TAMI-28. DIFFERENTIAL MIGRATION MECHANICS AND IMMUNE RESPONSES OF GLIOMA SUBTYPES
Ghaidan A. ShamsanChao LiuBrooke BramanSusan K. RatheAaron L. SarverNima GhaderiMariah McMahonRebecca KlankBarbara R. TschidaJoey McFarrenJann N. SarkariaH. Brent ClarkSteven S. RosenfeldDavid A. LargaespadaDavid J. Odde
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Abstract In Glioblastoma (GBM), tumor spreading is driven by tumor cells’ ability to infiltrate healthy brain parenchyma, which prevents complete surgical resection and contributes to tumor recurrence. GBM molecular subtypes, classical, proneural and mesenchymal, were shown to strongly correlate with specific genetic alterations (Mesenchymal: NF1; Classical: EGFRVIII; Proneural: PDGFRA). Here we tested the hypothesis that a key mechanistic difference between GBM molecular subtypes is that proneural cells are slow migrating and mesenchymal cells are fast migrating. Using Sleeping Beauty transposon system, immune-competent murine brain tumors were induced by SV40-LgT antigen in combination with either NRASG12V (NRAS) or PDGFB (PDGF) overexpression. Cross-species transcriptomic analysis revealed NRAS and PDGF-driven tumors correlate with human mesenchymal and proneural GBM, respectively. Similar to human GBM, CD44 expression was higher in NRAS tumors and, consistent with migration simulations of varying CD44 levels, ex vivo brain slice live imaging showed NRAS tumors cells migrate faster than PDGF tumors cells (random motility coefficient = 30µm2/hr vs. 2.5µm2/hr, p < 0.001). Consistent with CD44 function as an adhesion molecule, migration phenotype was independent of the tumor microenvironment. NRAS and human PDX/MES tumor cells were found to migrate faster and have larger cell spread area than PDGF and human PDX/PN tumors cells, respectively, in healthy mouse brain slices. Furthermore, traction force microscopy revealed NRAS tumor cells generate larger traction forces than PDGF tumors cells which further supports our theoretical mechanism driving glioma migration. Despite increased migration, NRAS cohort had better survival than PDGF which was attributed to enhanced antitumoral immune response in NRAS tumors, consistent with increased immune cell infiltration found in human mesenchymal GBM. Overall our work identified a potentially actionable difference in migration mechanics between GBM subtypes and establishes an integrated biophysical modeling and experimental approach to mechanically parameterize and simulate distinct molecular subtypes in preclinical models of cancer.Cite
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8574 Background: Mutations of NRAS and BRAF genes have been identified with high frequency in nevi, cutaneous melanomas, and melanoma metastases. Prevalence of such mutations during the disease progression phases and among the different types of metastasis still remains inconclusive. Methods: Paired samples of microdissected invasive primary melanomas (N=73) and synchronous or asynchronous metastases (N=164) from same patients underwent mutation analysis by automated DNA sequencing. Secondary lesions were from: regional (RN; N=49) or distant (DN; N=16) lymph nodes; regional (RS; N=16) or distant (DS; N=18) skin; visceral (VM; N=22) and brain (BM; N= 44) sites. Results: To date, mutations were identified in 44/73 (60%) primary melanomas [42% BRAF - 18% NRAS], 43/65 (66%) lymph nodes [49% BRAF - 17% NRAS], 21/34 (62%) subcutaneous metastases [38% BRAF - 24% NRAS], 13/22 (59%) visceral metastases [45% BRAF - 14% NRAS], and 31/44 (70%) brain metastases [48% BRAF - 23% NRAS]. Overall, a slight and not significant increase in mutation frequency after progression from primary melanoma was observed in our series: 108/164 (66%) mutated metastases [46% BRAF - 20% NRAS]. The only significant differences were found for subcutaneous metastases: a) DS presented a significantly higher mutation frequency than RS (78% vs. 44%); and b) a discontinuous pattern of BRAF/NRAS mutations was detected in primary melanomas vs. RS and DS lesions. These latter findings suggest that independent subclones may have been generated. Conclusions: Although collection and analysis of samples is still ongoing, our results may provide further clues about the impact of NRAS and BRAF mutations among the different stages of melanoma progression.
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Journal Article Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta‐analysis Get access J.‐H. Lee, J.‐H. Lee Department of Pathology, Korea University Ansan Hospital, 516, Gojan‐1 Dong, Danwon‐Gu, Ansan‐Si, Gyeonggi‐Do 425‐707, Korea Search for other works by this author on: Oxford Academic Google Scholar J.‐W. Choi, J.‐W. Choi Department of Pathology, Korea University Ansan Hospital, 516, Gojan‐1 Dong, Danwon‐Gu, Ansan‐Si, Gyeonggi‐Do 425‐707, Korea Search for other works by this author on: Oxford Academic Google Scholar Y.‐S. Kim Y.‐S. Kim Department of Pathology, Korea University Ansan Hospital, 516, Gojan‐1 Dong, Danwon‐Gu, Ansan‐Si, Gyeonggi‐Do 425‐707, Korea Young‐Sik Kim. E‐mail: apysk@korea.ac.kr Search for other works by this author on: Oxford Academic Google Scholar British Journal of Dermatology, Volume 164, Issue 4, 1 April 2011, Pages 776–784, https://doi.org/10.1111/j.1365-2133.2010.10185.x Published: 01 April 2011
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e14589 Background: To know the prevalence of mutations in the RAS family in our hospital, and to determine the effect of new mutations in KRAS and NRAS in overall survival and progression-free survival. Methods: This is a retrospective observational trial. We selected a group of 126 patients with metastatic colon cancer KRAS exon 2 wild type, who received first-line treatment in our center between 01/2007 and 12/2013. Determination was performed KRAS (exon 3 and 4) and NRAS mutation (exon 2, 3 and 4) by pyrosequencing. Results: We have made the determination of 90 patients (see table below). Of the patients studied, 79% of patients remained RAS wild type. 16% had new mutations: 54% NRAS codon 61, 38% NRAS codon 12 and 8% NRAS codon 13. Regarding the clinical impact, the new RAS wild type group (n = 73) has an overall survival of 30 months (95% CI 20 to 39.7) and a progression-free survival of 8 months (95% CI 7, 5 to 10.1). Mutated RAS group (n = 13) has an overall survival of 29 months (95% CI: 5-52) and a progression-free survival of 8 months (95% CI: 5.6 to 12.3). Conclusions: The frequency of mutations in KRAS/NRAS in our center was similar to clinical trials. In our institution, overall survival and progression-free survival in RAS wild type were similar to the previously reported. RAS mutated patients were not enough to extrapolate conclusions. Mutation Total patients (n = 90) Wild type samples 73 NRAS codon 12 mutant 5 NRAS codon 13 mutant 1 NRAS codon 61 mutant 7 Failed 4
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Abstract A majority of malignant melanomas harbor an oncogenic mutation in either BRAF or NRAS . If BRAF and NRAS transform melanoma cells by a similar mechanism, then additional genetic aberrations would be similar (or random). Alternatively, distinct mutation‐associated changes would suggest the existence of unique cooperating requirements for each mutation group. We first analyzed a panel of 52 melanoma cell lines ( n = 35, 11, 6 for BRAF*, NRAS* , and BRAF/NRAS wt/wt , respectively) by array‐based comparative genomic hybridization for unique alterations that associate with each mutation subgroup. Subsequently, those DNA copy number changes that correlated with a mutation subgroup were used to predict the mutation status of an independent panel of 43 tumors ( n = 17, 13, 13 for BRAF*, NRAS *, and BRAF/NRAS wt/wt , respectively). BRAF mutant tumors were classified with a high rate of success (74.4%, P = 0.002), whereas NRAS mutants were not significantly distinguished from wild types (26/43, P = 0.12). Copy number gains of 7q32.1‐36.3, 5p15.31, 8q21.11, and 8q24.11 were most strongly associated with BRAF* tumors and cell lines, as were losses of 11q24.2‐24.3. BRAF* melanomas appear to be associated with a specific profile of DNA copy number aberrations that is distinct from those found in NRAS* and BRAF/NRAS wt/wt tumors. These findings suggest that although both BRAF and NRAS appear to function along the same signal transduction pathway, each may have different requirements for cooperating oncogenic events. The genetic loci that make up this profile may harbor therapeutic targets specific for tumors with BRAF mutations. © 2009 Wiley‐Liss, Inc.
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Abstract In cutaneous melanoma, the most lethal form of skin cancer, BRAF is mutated in ∼45% of cases and NRAS in a further ∼20% of cases. The lack of known drivers in the remaining proportion of samples represents a challenge for personalized medicine. We hypothesize that the BRAF/NRAS double wild-type (WT) samples are a heterogeneous group driven by multiple events that arise independently. We performed whole exome sequencing (WES) and complimentary RNA-seq of 23 tumor tissue samples from 21 patients. Six of these tumors were WT for BRAF and NRAS, so we studied these cases further. The SNV load in the WT samples is more variable than the BRAF or NRAS driven melanomas. The median number of SNVs/Mb (± SD) for BRAF mutant (n = 9), NRAS mutant (n = 7) and BRAF WT/ NRAS WT (n = 6) samples were: 12.2 ± 8.0, 42.2 ± 82.7 and 12.6 ± 90.2 respectively. Next we analyzed the TCGA cutaneous melanoma cohort where there are 154 BRAF and 92 NRAS mutated and 76 BRAF WT/NRAS WT samples. The median numbers of SNVs (± SD) were 374 ± 638.3, 577 ± 518.3 and 146.5 ± 1221.6 respectively. The p-value for the comparisons BRAF vs. WT and NRAS vs. WT were 0.087 and 0.002 respectively (two-tailed Mann-Whitney test). We identified the somatic mutations that are specifically enriched for double wild-type samples and observed that the top two hits, NF1 (30.2% in double WT vs. 5.7% otherwise) and KIT (15.8% in double WT vs. 0.8% otherwise) are known driver gene candidates for wild-type melanomas, but we also find other novel candidate driver genes. Thus, we present a framework for identification of driver mutations and therapeutic targets in double wild-type melanomas and integration of these types of data with other large datasets such as those derived from RNA-seq and RPPA will assist in the development of approaches to stratify double wild-type patients for targeted or immune-therapies. Citation Format: Amit Mandal, Maria Romina Girotti, Amaya Viros, Gabriela Gremel, Elena Galvani, Rebecca Lee, Kok Haw Jonathan Lim, Simon J. Furney, Paul Lorigan, Richard Marais. Deciphering driver mechanisms for tumorigenesis in BRAF/NRAS double wild-type melanoma through integration of heterogeneous genome-wide datasets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1094. doi:10.1158/1538-7445.AM2015-1094
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Abstract Background: Mutations of NRAS and BRAF genes have been identified with high frequency in nevi, cutaneous melanomas, and melanoma metastases. Prevalence of such mutations during the disease progression phases and among the different types of metastasis still remains inconclusive. Methods: A total of 275 tumour tissues from 116 melanoma patients were screened for mutations; among them, paired samples of microdissected primary melanomas (N=92) and synchronous or asynchronous metastases (N=156) from same patients were included. Tissue samples underwent mutation analysis by automated DNA sequencing. Secondary lesions were from: lymph nodes (LM; N=77), skin (SM; N=36), visceral (VM; N=23) and brain (BM; N= 44) sites. The full coding sequences and splice junctions of p16CDKN2A (exons 1, 2, and 3) and NRAS (exons 2 and 3) genes as well as the entire sequence at the exon 15 of the BRAF gene were screened for mutations by direct sequencing on automated fluorescence-cycle sequencer (ABIPRISM 3130, Applied Biosystems, Foster City, CA). Results: Overall, mutations were identified in 56/95 (59%) primary melanomas [43% BRAF - 16% NRAS], 49/77 (64%) lymph nodes [48% BRAF - 16% NRAS], 22/36 (61%) subcutaneous metastases [53% BRAF - 8% NRAS], 13/23 (57%) visceral metastases [43% BRAF - 13% NRAS], and 31/44 (70%) brain metastases [48% BRAF - 23% NRAS]. Overall, a slight and not significant increase in mutation frequency after progression from primary melanoma was observed in our series: 115/180 (64%) mutated metastases [48% BRAF - 16% NRAS]. Considering the paired samples from the same patients, LM (92% consistency) and VM (96% consistency) presented a highly similar prevalence and distribution of BRAF/NRAS mutations in comparison with primary melanomas, whereas a discontinuous pattern of mutations was detected in BM (80% consistency) or, mostly, SM (75% consistency). Occurrence of distinct mutation distribution between primary melanomas and correspondent metastases suggests that independent subclones have been generated in a limited fraction of patients. The rate of mutations in p16CDKN2A gene was found to steadily increase moving from primary melanomas (5/69; 7%) to melanoma metastases (21/144; 15%), with the highest prevalence of p16CDKN2A alterations (18/29; 62%) in melanoma cell lines Conclusions: Our results provide further clues about the impact of NRAS and BRAF mutations among the different stages of melanoma progression. Moreover, we confirmed that p16CDKN2A mutation rates are increasing during disease progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3677. doi:1538-7445.AM2012-3677
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e20034 Background: Previous studies have investigated whether BRAF and NRAS mutation status in melanoma correlate with histologic parameters and overall survival (OS), but evaluation of mutation groups irrespective of specific mutation among histologic types of melanoma has led to variability in the reproducibility of results. We tested the hypothesis that different histologic types of melanoma (nodular [NM] and superficial spreading [SSM]) have distinct clinical associations with specific BRAF and NRAS mutations. Methods: Primary tumor histology, BRAF/NRAS mutation status, and clinical outcomes were collected for 195 patients (pts) with stage III or IV disease. Clinical associations with specific mutations were determined separately for patients with NM (n=105) and SSM (n=90) histologic types of primary cutaneous melanomas. Results: Mutational status in NM: 69 BRAF (66%), 19 NRAS (18%), & 17 wild-type (WT;16%). Specific BRAF mutations in NM: V600E 50 (75%), V600K 13 (19%), V600R 4 (6%). Specific NRAS mutations in NM: Q61K 6 (32%), Q61L 2 (11%), Q61R 8 (42%); other 3 (16%). Mutation status in SSM: 45 BRAF (50%), 24 NRAS (27%), 21 WT (23%). Specific BRAF mutations in SSM: V600E 32 (71%), V600K 12 (24%), V600R 0. Specific NRAS mutations in SSM: Q61K 2 (8%), Q61L 5 (21%), Q61R 12 (50%). The distribution of specific BRAF (p=0.21) and NRAS (p=0.29) mutations between NM and SSM was not significantly different. Among NM pts, pts with activating NRAS mutations had shorter OS from the diagnosis of Stage IV melanoma than WT (HR 3.42, p=0.02) and BRAF (HR 2.40, p=0.009). There was no significant difference for BRAF pts vs WT (HR 1.43, p=0.47). Among SSM patients, neither NRAS (HR 1.3, p=0.53) nor BRAF(HR 0.54, p=0.16) were significantly associated with OS compared to WT. Comparison of patients with BRAF V600E vs V600K showed significant association for OS from stage 4 in SSM pts (HR 0.24, p=0.01), but not in NM pts (HR 0.64, p=0.36). Conclusions: The prognostic significance of BRAF and NRAS mutations on OS from stage IV differed for pts with NM and SSM primaries. Further investigation of the histologic types of melanoma with specific BRAF and NRAS mutations in a larger series is necessary to validate these apparent impacts on patient outcomes.
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