bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3327: Sortilin controls the EGFR nuclear translocation
Authors: Akhrass Hussein Al , Naves Thomas , Jauberteau Marie-Odile , Vincent François , Lalloué Fabrice
Published: July 2017

The epidermal growth factor receptor (EGFR) deregulations play a central role in cancer progression. EGFR generates extra- and intracellular signals through various pathways, including its release in the tumor microenvironment through extracellular vesicles, as well as the nuclear EGFR signaling. That ultimately results in oncogenic signals promoting the tumor aggressiveness. Our previous report showed that sortilin, a member of the VPS10P sorting proteins, is associated with EGFR to allow its secretion through exosomes in non-small cell lung cancer (NSCLC) cells. Furthermore, sortilin is mainly present in the Golgi which is an essential compartment involved in the EGFR bypass towards the nucleus. Therefore, our aim is to investigate the potential role of sortilin in EGFR nuclear translocation.

EGFR and sortilin translocation into the nucleus has been validated by proximity ligation assay and confocal microscopy analysis. Moreover, to study the sortilin function in the EGFR nuclear translocation, we resorted to the NSCLC cell line A549 in which the sortilin expression was silenced using a shRNA delivery. Subcellular fractionations and nuclear protein extractions were performed to study the impact of the sortilin loss on the EGF-induced EGFR nuclear translocation.

EGF stimulation highly increases the nuclear translocation of the EGFR and sortilin complex. Interestingly, sortilin depletion significantly decreases the EGF-induced EGFR nuclear translocation in the A549 cells. Strikingly, sortilin-depleted cells exhibit an overexpression of EGFR target genes (MYC, ABCG2, AURKA and CCND1) compared to the control cells. These results suggest that the overexpression of the EGFR target genes is independent of EGFR nuclear translocation in sortilin-depleted cells. Thus, we analyze the activation of the known transcription factors directly involved in the control of EGFR up-regulated genes to determine whether they are activated in sortilin-depleted cells. Moreover, to better understand the sortilin-dependent EGFR nuclear translocation, we compare functional properties (proliferation,

migration and angiogenesis assays) of sortilin-depleted and control cells.

Altogether, our findings suggest a key role of sortilin in the regulation of the EGFR nuclear transport, towards the control of target genes that are widely involved in cancer progression.

Citation Format: Hussein Al Akhrass, Thomas Naves, Marie-Odile Jauberteau, François Vincent, Fabrice Lalloué. Sortilin controls the EGFR nuclear translocation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3327. doi:10.1158/1538-7445.AM2017-3327
Abstract 3333: The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor
Authors: Brand Toni M. , Iida Mari , Corrigan Kelsey L. , Braverman Cara M. , Coan John P. , Flanigan Bailey G. , Stein Andrew P. , Salgia Ravi , Rolff Jana , Kimple Randall J. , Wheeler Deric L.
Published: July 2017

The epidermal growth factor receptor (EGFR) is an important therapeutic target in patients with various cancers. Unfortunately, resistance to anti-EGFR therapeutics is common. Previous studies in our laboratory identified two mechanisms of resistance to the EGFR monoclonal antibody cetuximab: First, the nuclear translocation of EGFR bypasses the inhibitory effects of cetuximab; and second, the receptor tyrosine kinase AXL mediates cetuximab resistance by maintaining EGFR activation and downstream signaling. On the basis of these findings, we hypothesized that AXL could mediate the nuclear translocation of EGFR in the setting of cetuximab resistance. We found that NSCLC cetuximab-resistant clones and patient-derived xenografts (PDXs) had increased abundance of nuclear EGFR (nEGFR) and AXL. Cellular fractionation techniques, super resolution microscopy, and electron microcopy revealed that genetic loss of AXL diminished the nuclear translocation and accumulation of EGFR. Building on previous studies indicating that SRC family kinases (SFKs) and HER family ligands mediate the nuclear translocation of EGFR, we found that AXL knockdown down-regulated the expression of the SFKs YES and LYN, and the ligand neuregulin-1 (NRG1). Furthermore, AXL knockdown decreased the interaction between EGFR and HER3 and the nuclear abundance of HER3. Nuclear localization of EGFR could be rescued only upon simultaneous overexpression of Lyn and NRG1 in cells depleted of AXL. Collectively, these data uncover a previously unrecognized role for AXL in regulating the nuclear translocation of EGFR and suggest that AXL-mediated SFK activation and NRG1 expression are necessary and sufficient to regulate this process.

Citation Format: Toni M. Brand, Mari Iida, Kelsey L. Corrigan, Cara M. Braverman, John P. Coan, Bailey G. Flanigan, Andrew P. Stein, Ravi Salgia, Jana Rolff, Randall J. Kimple, Deric L. Wheeler. The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3333. doi:10.1158/1538-7445.AM2017-3333
Abstract 3353: An epigenetic biomarker panel in HPV related oropharyngeal squamous cell carcinoma
Authors: Ren Shuling , Gaykalova Daria A. , Guo Theresa , Khan Zubair , Ando Mizuo , Haft Sunny , Califano Joseph A.
Published: July 2017

HPV related head and neck squamous cell carcinoma (HNSCC) is known to occur in a separate patient population in comparison to tobacco related HNSCC, and also exhibits less mutational, genetic, and chromosomal alterations, and a different composition of epigenetic alterations. In particular, HPV is present in up to 70% of oropharyngeal squamous cell carcinoma (OPSCC), and is driving the increase in incidence rates of OPSCC. Given the precedent for successful screening for cervical cancer, it is attractive to consider similar population based screening for HPV related OPSCC. Here, we developed an epigenetic biomarker panel that can be combined with high risk HPV DNA detection with potential utility in screening. We performed high throughput qMBD-seq analysis on 50 primary HPV positive OPSCC samples and 25 UPPP normal samples. Subsequently, we performed an outlier analysis and ranking strategy of 100 bp segmented regions within this discovery cohort and defined 20 differentially methylated regions to be used as biomarkers for detection. Simultaneously, we validated these 20 biomarker candidates using TCGA methylation array dataset and found that the methylation levels of these 20 candidates in HPV positive OPSCC were significantly higher than those in normal samples. Moreover, we created QMSP assays for these genes, and validated EMBP1 and KCNA3 in a separate validation cohort including tissue and saliva from 46 HPV related OPSCC and 25 normal controls. The sensitivity and specificity values for EMBP1, KCNA3 or both in tissue and saliva are shown. EMBP1 has a high sensitivity in tissue and a high specificity in saliva, while KCNA3 has a high specificity in both tissue and saliva. A panel including both genes has an extremely high sensitivity. Ultimately, we defined a panel of two differentially methylation regions that has a high sensitivity in tissue. This study might serve as a diagnostic biomarker development trial for HPV OPSCC detection with potential application for development of a population based screening test.

Sensitivity and specificity for HPV related OPSCC in tissue and salivaTissueTissueSalivaSalivaGeneSensitivitySpecificitySensitivitySpecificityEMBP197.7%83.3%13.3%94.7%KCNA376.7%92.0%13.3%94.7%EMBP1& KCNA3100.0%84.0%13.0%84.2%

Citation Format: Shuling Ren, Daria A. Gaykalova, Theresa Guo, Zubair Khan, Mizuo Ando, Sunny Haft, Joseph A. Califano. An epigenetic biomarker panel in HPV related oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3353. doi:10.1158/1538-7445.AM2017-3353
Abstract 3357: Heritable methylation marks associated with breast cancer risk
Authors: Southey Melissa C. , Joo Jihoon E. , Dowty James G. , Milne Roger L. , Wong EE Ming , Dugué Pierre-Antoine , English Dallas , Hopper John L. , Goldgar Daivd E. , Giles Graham G.
Published: July 2017

While most epigenetic marks are reprogrammed during early embryogenesis, some studies have reported Mendelian-like inheritance of germline DNA methylation in particular cancer susceptibility genes. We aimed to identify heritable methylation marks associated with breast cancer susceptibility by studying 25 Australian multi-generational families with multiple cases of breast cancer who were not known to carry mutations in breast cancer susceptibility genes. Peripheral blood DNA methylation was measured at approximately 480,000 genetic loci (methylation sites) for 210 members of these families using the Infinium HumanMethylation450 BeadArray. We developed and applied a new statistical method to identify heritable methylation marks based on complex segregation analysis. Carrier probabilities were estimated for the 1000 most heritable methylation marks, based on family structure but not disease status, and Cox proportional hazards survival analysis was used to test for associations between these carrier probabilities and breast cancer. After correcting for multiple testing, we identified 11 methylation marks whose corresponding carriers probabilities were significantly associated with breast cancer. Three marks clustered within 200 base pairs of the noncoding RNA miR886. We found evidence for these marks to be associated with aggressive (but not non-aggressive) prostate cancer risk in a population-based prostate cancer study. The association with breast cancer risk was replicated for three of the eleven marks in an independent population-based breast cancer study. Our work demonstrates the efficacy of our novel approach for identifying heritable methylation marks associated with breast cancer. This methodology may provide insights into other disease settings where known lifestyle exposures and genetic risk variants do not fully explain the familial relative risk.

Citation Format: Melissa C. Southey, Jihoon E. Joo, James G. Dowty, Roger L. Milne, EE Ming Wong, Pierre-Antoine Dugué, Dallas English, John L. Hopper, Daivd E. Goldgar, Graham G. Giles. Heritable methylation marks associated with breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3357. doi:10.1158/1538-7445.AM2017-3357
Abstract 3361: Methylome differences in differentiated thyroid cancers and benign adenomas
Authors: Stephen Josena K. , Chen Kang Mei , Merritt Jason , Datta Indrani , Chitale Dhananjay , Divine George , Worsham Maria J.
Published: July 2017

Many recent studies examining aberrant DNA methylation in thyroid cancer are restricted to either candidate genes or genome-wide methylation in specific thyroid tumor subtypes. The goal of this study was to identify differentially methylated genes globally and their association with molecular pathways and signaling networks. Common mutations in papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), follicular adenoma (FA) and normal thyroid were also examined. Genome-wide methylation profiling using the Infinium HumanMethylation450FFPE (formalin fixed paraffin embedded) BeadChip Array was performed on 24 thyroid cases (8 PTC, 8 FTC, 4 FA and 4 normal thyroid). Ingenuity Pathway Analysis (IPA) was utilized to assess the roles of significantly differentially methylated genes in biological functions, signaling/metabolic pathways, and networks. Common mutations in 4 genes (BRAF, NRAS, HRAS, and KRAS) were assessed using TaqMan Mutation Detection assay. Twelve genes were significantly differentially methylated among 4 comparison groups: cancer vs normal, cancer vs adenoma, PTC vs normal, and PTC follicular variant (PTC-FV) vs PTC-Classic. CTU1 and HLA-DPB1 were significantly hypermethylated and AARS2, TMSB10, RNF216L, KIF15, KIAA1143, and SLC2A13 were significantly hypomethylated between cancer and normal. Significant differential hypermethylation was noted for PNPLA7 and NPC1L1 in cancer vs adenoma and SNX6 in PTC-FV vs PTC-Classic. NT5C1B was hypermethylated and AARS2 was hypomethylated in PTC vs normal. IPA identified 2 gene networks, involving 11/12 genes, characterized by 1) Cellular development, Cellular growth and proliferation, Connective tissue development and function and 2) Drug metabolism, Cell-mediated immune response, Cellular development. NT5C1B was involved in all 4 highly ranked canonical nucleotide degradation pathways. Several significant bio-functions involved NPC1L1. Mutations of NRAS codon 61 were identified in 1 sample each of FTC-Classic, PTC-FV and FA and BRAF V600E in one PTC-Classic sample. Differential methylation of AARS2, CTU1, HLA-DPB1, SLC2A13, PNPLA7, NPC1L1, NT5C1B and SNX6 suggest potential markers for discriminating thyroid cancers from adenomas and normal. NT5C1B was noted in highly ranked canonical pathways, suggesting a role in nucleotide degradation. Pathway analysis of differentially methylated genes support important biological processes in thyroid cancer pathogenesis. Supported by JFCI/CRAG A20038

Citation Format: Josena K. Stephen, Kang Mei Chen, Jason Merritt, Indrani Datta, Dhananjay Chitale, George Divine, Maria J. Worsham. Methylome differences in differentiated thyroid cancers and benign adenomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3361. doi:10.1158/1538-7445.AM2017-3361
Abstract 3362: PBRM1 regulates the transcription of cell adhesion genes in ccRCC
Authors: Chowdhury Basudev
Published: July 2017

Advances in parallel sequencing have facilitated the recent identification of Polybromo-1 (PBRM1), a component of the PBAF (Polybromo-associated-BRG1- or BRM-associated factors) chromatin remodeling complex, as the second most frequently mutated gene in clear-cell renal cell Carcinoma (ccRCC). While several studies have described the diversity of PBRM1 mutations in ccRCC and established the mutation of PBRM1 as a “driver” of ccRCC carcinogenesis, little is known about the biological significance of PBRM1. With the intent of shedding light on the mechanistic roles of PBRM1, we have employed Next Generation Sequencing to delineate the transcriptional profile of cellular models of ccRCC with or without PBRM1 expression and identified genes associated with cell adhesion to be regulated by PBRM1 in 2D monolayer cultures. Since 2D cultures lack the architectural complexities to support studies pertaining to cellular adhesion, we evaluated the role of PBRM1 in 3D culture on Basement Membrane Matrix to provide a physiologically relevant analysis of gene expression and the resulting cellular phenotype. The results showed that of the 97 cell adhesion genes identified to be upregulated by PBRM1 in 2D cultures, 45 were also upregulated in 3D cultures and a subset of 32/45 genes underwent increased gene expression in 3D with respect to 2D cultures. The gene expression profile of candidate genes were verified by qPCR and Western Blotting. Mining The Cancer Genome Atlas (TCGA) publicly available data, we were able to correlate the gene expression profiles of cell adhesion genes identified in 3D cultures with those of ccRCC patients bearing at least 1 copy of the PBRM1 gene. Furthermore, we performed a PBRM1-ChIP-seq to map the genome-wide targets of PBRM1 and revealed the association of PBRM1 occupancy at proximal regulatory regions of 8/45 cell adhesion genes. All of these taken together, indicate a role for PBRM1 in regulating the gene expression of cell adhesion genes which act as important players in cellular morphogenesis and responsiveness to changes in the external environment. Disruption of cell adhesion is believed to affect signal transduction and pave the way facilitating metastasis in neoplastic cells. Currently we are investigating the effect of loss-of-PBRM1 on Epithelial to Mesenchymal Transition (EMT) and metastasis in ccRCC.

Note: This abstract was not presented at the meeting.

Citation Format: Basudev Chowdhury. PBRM1 regulates the transcription of cell adhesion genes in ccRCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3362. doi:10.1158/1538-7445.AM2017-3362
Abstract 3368: Omics data integration analysis in high grade serous ovarian cancer: results from three studies
Authors: Natanzon Yanina , Earp Madalene , Cunningham Julie M. , Kalli Kimberly R. , Winham Stacey J. , Armasu Sebastian M. , Larson Melissa C. , Wang Chen , Bowtell David D. , Garsed Dale W. , Goode Ellen
Published: July 2017

High grade serous ovarian cancer (HGSOC) is a complex disease in which initiation and progression have been associated with gene mutation, DNA methylation changes, genetic variation, and epigenetic processes. Variation in several susceptibility regions and cancer-typical global methylation patterns have been observed in HGSOC; however, this knowledge has not been compelling in understanding HGSOC intiation or progression. As ingetration of genomic, epigenomic, and transcriptomic data has increased mechanistic understanding in other cancers, we hypothesized that tumor methylation alone or in combination with germline genetic variation influences tumor gene expression in HGSOC. We examined three nested models using an Elastnic Net (ENET) penalized regression method while adjusting for somatic copy number (CNV): a) germline genotype and tumor DNA methylation (full model), b) genotype only, and c) DNA methylation only. We included 339 cases from The Cancer Genome Atlas (TCGA), 54 cases from Mayo Clinic, and 78 cases from the Australian Ovarian Cancer Study (AOCS). Genotyping and copy number calls on germline DNA, expression, methylation and copy number on somatic samples were collected and analyzed on different platforms separately at each study site. We excluded genes with low overall expression and thus analyzed a total of 11,922 genes available in three datasets ( Ensembl IDs, 500kb window up- and downstream). In general, combining genomic data in HGSOC did not reveal a role for germline genetic variation in altering gene expression. However, in methylation only models 79 genes were associated with differential expression in the TCGA cases (permutation multiple testing adjusted p-val <0.05), in the Mayo cases (unadjusted p-val <0.05) and AOCS cases (unadjusted p-val <0.05). A known tummor suppressor (FBXW7) was associated with differential expression in the three datasets at p-val <0.01. This work demonstrates the feasibility, utility, and statistical power of ENET gene-level analyses incoporating maximal genomic information.

Citation Format: Yanina Natanzon, Madalene Earp, Julie M. Cunningham, Kimberly R. Kalli, Stacey J. Winham, Sebastian M. Armasu, Melissa C. Larson, Chen Wang, David D. Bowtell, Dale W. Garsed, Ellen Goode. Omics data integration analysis in high grade serous ovarian cancer: results from three studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3368. doi:10.1158/1538-7445.AM2017-3368
Abstract 3373: The use of long-read sequencing to determine the structure of the Her2 amplicon in breast cancer
Authors: Rosenfeld Jeffrey , Goodwin Sara , Ganesan Shridar
Published: July 2017

HER2 amplification is present in ~15% of breast cancers, and is present in small subsets of other cancer types including gastric and lung cancers. Therapy targeting HER2 is now routinely used in treatment of HER2-amplificed breast cancer and has changed the natural history of this disease. The region of chromosome 17q12 containing Her2 contains many genes, and little is known about the actual genomic alterations that underlie the amplification event. While it is known that parts of the amplicon exist in multiple copies in a tumor, the exact structure of the duplicated region is not known and their relative orientation and positions in the genome is not clear. There is the potential for rearrangements between the genes and for different segments of the amplicon to exist and varied copy numbers. There is no clear way to assemble the complete structure of this region using short reads, as they cannot resolve repetitive regions and identify junction reads. Long read sequencing has the potential to overcome these limitations, however the sequencing depth is limited and difficult to apply to large genomes. To apply long read sequencing to a limited part of the cancer genome, we have used Nimblegen target enrichment coupled with Oxford Nanopore and Pacific Bioscience sequencing in an attempt to sequence and assemble the Her2 amplicon. The error rate of the sequencers are not of concern since we are not looking at base-pair level variation. Instead, our need is for long reads that can span the gene boundaries and repetitive elements to give complete structure of the amplicon. Nimblegen custom capture probes were designed for the 2mb chromosomal region containing Her2. DNA was extracted from 3 breast cancer cell lines and enriched using the Nimblegen kit. This DNA was then sequenced with long reads on the MinIon and the PacBio to allow for complete assembly of the region. The DNA was assembled to determine the structure of the HER2 amplicon in each cell line. These results may give new insight into the molecular events that led to formation of the HER2 amplicon.

Citation Format: Jeffrey Rosenfeld, Sara Goodwin, Shridar Ganesan. The use of long-read sequencing to determine the structure of the Her2 amplicon in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3373. doi:10.1158/1538-7445.AM2017-3373
Abstract 3374: APA isoform diversity in triple negative breast cancers
Authors: Erson-Bensan Ayse Elif , Akman Begum H. , Oyken Merve , Agus Hizlan H. , Yavuz Esra , Erdem Murat , Can Tolga
Published: July 2017

Alternative polyadenylation (APA) plays a role in gene expression regulation generally by shortening of 3'UTRs and relieving microRNA-mediated repression. Therefore, APA is gaining increased attention as a potential mechanism to activate oncogenes. Owing to high proliferative indices of triple negative breast cancers (TNBCs), we hypothesized APA to cause 3'UTR length changes in this aggressive subgroup of breast cancers. Our probe-based meta-analysis approach identified 3'UTR length alterations where the significant majority was shortening events (70%, 113 of 165) of mostly proliferation-related transcripts in over 500 TNBC patients compared with normal breast tissue. Representative shortening events correlated with increased protein levels and relapse free survival of patients, suggesting functional significance of isoform variability. To begin addressing the underlying mechanisms of 3’UTR shortening, we turned to APA machinery proteins. We detected variable expression of APA machinery proteins in different breast cancer subtypes but CSTF2 (cleavage stimulation factor 2) has the most prominent overexpression in breast cancer cells. Therefore, among potential regulators of 3’UTR shortening, we further investigated the role of CSTF2 in proximal polyA signal selection. Because some of the TNBC patients are EGFR positive, we found EGF treatment to cause increased CSTF2 levels. Higher CSTF2 levels indeed correlated with further shortening of the 3'UTRs. Accordingly, RNAi-induced silencing of CSTF2 decreased the proliferative rate of cancer cells. Therefore, our integrated approach revealed a pattern of 3'UTR length changes in TNBC patients and a potential link between APA and EGF signaling. Further studies are underway to investigate the mechanistic link between EGF signaling and regulation of 3’UTR lengths. (This work is funded by TUBITAK 112S478 and 114Z884)

Citation Format: Ayse Elif Erson-Bensan, Begum H. Akman, Merve Oyken, Hizlan H. Agus, Esra Yavuz, Murat Erdem, Tolga Can. APA isoform diversity in triple negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3374. doi:10.1158/1538-7445.AM2017-3374
Abstract 3387: Mutational analysis in metastatic melanoma to the breast and ovary: correlation with clinic-pathological and immunohistochemical findings
Authors: Giubellino Alessio , Moreno Vanessa , Naizhen Xu , Xi Liqiang , Raffeld Mark , Merino Maria
Published: July 2017

Metastatic malignant melanoma or stage IV melanoma is the most deadly skin cancer, claiming an estimated 10,000 deaths every year. The occurrence of metastatic malignant melanoma to the breast and ovary is uncommon, and can represent a challenge for the diagnosis and clinical management. We investigated and correlated the morphologic, immunohistochemical and molecular aspects of these tumors.

Morphologic analysis of the cases revealed a predominance of epithelioid pattern, and few of these were associated with more spindle cell areas. Most of the tumor cells aberrantly expressed high levels of PD-L1, especially in the metastatic melanoma to the breast. Other relevant melanoma targets for immunotherapy were also explored by immunohistochemistry. Of note, most of the metastatic tumors were negative for NY-ESO.

The mutational analysis of these metastatic lesions, using next generation sequencing for the detection of cancer gene mutations including rare mutations at low frequency, found an heterogeneous mutational landscape, reinforcing the importance of personalized treatments. Mutations in RAS gene family members in these metastatic tumors were the most frequent in our samples, followed by CDKN2A and CTNNB1 mutations. Other less frequent mutations were also detected, including ATM and KIT. Interestingly, one rapidly progressive melanoma metastatic to the breast in a young patient had areas of spindle cell morphology and no common melanoma gene mutations.

Overall, our cases illustrate the pathologic and molecular variability of metastatic malignant melanoma to uncommon organs of involvement, such as breast and ovary, and provide novel insights into their diagnostic and clinical management algorithm.

Note: This abstract was not presented at the meeting.

Citation Format: Alessio Giubellino, Vanessa Moreno, Xu Naizhen, Liqiang Xi, Mark Raffeld, Maria Merino. Mutational analysis in metastatic melanoma to the breast and ovary: correlation with clinic-pathological and immunohistochemical findings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3387. doi:10.1158/1538-7445.AM2017-3387
Abstract 3389: Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma
Authors: Liang Winnie S. , Hendricks William , Kiefer Jeffrey , Schmidt Jessica , Sekar Shobana , Carpten John , Craig David W. , Adkins Jonathan , Cuyugan Lori , Manojlovic Zarko , Halperin Rebecca F. , Helland Adrienne , Nasser Sara , Legendre Christophe , Hurley Laurence H. , Sivaprakasam Karthigayini , Johnson Douglas B. , Crandall Holly , Busam Klaus J. , Zismann Victoria , Luca Valerie De , Lee Jeeyun , Sekulic Aleksandar , Ariyan Charlotte E. , Sosman Jeffrey , Trent Jeffrey
Published: July 2017

Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited. Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms. We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients. Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures. Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations. Contrasting with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients. We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells. These findings provide insight into the role of TERT in ALM tumorigenesis, and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM.

Citation Format: Winnie S. Liang, William Hendricks, Jeffrey Kiefer, Jessica Schmidt, Shobana Sekar, John Carpten, David W. Craig, Jonathan Adkins, Lori Cuyugan, Zarko Manojlovic, Rebecca F. Halperin, Adrienne Helland, Sara Nasser, Christophe Legendre, Laurence H. Hurley, Karthigayini Sivaprakasam, Douglas B. Johnson, Holly Crandall, Klaus J. Busam, Victoria Zismann, Valerie De Luca, Jeeyun Lee, Aleksandar Sekulic, Charlotte E. Ariyan, Jeffrey Sosman, Jeffrey Trent. Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3389. doi:10.1158/1538-7445.AM2017-3389
Abstract 3391: Next generation sequencing paves the way for personalized medicine in pheochromocytoma and paraganglioma patients and their families
Authors: Gieldon Laura , Richter Susan , Rump Andreas , Hackmann Karl , Tzschach Andreas , Eisenhofer Graeme , Pęczkowska Mariola , Prejbisz Aleksander , Schröck Evelin , Klink Barbara
Published: July 2017

More than 35% of pheochromocytomas and paragangliomas (PPGL) are thought to occur due to an underlying genetic predisposition. 18 susceptibility genes have been identified for PPGL, which explain app. 30% of familial cases. Further PPGL predisposition genes, however, remain to be discovered. In addition, identification of biomarkers for malignancy also remains a current challenge since histologic assessment does not suffice discrimination of malignant PPGLs. Personalized screening protocols, risk stratification measures and surveillance guidelines are needed for PPGL patients and their family members at risk.

We performed panel sequencing for 94 tumor predisposition syndrome (TPS) genes and identified 12 pathogenic germline mutations in 60 PPGL patients. Two of these patients were identified with NF1-mutations although they did not meet the diagnostic criteria for Neurofibromatosis. Thus, TPS gene panel sequencing proved superior to routine Sanger sequencing since mutations were identified in genes that would have routinely been excluded from analysis.

Combining results of immunohistochemistry as well as biochemical profiling by measuring Krebs-cycle metabolites and genetic analyses of tumors we were able to reclassify variants of uncertain significance (VUS) as either pathogenic mutations or polymorphisms in several patients.

Three incidentally found pathogenic mutations in CHEK2 and MSH2 prompted further analyses such as testing for microsatellite instability in order to evaluate possible effects on PPGL development.

For patients in whom no underlying mutation could be detected we performed custom candidate gene panel sequencing of tumor derived-DNA. Whole exome sequencing (WES) was carried out in 15 cases highly suspicious for a genetic background in order to identify new candidate predisposition genes that will further be evaluated in the patient cohort and in functional studies.

We will also present guidelines for diagnostics, surveillance and prevention of PPGL for patients and their family members.

Citation Format: Laura Gieldon, Susan Richter, Andreas Rump, Karl Hackmann, Andreas Tzschach, Graeme Eisenhofer, Mariola Pęczkowska, Aleksander Prejbisz, Evelin Schröck, Barbara Klink. Next generation sequencing paves the way for personalized medicine in pheochromocytoma and paraganglioma patients and their families [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3391. doi:10.1158/1538-7445.AM2017-3391
Abstract 3395: Alternative splicing of the MEAF6 gene promotes neuroendocrine prostate cancer development
Authors: Lee Ahn R. , Li Yinan , Xie Ning , Collins Colin C. , Dong Xuesen
Published: July 2017

In response to the selection pressures exerted by potent androgen receptor (AR) pathway inhibitors (ARPI), adenocarcinoma prostate cancer (PCa) cells can undergo an adaptive process of cellular phenotype reprogramming termed neuroendocrine trans-differentiation. With this AR-bypass mechanism of survival emerges a lethal treatment-resistant PCa subtype called treatment-induced neuroendocrine PCa (t-NEPC). t-NEPC is becoming a major clinical issue as it is estimated to affect >25% of advanced-stage PCa patients with the level of incidences predicted to rise as a result of the extensive applications of ARPI in the clinic. This underscores the gravity of our aims to delineate the molecular underpinnings of t-NEPC to inform future therapies that prevent or mitigate t-NEPC development.

In this study, we have identified a splice variant of the MYST/Esa1-associated factor 6 (MEAF6) gene, MEAF6-1, that is highly expressed in t-NEPC tumor biopsies as well as neuroendocrine cell lines of prostate and lung cancers. We show that the neuronal RNA splicing factor, SRRM4, stimulates MEAF6-1 splicing. Enhanced MEAF6-1 expression in prostate adenocarcinoma cell lines does not induce neuroendocrine trans-differentiation of these cells. Rather, it stimulates cell proliferation, anchorage-independent cell growth, invasion, and xenograft tumor growth. Gene microarray identified that these MEAF6-1 actions are in part mediated by the ID1 and ID3 genes. These findings suggest that the MEAF6-1 variant does not induce neuroendocrine differentiation of prostate cancer cells, but facilitates t-NEPC progression through accelerating proliferation of cells that have acquired neuroendocrine phenotypes.

Citation Format: Ahn R. Lee, Yinan Li, Ning Xie, Colin C. Collins, Xuesen Dong. Alternative splicing of the MEAF6 gene promotes neuroendocrine prostate cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3395. doi:10.1158/1538-7445.AM2017-3395
Abstract 340: SRMS regulates normal and oncogenic KIT signaling
Authors: Nagaraj Sachin Raj M , Kazi Julhash U. , Rönnstrand Lars
Published: July 2017

SRMS (Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) belongs to a family of non-receptor tyrosine kinases, which harbours a Src homology 3 and a Src homology 2, as well as a protein kinase domain. SRMS was first identified in a screen for the genes that regulate the growth and differentiation of neuroepithelial cells. SRMS, however, is an understudied member of this family. The present study was undertaken in order to explore the role of SRMS in signaling downstream of KIT. The receptor tyrosine kinase KIT, also known as the stem cell factor receptor, plays a key role in several developmental processes and have been implicated in many human cancers such as gastrointestinal stromal tumors, acute myeloid leukemia and testicular carcinoma. To understand the role of SRMS in KIT signaling, we generated Ba/F3 cell lines overexpressing KIT and SRMS. We observed that SRMS regulates normal and oncogenic KIT signaling differentially with respect to cell proliferation and apoptosis. SRMS association triggers KIT ubiquitination which in turn downregulates the receptor. Further, expression of SRMS downregulates wild-type KIT-mediated phosphorylation of AKT, ERK1/2, p38. Taken together the data demonstrates that SRMS regulates both normal KIT and an oncogenic mutant of KIT, and have differential impact on receptor downstream signaling.

Citation Format: Sachin Raj M Nagaraj, Julhash U. Kazi, Lars Rönnstrand. SRMS regulates normal and oncogenic KIT signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 340. doi:10.1158/1538-7445.AM2017-340
Abstract 3410: Phenotypic characterization of allelic variation within the HEAT repeats of the mechanistic target of rapamycin
Authors: Wiley Patricia A. , Gary Joy , Phyo Zaw , Zhang Shuling , Sciorillo Amanda , Fraizer Dorian
Published: July 2017

The mammalian target of rapamycin (mTOR), a protein in the phosphoinositide 3-kinase (PI3K) pathway important to cell cycle regulation, is frequently activated in cancer. This signaling pathway regulates cell growth and metabolism, and when dysregulated in cancer, it contributes to tumor angiogenesis and growth. mTOR contains two distinct complexes, MTORC1, and MTORC2. MTORC1 plays a role in biosynthesis, while MTORC2 helps with cellular metabolism. It has been shown that mTOR knockout models are embryonically lethal to mice. However, lower levels of functional mTOR have been shown to be beneficial and slow cancer progression. Allelic variation occurs within inbred strains of mice, including BALB/c mice. One allelic variant in mTOR, R628C, which contributes to tumor susceptibility has been identified in BALB/c mice. This single amino acid change occurs in the HEAT repeats of mTOR. HEAT repeats have been shown to facilitate binding partner interactions, dimerization, and localization. Studies on mutations found within the HEAT repeats in several proteins have altered protein function and emphasize the structural importance of HEAT repeats. The goal of this study is to further elucidate the functional significance of HEAT repeats in TOR function and how allelic variation in the HEAT repeats may contribute to tumor susceptibility. Characterization of this allelic variant has shown stress inputs, such as irradiation, lead to a more severe phenotype and reduced mTORC1/2 signaling. Subsequent in vitro studies using a tetracycline inducible expression system (T-RExTM) have shown differential binding to mTORC1/2 complex partners in cells expressing the Balb/c allele. Additional studies are being performed to assess other complex binding partners, dimerization state and localization of the allelic variant. Our findings will model mTOR signaling activation mechanisms in humans to aid in development of new cancer treatments.

Note: This abstract was not presented at the meeting.

Citation Format: Patricia A. Wiley, Joy Gary, Zaw Phyo, Shuling Zhang, Amanda Sciorillo, Dorian Fraizer. Phenotypic characterization of allelic variation within the HEAT repeats of the mechanistic target of rapamycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3410. doi:10.1158/1538-7445.AM2017-3410
Abstract 3415: RNF43 somatic mutations in endometrioid ovarian cancers occur in the setting of synchronous endometrioid endometrial cancers
Authors: Levine Defne L. , Dao Fanny , Olvera Narciso , LaVigne Katherine , Solit David B. , Jelinic Petar
Published: July 2017

Introduction: Somatic frameshift mutations in RNF43 have previously been filtered out in cancer genome sequencing projects given that they occur in homopolymer tracts and resemble polymerase slippage errors. Two RNF43 hotspot mutations have been identified and validated in endometrioid endometrial cancer (EEC) and appear to negatively regulate Wnt signaling. These frameshift mutations occur at codons R117 and G659. Due to shared clinical and morphologic features of EEC and endometrioid ovarian cancer (EmOC), we determined the frequency of RNF43 somatic mutations in EmOC.

Methods: We reviewed the clinical and pathologic features of EmOC samples diagnosed from 2006 to 2015, retrieved from laboratory databases and institutional archives. DNA was extracted from formalin-fixed, paraffin-embedded tumor samples using standard protocols. Sanger and next-generation sequencing (NGS) were used to screen for hotspot mutations at codons R117 and G659 with custom designed primers. NGS at each hotspot was covered with a minimum sequencing depth of 400X.

Results: Forty-seven EmOC patients with available tumor specimens were identified and included in the analysis. The median age at diagnosis was 55 years old (range 34-84). The majority of patients had FIGO stage I or stage II disease (n=38, 81%). Thirteen (28%) patients had synchronous endometrial endometrioid or mixed histology tumors. Two (4.3%) RNF43 somatic mutations at codon G659 were identified in both Sanger and NGS from the EmOC tumor specimens. No mutations at codon R117 were identified. The allele fractions of the G659 mutations were 4.3% and 2.8% seen in 33 and 18 reads with a coverage of 775X and 646X, respectively. A review of pathology reports indicated that both mutated samples had synchronous EECs. These two mutated samples represent 15.4% of cases diagnosed with synchronous EmOC and EEC in this cohort.

Conclusions: RNF43 somatic mutations are uncommon in EmOCs and associated with synchronous EECs. Recent massively parallel sequencing data suggests that EmOCs in the setting of synchronous EECs are clonally related and disseminated cells are related to nearby anatomic structures. Our data supports that RNF43 somatic mutations in EmOC are clonally expanded from EECs likely through trans-tubal migration.

Citation Format: Defne L. Levine, Fanny Dao, Narciso Olvera, Katherine LaVigne, David B. Solit, Petar Jelinic. RNF43 somatic mutations in endometrioid ovarian cancers occur in the setting of synchronous endometrioid endometrial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3415. doi:10.1158/1538-7445.AM2017-3415
Abstract 3416: Clarifying the biological significance of the CHK2 K373E somatic mutation discovered in The Cancer Genome Atlas database
Authors: Higashiguchi Masayoshi , Nagatomo Izumi , Kijima Takashi , Morimura Osamu , Miyake Kotaro , Minami Toshiyuki , Koyama Shohei , Hirata Haruhiko , Iwahori Kota , Takimoto Takayuki , Takeda Yoshito , Kida Hiroshi , Kumanogoh Atsushi
Published: July 2017

Identification of somatic mutations that contribute to cancer development leads not only to more precise understanding of cancer pathogenesis, but also development of novel molecular targeted therapies. Analyses of cancer genomes using high-throughput sequencing technology are currently conducted as international collaborative research projects, and the results of those analyses are deposited in public databases, allowing everyone in the world to access to these data.

To identify a somatic mutation that plays an important role in cancer pathogenesis, we counted the occurrences of each somatic mutation found in the TCGA lung adenocarcinoma dataset and became interested in the CHK2 K373E mutation (c1117A>G), which was present in 31 of 542 patients. The K373E mutation impaired CHK2 autophosphorylation at Thr383 and Ser516. In vitro kinase assay revealed that the K373E mutation markedly attenuates CHK2 kinase activity. Growth curves showed that wild-type CHK2 substantially suppressed cell proliferation, but this effect was lost in the K373E mutant in HCT-15 harboring tetracycline-inducible CHK2. Clonogenic assays revealed that wild-type CHK2 promoted survival after ionizing radiation and this pro-survival function was impaired in K373E CHK2. The results of western blotting and RT-PCR suggested that the p53-independent induction of p21 by CHK2 might underlie these effects.

Therefore, we identified a somatic mutation that contributes to cancer pathogenesis, using information in a public database. This kind of attempt is expected to lead to discovering new 'driver' mutations in the future.

Citation Format: Masayoshi Higashiguchi, Izumi Nagatomo, Takashi Kijima, Osamu Morimura, Kotaro Miyake, Toshiyuki Minami, Shohei Koyama, Haruhiko Hirata, Kota Iwahori, Takayuki Takimoto, Yoshito Takeda, Hiroshi Kida, Atsushi Kumanogoh. Clarifying the biological significance of the CHK2 K373E somatic mutation discovered in The Cancer Genome Atlas database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3416. doi:10.1158/1538-7445.AM2017-3416
Abstract 3418: Additional mutation in PTPN11 gene promotes tumorigenesis of the NF1 gene mutated cells
Authors: Arima Yoshimi , Harigai Ritsuko , Sato Ryo , Takenouchi Toshiki , Kosaki Kenjiro , Saya Hideyuki
Published: July 2017

The NF1 tumor suppressor gene encodes neurofibromin and is a functional Ras GTPase-activating protein (RasGAP) involved in negatively regulating the Ras signal by accelerating the conversion of activated Ras-GTP to inactive Ras-GDP. NF1 gene germline mutations cause Neurofibromatosis type 1 (NF1, von Recklinghausen disease). We hypothesized that additional genetic alterations promote the malignancy of NF1-associated tumors. To test our hypothesis, we inoculated a GFP-labeled human NF1-deficient cell line, sNF96.2-GFP, which has a frame-shift mutation (c.3683delC, p.Asn1229MetfsTer11) in the NF1 gene, into the renal sub-capsules of immunodeficient mice. A subclonal cell line, the A-1 cell, was established from the developed tumor. We found that A-1 cells show much higher tumorigenic activity and phosphorylation status of MEK and Akt than the parental sNF96.2-GFP cells. We analyzed the genomic DNA of both the sNF96.2 and the A-1 cells by using the next-generation sequencing and our medical exome panel of 4813 genes, which are known to be responsible for most human genetic disorders. We identified 18 heterozygous variants within coding regions of 17 genes that were present in the A-1 cells, but not in the original sNF96.2 cells. We found a single base substitution (c.1508G>T, p.Gly503Val) in the PTPN11 gene, which encodes the tyrosine phosphatese SHP-2, and is associated with the regulation of the Ras signaling pathway. It is critical to note that constitutional gain-of-function mutations in the PTPN11 gene cause Noonan Syndrome in humans due to activation of the Ras pathway. To determine the role of PTPN11 mutation in NF1-associated tumors, we established a cell line overexpressing PTPN11mut in sNF96.2-GFP cells. We inoculated parental cells and PTPN11mut cells into the subcutaneous of nude mice, and we found that PTPN11mut cells show much higher tumorigenic activity than the parental sNF96.2-GFP cells. Our data suggests that this additional gene mutation in PTPN11 promotes the malignant characteristics of NF1-associated tumors.

Citation Format: Yoshimi Arima, Ritsuko Harigai, Ryo Sato, Toshiki Takenouchi, Kenjiro Kosaki, Hideyuki Saya. Additional mutation in PTPN11 gene promotes tumorigenesis of the NF1 gene mutated cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3418. doi:10.1158/1538-7445.AM2017-3418