic sarcoma cases and report a clinical response to a MEK inhibitor (Cobimetinib) in a patient with a NF1-mutated histiocytic sarcoma. A smaller subset of cases (21%) also showed mutations resulting in activation of the PI3K signaling pathway (PTEN, MTOR, PIK3R1, PIK3CA). In addition, the tumor-suppressor gene CDKN2A was the most frequently altered gene (46%). Further, a subset of histiocytic sarcoma cases shows striking molecular genetic similarities to B cell lymphomas, supporting a clonal relationship between B cell neoplasms and a subset of histiocytic sarcomas. These findings support a cooperative role for MAPK, PI3K, and cyclin-CDK4/6-INK4 signaling in the pathogenesis of histiocytic sarcoma and provide a rational basis for targeting these pathways.

RAS activation and CDKN2A deletion to predict prognosis in cancer of unknown primary.

e13026 Background: Cancer of unknown primary (CUP) denotes a malignancy with histologically confirmed metastatic spread while the primary tumor remains elusive. Given the typically dismal response to empiric chemotherapy, the deciphering of molecular profiles holds a particular promise for targeted therapy. Here, we address the prognostic implications of mutations and copy number variations (CNVs) detected in tumor tissue in the context of a comprehensive clinical risk assessment. Methods: We performed targeted panel sequencing in a series of 252 CUP patients. Median age was 60.3 years and 59.1% were female. 71.2% of patients had unfavorable CUP. Lymph nodes, bone marrow, lung, peritoneum and pleura were the most frequent metastatic sites. Among histologic subtypes, adeno- (74.7%) and squamous cell carcinomas (13.7%) prevailed. DNA was extracted from micro-dissected formalin-fixed, paraffin-embedded tissue samples. For library preparation mostly multiplex PCR-based Ion Torrent AmpliSeq technology (Life Technologies) with Oncomine comprehensive assays v1 and v3 was used. Results: The prevailing genetic alterations were deletions in tumor suppressor genes TP53 (49.6%), CDKN2A (19.0%) and NOTCH1 (14.1%) as well as oncogenic activation of KRAS (23.4%), FGFR4 (14.9%) and PIK3CA (10.7%). KRAS activation was predominantly found in adenocarcinomas (p = 0.01) and PIK3CA activation in squamous cell carcinomas (p = 0.03). Regarding prognosis, male sex, high ECOG score, unfavorable CUP subtype, a higher number of involved organs as well as RAS activation predicted decreased event-free and overall survival in multivariate analysis. Deletions in CDKN2A were a poor prognostic factor regarding overall survival. Markedly, TP53 mutations did not significantly influence prognosis. Since July 2015, 17/198 (8.6%) patients received a recommendation for molecularly guided therapy. However, due to lack of coverage by insurers or rapid clinical deterioration only 7 of these patients (3.5%) actually received targeted treatment. Conclusions: Besides offering targets for therapy, targeted panel sequencing in CUP is prognostically relevant, with RAS activation and CDKNA2 deletion emerging as independent risk factors in a comprehensive assessment with clinical risk factors.

A novel pancreatic cancer model originated from transformation of acinar cells in adult tree shrew, a primate-like animal.

ABSTRACTPancreatic cancer is one of the most lethal common cancers. The cell of origin of pancreatic ductal adenocarcinoma (PDAC) has been controversial, and recent evidence suggested acinar cells as the most probable candidate. However, the genetic alterations driving the transformation of pancreatic acinar cells in fully mature animals remain to be deciphered. In this study, lentivirus was used as a tool to introduce genetic engineering in tree shrew pancreatic acinar cells to explore the driver mutation essential for malignant transformation, establishing a novel tree shrew PDAC model, because we found that lentivirus could selectively infect acinar cells in tree shrew pancreas. Combination of oncogenic KRASG12D expression and inactivation of tumor suppressor genes Tp53, Cdkn2a and Cdkn2b could induce pancreatic cancer with full penetrance. Silencing of Cdkn2b is indispensable for Rb1 phosphorylation and tumor induction. Tree shrew PDAC possesses the main histological and molecular features of human PDAC. The gene expression profile of tree shrew PDAC was more similar to human disease than a mouse model. In conclusion, we established a novel pancreatic cancer model in tree shrew and identified driver mutations indispensable for PDAC induction from acinar cells in mature adults, demonstrating the essential roles of Cdkn2b in the induction of PDAC originating from adult acinar cells. Tree shrew could thus provide a better choice than mouse for a PDAC model derived from acinar cells in fully mature animals.