Loss Of Chromosome 3p Research Articles

Tumour suppressor gene (CDKNA2) status on chromosome 9p in resected renal tissue improves prognosis of localised kidney cancer.

BackgroundGenetic alterations on chromosome 9p, including inactivation of the tumour suppressor gene, CDKN2A, result in cellular proliferation and growth of tumours. Our aim was to use microsatellite analysis and fluorescence in situ hybridization (FISH) to characterise the architecture of this region.ResultsSeventy-five out of 77 clear cell renal cell cancers (tumour/normal pairs) were interpretable for LOH analysis on chromosome 9p (two tumours were excluded, as all five primers were uninformative). Twenty out of 75 (26.6%) tumours showed LOH in at least one of the five primers employed. Most allelic deletions were detected, telomeric to the CDKN2A region at D9S916, with 11 out of 52 informative tumours (21%) displaying LOH. The LOH in the coding region of CDKN2A, at D9S974 and D9S942, was associated with a higher pT-stage (p = 0.004) and metastasis (p = 0.006, both markers). The rate of chromosome 9p deletion in ccRCC was 44% (35/80 cases) according to FISH. Somatic copy number loss of chromosome 9p was associated with a larger tumour size (p = 0.002), higher pathological tumour stage (p = 0.021), presence of tumour necrosis (p = 0.019) and microvascular invasion (p = 0.032). The cases with copy number loss, loss of heterozygosity and copy number neutral (n = 42) were at a higher risk of cancer-specific death when compared to tumours in category D (n = 32) (Log-rank: p = 0.001). Seventeen patients with localised ccRCC developed recurrence, and fourteen of those showed either LOH or somatic copy number loss at CDKN2A (Log-rank: p = 0.005). Multivariate analysis showed that LOH or copy number loss at CDKN2A retained its independent prognostic effect, improving the predictive accuracy of stage and SSIGN score by concordance Index C from 0.823 to 0.878 (p = 0.001).Materials and MethodsCytogenetics data, microsatellite analysis and FISH were acquired for a cohort of patients undergoing resection for clinically localised renal cancer between January 2001 and December 2005. Five microsatellite markers (D9S916, D9S1814, D9S974, D9S942 and D9S171) assessed loss of heterogeneity (LOH) using DNA samples and in the same cohort FISH analysis was accomplished on tissue microarray slides. The FISH data were scored by two observers blinded to the histological data of the patients. Cytogenetic aberrations were correlated with histological and clinical outcomes by univariate and multivariate analyses using different prognostic models. Disease specific and recurrence free survival based on cytogenetic changes were assessed by Kaplan Meier methods.ConclusionsA comprehensive cytogenetic analysis using microsatellite analysis and FISH of the CDKN2A region on chromosome 9p improves the predictive accuracy of known prognostic factors in clinically localised renal cell carcinoma undergoing surgical resection.

Targeted next-generation sequencing of CIC-DUX4 soft tissue sarcomas demonstrates low mutational burden and recurrent chromosome 1p loss

Gene fusions between CIC and DUX4 define a rare class of soft tissue sarcomas poorly understood at the molecular level. Previous karyotyping and fluorescence in situ hybridization studies support chromosome 8 trisomy as a recurrent alteration; however, other driving alterations are largely unknown. Thus, we analyzed 11 formalin-fixed, paraffin-embedded CIC-DUX4 sarcoma tissue samples (including 3 sample pairs) using targeted Ion Torrent-based multiplexed polymerase chain reaction next-generation sequencing to characterize potential somatic driver alterations in 409 genes. Although we did not identify recurrent somatic mutations (point mutations or insertions/deletions), copy number analysis showed recurrent, broad copy number alterations, including gain of chromosome 8 and loss of 1p. In one sample pair (untreated primary and local recurrence resections), we identified similar copy number profiles and a somatic ARID1A R963X nonsense mutation exclusively in the local recurrence sample. In another sample pair (pre- and post-radiation treatment specimens), we observed single-copy loss of chromosome 7q exclusively in the posttreatment recurrence sample, supporting it as an acquired event after radiation treatment. In the last sample pair (near-concurrent, postchemotherapy primary and distant metastasis), molecular profiles were highly concordant, consistent with limited intertumoral heterogeneity. In summary, next-generation sequencing identified limited somatic driver mutations in CIC-DUX4 sarcomas. However, we identified novel, recurrent copy number alterations, including chromosome 1p, which is also the locus of ARID1A. Additional functional work and assessment of larger cohorts are needed to determine the biological and clinical significance of the alterations identified herein.

TERT promoter mutations and chromosome 8p loss are characteristic of nonalcoholic fatty liver disease-related hepatocellular carcinoma.

The number of patients with nonalcoholic fatty liver disease (NAFLD)-related hepatocellular carcinoma (HCC) is increasing. To understand the molecular features of the tumor phenotype, we aimed to clarify the overall landscape of genetic aberrations accumulated in NAFLD-related HCC. Of 247 HCC patients who underwent hepatectomy during 2010 to 2014 at a single center in Japan, 10 were diagnosed with NAFLD-HCC based on strict clinical and pathologic criteria. We analyzed the genetic aberrations of 11 NAFLD-HCC tumor samples from these 10 patients by whole-exome sequencing, targeted sequencing of the selected genes, and copy number variation studies. Whole-exome sequencing revealed a mean somatic mutation rate of 1.86 per megabase, and 12 genes were recurrently mutated in NAFLD-HCCs. Targeted sequencing of the 26 selected genes (12 recurrently mutated genes in whole-exome sequencing and 14 representative HCC-associated genes) revealed that TERT promoter mutations occurred in 9 of 11 HCCs (82%), followed by CTNNB1 (45%) and TP53 (36%) mutations. Array-based copy number variation studies identified recurrent gains at 1q and 8q, and recurrent losses at 1p, 4q, 6q, 8p, 13q, 16p, 17p, and 18q. Notably, chromosome 8p loss occurred in all of the NAFLD-HCC samples. The current study provided the characteristics of genetic aberrations in NAFLD-HCC and suggested that TERT promoter mutations and chromosome 8p loss mainly contribute to NAFLD-related liver carcinogenesis.

Abstract 4707: Investigating the role of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC)

Abstract Enhancer of zeste homolog 2 (EZH2) is a key component of the polycomb repressive complex 2 (PRC2). EZH2 is frequently overexpressed in a wide variety of human malignancies including non-Hodgkin lymphoma, gastric cancer, pancreatic cancer, and lung cancer. Thus it has potential to become a therapeutic target. Characterization of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC) has not been fully explored. ccRCC have been defined by mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in combination with chromosome 3p loss. Recent sequencing efforts have revealed that several chromatin remodeling genes encoded on chromosome 3p are often mutated, of which PBRM1 is the most frequent (41%). The PBRM1 gene codes for the BAF180 protein, a SWI/SNF chromatin remodeling complex subunit. Loss of BAF180 in ccRCC may disrupt the PBAF variant of the SWI/SNF complex. The SWI/SNF complex remodels the chromatin landscape by either sliding or evicting the nucleosomes from the chromatin. This chromatin remodeling modulates the accessibility to promoter regions by transcriptional machinery. It is through this mechanism that the SWI/SNF complex can regulate a range of cellular processes. It has been demonstrated that the SWI/SNF complex can act antagonistically to the PRC2 complex by evicting PRC2 complex from the promoters of tumor suppressors such as CDKN2A/p16. Disruption of the SWI/SNF complex would impede the eviction of the PRC2 complex, similarly observed in SNF5-deficient malignant rhabdoid tumors. Therefore, we hypothesize that PBRM1 inactivation disrupts specific SWI/SNF complexes allowing EZH2 to bind and repress target tumor suppressor genes. Thus inhibition of EZH2 in ccRCC may present as a targeted therapeutic option in tumors with PBRM1 mutations. We have investigated EZH2 in ccRCC cell lines with PBRM1 mutations and observed that these cells lines have overexpression of EZH2 in comparison to RPTEC (renal cortex proximal tubule epithelium cell line). We examined the effects on two EZH2 inhibitors (GSK126 and EPZ6438) on ccRCC cell lines both in vitro and in vivo. Our preliminary data suggests EZH2 inhibition results in reduced growth of ccRCC cell lines with PBRM1 mutations. Citation Format: Darmood Wei, Christopher J. Ricketts, Laura S. Schmidt, Youfeng Yang, Cathy D. Vocke, William M. Linehan. Investigating the role of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4707.

Chromosome 8p tumor suppressor genes SH2D4A and SORBS3 cooperate to inhibit interleukin-6 signaling in hepatocellular carcinoma.

Several chronic inflammatory liver diseases, e.g., chronic hepatitis B or C viral infection and steatohepatitis, have been shown to predispose to the development of hepatocellular carcinoma (HCC). In patients with chronic liver disease, interleukin-6 (IL-6) serum levels are elevated and increase even more when HCC develops. However, the impact and regulatory mechanisms of IL-6 signaling during hepatocarcinogenesis are still poorly defined. Here, we show that gene expression profiles of patients with chromosome 8p loss correlate with increased IL-6 signaling. In addition, the chromosome 8p tumor suppressor genes Src homology 2 domain containing 4A (SH2D4A) and Sorbin and Src homology 3 domain containing 3 (SORBS3) together exerted greater inhibition of cell growth and clonogenicity compared to a single gene. Overexpression of SH2D4A and SORBS3 in HCC cells led to decreased IL-6 target gene expression and reduced signal transducer and activator of transcription 3 (STAT3) signaling. In situ and in vitro coimmunoprecipitation assays revealed that SH2D4A directly interacts with STAT3, thereby retaining STAT3 in the cytoplasm and inhibiting STAT3 transcriptional activity. On the other hand, SORBS3 coactivated estrogen receptor α signaling, leading indirectly to repression of STAT3 signaling. In human HCC tissues, SH2D4A was positively associated with infiltrating regulatory and cytotoxic T-cell populations, suggesting distinct immunophenotypes in HCC subgroups with chromosome 8p loss. Thus, the genetically linked tumor suppressors SH2D4A and SORBS3 functionally cooperate to inhibit STAT3 signaling in HCC. The chromosome 8p tumor suppressor genes SORBS3 and SH2D4A are physically and functionally linked and provide a molecular mechanism of inhibiting STAT3-mediated IL-6 signaling in HCC cells. (Hepatology 2016;64:828-842).

Abstract LB-165: Multiple mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma

Abstract The let-7 microRNA family are known tumor suppressors often deregulated in cancer, yet the underlying mechanisms of let-7 disruption remain poorly understood. Neuroblastoma, a neural crest derived tumor, is defined in part by poor prognosis associated with genetic amplification of MYCN, itself a let-7 target. The let-7 biogenesis inhibitor LIN28B has recently been implicated as a critical regulator of MYCN, but through CRISPR-mediated gene disruption we show that LIN28B is dispensable for both MYCN protein expression and growth of MYCN-amplified neuroblastoma cell lines despite robust de-repression of let-7, prompting us to explore additional mechanisms for let-7 disruption. Consequently, we have found a novel non-coding role for amplified MYCN mRNA as a potent let-7 sponge that through exceptionally high expression defines a sub-class of self-sponging amplified-competing-endogenous-RNA (aceRNA) and reconciling the dispensability of LIN28B in neuroblastoma cell lines. Furthermore, by analyzing a large cohort of tumor samples from patients, we observe frequent genomic loss of let-7 that inversely associates with MYCN-amplification, providing a functional explanation for the known MYCN-amplification-independent pattern of chromosome 3p and 11q loss, which harbor let-7g and let-7a2, respectively. We thus propose a model whereby let-7 disruption by genetic loss, LIN28B expression, or aceRNA sponging is a unifying mechanism of neuroblastoma pathogenesis. Indeed, our data show that the majority of neuroblastomas have at least one let-7 disruption event and that genetic loss in non-MYCN-amplified tumors marks decreased survival, further underscoring its importance. The inverse selective relationship between allelic loss and sponging of let-7 from highly expressed or amplified oncogenes may have broad implications for oncogenesis. Citation Format: John T. Powers, Kaloyan Tsanov, Frederik Roels, Catherine Spina, Richard Ebright, Marc Seligson, Yvanka de Soysa, Patrick Cahan, Daniel Pearson, Jessica Theißen, Ho-Chou Tu, Grace LaPier, Jihan Osborne, Samantha Ross, James Collins, Frank Berthold, George Daley. Multiple mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-165.

The non-coding landscape of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease marked by frequent recurrence and metastasis and stagnant survival rates. To enhance molecular knowledge of HNSCC and define a non-coding RNA (ncRNA) landscape of the disease, we profiled the transcriptome-wide dysregulation of long non-coding RNA (lncRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) using RNA-sequencing data from 422 HNSCC patients in The Cancer Genome Atlas (TCGA). 307 non-coding transcripts differentially expressed in HNSCC were significantly correlated with patient survival, and associated with mutations in TP53, CDKN2A, CASP8, PRDM9, and FBXW7 and copy number variations in chromosomes 3, 5, 7, and 18. We also observed widespread ncRNA correlation to concurrent TP53 and chromosome 3p loss, a compelling predictor of poor prognosis in HNSCCs. Three selected ncRNAs were additionally associated with tumor stage, HPV status, and other clinical characteristics, and modulation of their expression in vitro reveals differential regulation of genes involved in epithelial-mesenchymal transition and apoptotic response. This comprehensive characterization of the HNSCC non-coding transcriptome introduces new layers of understanding for the disease, and nominates a novel panel of transcripts with potential utility as prognostic markers or therapeutic targets.

MB-14MOLECULAR AND PROGNOSTIC HETEROGENEITY WITHINMYCANDMYCNAMPLIFIED MEDULLOBLASTOMAS

MYC and MYCN are the most commonly amplified oncogenes in medulloblastoma; their association with poor prognosis in disease-wide studies has supported their adoption as high-risk disease biomarkers in current clinical trials. However, recent observations suggest some patients with MYC/MYCN amplified tumours achieve long term survival and may suffer unnecessary side effects associated with intensified therapies. To further understand this heterogeneity, we characterised the molecular, clinical and pathological features of focussed cohorts of MYC (n = 37) and MYCN (n = 57) amplified tumours (identified by FISH and/or copy number profiling), and assessed their associations with disease outcome. Within the MYCN cohort (24 MYCNSHH; 24 MYCNGroup4; 3 MYCNGroup3; 6 NA), patient survival was subgroup-dependent; patients with MYCNGroup4 and no other clinico-pathological risk factors (subtotal resection, metastatic disease or LCA pathology) had a favourable event free survival (EFS). In contrast, MYCNSHH was associated with LCA (MYCNSHH vs MYCNGroup4, p< 0.0001) and a dismal EFS regardless of additional risk factors. TP53 mutation was a frequent feature of MYCNSHH (12/23), usually in conjunction with Chromosome 17p loss and GLI2 amplification, and conferred a quicker time to progression within MYCNSHH (p = 0.05). LCA pathology was the poorest prognostic factor in MYC-amplified tumours. The majority (20/30) of sub-grouped MYC-amplified tumours were MYCGroup3. Rare MYCGroup4 tumours (5/30), had fewer (<50%) amplified tumour cells and a better EFS than MYCGroup3 (p = 0.04). These data highlight the importance of subgroup identification as a basis for refined stratification of medulloblastoma risk using MYC/MYCN amplification, and suggest that MYC/MYCNGroup4 as isolated risk-factors do not confer high-risk disease.

Integrated Genomics for Pinpointing Survival Loci within Arm-Level Somatic Copy Number Alterations

The identification of driver loci underlying arm-level somatic copy number alterations (SCNAs) in cancer has remained challenging and incomplete. Here, we assess the relative impact and present a detailed landscape of arm-level SCNAs in 10,985 patient samples across 33 cancer types from The Cancer Genome Atlas (TCGA). Furthermore, using chromosome 9p loss in lower grade glioma (LGG) as a model, we employ a unique multi-tiered genomic dissection strategy using 540 patients from three independent LGG datasets to identify genetic loci that govern tumor aggressiveness and poor survival. This comprehensive approach uncovered several 9p loss-specific prognostic markers, validated existing ones, and redefined the impact of CDKN2A loss in LGG.

Loss of Chromosome 8p Governs Tumor Progression and Drug Response by Altering Lipid Metabolism

Large-scale heterozygous deletions are a hallmark of cancer genomes. The concomitant loss of multiple genes creates vulnerabilities that are impossible to reveal through the study of individual genes. To delineate the functional outcome of chromosome 8p loss of heterozygosity (LOH), a common aberration in breast cancer, we modeled 8p LOH using TALEN-based genomic engineering. 8p LOH alters fatty acid and ceramide metabolism. The shift in lipid metabolism triggers invasiveness and confers tumor growth under stress conditions due to increased autophagy. The resistance of 8p-deleted cells to chemotherapeutic drugs concurs with poorer survival rates of breast cancer patients harboring an 8p LOH. The autophagy dependency of 8p-deleted cells provides the rational basis for treatment of 8p LOH tumors with autophagy inhibitors.

Reprofiling Metastatic Samples for Chromosome 9p and 14q Aberrations as a Strategy to Overcome Tumor Heterogeneity in Clear-cell Renal Cell Carcinoma.

Losses of chromosomes 9p and 14q are associated with worse outcomes in patients affected by clear-cell renal cell carcinoma (RCC) and are helpful for prognostic risk stratification. Both chromosomal loci harbor several hot-spot molecular pathways suitable for targeted therapeutic interventions. Intratumor heterogeneity may foster tumor adaptation and therapeutic failure. We sought to investigate the presence of losses of the hot spots of chromosomal loci 9p and 14q in primary clear-cell RCC and matched metastatic tissues. CD10 and CD13 were performed on 7 cases of clear-cell RCC with hematogenous tissue metastases. Cytogenetic fluorescence in situ hybridization analysis was performed on primary and matched metastatic tissues using specific probes mapping the 9p and the 14q loci. The loss of chromosome 9p was observed in 85% of both primary clear-cell RCCs and in matched metastases; 14% showed discordance between primary and matched metastases showing gains. The loss of chromosome 14q was observed in 58% of both primary and matched metastases. Only 3/7 (42%) did show an equal status of loss of chromosome 14q. Heterogeneity of the cytogenetic status between metastatic and primary clear-cell RCCs is observed for the loss of chromosome 14q rather than chromosome 9p. The impact of chromosome 14q cytogenetic status, harboring the HIF1 gene, a major driver for the angiogenenic switch, may drive the efficacy of targeted inhibitors, whereas the loss of chromosome 9p, harboring other hot-spot genes, seems to be related to the metastatic behavior per se, without cytogenetic modulation. Reprofiling the metastatic tissue, as compared with the primary tumor, in patients affected by metastatic RCC could be a novel approach to overcome resistance to VEGF(Rs)-targeting agents.

Management of patients with recurrence of diffuse low grade glioma: A systematic review and evidence-based clinical practice guideline.

These recommendations apply to adult patients with recurrent low-grade glioma (LGG) with initial pathologic diagnosis of a WHO grade II infiltrative glioma (oligodendroglioma, astrocytoma, or oligo-astrocytoma). Do pathologic and molecular characteristics predict outcome/malignant transformation at recurrence? IDH STATUS AND RECURRENCE: (Level III) IDH mutation status should be determined as LGGs with IDH mutations have a shortened time to recurrence. It is unclear whether knowledge of IDH mutation status provides benefit in predicting time to progression or overall survival. TP53 STATUS AND RECURRENCE: (Level III) TP53 mutations occur early in LGG pathogenesis, remain stable, and are not recommended as a marker of predisposition to malignant transformation at recurrence or other measures of prognosis. MGMT STATUS AND RECURRENCE: (Level III) Assessment of MGMT status is recommended as an adjunct to assessing prognosis as LGGs with MGMT promoter methylation are associated with shorter PFS (in the absence of TMZ) and longer post-recurrence survival (in the presence of TMZ), ultimately producing similar overall survival to LGGs without MGMT methylation. The available retrospective reports are conflicting and comparisons between reports are limited CDK2NA STATUS AND RECURRENCE: (Level III) Assessment of CDK2NA status is recommended when possible as the loss of expression of the CDK2NA via either methylation or loss of chromosome 9p is associated with malignant progression of LGGs. PROLIFERATIVE INDEX AND RECURRENCE: (Level III) It is recommended that proliferative indices (MIB-1 or BUdR) be measured in LGGs as higher proliferation indices are associated with increased likelihood of recurrence and shorter progression free and overall survival. 1P/19Q STATUS AND RECURRENCE: There is insufficient evidence to make any recommendations. What role does chemotherapy have in LGG recurrence? TEMOZOLOMIDE AND RECURRENCE: (Level III) Temozolomide is recommended in the therapy of recurrent LGG as it may improve clinical symptoms. Oligodendrogliomas and tumors with 1p/19q co-deletion may derive the most benefit. PCV AND RECURRENCE: (Level III) PCV is recommended in the therapy of LGG at recurrence as it may improve clinical symptoms with the strongest evidence being for oligodendrogliomas. CARBOPLATIN AND RECURRENCE : (Level III) Carboplatin is not recommended as there is no significant benefit from carboplatin as single agent therapy for recurrent LGGs. OTHER TREATMENTS (NITROSUREAS, HYDROXYUREA/IMANITIB, IRINOTECAN, PACLITAXEL) AND RECURRENCE: There is insufficient evidence to make any recommendations. It is recommended that individuals with recurrent LGGs be enrolled in a properly designed clinical trial to assess these chemotherapeutic agents. What role does radiation have in LGG recurrence? RADIATION AT RECURRENCE WITH NO PREVIOUS IRRADIATION: (Level III) Radiation is recommended at recurrence if there was no previous radiation treatment. RE-IRRADIATION AT RECURRENCE: (Level III) It is recommended that re-irradiation be considered in the setting of LGG recurrence as it may provide benefit in disease control. There is insufficient evidence to make any specific recommendations. It is recommended that individuals with recurrent LGGs be enrolled in a properly designed clinical trial to assess the role of surgery at recurrence.

Clear Cell Renal Cell Carcinoma With Borderline Features of Clear Cell Papillary Renal Cell Carcinoma: Combined Morphologic, Immunohistochemical, and Cytogenetic Analysis.

Clear cell papillary renal cell carcinoma is increasingly recognized as a distinct tumor with unique morphology, immunohistochemistry, and cytogenetics. Histopathology often mimics clear cell renal cell carcinoma; however, metastasis has not been reported, emphasizing the clinical value of recognizing these likely nonaggressive tumors. We studied tumors with borderline morphology of clear cell papillary renal cell carcinoma, utilizing immunohistochemistry and fluorescence in situ hybridization or karyotyping. Tumors from 22 patients (ages 33 to 82 y) were analyzed. Clear cell papillary renal cell carcinoma-like morphology varied from 10% to 90% of the tumor (median 25%). Sources of resemblance included: branched glands (95%), nuclear alignment (68%), small papillary tufts (32%), focal branching papillae (27%), and prominent papillary structures (9%). Carbonic anhydrase IX uniformly revealed diffuse positivity. Staining for cytokeratin 7 (CK7) was focal (64%) or negative (18%) in most tumors (82%); however, >50% labeling was present in 4 (18%). Reactivity for both CD10 and α-methyl-acyl-CoA-racemase (AMACR) was usually present (median 80% and 60% of cells). Seven tumors showed reactivity for high-molecular weight keratin (32%). Chromosome 3p loss was confirmed in 15 tumors (68%), including 4/7 with labeling for high-molecular weight keratin or >50% reactivity for CK7. A discordant immunohistochemical pattern typically correlates with loss of material from chromosome 3p in tumors with incomplete morphology of clear cell papillary renal cell carcinoma, supporting classification as clear cell renal cell carcinoma. Diffuse labeling for CK7 can uncommonly be observed in clear cell renal cell carcinomas confirmed to have chromosome 3p loss, although these do not exhibit the expected staining pattern of clear cell papillary renal cell carcinoma, including positivity for CD10 and AMACR.

How should clinicians address intratumour heterogeneity in clear cell renal cell carcinoma?

Despite the availability of multiple targeted therapies, the 5-year survival rate of patients with metastatic clear cell renal cell carcinoma (ccRCC) rarely exceeds 10%. Recent insights into the mutational landscape and evolutionary dynamics of ccRCC have offered up a plausible explanation for these outcomes. The purpose of this review is to link the research findings to potential changes in clinical practice. Intratumour heterogeneity (ITH) dominates the evolutionary landscape in ccRCC at the genetic, transcriptomic and proteomic level. Spatial and temporal separation of tumour subclones within the primary tumour as well as between primary and metastatic sites has been demonstrated at single nucleotide resolution. In the cases analysed to date, approximately two-thirds of somatic mutations are not shared between multiple biopsies from the same primary tumour. Very few of the key disease-driving events are shared across all primary tumour regions (with the exception of VHL and loss of chromosome 3p), whereas the majority are restricted to one or more tumour regions (TP53, SETD2, BAP1, PTEN, mTOR, PIK3CA and KDM5C). ITH must be considered in the management of ccRCC with respect to diagnostic procedures, prognostic and predictive biomarkers and drug development.

Abstract 2978: An integrated comparative analysis of TCGA lung adenocarcioma and lung squamous cell carcinoma copy number and RNA-Seq expression data

Abstract The Cancer Genome Atlas (TCGA) contains various types of genomic data from a wide variety of cancers, several of which affect the same tissue site. Here we analyze copy number and RNA-Seq data from TCGA for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Level 1 (raw) data, rather than Level 3 (segmented) data, from TCGA was used for an integrated analysis of copy number and gene expression profiles of the two cancers. Using the copy number and SNP- probes, re-processed tumor profiles were more consistent with a control set in terms of median number of copy number events, sample ploidy, and breakpoint genes than with the published “level 3” TCGA data. Probe-level data were analyzed using Nexus Copy Number SNP-FASST2 (a multi-state HMM algorithm that uses both SNP and copy number probes in making state assignments), with systematic correction applied to correct for GC biases. Additionally we performed manual baseline adjustment to correct for sample ploidy based on whole-genome B-alelle frequency data for each sample. Overall, the median number of copy number events in the LUAD TCGA data set was reduced from 371 (in the level 3 set) to 299, and from 681 (in the level 3 set) to 177 in the LUSC TCGA data set. After manual inspection, more than 38% of the TCGA LUAD samples and 50% of the TCGA LUSC samples available at level 3 were found to have incorrect baseline ploidy assignments. The resultant re-analyzed copy number data sets were used for an integrated analysis between the two tumor types. Comprehensive comparative analysis using Fisher's Exact Test revealed statistically significant differences (percent differential = 25%, p&amp;lt;0.001) in copy number profiles between the two lung tumor types; copy number changes include differential loss of chromosome 1p, loss of chromosome 3p, gain of chromosome 3q, loss of chromosome 4 and loss of chromosome 5. These correlated to changes in overall survival: individuals with loss of chromosome 1p and/or loss of 5q resulted in a significantly poorer prognosis (p&amp;lt;0.05). While more frequent in the LUSC sample population, this change in overall survival outcome was extended to samples with chromosome 1p loss in LUAD samples as well. Integration with RNA-Seq expression data from each tumor type revealed statistically significant correlations (p&amp;lt;0.05) with these copy number alterations, identifying potential driver genes of interest among each subtype and lung tumors in general. Citation Format: Andrea J. OHara, Raja Keshavan, Zhiwei Che, Soheil Shams. An integrated comparative analysis of TCGA lung adenocarcioma and lung squamous cell carcinoma copy number and RNA-Seq expression data. [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 2978. doi:10.1158/1538-7445.AM2015-2978

Abstract LB-169: Multidimensional genomic dissection of chromosome 9p in glioma

Abstract Background: Characterization of focal somatic copy number alterations (SCNAs) has led to the identification of many cancer genes, yet similar investigations of arm-level SCNAs remain challenging. The identity of driver genes within these broad SCNAs remains a critical unanswered question in cancer genetics. One of the most frequent arm-level SCNAs is 9p loss, which contains the tumor suppressor gene (TSG) CDKN2A. It is also believed that other TSGs exist on 9p, though their identity has yet to be revealed. Methods: We analyzed every arm-level SCNA in 28 cancer types from The Cancer Genome Atlas (TCGA) to clarify the relative impact of 9p loss across cancer. We also performed a multi-tiered genomic dissection of chromosome 9p in 540 patients from 3 independent lower grade glioma (LGG) datasets (TCGA, REMBRANDT, MSKCC) to pinpoint genetic loci that are tied to tumor aggressiveness and poor survival. Focal and arm-level SCNAs were determined by GISTIC2.0. As per recently proposed criteria, 3 LGG subtypes were clustered by IDH mutation and 1p/19q deletion status. Survival analyses were performed using log-rank tests or Cox regression. Results: We found that chromosome 9p loss is one of the most frequent and prognostic arm-level events across 28 TCGA cancer types. Of these, the strongest 9p survival association was found in LGG. We performed a large-scale associations test of 376 important clinical and molecular variables and revealed that 9p loss was only associated with 6q or 9q loss, and 9p loss alone was sufficient to predict poor prognosis. On subtype-specific analysis, 9p loss predicted worse overall survival (OS) in both IDH mutant LGG subtypes but not IDH wild-type (IDHwt). To identify underlying drivers on 9p, we identified 87 genes most frequently targeted by 9p loss. Additional genetic events were rare except for homozygous deletion (HD) at 9p21.3, which contains CDKN2A. In contrast, mRNA/miRNA expression at all gene loci was much more variable and poorly correlated to copy number status. Therefore, pan-LGG and subtype-specific gene expression analyses were used to identify several drivers of tumor aggressiveness, notably KLHL9, MTAP, PLAA, and PTPRD. Although CDKN2A HD was linked to worse OS in a pan-LGG analysis, this event significantly co-occurred with the LGG IDHwt subtype, a group with GBM-like survival outcomes. Further, CDKN2A copy number status and expression did not predict worse OS in any subtype. Discussion: We characterize the nature of 9p loss in LGG, pinpoint genes involved in worse survival, and redefine the role of the tumor suppressor CDKN2A. These results provide new critical insight into long-standing questions about the nature of chromosome 9p loss and establish a framework for examining other arm-level SCNAs in cancer. Citation Format: David M. Roy, Logan A. Walsh, Alexis Desrichard, Jianjiong Gao, Promita Bose, Jason T. Huse, William Lee, Timothy A. Chan. Multidimensional genomic dissection of chromosome 9p in glioma. [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 LB-169. doi:10.1158/1538-7445.AM2015-LB-169

Abstract LB-290: Multiple distinct mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma

Abstract The let-7 microRNA family are known tumor suppressors often deregulated in cancer, yet the underlying mechanisms of let-7 disruption remain poorly understood. Neuroblastoma, a neural crest derived tumor, is defined in part by poor prognosis associated with genetic amplification of MYCN, itself a let-7 target. The let-7 biogenesis inhibitor LIN28B has recently been implicated as a critical regulator of MYCN, but we have employed siRNA and CRISPR-mediated gene disruption to show that LIN28B is dispensable for both MYCN protein expression and growth of MYCN-amplified neuroblastoma cell lines despite robust de-repression of let-7, which prompted us to explore additional mechanisms for let-7 disruption. Consequently, we have found that amplified MYCN mRNA is a potent let-7 sponge that through exceptionally high expression defines a sub-class of self-sponging amplified-competing-endogenous-RNA (aceRNA), which reconciles the dispensability of LIN28B in NB cell lines. In addition, by analyzing a large cohort of tumor samples from patients, we observe frequent genomic loss of let-7 that inversely associates with MYCN-amplification, providing a functional explanation for the known MYCN-amplification-independent pattern of chromosome 3p and 11q loss, which harbor let-7g and let-7a2, respectively. We thus propose a model whereby let-7 disruption by genetic loss, LIN28B expression, or aceRNA sponging is a unifying mechanism of neuroblastoma pathogenesis. Indeed, our data show that the majority of neuroblastomas have at least one let-7 disruption event and that genetic loss in non-MYCN-amplified tumors marks decreased survival, further underscoring its importance. The inverse selective relationship between allelic loss and sponging of let-7 from highly expressed or amplified oncogenes may have broad implications for oncogenesis. Note: This abstract was not presented at the meeting. Citation Format: John T. Powers, Kaloyan M. Tsanov, Frederik Roles, Richard Ebright, Marc Seligson, Yvanka de Soysa, Patrick Cahan, Jessica Theissen, Grace S. LaPier, Dan S. Pearson, Frank Berthold, George Q. Daley. Multiple distinct mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma. [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 LB-290. doi:10.1158/1538-7445.AM2015-LB-290

Clinical, histopathologic, and genomic features of Spitz tumors with ALK fusions.

Activating kinase fusions have recently been described as early oncogenic events that are mutually exclusive with HRAS and BRAF mutations in Spitz tumors. Here, we report a series of 32 Spitz tumors with ALK fusions (6 Spitz nevi, 22 atypical Spitz tumors, and 4 spitzoid melanomas) in patients ranging from 5 months to 64 years (median=12 y) of age. The tumors typically presented as exophytic papules on the extremities and were occasionally darkly pigmented. In addition to ALK fusions previously described in other tumor types (NPM1-ALK, TPR-ALK), we identified 2 novel ALK fusions (CLIP1-ALK and GTF3C2-ALK) in our cohort of Spitz tumors. Array comparative genomic hybridization of 19 of these tumors demonstrated a high frequency of chromosome 2 aberrations (where ALK resides, 63%) and chromosome 1p loss in 37% of the cases. Spitz tumors with ALK fusions demonstrated unique histopathologic features. Clefts and small vesicle-like spaces were arrayed between plump spindled melanocytes with fibrillar cytoplasm and enlarged nuclei. These melanocytes were typically arrayed in elongated and fusiform nests with radial orientation. The tumors often had extension into the dermis or subcutis with a wedge-shaped or bulbous lower border (45% and 17%, respectively). An infiltrative growth pattern was often present at the periphery of the tumor and was highlighted by ALK immunohistochemistry. In conclusion, Spitz tumors with ALK rearrangement show distinct histopathologic features that should aid in improving classification of these diagnostically challenging tumors.

Abstract 3821: A prognostic model of head and neck cancer ties TP53 mutation to 3p loss

Abstract Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by aggressive clinical behavior with a propensity for metastasis and recurrence. Current understanding of HNSCC pathogenesis remains limited, and there are few biomarkers that reliably predict patient outcomes such as survival or response to therapy. Here we report a comprehensive analysis of the molecular and clinical features of HNSCC that define patient survival. Building on data and infrastructure established by The Cancer Genome Atlas (TCGA), we develop an approach that integrates molecular events across multiple tiers of ‘omics data to progressively factor patients into subgroups with the greatest survival differences. We show that in HPV negative patients, the detrimental impact of TP53 mutation occurs only in combination with loss of chromosome 3p, leading to a marked decrease in median survival from &amp;gt;5 years for TP53 mutation only to 1.7 years for both events. Conditioned on TP53/3p, patients are stratified by a hierarchy of events incorporating MUC5B mutation, mir-548k expression, and mutation and expression of Abnormal Spindle Protein (ASPM), while in the absence of the TP53/3p event RAS signaling is an important driver. We also show in HPV positive patients that HPV-mediated inactivation of TP53 acts in synergy with 3p deletion. The major findings are replicated in an independent HNSCC cohort as well as a pan-cancer TCGA cohort of over 3000 patients. Together, the identified markers enable a new multi-tiered molecular classification of HNSCC. Citation Format: Andrew M. Gross, Ryan K. Orosco, John P. Shen, Ann Marie Egloff, Hannah Carter, Matan Hoffree, Michel Choueiri, Charles S. Coffey, Scott M. Lippman, David Neil Hayes, Ezra E. Cohen, Jennifer R. Grandis, Quyen T. Nguyen, Trey Ideker. A prognostic model of head and neck cancer ties TP53 mutation to 3p loss. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3821. doi:10.1158/1538-7445.AM2014-3821

820P - Loss of Chromosomes 9P and 14Q: Re-Profiling Metastatic Tissue for Re-Targeting Patients with Metastatic Renal Cell Carcinoma

ABSTRACT Aim: Loss of chromosomes 9p and 14q correlates with poor prognosis in patients (pts) with clear cell renal cell carcinoma (ccRCC). Both chromosomal loci also do harbor several hot spot molecular pathways suitable for targeted therapies. We sought to investigate the presence of losses of the hot spots chromosomal loci 9p and 14q in primary ccRCC and matched metastatic cancer tissue in pts treated with angiogenic inhibitors. Methods: 7 pts with ccRCC, with available metastatic tissue were recruited. CD10 and CD13 was performed at immunophenotypical level. Molecularly, cytogenetic interphase fluorescente in situ hybridization analysis was performed on formalin-fixed and paraffin embedded primary and matched metastatic tissues by using the locus specific probes mapping the 9p and 14q loci. Cases were scored as having loss of chromosome if >40% nuclei presented single signals and gains when >15% showed three or more signals. Results: Loss of chromosome 9p was observed in 6/7 (85%) primary ccRCC and in 6/7 (85%) matched metastases; one case showed discordance between primary tumor and metastases. Loss of chromosome 14q was detected in 4/7 (57%) primary ccRCC and in 4/7 matched metastases. The concordance of the 14q chromosomal status between primary tumors and corresponding tissue metastases was not demonstrated in 3/7 cases (42%). 3/4 cases (75%) with concordance in cytogenetic status for chromosome 14q developed metastases after at least 3 years of follow-up, the remaining within 1 year. Conclusions: Heterogeneity of cytogenetic status between metastatic and primary ccRCC is observed for loss of chromosome 14q rather than chromosome 9p. Chromosome 14q cytogenetic status, harboring the HIF-1 gene, may affect the efficacy of targeted inhibitors, whereas loss of chromosome 9p, harboring the p16 gene, seems to influence the metastatic behavior per se, without cytogenetic modulation. Those pts showing an homogeneous status for chromosome 14q loss developed metastases after longer years of follow-up. Re-profiling metastatic tissue for re-targeting pts affected by metastatic renal cell carcinoma may be proposed to overcome resistance to anti-angiogenic agents. Disclosure: All authors have declared no conflicts of interest.