Molecular Inversion Probe Array Research Articles

Abstract 2492: Identification of RBMS1 in the amplified region 2q24 as a major driver of cellular growth in childhood hepatoblastoma

Abstract Hepatoblastoma represents the most common primary malignancy of the liver in childhood. Pathological activation of the WNT signaling pathway is characteristic for hepatoblastoma caused by mutations of CTNNB1 or other genes encoding components of the pathway. Genome-wide chromosomal analyses uncovered recurrent chromosomal alterations, in particular gain of chromosome 2q, and in some cases amplification of the region 2q24 that suggests the presence of a so far unidentified oncogene in this chromosomal region. The aim of this study was to identify and further characterize this oncogene. In the framework of the clinical hepatoblastoma study of the German Society for Pediatric Oncology and Hematology, we had the opportunity to study samples of 76 hepatoblastoma patients. Using molecular inversion probe (MIP) array technology, genome-wide, high-resolution, quantitative chromosomal copy number profiles were generated and the smallest overlapping amplified region was identified. RNA and protein expression in normal and diseased tissues as well as ascribed functions of the genes located in this area were evaluated in silico in available databases. RNA expression levels of these genes were analyzed by Nanostring method and Affymetrix arrays and compared between 2q24-amplified tumors versus non-amplified tumors and normal liver tissue. The most promising candidate gene regarding its expression pattern and described function was selected for further analysis. Its protein expression was studied in situ by immunohistochemistry in hepatoblastoma tissue microarrays. The biological function was analyzed in vitro by targeted siRNA-mediated knockdown experiments in 3 hepatoblastoma cell lines (HepT1, HepG2, HuH6) und subsequent cell growth assays (MTT) and WNT signaling pathway specific luciferase reporter assays. Gain of chromosome 2q was present in 44.7% of the cases. Amplification of the 2q24.2-24.3 region was detected in 11.8%. In the smallest overlapping amplified region of 5.6 Mbp, we could accurately map 20 protein coding genes, three genes encoding long noncoding RNAs and one gene encoding a micro-RNA. The RBMS1 gene encoding a single-stranded DNA/RNA binding protein showed significant RNA overexpression in 2q24 amplified tumors. This overexpression was validated by immunohistochemical studies at the protein level. RBMS1 knockdown by specific siRNA transfection resulted in a significantly reduced proliferation of hepatoblastoma cells. RBMS1 knockdown also resulted in a marked reduction of the WNT pathway activity in these cells. We identified RBMS1 located within the amplicon on chromosome 2q24 as a potential oncogenic driver in hepatoblastoma. Initial data suggest that RBMS1 may exert this function by interaction with the WNT signaling pathway that is pathologically activated in hepatoblastoma. Citation Format: Martin Rodemann, Verena Dreschmann, Evelyn Dörner, Dietrich von Schweinitz, Christian Vokuhl, Torsten Pietsch. Identification of RBMS1 in the amplified region 2q24 as a major driver of cellular growth in childhood hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2492.

Abstract P3-09-05: Genomic and clinical characterization of breast tumors with unusual HER2 FISH pattern (ratio < 2, HER2 copy number ≥ 6): Are they mostly HER2 “positive?”

Abstract Background: Breast tumors with HER2/CEP17 fluorescent in situ hybridization (FISH) ratio < 2 and HER2 copy number ≥ 6, defined as Group 3 FISH pattern by the 2018 ASCO/CAP HER2 testing guidelines, are clinically rare. Their biologic and molecular characteristics are under-characterized. They require only a concomitant HER2 immunohistochemistry score of at least 2+ to merit HER2 “positive” status by the ASCO/CAP guidelines. We seek to characterize the genomic and tumor microenvironment landscape of breast tumors with this unique HER2 FISH pattern. Our second aim is to assess the clinicopathologic features with emphasis on HER2-targeted therapy response.Method: Breast cancers with Group 3 FISH pattern were evaluated by the following methods: 1) High-resolution genome-wide copy number alterations by molecular inversion probe (MIP) array; 2) molecular profiling of tumor immune microenvironment, tumor signaling pathways, and PAM50-based intrinsic subtypes by Nanostring nCounter Breast Cancer 360 Panel; 3) tumor infiltrating lymphocytes (TIL) histologic quantitation, and 4) clinical chart review. Classically amplified HER2 breast tumors (Group 1 FISH pattern; ratio ≥ 2 and HER2 copy number ≥ 2) were used as comparison. Results: Thirty-five (1.3%) cases were identified from 2731 consecutive clinical cases that underwent HER2 FISH testing from 2014 to 2019. Of those, thirteen consecutive cases (spanning 2014 - 2017) with sufficient genomic material were analyzed using MIP array. Group 3 tumors had a more complex karyotype and greater chromosomal instability, compared to classically amplified HER2 breast tumors. None of the Group 3 tumors showed HER2 locus amplification at 17q12. Instead, most showed gain of the 17q arm. Six of the Group 3 tumors were profiled by Nanostring nCounter. Compared to HER2 classically amplified tumors, Group 3 tumors were more immune cold, enriched in ER signaling and TGF-beta signaling pathways. In contrast, HER2 classically amplified tumors were enriched in immune infiltration, cytokine and chemokine signaling, PI3K and MAPK signaling, epithelial-mesenchymal transition signaling, and proliferation (P < 0.5 for all). PAM50 analysis showed that classically amplified tumors were more enriched for HER2-subtype (2/4; 50%), while the majority of the Group 3 tumors were enriched for Luminal B-subtype (5/6; 83%). TIL percentage was statistically higher in HER2 classically amplified tumors compare to Group 3 tumors (avg 53% vs 3%; P = 0.02). Clinicopathologic correlation revealed a high rate of ER positivity and high tumor grade in Group 3 tumors. Group 3 FISH pattern can occur as de novo or in the context of FISH status change following therapy. In the 17 evaluable patients for HER2-targeted treatment efficacy, none of the eight patients who received HER2-targeted neoadjuvant therapy achieved complete pathologic response. Nine of ten patients who received TDM-1 in the metastatic setting progressed with minimal treatment response. Significantly, most of these patients (16/17; 94%) were considered overall HER2 positive by the latest ASCO/CAP guideline. Conclusion: Breast tumors with Group 3 HER2 FISH pattern are molecularly and clinically dissimilar from classically amplified HER2 positive breast tumors. HER2-targeted therapy did not appear efficacious in either the neoadjuvant or metastatic/recurrent settings. The lack of apparent efficacy of HER2-targeted therapy, in the context of their HER2 positive status by the current HER2 guideline assessment, warrants further investigation of this HER2 FISH subtype. Citation Format: Christina H Wei, Lixin Yang, Daphne Stewart, Victoria Bedell, Daniel Schmolze, Sophia Apple, Joyce L. Murata-Collins, Raju Pillai, Joanne E. Mortimer. Genomic and clinical characterization of breast tumors with unusual HER2 FISH pattern (ratio < 2, HER2 copy number ≥ 6): Are they mostly HER2 “positive?” [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-05.

EMBR-25. GENOME-WIDE GENETIC AND EPIGENETIC ASSESSMENT OF GROUP 4 MEDULLOBLASTOMA FOR IMPROVED, BIOMARKER DRIVEN, PROGNOSTICATION AND RISK-STRATIFICATION

IntroductionMedulloblastoma (MB) is the most common malignant brain tumour in children. The most frequent molecular subgroup, Group 4 (MBGrp4) accounts for ~35/40% of cases, however it has the least understood underlying biology. Clinical outcomes are heterogeneous in MBGrp4 and are not accounted for by established clinico-pathological risk factors. There is now a requirement for a comprehensive study of MBGrp4, considering established clinico-pathological features and novel molecular biomarkers to enhance risk-stratification and identify novel therapeutic targets. MethodsA clinically-annotated, retrospective MBGrp4 discovery cohort (n = 420) was generated from UK CCLG institutions, collaborating European centres and SIOP-UKCCSG-PNET3 and HIT-SIOP-PNET4 clinical trials. Contemporary, multi-omics profiling was performed. Focal and arm level copy number aberrations (CNAs) were determined from molecular inversion probe (MIP) or DNA methylation array which additionally provided next generation non-WNT/non-SHH (Grp3/Grp4) subtype classifications. Targeted next-generation DNA sequencing was performed to overlay the mutational landscape. Survival modelling was carried out with patients >3 years old who received craniospinal irradiation. ResultsMBGrp4 subtypes were assigned to 88% of tumours with available data. Subtype VIII was strongly associated with i17q (p<0.0001). The favourable-risk cytogenetic signature (2 or 3 of; chromosome 7 gain, chromosome 8 loss and/or chromosome 11 loss) associated with both subtypes VI and VII (p<0.0001). MYCN amplifications were strongly associated with subtype V (p<0.0001) in addition to 16q loss (p<0.0001). The high-risk CNA group was enriched for mutations in genes involved in chromatin remodelling (p<0.0001). Risk factors were identified from multivariate survival modelling. Subtype and CNA groups contributed to improved risk-stratification models that outperformed current clinical schemes. ConclusionComprehensive genetic and epigenetic profiling in this large retrospective cohort has improved our understanding of the molecular and clinical heterogeneity within MBGrp4. Incorporation of molecular biomarkers improved risk-stratification for MBGrp4.

Medulloblastoma in Adults: Cytogenetic Phenotypes Identify Prognostic Subgroups.

Adult medulloblastomas (MB) are rare. We investigated the genetic landscape and prognostic impact of genetic aberrations in a cohort of 117 adult medulloblastomas. Histological features and pathway activation were evaluated at the protein level; 14.5% showed wingless-type activation, 63.3% SHH activation, and 22.2% were classified as non-WNT/non-SHH-MB. Genome-wide copy number analysis was performed by molecular inversion probe array technology. MB-related genes were sequenced in WNT- and SHH-activated MBs. 79.7% of SHH-MBs showed desmoplastic/nodular histology; all other MBs had classic histology. WNT-MBs carried oncogenic CTNNB1 mutations in 88.2% and had monosomy 6 in 52.9%. In SHH-MBs, TERT promoter mutations occurred in 97%, mutations in PTCH1 in 38.2%, SMO in 15.5%, SUFU in 7.4%, and TP53-mutations in 4.1%. In all, 84.6% of non-WNT/non-SHH-MBs had an isochromosome 17q. A whole chromosomal aberration (WCA) signature was present in 45.1% of SHH-TP53-wild type (wt)-MBs and 65.4% of non-WNT/non-SHH-MBs. In 98 cases with survival data, WNT-MBs had a 5-year overall survival (OS) of 68.6%. SHH-MBs TP53wt type and non-WNT/non-SHH-MBs showed 5-year OS of 80.4% and 70.8%, respectively. TP53-mutant SHH-MBs represented a prognostically unfavorable entity; all patients died within 5 years. Patients with a WCA signature showed significantly increased OS (p = 0.011 for SHH-TP53wt-MBs and p = 0.048 for non-WNT/non-SHH-MBs).

EPEN-27. CDKN2A DELETION IN SUPRATENTORIAL EPENDYMOMA WITH RELA ALTERATION INDICATES A DISMAL PROGNOSIS – A RETROSPECTIVE ANALYSIS OF THE HIT EPENDYMOMA TRIAL COHORT

INTRODUCTIONSince supratentorial RELA-fusion positive ependymomas are considered a biologically distinct disease, we aimed to identify histological and genetic predictors of outcome in a defined cohort of pediatric patients.MATERIALS AND METHODSWe analyzed 54 RELA ependymomas in pediatric patients treated according to HIT2000-E protocols. All cases underwent central neuropathological review. Genome-wide copy number alterations were assessed by molecular inversion probe or SNP array. RELA alterations were detected by RT-PCR, sequencing and assessment of nuclear p65-RelA protein. Copy number alteration of the CDKN2A (cyclin dependent kinase inhibitor 2A) locus and concordant p16 protein expression were analyzed.RESULTSFifty-two tumors were classified as WHO-grade III (96.3%) with high mitotic activity in 39 cases (72.2%), vascular proliferation in 47 (87.0%), necrosis in 43 (79.6%) and clear cell morphology in 19 (35.2%). All tumors harbored RELA alterations. Homo- or heterozygous CDKN2A deletions were detected in 9 (16.7%) and 14 (25.9%) cases, respectively. p16 protein expression was lost in all cases with homozygous deletion. Median follow-up was 5.4 years with 5-years EFS and OS of 74.1% and 92.6%. In Kaplan-Meier analysis high mitotic activity was related to shorter EFS (p=0.016) and clear cell morphology to longer OS (p=0.039); CDKN2A deletion was associated with shorter OS (homozygous deletion, p=0.009; homo-or heterozygous deletion, p=0.034). No correlation between CDKN2A deletion and high mitotic activity was found but with higher age at diagnosis (p=0.001).CONCLUSIONDeletion of CDKN2A occurred in 42.6% of supratentorial ependymomas with RELA alteration and represented a genetic predictor of worse overall outcome in pediatric patients.

PATH-27. GIANT CELL GLIOBLASTOMA DOES NOT REPRESENT A DISTINCT TUMOUR ENTITY BUT STRATIFIES INTO DIFFERENT GENETICALLY DEFINED SUBTYPES

Abstract BACKGROUND Giant cell glioblastoma (G-GBM) is a rare variant of glioblastoma (GBM) characterized by large often multinucleated tumour cells. G-GBM occurs most frequently de novo; in contrast to IDH wildtype GBM they carry TP53 mutations in high frequency. METHODS We collected 58 G-GBM from 25 children and 33 adults diagnosed at the Brain Tumor Reference Center of the German Society of Neuropathology and Neuroanatomy (DGNN) in Bonn, and performed a systematic histological, immunohistochemical and genetic analysis by molecular inversion probe array assay, panel next generation sequencing and pyrosequencing. RESULTS 78.4% of G-GBM carried mutations of TP53. Five G-GBM represented IDH-mutated gliomas, 5 cases had H3F3A-G34, and 3 had H3-K27M mutations. The vast majority (75%) lacked IDH or histone gene mutations. In these, chromosome 7 showed a copy number gain in 2/3 of the cases in contrast to the other molecular types. TERT promotor mutations occurred only in adult patients at a frequency of 35.5%. Similarly, chromosome 1q gain, 10 and 13 loss was more frequent in tumours of adult patients. MGMT promotor was methylated in 30.6 % of G-GBM. As a potential therapeutic target, PDGFR-alpha was found expressed in the vast majority of cases and its gene showed copy number gain in 34.5%. CONCLUSIONS In summary, G-GBM is a histological phenotype most likely related to p53 dysfunction but can occur in different genetically defined GBM entities (IDH-, histone gene mutated or in IDH/histone wildtype GBM). (supported by grants from the Deutsche Kinderkrebsstiftung and the Medical Faculty of the University of Bonn, BONFOR)

PATH-32. GENOMIC LANDSCAPE AND BIOLOGY OF MEDULLOBLASTOMA IN ADULTS

Abstract BACKGROUND Medulloblastoma (MB) is a rare disease in adults. Only few cohorts have been studied so far. METHODS Histological features were evaluated and tumors were annotated to WNT-MB, SHH-MB, and non-WNT/non-SHH-MB by immunohistochemistry. Copy number alterations were analyzed by genome-wide molecular inversion probe array. MB-related genes were screened by NGS panel and Sanger sequencing in WNT- and SHH-MB. RESULTS The cohort of 117 tumors contained 14.5% WNT-MB, 63.2% SHH-MB, and 22.2% non-WNT/non-SHH-MB. Classic histology was found in WNT-MB, non-WNT/non-SHH-MB and 22% of SHH-MB; 78% of SHH-MB showed desmoplastic/nodular histology. In WNT-MB, CTNNB1 mutations were found in 88.2% and monosomy 6 in 52.9% of cases. In SHH-MB, PTCH1 mutations were present in 40% of cases and chromosome 9q loss including the PTCH1 locus was the most frequent copy number event in SHH-MB (50%), while SMO and SUFU mutations were found only in 15.4% and 7.7%, respectively. TERT promoter mutations were present in 92.3% of SHH-MBs. Only 2 (3%) of SHH-MB were TP53-mutated (1.7% of whole cohort). In non-WNT/non-SHH-MB, isochromosome 17q was the most frequent chromosomal alteration (84.6%). A previously published whole chromosomal aberration (WCA) signature with ≥1 WCA was found in 69.2% of cases. For 87 cases, survival data were available. WNT-MB, SHH-MB with wildtype TP53 and non-WNT/non-SHH-MB showed similar outcomes (5-year OS: 75%, 78.1% and 69.1%, respectively). Both SHH-MB patients with mutant TP53 died of disease. Patients with non-WNT/non-SHH-MB characterized by the cytogenetic WCA phenotype showed – as described in pediatric standard-risk MB – significant better overall survival compared to patients with tumors lacking WCA (p=0.02). CONCLUSIONS Adult MB represents four defined biological/genomic entities. While in our cohort WNT-MB and SHH-MB-TP53wt were standard risk, non-WNT/non-SHH-MB patients could be divided into two risk-groups according to the presence or absence of WCA in the tumors as previously published for childhood standard-risk MB.

OS9.8 Genomic landscape of medulloblastoma in adults

Abstract BACKGROUND Medulloblastoma (MB) is a rare disease in adults. Therefore, only few cohorts have been studied so far. METHODS Histological features were evaluated and annotation of the tumors to WNT-MB, SHH-MB, and non-WNT/non-SHH-MB was performed by immunohistochemistry. Systematic analysis of tumor samples for genome-wide copy alterations was done by molecular inversion probe array. WNT- and SHH-activated MB were screened by NGS panel and Sanger sequencing for known MB-related genes. RESULTS The cohort of tumors from 117 patients contained 14.5% WNT-MB, 63.2% SHH-MB, and 22.2% non-WNT/non-SHH MB. Classic histology was found in WNT-MB, non-WNT/non-SHH-MB and 22% of SHH-MB; 78% of SHH-MB showed desmoplastic/nodular histology. In WNT-MB, CTNNB1 mutations were found in 88.2% and monosomy 6 in 52.9% of cases. In SHH-MB, PTCH1 mutations were present in 40% of cases and chromosome 9q loss including the PTCH1 locus was the most frequent copy number event in SHH-MB (50%), while SMO and SUFU mutations were found only in 15.4% resp. 7.7%. Mutations in the TERT promoter region were found in 92.3% of SHH-MBs. Only 2 (3%) of the SHH-MB were TP53-mutated (1.7% of the whole cohort). In non-WNT/non-SHH-MB, isochromosome 17q was the most frequent chromosomal alteration (84.6%). A previously published whole chromosomal aberration (WCA) signature with ≥1 WCA was found in 69.2% of cases. For 85 cases, survival data were available. WNT-MB presented no relapses (5-year OS: 100%), while SHH-MB with wildtype TP53 and non-WNT/non-SHH-MB showed similar outcomes (5-year OS: 78.1% and 69.1%, respectively). Both SHH-MB patients with mutant TP53 died of disease. Patients with non-WNT/non-SHH-MB characterized by the cytogenetic WCA phenotype showed - as described in pediatric standard-risk MB - significant better overall survival compared to patients with tumors lacking any WCA (p=0.01). CONCLUSIONS Adult MB represent four defined biological/genomic entities. In contrast to previously published data adult patients with WNT-MBs showed excellent survival. However, the number of patients with WNT-MB was limited so that this result has to be interpreted with caution. While in our cohort SHH-MB-TP53wt were standard risk independent of their mutational or chromosomal status, non-WNT/non-SHH-MB patients could be divided into two risk-groups according to the presence or absence of WCA in the tumors as previously published for childhood standard-risk MB by our group.

Prognostic effect of whole chromosomal aberration signatures in standard-risk, non-WNT/non-SHH medulloblastoma: a retrospective, molecular analysis of the HIT-SIOP PNET 4 trial.

SummaryBackgroundMost children with medulloblastoma fall within the standard-risk clinical disease group defined by absence of high-risk features (metastatic disease, large-cell/anaplastic histology, and MYC amplification), which includes 50–60% of patients and has a 5-year event-free survival of 75–85%. Within standard-risk medulloblastoma, patients in the WNT subgroup are established as having a favourable prognosis; however, outcome prediction for the remaining majority of patients is imprecise. We sought to identify novel prognostic biomarkers to enable improved risk-adapted therapies.MethodsThe HIT-SIOP PNET 4 trial recruited 338 patients aged 4–21 years with medulloblastoma between Jan 1, 2001, and Dec 31, 2006, in 120 treatment institutions in seven European countries to investigate hyperfractionated radiotherapy versus standard radiotherapy. In this retrospective analysis, we assessed the remaining tumour samples from patients in the HIT-SIOP PNET 4 trial (n=136). We assessed the clinical behaviour of the molecularly defined WNT and SHH subgroups, and identified novel independent prognostic markers and models for standard-risk patients with non-WNT/non-SHH disease. Because of the scarcity and low quality of available genomic material, we used a mass spectrometry-minimal methylation classifier assay (MS-MIMIC) to assess methylation subgroup and a molecular inversion probe array to detect genome-wide copy number aberrations. Prognostic biomarkers and models identified were validated in an independent, demographically matched cohort (n=70) of medulloblastoma patients with non-WNT/non-SHH standard-risk disease treated with conventional therapies (maximal surgical resection followed by adjuvant craniospinal irradiation [all patients] and chemotherapy [65 of 70 patients], at UK Children's Cancer and Leukaemia Group and European Society for Paediatric Oncology (SIOPE) associated treatment centres between 1990 and 2014. These samples were analysed by Illumina 450k DNA methylation microarray. HIT-SIOP PNET 4 is registered with ClinicalTrials.gov, number NCT01351870.FindingsWe analysed methylation subgroup, genome-wide copy number aberrations, and mutational features in 136 assessable tumour samples from the HIT-SIOP PNET 4 cohort, representing 40% of the 338 patients in the trial cohort. This cohort of 136 samples consisted of 28 (21%) classified as WNT, 17 (13%) as SHH, and 91 (67%) as non-WNT/non-SHH (we considered Group3 and Group4 medulloblastoma together in our analysis because of their similar molecular and clinical features). Favourable outcomes for WNT tumours were confirmed in patients younger than 16 years, and all relapse events in SHH (four [24%] of 17) occurred in patients with TP53 mutation (TP53mut) or chromosome 17p loss. A novel whole chromosomal aberration signature associated with increased ploidy and multiple non-random whole chromosomal aberrations was identified in 38 (42%) of the 91 samples from patients with non-WNT/non-SHH medulloblastoma in the HIT-SIOP PNET 4 cohort. Biomarkers associated with this whole chromosomal aberration signature (at least two of chromosome 7 gain, chromosome 8 loss, and chromosome 11 loss) predicted favourable prognosis. Patients with non-WNT/non-SHH medulloblastoma could be reclassified by these markers as having favourable-risk or high-risk disease. In patients in the HIT-SIOP PNET4 cohort with non-WNT/non-SHH medulloblastoma, with a median follow-up of 6·7 years (IQR 5·8–8·2), 5-year event-free survival was 100% in the favourable-risk group and 68% (95% CI 57·5–82·7; p=0·00014) in the high-risk group. In the validation cohort, with a median follow-up of 5·6 years (IQR 3·1–8·1), 5-year event-free survival was 94·7% (95% CI 85·2–100) in the favourable-risk group and 58·6% (95% CI 45·1–76·1) in the high-risk group (hazard ratio 9·41, 95% CI 1·25–70·57; p=0·029). Our comprehensive molecular investigation identified subgroup-specific risk models which allowed 69 (51%) of 134 accessible patients from the standard-risk medulloblastoma HIT-SIOP PNET 4 cohort to be assigned to a favourable-risk group.InterpretationWe define a whole chromosomal signature that allows the assignment of non-WNT/non-SHH medulloblastoma patients normally classified as standard-risk into favourable-risk and high-risk categories. In addition to patients younger than 16 years with WNT tumours, patients with non-WNT/non-SHH tumours with our defined whole chromosomal aberration signature and patients with SHH-TP53wild-type tumours should be considered for therapy de-escalation in future biomarker-driven, risk-adapted clinical trials. The remaining subgroups of patients with high-risk medulloblastoma might benefit from more intensive therapies.FundingCancer Research UK, Swedish Childhood Cancer Foundation, French Ministry of Health/French National Cancer Institute, and the German Children's Cancer Foundation.

Abstract LB-324: A whole chromosome aberration phenotype in non-WNT/non-SHH tumors predicts outcome within standard-risk medulloblastomas from the HIT-SIOP-PNET4 clinical trial

Abstract Introduction: Standard-risk medulloblastoma (SR-MB; 50-60% of patients) is currently defined by the absence of high-risk (e.g. metastatic disease, large-cell/anaplastic histology, MYC amplification) disease features. 75-85% survival rates are achieved, however the identification and validation of novel prognostic biomarkers will be essential to improve risk-adapted therapies, aimed at increased survival and reduced treatment-related late-effects. Experimental procedures: We undertook comprehensive analysis of the pan-European HIT-SIOP-PNET4 prospective clinical trial (2001-2006; age 4-21 years at diagnosis), encompassing central clinical and radiological review, and annotation of molecular pathological features to the WHO (2016) classification. Methods were developed and/or adapted to assess methylation-dependent molecular subgroup (MassArray), copy number aberrations (molecular inversion probe array) and mutational status in scant archival material previously refractory to analysis (n=136). Independent prognostic markers/predictive models for non-WNT/non-SHH patients (n=91) were identified by multivariate analyses, and validated in a representative independent cohort (n=70). Results: WNT (n=28; 21%) and Group 4 (n=76; 56%) tumors were enriched in SR-MB compared to published disease-wide estimates. Favorable-risk WNT disease was confirmed in patients &amp;lt;16.0 years at diagnosis. All events in SHH (4 of 17) occurred in tumors with TP53 mutation and/or chr17p loss. In non-WNT/non-SHH tumors, a novel whole chromosomal aberration (WCA) phenotype characterized by chr7 gain, chr8 loss, and/or chr11 loss, was identified in 42% of patients (38 of 91). This phenotype predicted an excellent prognosis (100% 5-year PFS) and its incorporation into novel survival models out-performed current risk-stratification schemes for SR-MB. Conclusion: A favorable-risk WCA phenotype identifies a large proportion (42%) of non-WNT/non-SHH SR-MB patients for whom therapy de-escalation should be considered in future biomarker-driven risk-adapted clinical trials; remaining patients (58%; 68% 5-year PFS) might benefit from more intensive and/or novel therapies. Citation Format: Tobias Goschzik, Ed C. Schwalbe, Debbie Hicks, Dominique Figarella-Branger, Francois Doz, Stefan Rutkowski, Goran Gustafsson, Birgitta Lannering, Torsten Pietsch, Steve C. Clifford. A whole chromosome aberration phenotype in non-WNT/non-SHH tumors predicts outcome within standard-risk medulloblastomas from the HIT-SIOP-PNET4 clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-324.

Abstract PD1-12: Identification of copy number alterations associated with the progression of high risk premalignant breast lesions to breast carcinoma

Abstract Background: Atypical ductal hyperplasia (ADH) is common finding in the mammographic era, but has wide variation in diagnosis and treatment. ADH is a high risk factor for invasive breast ductal carcinoma (IDC), and is also considered to be a non-obligate precursor to IDC. Although a few studies have revealed some of the common genomic characteristics of ADH, a clear understanding of the molecular changes associated with breast cancer progression has been limited by inadequately powered studies and low resolution methodology (Lopez-Garcia et al., 2010, Histopathology 57: 171-192). Copy number alterations (CNAs), one of the major genetic alterations in cancer, are present in IDC and ductal carcinoma in situ (DCIS) and given a suitable detection method could be used in research and potentially in the clinical setting to predict patient prognosis. Method: We optimised a robust, cost effective low-coverage whole genome sequencing (LCWGS) method for CNA detection, using as little as 5 ng of formalin-fixed paraffin-embedded tissue derived DNA. We applied this novel method to 21 cases of pure ADH (not associated with cancer) as well as 20 ADH with synchronous DCIS and/or IDC. Cases were reviewed independently by two pathologists, followed by micro-dissection and DNA extraction. CNA were analysed using the LCWGS method, using 5-10 ng total DNA input per sample. Results: Highly accurate copy number profiles produced by low input LCWGS are comparable to those obtained from an alternative method, Molecular Inversion Probe arrays, while requiring 10 fold less input DNA (Kader et al., 2016, Genome Medicine 8: 121). Genetic analysis of pure ADH found that 95% had at least one copy number event and the median fraction of the genome altered was 8% (0-19%). Surprisingly, while 29% of pure ADH showed 16q loss and 1q gain (CNAs common in low-grade (LG) breast cancer), 29% of pure ADH showed 8q gain, an event more frequently observed in high grade (HG) cancer. Analysis of ADH with synchronous DCIS and/or IDC showed that 67% of ADH was clonally related with both LG (6/8) and HG carcinoma (4/7). The final analysis of 20 synchronous cases (9 LG, 11 HG) will be presented. Conclusion: Here we report the most detailed molecular taxonomy of this high risk pre-malignant breast lesion with the largest cohort undertaken worldwide. Our observation that 67% of ADH is clonal to breast carcinoma suggests that both LG and HG carcinoma can evolve from a similar ancestor lesion. As ADH has the potential to develop both LG and HG carcinoma, a biomarker signifying breast cancer progression from pre-malignant lesions is highly desirable for the more precise treatment of individual patients diagnosed with ADH. Citation Format: Kader T, Hill P, Opeskin K, Goode DL, Elder K, Pang J-M, Fox SB, Mann GB, Campbell IG, Gorringe KL. Identification of copy number alterations associated with the progression of high risk premalignant breast lesions to breast carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-12.

Abstract P3-04-08: Genomic analysis of breast papillomas

Abstract Background: Papillomas are often found co-existing with breast carcinoma yet they are not considered to be a true precursor of the disease. Previous studies have shown that some cases may carry copy number alterations (CNA) or mutations in AKT1/PIK3CA (Troxell et al., 2010, Modern Pathology 23: 27-37) suggesting this lesion may have malignant potential. To date, a detailed study of both pure papillomas (not associated with cancer) and those seen in the same breast as a carcinoma has not been undertaken. Therefore, we set out to investigate the molecular changes associated with this lesion and whether papillomas can be clonally related to synchronous breast carcinoma. Method: Papilloma cases were identified from a hospital database and independently reviewed by consultant pathologists followed by micro-dissection of formalin-fixed paraffin-embedded tumour tissue and DNA extraction. For CNA detection either Affymetrix Molecular inversion Probe (MIP) 330K arrays were used or low-coverage whole genome sequencing using 5-10 ng of DNA (Kader et al., 2016, Genome Medicine 8: 121) where there was insufficient DNA for MIP arrays. We applied either of these 2 methods to 24 cases of pure papilloma as well as 20 papilloma with synchronous ductal carcinoma in situ (DCIS) and/or invasive ductal carcinoma (IDC). Additionally, targeted exon sequencing of breast cancer driver genes was performed for a subset of cases. Results: Among the pure papillomas 31% (5/16) showed CN change with, the most frequent change being 16q loss (2/16). Of the papillomas synchronous with DCIS/IDC analysed to date, 2/5 were shown to be clonal with the co-existing carcinoma. Final CNA analysis will be presented for 24 pure papilloma cases and 20 synchronous cases. Targeted sequencing revealed that all for pure papillomas analysed to date harboured somatic mutations in PIK3CA (3/4 cases) and PIK3R1 (1/4,) suggesting that most papillomas are driven by alterations in the PI3-kinase/AKT pathway. The final sequencing data to be presented will include an additional 5 pure papillomas and 10 synchronous cases. Conclusion: Our observation that 40% of papillomas are clonal to breast carcinoma suggests that DCIS or IDC can arise from a common ancestor as co-existing papillomas, however, most papillomas co-existing with carcinoma are likely to be independent in our cohort. Citation Format: Elder KJ, Kader T, Hill P, Opeskin K, Goode DL, Pang J-M, Fox SB, Mann GB, Campbell IG, Gorringe KL. Genomic analysis of breast papillomas [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-08.

Molecular subtyping of tumors from patients with familial glioma.

Single-gene mutation syndromes account for some familial glioma (FG); however, they make up only a small fraction of glioma families. Gliomas can be classified into 3 major molecular subtypes based on isocitrate dehydrogenase (IDH) mutation and 1p/19q codeletion. We hypothesized that the prevalence of molecular subtypes might differ in familial versus sporadic gliomas and that tumors in the same family should have the same molecular subtype. Participants in the FG study (Gliogene) provided samples for germline DNA analysis. Formalin-fixed, paraffin-embedded tumors were obtained from a subset of FG cases, and DNA was extracted. We analyzed tissue from 75 families, including 10 families containing a second affected family member. Copy number variation data were obtained using a first-generation Affymetrix molecular inversion probe (MIP) array. Samples from 62 of 75 (83%) FG cases could be classified into the 3 subtypes. The prevalence of the molecular subtypes was: 30 (48%) IDH-wildtype, 21 (34%) IDH-mutant non-codeleted, and 11 (19%) IDH-mutant and 1p/19q codeleted. This distribution of molecular subtypes was not statistically different from that of sporadic gliomas (P = 0.54). Of 10 paired FG samples, molecular subtypes were concordant for 7 (κ = 0.59): 3 IDH-mutant non-codeleted, 2 IDH-wildtype, and 2 IDH-mutant and 1p/19q codeleted gliomas. Our data suggest that within individual families, patients develop gliomas of the same molecular subtype. However, we did not observe differences in the prevalence of the molecular subtypes in FG compared with sporadic gliomas. These observations provide further insight into the distribution of molecular subtypes in FG.

Genomic analysis of follicular dendritic cell sarcoma by molecular inversion probe array reveals tumor suppressor-driven biology.

Follicular dendritic cell sarcoma is a rare malignant neoplasm of dendritic cell origin that is currently poorly characterized by genetic studies. To investigate whether recurrent genomic alterations may underlie the biology of follicular dendritic cell sarcoma and to identify potential contributory regions and genes, molecular inversion probe array analysis was performed on 14 independent formalin-fixed, paraffin-embedded samples. Abnormal genomic profiles were observed in 11 out of 14 (79%) cases. The majority showed extensive genomic complexity that was predominantly represented by hemizygous losses affecting multiple chromosomes. Alterations of chromosomal regions 1p (55%), 2p (55%), 3p (82%), 3q (45%), 6q (55%), 7q (73%), 8p (45%), 9p (64%), 11q (64%), 13q (91%), 14q (82%), 15q (64%), 17p (55%), 18q (64%), and 22q (55%) were recurrent across the 11 samples showing abnormal genomic profiles. Many recurrent genomic alterations in follicular dendritic cell sarcoma overlap deletions that are frequently observed across human cancers, suggesting selection, or an active role for these alterations in follicular dendritic cell sarcoma pathogenesis. In support of a tumor suppressor-driven biology, homozygous deletions involving tumor suppressor genes CDKN2A, RB1, BIRC3, and CYLD were also observed. Neither recurrent gains nor amplifications were observed. This genomic characterization provides new information regarding follicular dendritic cell sarcoma biology that may improve understanding about the underlying pathophysiology, provide better prognostication, and identify potential therapeutic markers for this rare disease.

Abstract NTOC-112: GENOMIC SCARS AND CLINICAL RESPONSE TO COMBINATION THERAPY OF PARP AND ANGIOGENESIS INHIBITORS IN OVARIAN CANCER

Abstract BACKGROUND: Genomic instability, frequently resulting in chromosomal allelic deletion with allelic imbalance (AI)/loss of heterozygosity (LOH), is characteristic of high-grade serous ovarian cancer (HGSOC). Frequent allelic deletion is thought to arise from deficiency in DNA repair by homologous recombination (HR) resulting in the so called “genomic scars” of HR deficiency. Quantification of AI/LOH events in the tumor genome has previously been shown to predict response to therapy using platinum compounds. Recently, PARP inhibitors have proved useful in treating a sub-set of patients with HGSOC, particularly tumors harboring BRCA1/2 mutations. Combination with an angiogenesis inhibitor significantly improved the outcome. This study explores the potential of using AI/LOH scores to predict clinical response of HGSOC to PARP inhibition alone or in combination with an angiogenesis inhibitor. MATERIALS AND METHODS: Molecular inversion probe array data were generated using tumors from a sub-set of patients (n=37) enrolled in a clinical trial comparing the PARP inhibitor Olaparib to the combination of Olaparib with the anti-angiogenic agent Cediranib (NCT01116648). AI/LOH regions were identified using an ASCAT based algorithm. Markers of genomic instability associated with DNA repair deficiency were scored. These quantify AI regions (NAI), telomeric AI (NtAI), large scale transition (LST), fraction of LOH (FLOH), and HRD-LOH. dChip was used for copy number analysis. The best overall response to therapy was determined using the RECIST 1.1 criteria for complete and partial response (CR, n = 3 and PR, n = 18), and stable disease without objective response (SD, n = 16). RESULTS: A high tumor NAI-score was positively correlated with the degree of clinical response to therapy (either olaparib alone or in combination with cediranib) (Chi-square test for trend, p = 0.036). This association remains statistically significant in the subgroup carrying BRCA mutations (n = 22, Chi-square test for trend, p = 0.0488). In this limited sample, the objective response rate of high NAI tumors to the combination therapy was high (7 out of 8), especially in patient carrying wild-type BRCA1/2 genes (2 out of 2, p = 0.045). The results suggest NAI may be a potential genomic marker for response to the therapy combining PARP and angiogenesis inhibitors. However, no significant association was observed between the degree of objective response and scores of other genomic measurements NtAI, LST, or HRD-LOH. SUMMARY: High NAI-score was associated with objective response to olaparib, alone or in combination with cediranib, supporting NAI as a candidate of genomic marker for predicting response to PARP inhibitor-based therapy in HGSOC. A larger cohort would be required to further evaluate predictive value of NAI for response to the combinational therapy. Citation Format: Zhigang C. Wang, Nicolai Juul Birkbak, William T. Barry, Thomas M. Roberts, Eric P. Winer, J Dirk Iglehart, Ursula A. Matulonis, S. Percy Ivy, and Joyce F. Liu. GENOMIC SCARS AND CLINICAL RESPONSE TO COMBINATION THERAPY OF PARP AND ANGIOGENESIS INHIBITORS IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-112.

Validation of quantitative PCR-based assays for detection of gene copy number aberrations in formalin-fixed, paraffin embedded solid tumor samples

Gene copy number changes are important somatic alterations in cancers. A number of high throughput methods, such as next generation sequencing, are capable of detecting copy number aberrations, but their use can be challenging and cost prohibitive for screening a small number of markers. Furthermore, detection of CNAs by high throughput platforms needs confirmation by an orthogonal technique, especially in cases with low level CNAs. Here, we have validated TaqMan based quantitative PCR (qPCR) assays to detect CNAs in genes of high clinical importance in formalin-fixed, paraffin-embedded (FFPE) samples. A cohort of 22 tumors of various types that harbor 67 CNAs in 13 genes was assessed. The abnormalities in these tumors were detected by using a NGS-based 50 gene hotspot panel on Ion Torrent PGM and molecular inversion probe (MIP) array. The CNAs included ERBB2 (n = 6), PDGFRA (n = 6), KIT (n = 7), NRAS (n = 3), PIK3CA (n = 6), MYC (n = 7), MET (n = 4), FLT3 (n = 6), FGFR3 (n = 3), FGFR2 (n = 3), EGFR (n = 7), KRAS (n = 6) and FGFR1 (n = 5). Different amounts of input DNA were tested and 5 ng FFPE DNA was found to be adequate without limiting detection sensitivity. All 22 (100%) positive tumor samples revealed by MIP array were confirmed by real time qPCR and 17 of 22 (77.2%) samples tested by NGS were confirmed. The limit of detection of the qPCR assay was determined by serial dilution of SKBR3 cell line DNA (with amplified ERBB2) and showed an ability to detect 3 copies consistently up to 0.75% dilution. The ability to use low input of FFPE DNA, high sensitivity, and short turnaround time makes qPCR a valuable and economically viable platform for detecting single gene CNAs as well as for confirmation of CNAs detected by high throughput screening assays.

Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue.

Unlocking clinically translatable genomic information, including copy number alterations (CNA), from formalin-fixed paraffin-embedded (FFPE) tissue is challenging due to low yields and degraded DNA. We describe a robust, cost-effective low-coverage whole genome sequencing (LC WGS) method for CNA detection using 5 ng of FFPE-derived DNA. CN profiles using 100 ng or 5 ng input DNA were highly concordant and comparable with molecular inversion probe (MIP) array profiles. LC WGS improved CN profiles of samples that performed poorly using MIP arrays. Our technique enables identification of driver and prognostic CNAs in archival patient samples previously deemed unsuitable for genomic analysis due to DNA limitations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0375-z) contains supplementary material, which is available to authorized users.

Genomewide copy number analysis of Müllerian adenosarcoma identified chromosomal instability in the aggressive subgroup

AbstractMüllerian adenosarcomas are malignant gynecologic neoplasms. Advanced staging and sarcomatous overgrowth predict poor prognosis. Because the genomic landscape remains poorly understood, we conducted this study to characterize the genomewide copy number variations in adenosarcomas. Sixteen tumors, including eight with and eight without sarcomatous overgrowth, were subjected to a molecular inversion probe array analysis. Copy number variations, particularly losses, were significantly higher in cases with sarcomatous overgrowth. Frequent gains of chromosomal 12q were noted, often involving cancer-associated genes CDK4 (six cases), MDM2, CPM, YEATS4, DDIT3, GLI1 (five each), HMGA2 and STAT6 (four), without association with sarcomatous overgrowth status. The most frequent losses involved chromosomes 13q (five cases), 9p, 16q and 17q (four cases each) and were almost limited to cases with sarcomatous overgrowth. MDM2 and CDK4 amplification, as well as losses of RB1 (observed in two cases) and CDKN2A/B (one case), was verified by FISH. By immunohistochemistry, all MDM2/CDK4-coamplified cases were confirmed to overexpress both encoded proteins, whereas all four cases with (plus an additional four without) gain of HMGA2 overexpressed the HMGA2 protein. Both cases with RB1 loss were negative for the immunostaining of the encoded protein. Chromothripsis-like copy number profiles involving chromosome 12 or 14 were observed in three fatal cases, all of which harbored sarcomatous overgrowth. With whole chromosome painting and deconvolution fluorescent microscopy, dividing tumor cells in all three cases were shown to have scattered extrachromosomal materials derived from chromosomes involved by chromothripsis, suggesting that this phenomenon may serve as visual evidence for chromothripsis in paraffin tissue. In conclusion, we identified frequent chromosome 12q amplifications, including loci containing potential pharmacological targets. Global chromosomal instability and chromothripsis were more frequent in cases with sarcomatous overgrowth. To our knowledge, this is the first time that evidence of chromothripsis has been demonstrated in paraffin-embedded clinical tissues and in adenosarcomas.

Ability to Generate Patient-Derived Breast Cancer Xenografts Is Enhanced in Chemoresistant Disease and Predicts Poor Patient Outcomes

BackgroundBreast cancer patients who are resistant to neoadjuvant chemotherapy (NeoCT) have a poor prognosis. There is a pressing need to develop in vivo models of chemo resistant tumors to test novel therapeutics. We hypothesized that patient-derived breast cancer xenografts (BCXs) from chemo- naïve and chemotherapy-exposed tumors can provide high fidelity in vivo models for chemoresistant breast cancers.MethodsPatient tumors and BCXs were characterized with short tandem repeat DNA fingerprinting, reverse phase protein arrays, molecular inversion probe arrays, and next generation sequencing.ResultsForty-eight breast cancers (24 post-chemotherapy, 24 chemo-naïve) were implanted and 13 BCXs were established (27%). BCX engraftment was higher in TNBC compared to hormone-receptor positive cancer (53.8% vs. 15.6%, p = 0.02), in tumors from patients who received NeoCT (41.7% vs. 8.3%, p = 0.02), and in patients who had progressive disease on NeoCT (85.7% vs. 29.4%, p = 0.02). Twelve patients developed metastases after surgery; in five, BCXs developed before distant relapse. Patients whose tumors developed BCXs had a lower recurrence-free survival (p = 0.015) and overall survival (p<0.001). Genomic losses and gains could be detected in the BCX, and three models demonstrated a transformation to induce mouse tumors. However, overall, somatic mutation profiles including potential drivers were maintained upon implantation and serial passaging. One BCX model was cultured in vitro and re-implanted, maintaining its genomic profile.ConclusionsBCXs can be established from clinically aggressive breast cancers, especially in TNBC patients with poor response to NeoCT. Future studies will determine the potential of in vivo models for identification of genotype-phenotype correlations and individualization of treatment.

Abstract LB-048: Copy number and loss of heterozygosity (LOH) analysis in 52 breast cancer FFPE samples using molecular Inversion probe array: detailed analysis of reproducibility and performance compared to NGS platforms

Abstract Introduction: Somatic mutations are routinely identified using NGS cancer panels but these panels lack genome-wide coverage for copy number (CN) and LOH analysis. To investigate mutation, CN, and LOH in late-stage breast cancer we tested &amp;gt;50 samples using the Oncoscan molecular inversion probe (MIP) array and evaluated its reproducibility and performance compared to NGS platforms. Methods: 52 breast cancer samples (stage IIIA - IV) were analyzed using MIP array (Oncoscan, Affymetrix). Four samples were tested in technical triplicates to determine assay reproducibility. In addition, 28 samples were sequenced by amplicon-based NGS and five of these samples were also tested using a capture-based NGS platform for mutation, CN, and LOH comparison. Results: MIP array provided highly reproducible results for CN and LOH, with &amp;gt;98% of calls showing CN range in the technical triplicates of &amp;lt; 0.5 copy for 891 cancer genes analyzed. Variability in CN seems to be proportional to absolute copy number at the tested locus, with CN range in technical triplicates of &amp;gt;2 copies seen in only two cases, once for ERBB2 (CN range 32 - 35 copies) and once for FLOT2 (CN range 22 - 25 copies). Gene level results were then categorized in five groups: homozygous deletion, single copy loss, diploid, low grade amplification (≤6 copies), or high grade amplification (&amp;gt;6 copies). Using these predetermined cut points, we saw &amp;gt;99% concordance rate among the technical replicates in the MIP array. We found a 93% concordance rate between MIP array and CN/LOH calls by capture-based NGS. Discordant calls between NGS and MIP array were either LOH calls or single copy number change (diploid vs. single copy loss or gain). MIP array mutation analysis of 28 samples showed good sensitivity, correctly detecting the 17 PIK3CA mutations and one TP53 mutation identified by NGS in this cohort. There were seven false positive calls by MIP array, five of them occurring in two genotypes (2x NRAS G12S/C, and 3x EGFR L858R). The other two false positives occurred in PIK3CA, with one false positive (H1047L) occurring in association with a high-grade PIK3CA amplification (7 copies). Increasing CN at the mutation locus was associated with a higher mutation score provided by MIP array (p&amp;lt;0.0001), which may explain some false positive calls. Conclusions: MIP array platform provides a great alternative for assessing CN and LOH in FFPE samples at lower cost and using less input DNA than NGS (80ng vs. 250ng). There was good correlation between CN and LOH results from MIP array and capture-based NGS, with discordant results limited to small CN differences or LOH calls. Mutation analysis by MIP array showed no false negatives when compared to NGS, while false positives seem to occur either due to probe-specific issues or in association with amplifications at the genotyping locus. Citation Format: Candice L. Horn, Fabio Nunes, John Calley, Steven Bray, Isabella Wulur, Mark Farmen, Robert Gallavan, Iris Halfpenny, Paul Medlow, Keith McGreeghan-Crosby, Gera Jellema. Copy number and loss of heterozygosity (LOH) analysis in 52 breast cancer FFPE samples using molecular Inversion probe array: detailed analysis of reproducibility and performance compared to NGS platforms. [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-048. doi:10.1158/1538-7445.AM2015-LB-048