Genomic Mutational Status Research Articles

Construction and verification of a histone deacetylases-related prognostic signature model for colon cancer

Histone deacetylases (HDACs) contribute significantly to the initiation, progression, and prognosis of colorectal adenocarcinoma (COAD). Additionally, HDACs regulate the tumor microenvironment, immune escape, and tumor stem cells, and are closely linked to COAD prognosis. We developed a prognostic model for COAD that incorporates HDACs to evaluate their specific roles. The COAD dataset containing clinical and mutation data was collected using the TCGA and GEO databases to obtain genes associated with HDAC. LASSO analysis and univariate and multivariate Cox regression analysis were used to determine the presence of prognostic genes. Multivariate Cox analysis was also used to determine risk scores for HDAC-related features. Furthermore, genomic alterations, immune infiltration, and drug response were compared between high- and low-risk groups. Cellular experiments validated the potential regulatory role of BRD3 on COAD proliferation, migration, and apoptosis. The median risk scores, calculated based on the characteristics, demonstrated a more significant prognostic improvement in patients in the low-risk group. Furthermore, HDAC-related features were identified as important independent prognostic factors for patients with COAD. Additionally, genomic mutation status, immune infiltration, and function, as well as response to immunotherapy and chemotherapy, were found to be associated with risk scores. Subgroup analyses indicate that anti-PD-1 therapy may be beneficial for patients in the low-risk group. Additionally, a decrease in risk score was associated with a decrease in immune infiltration. Finally, HCT116 and HT29 cells exhibited inhibition of BRD3 gene proliferation and migration, as well as promotion of apoptosis. In patients with COAD, HDAC-related characteristics may be useful in predicting survival and selecting treatment.

Abstract P3-05-50: Comprehensive Analysis of Pyroptotic Gene Prognostic Signatures Associated with Tumor Immune Microenvironment and Genomic Mutation in Breast Cancer

Abstract Background: Breast cancer is becoming a tumor with the highest morbidity rate, and inflammation-induced cell death namely pyroptosis reportedly plays dual roles in cancers. However, the specific mechanism between pyroptosis and the clinical prognosis of breast cancer patients is indistinct. Hence, novel pyroptosis-related biomarkers and their contributing factors deserve further exploration to predict the prognosis in breast cancer. Methods: Pearson’s correlation analysis, and univariate and multivariate Cox regression analysis were utilized to obtain six optimal pyroptosis-related gene prognostic signatures (Pyro-GPS). The risk score of each breast cancer patient was calculated. Next, a Pyro-GPS risk model was constructed and verified in TCGA cohort (n=1,089) and GSE20711 cohort (n=88). Then analyses of immune microenvironment, genomic variation, functional enrichment, drug response and clinicopathologic feature stratification associated with the risk score of Pyro-GPS were performed. Ultimately, a nomogram based on the risk score and several significant clinicopathologic features was established. Results: The low-risk breast cancer patients have better survival outcomes than the high-risk patients. The low-risk patients also show higher immune cell infiltration levels and immune-oncology target expression levels. There is no significant difference in genetic variation between the two risk groups, but the frequency of gene mutations varies. Functional enrichment analysis shows that the low-risk patients are prominently correlated with immune-related pathways, whereas the high-risk patients are enriched in cell cycle, ubiquitination, mismatch repair, homologous recombination and biosynthesis-related pathways. Pyro-GPS is positively correlated with the IC50 of anti-tumor drugs. Furthermore, comprehensive analyses based on risk score and clinicopathological features were performed to predict the prognosis of breast cancer patients. Conclusions: The risk score of Pyro-GPS can serve as a promising hallmark to predict the prognosis of BRCA patients. Risk score evaluation may assist to acquire relevant information of tumor immune microenvironment, genomic mutation status, functional pathways and drug sensitivity, and thus provide more effective personalized strategies. Citation Format: Hongfei Zhang, Xiafei Yu, Junzhe Yang, Xiaoan Liu, Yanhui Zhu. Comprehensive Analysis of Pyroptotic Gene Prognostic Signatures Associated with Tumor Immune Microenvironment and Genomic Mutation in Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-50.

Clinicopathological and computed tomography features of patients with early-stage non-small-cell lung cancer harboring ALK rearrangement

BackgroundAlthough some studies have assessed the correlation between computed tomography (CT) features and anaplastic lymphoma kinase (ALK) rearrangement in patients with non-small-cell lung cancer (NSCLC), few have focused on early-stage patients. The results of some previous studies are inconsistent and contradictory. Therefore, this study aimed to analyze the clinicopathological and CT features of patients with early-stage NSCLC harboring ALK rearrangement.MethodsThis retrospective analysis included 65 patients with ALK rearrangement and 629 ALK-negative patients. All patients had surgically resected NSCLC and were diagnosed with stage IA or stage IIB NSCLC. Clinicopathological features and CT signs, including tumor size and density, consolidation tumor ratio (CTR), lesion location, round or irregular shape, lobulated or spiculated margins, air bronchograms, bubble-like lucency or cavities, and pleural retraction, were investigated according to different genotypes.ResultsThe prevalence of ALK rearrangement in patients with early-stage NSCLC was 9.3% (65/694). Patients with ALK rearrangement were significantly younger than those without ALK rearrangement (P = 0.033). The frequency of moderate cell differentiation was significantly lower in tumors with ALK rearrangement than in those without ALK rearrangement (46.2% vs. 59.8%, P = 0.034). The frequency of the mucinous subtype was significantly higher in the ALK-positive group than in the ALK-negative group (13.8% vs. 5.4%, P = 0.007). No significant differences were found in any CT signs between the ALK-positive and ALK-negative groups.ConclusionsPatients with ALK-positive lung cancer may have specific clinicopathological features, including younger age, lower frequency of moderate cell differentiation, and higher frequency of the mucinous type. CT features may not correlate with ALK rearrangement in early-stage lung cancer. Immunohistochemistry or next-generation sequencing is needed to further clarify the genomic mutation status.

Comprehensive analysis of pyroptotic gene prognostic signatures associated with tumor immune microenvironment and genomic mutation in breast cancer.

BackgroundBreast cancer is becoming a tumor with the highest morbidity rate, and inflammation-induced cell death namely pyroptosis reportedly plays dual roles in cancers. However, the specific mechanism between pyroptosis and the clinical prognosis of breast cancer patients is indistinct. Hence, novel pyroptosis-related biomarkers and their contributing factors deserve further exploration to predict the prognosis in breast cancer.MethodsPearson’s correlation analysis, and univariate and multivariate Cox regression analysis were utilized to obtain six optimal pyroptosis-related gene prognostic signatures (Pyro-GPS). The risk score of each breast cancer patient was calculated. Next, a Pyro-GPS risk model was constructed and verified in TCGA cohort (n=1,089) and GSE20711 cohort (n=88). Then analyses of immune microenvironment, genomic variation, functional enrichment, drug response and clinicopathologic feature stratification associated with the risk score of Pyro-GPS were performed. Subsequently, a nomogram based on the risk score and several significant clinicopathologic features was established. Ultimately, we further investigated the role of CCL5 in the biological behavior of MDA-MB-231 cell line.ResultsThe low-risk breast cancer patients have better survival outcomes than the high-risk patients. The low-risk patients also show higher immune cell infiltration levels and immune-oncology target expression levels. There is no significant difference in genetic variation between the two risk groups, but the frequency of gene mutations varies. Functional enrichment analysis shows that the low-risk patients are prominently correlated with immune-related pathways, whereas the high-risk patients are enriched in cell cycle, ubiquitination, mismatch repair, homologous recombination and biosynthesis-related pathways. Pyro-GPS is positively correlated with the IC50 of anti-tumor drugs. Furthermore, comprehensive analyses based on risk score and clinicopathological features were performed to predict the prognosis of breast cancer patients. Additionally, in vitro experiments confirmed that breast cancer cells with high expression of CCL5 had weaker proliferation, invasion and metastasis abilities as well as stronger apoptosis and cell cycle arrest abilities.ConclusionsThe risk score of Pyro-GPS can serve as a promising hallmark to predict the prognosis of BRCA patients. Risk score evaluation may assist to acquire relevant information of tumor immune microenvironment, genomic mutation status, functional pathways and drug sensitivity, and thus provide more effective personalized strategies.

Intra-Tumoral Genomic Heterogeneity in Rectal Cancer: Mutational Status Is Dependent on Preoperative Biopsy Depth and Location.

Simple SummaryA subset of patients with rectal cancer are treated before surgery with chemoradiation. Unfortunately, this neoadjuvant chemoradiotherapy does not have the preferred effect of tumor downstaging in all patients, but does bring substantial side effects and possible complications. A pre-treatment prediction based on available parameters might provide a means to better select therapy for individual patients. Genomic mutational status of pre-treatment biopsies may provide prognostic information, however, it also might be influenced by tumor heterogeneity. This study investigates whether pre-treatment biopsy material is a reliable way of defining mutational status in rectal cancer.Neoadjuvant therapy before surgical resection is indicated for patients with locally advanced rectal cancer. However, a significant number of patients show minimal or no response to neoadjuvant therapy. Unfortunately, we are currently unable to predict response and identify non-responding patients before neoadjuvant treatment is given. Genomic mutational status might provide valuable prognostic information. However, it is unclear whether predictions based on genomic mutational status in single preoperative biopsies are reliable due to intra-tumoral heterogeneity. In this study we aim to investigate the reliability of genomic mutations found in single pre-operative biopsies by comparing genomic mutations to four other locations within the same tumor using next generation sequencing. Rectal cancer patients undergoing primary resection without neoadjuvant therapy were included. From each patient, one biopsy, two deep and two superficial samples were obtained and sequenced using a targeted next generation sequencing gene panel. Concordance between these five samples was assessed. In this feasibility study we included 11 patients. In 7 out of 11 (64%) patients, all 5 samples showed concordant mutations. In 4 out of 11 patients (36%) discordant mutations were observed. In conclusion, assessment of mutational status on a single pre-operative biopsy shows discordance with tumor tissue from other locations in 36% of cases. These results warrant careful interpretation of biopsy material analysis, as these might be influenced by tumor heterogeneity.

Molecular correlates of response to nivolumab at baseline and on treatment in patients with RCC

BackgroundNivolumab is an immune checkpoint inhibitor targeting the programmed death-1 receptor that improves survival in a subset of patients with clear cell renal cell carcinoma (ccRCC). In contrast to other...

Abstract 5885: The impact of genomic mutational status and correlation with tumor mutation burden in non-small cell lung cancer of Xuanwei, Yunnan Province, China

Abstract Background: Xuanwei County in Yunnan Province is one of the regions with the highest lung cancer mortality rates in China, the mortality rate in female lung cancer ranks first in China. Our previous study identified a higher mutation frequency of KRAS (25%) and EGFR uncommon mutations (56.4%) in a Xuanwei cohort compared to a large cohort of Chinese patients(pts) with non-small cell lung cancer (NSCLC) (n=1200). Tumor mutation burden (TMB) is a predictive biomarker for immune checkpoint inhibitors in NSCLC. The aim of this study was to explore gene mutations and the correlation with TMB in Xuanwei NSCLC pts. Materials and Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor samples and matched blood samples of 111 Xuanwei NSCLC pts were collected for 450 cancer gene next-generation sequencing (NGS) targeted panel assay. Genomic alterations (GAs), including single base substitution, short and long insertion/deletions, copy number variations, gene fusions, and rearrangements, and TMB values were assessed with a mean coverage of 1000X. Results: Tumor samples were lung adenocarcinoma (LUAD) (n=99, 89.2%), lung squamous cell carcinoma (LUSC) (n=6, 5.4%), or others (n=6, 5.4%). The cohort included 58 males (52.3%) and 53 females (47.7%) with a median age of 54 years (range 36-78). In total, 73.8% of the pts had one or more actionable GAs. The most common were TP53 (51%), EGFR (49%), KRAS (28%), LRP1B (26%), SPTA1 (23%), NF1 (18%), MED12 (14%), RBM10 (14%), and KMT2C (13%). TMB high (TMB-H) (≥10 muts/Mb) was seen in 58.5% (65/111) of the Xuanwei cohort compared with 29.4% of the Chinese cohort(P <0.001), and the Xuanwei cohort had a higher median TMB than the Chinese cohort (13.1 vs 4.6 muts/Mb, respectively, P <0.001). A higher TMB was associated with higher age, and TMB was higher in males and current/former smokers. Genomic alterations associated with TMB were analysised. Among the common actionable mutations. KRAS mutation cases had a higher median TMB than KRAS wildtype (21.1 vs 9.6 muts/Mb, respectively, P <0.01). TMB-H was seen in 80.6% (25/31) of the Xuanwei KRAS mutation cohort. KRAS G12C (61.3%, 19/31) was the most common mutation and these cases showed lower TMB than non-KRAS G12C cases (P<0.05). EGFR mutations were significantly adversely correlated with TMB (P <0.001). Tumors with the common mutations in EGFR showed significantly lower TMB than those with the uncommon mutations in EGFR (P <0.01). TP53, LRP1B, SPTA1, NF1, and KMT2C mutations frequently occurred in NSCLC pts and were significantly positively correlated with TMB(p<0.001, p<0.001, p<0.001, p<0.001, and p<0.001, respectively). Conclusions: The Xuanwei NSCLC cohort had a higher median TMB than the Chinese NSCLC cohort. KRAS, TP53, LRP1B, SPTA1, NF1, and KMT2C mutations positively correlated with TMB while EGFR mutations were significantly adversely correlated with TMB. NGS revealed potential immunotherapy biomarkers for Xuanwei NSCLC pts. Citation Format: Yinqiang Liu, Jin Liang, Juan Zhao, Shuirong Zhang, Shiyue Zhang, Yunfei Shi, Ruoshan Huang, Xiaojie Wang. The impact of genomic mutational status and correlation with tumor mutation burden in non-small cell lung cancer of Xuanwei, Yunnan Province, China [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5885.

Beyond the Genomic Mutation: Rethinking the Molecular Biomarkers of K-RAS Dependency in Pancreatic Cancers.

Oncogenic v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-RAS) plays a key role in the development and maintenance of pancreatic ductal adenocarcinoma (PDAC). The targeting of K-RAS would be beneficial to treat tumors whose growth depends on active K-RAS. The analysis of K-RAS genomic mutations is a clinical routine; however, an emerging question is whether the mutational status is able to identify tumors effectively dependent on K-RAS for tailoring targeted therapies. With the emergence of novel K-RAS inhibitors in clinical settings, this question is relevant. Several studies support the notion that the K-RAS mutation is not a sufficient biomarker deciphering the effective dependency of the tumor. Transcriptomic and metabolomic profiles of tumors, while revealing K-RAS signaling complexity and K-RAS-driven molecular pathways crucial for PDAC growth, are opening the opportunity to specifically identify K-RAS-dependent- or K-RAS-independent tumor subtypes by using novel molecular biomarkers. This would help tumor selection aimed at tailoring therapies against K-RAS. In this review, we will present studies about how the K-RAS mutation can also be interpreted in a state of K-RAS dependency, for which it is possible to identify specific K-RAS-driven molecular biomarkers in certain PDAC subtypes, beyond the genomic K-RAS mutational status.

Title: SNP Microarray Reveals Predicted Outcomes of a Novel High Risk AML Subgroup with ERG Amplification

Increased expression of ERG (ETS-related gene), a proto-oncogene within the ETS (erythroblast transformation specific) family, is associated with an unfavorable clinical outcome in AML patients. A mechanism for ERG over-expression is ERG amplification. ERG amplification is present in 4-6% of adult AML. This study investigates the genetic profile of ERG-amplified AML by assessment of genomic copy number changes and mutational status of AML-related genes and how this correlates to treatment response and overall outcome. Currently copy number analysis is not recommended by ELN or WHO as a diagnostic standard in AML. An institutional retrospective review of 205 adult AML cases from 2015 to 2018 provided two cohorts: a discovery cohort and control cohort. The discovery cohort includes 11 cases (4%) with ERG amplification. All cases in the discovery cohort include either TP53 mutation and/or loss/LOH of 17p. Thus, we developed a control cohort of 39 cases with TP53 abnormalities without ERG amplifications to determine the role TP53 plays in the observed outcomes. The 179 adult AML cases from the TCGA public database comprises the validation cohort, which incorporates both copy number and molecular sequencing data. Approximately 4% of AML cases in the TCGA dataset show ERG amplification. For our discovery and control cohorts, genomic DNA was extracted (Maxwell, Promega, Madison, WI) from fresh bone marrow aspirates. Copy number/LOH analysis was performed using the CytoSNP microarray (Illumina, San Diego, CA). Sequence variants were evaluated in hotspot regions of 49 genes using the ThunderBolts Myeloid Panel library preparation (RainDance Technologies, Billerica, MA) and paired-end sequencing (MiSeq, Illumina, San Diego, CA). Alignment and variant calling was performed with MiSeq Reporter (Illumina) and NextGene (Soft Genetics, State College, PA) and sequences were annotated using VarSeq (Golden Helix, Bozman, MT). SNP microarray analysis of the discovery cohort revealed a distinct pattern of complex cytogenetics including chromothripsis, TP53 loss/LOH and loss of 5q. Molecular sequencing was available for 10 of 11 patients. All specimens analyzed for mutational status demonstrated TP53 loss of function mutations. The majority of cases (9/11) demonstrated complete loss of function of TP53 by mutation in conjunction with a "second hit" in the form of loss due to deletion or LOH of 17p. These nine patients failed to achieve any response with chemotherapy with a median survival time of 3.8 months. The two surviving patients had a single TP53 mutation with an intact second allele. Both of these patients are post-allogeneic stem cell transplant with no evidence of relapse. There is an association of ERG amplification with MDS as 8 of the 11 patients transformed from very-high risk MDS. The interaction between ERG and TP53 allows one to predict treatment response and outcomes based on the presence or absence of an intact TP53 allele. Within the control group, there were no significant differences in survival or response to treatment based on number of intact TP53 alleles. The validation cohort of TCGA patients confirmed the strong positive correlation between ERG amplification and TP53 mutations. There were 8 cases with ERG amplification. Of these, 6 cases had a similar genetic pattern found in the discovery cohort including TP53 mutations, loss of 5q and complex cytogenetics. The median overall survival (OS) of this cohort of ERG amplified + TP53 mutation was 2.5 months. ERG amplification classifies a high-risk population of AML detected by copy number/LOH analysis with a distinct genetic signature including loss of 5q, complex cytogenetics including chromothripsis and loss of function TP53 mutation. The successive loss of the second TP53 allele in the setting of ERG amplification identifies a subset of patients with resistance to cytarabine and a very poor prognosis. These patients are unlikely to benefit from cytotoxic chemotherapy and may have improved outcomes with alternative regimens and/or clinical trial. ERG amplification is associated with AML that transforms from very-high risk MDS. Recognizing these patients before transforming to AML could allow for early transplant. This high-risk subgroup is currently un-recognized by standard cytogenetics and genetic mutation profiling; therefore, we recommend that copy number/LOH analysis become part of the standard diagnostic testing for MDS and AML. Disclosures No relevant conflicts of interest to declare.

Abstract 1691: The impact of genomic mutational status on PD-L1 expression and tumor mutation burden in non-small cell lung cancer

Abstract Background: Immune checkpoint inhibitors have provided remarkable antitumor effects in non-small cell lung cancer (NSCLC), while their efficacy in patients harboring driver mutations is still controversial and the correlation between driver gene mutations and PD-L1 expression remains unclear. In this study, we explored the association of PD-L1 expression, tumor mutation burden (TMB) and genomic mutation in NSCLC patients. Methods: FFPE tumor and matched blood samples of 244 NSCLC patients were collected for NGS-based targeted panel sequencing from July 2017 to July 2018. Genomic alterations including single nucleotide variations, short and long insertions/deletions, copy number variations and gene rearrangements were assessed. PD-L1 expression, TMB and microsatellite instability status (MSI) were assessed in 244 (100%), 188 (77%) and 198 (81%) patients, respectively. Results: There were 145 males (59.4%) and 99 females (40.6%) with a median age of 62 (31-84) years were involved, including 183 adenocarcinomas and 61 squamous carcinomas. PD-L1 expression of negative, 1-49% and ≥50% were 63.5%, 20.9% and 15.6%, respectively. The median value of TMB was 9.2 (0.8-68.9) muts/Mb and TMB≥10 muts/Mb was seen in 30.9% tumors. All the tumors but one were MSS status. The demographic features were comparable among different PD-L1 expression and TMB level (cutoff is 10 muts/Mb) group. We found recurrent mutations (>10%) in 6 genes, including TP53 (67.6%), EGFR (60.7%), CDKN2A (15.2%), KRAS (13.9%), PI3KCA (11.1%) and LRP1B (10.7%). AKT2 (2.5%) amplification was associated with higher TMB (p=0.011). For the relationship between driver genes and immunotherapeutic biomarkers, TP53 were positively associated with higher PD-L1 expression (p<0.001), while BRAF associated with lower PD-L1 (p<0.001); TP53 and BRAF mutations were correlated with higher TMB (p<0.001, p<0.001; respectively), while EGFR, KRAS, ERBB2 and ALK correlated with lower TMB (p<0.001, p<0.001, p<0.001,p=0.046; respectively). Interestingly, in terms of double mutants, NSCLC patients with EGFR and NOTCH1 co-mutations had lower level of TMB than either single mutant alone. EGFR and RBM10 double mutant was associated with lower PD-L1 expression than either single mutant. Conclusion: This study revealed the impact of genomic mutational status on immunotherapeutic biomarkers in NSCLC. TP53 mutation was associated with higher PD-L while TP53, BRAF mutations and AKT2 amplification correlated with higher TMB. EGFR with NOTCH1 or RBM10 co-mutation is associated with lower TMB or PD-L1, which needs further investigation in their impact on immunotherapy. Our study indicated that NGS panel sequencing and co-mutation analysis could provide potentially insights to precise immunotherapy. Citation Format: Qing Zhou, Weiguang Gu, WenFan Fu, Shijie Mai, Daren Lin, Shiyue Zhang, Wenjing Wang, Peng Zhang. The impact of genomic mutational status on PD-L1 expression and tumor mutation burden in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1691.

CT-based radiogenomic signature to identify isocitrate dehydrogenase (IDH)1/2 mutations in advanced intrahepatic cholangiocarcinoma.

4081 Background: IDH1/2 mutations have a high prevalence (20%) in intrahepatic cholangiocarcinoma (iCCA) and can be associated with therapeutic benefit from IDH inhibitors. Radiomics, a developing field within imaging, has shown its ability to discriminate between tumors of distinct genomic profiles and mutational status. Methods: We developed a radiogenomic signature to robustly predict IDH1/2 mutation status (mutated versus wild-type [WT]) in 22 patients with iCCA using the pretreatment CT scans. The triphasic hepatic CT scan was used to segment the lesion. After semiautomatic segmentation of the tumor, the extracted volume of interest (VOI) was imported into our in-house radiomic pipeline and 610 radiomic features were extracted. The least absolute shrinkage and selection operator regression (LASSO) and minimum redundancy and maximum relevance (mRMR) were used for feature selection. Selected features were used to build a classification model for prediction of IDH1/2 mutation status (XGboost). The performance of the radiomics model was assessed using leave-one-out cross-validation (LOOCV). Results: Of 22 patients, 16 patients (male, 6; female, 10; average age, 55.5 years) had IDH1 (N = 14) or IDH2 (N = 2) mutations and 6 patients (male, 4; female, 2; average age = 55.5 years) had IDH1/2 WT.The CT-derived radiomic signature robustly predicted presence of IDH1/2 mutations versus WT with an area under the curve (AUC), sensitivity and specificity of 98.4%, 83.3% and 93.8%, respectively ( P = 0.037) and in a subgroup analysis presence of IDH1 mutation versus WT with an AUC, sensitivity and specificity of 98.2%, 83.3% and 92.8%, respectively ( P = 0.035). Conclusions: To our knowledge, this is the first study investigating the ability of radiogenomics as a potential method to predict the IDH1/2 mutation status in iCCA patients. Our data suggest that radiogenomic signature may correlate with IDH1/2 mutations and represent a promising non-invasive tool to stratify the patients based on molecular alterations.

2. Distinct genetic signature of a high risk AML subgroup with ERG amplification

Recurrent amplification of 21q22 of the ERG locus has been detected in 6–9% of AML cases and reportedly causes increased gene expression which has been associated with poor overall survival and resistance to cytarabine. The purpose of the study was to determine a genetic profile of ERG-amplified AML by assessment of genomic copy number changes and mutational status of AML related genes.

Silent Sentence Completion Shows Superiority Localizing Wernicke\u2019s Area and Activation Patterns of Distinct Language Paradigms Correlate with Genomics: Prospective Study

Preoperative mapping of language areas using fMRI greatly depends on the paradigms used, as different tasks harness distinct capabilities to activate speech processing areas. In this study, we compared the ability of 3 covert speech paradigms: Silent Sentence Completion (SSC), category naming (CAT) and verbal fluency (FAS), in localizing the Wernicke’s area and studied the association between genomic markers and functional activation. Fifteen right-handed healthy volunteers and 35 mixed-handed patients were included. We focused on the anatomical areas of posterosuperior, middle temporal and angular gyri corresponding to Wernicke’s area. Activity was deemed significant in a region of interest if P < 0.05. Association between fMRI activation and genomic mutation status was obtained. Results demonstrated SSC’s superiority at localizing Wernicke’s area. SSC demonstrated functional activity in 100% of cancer patients and healthy volunteers; which was significantly higher than those for FAS and CAT. Patients with 1p/19q non-co-deleted had higher extent of activation on SSC (P < 0.02). Those with IDH-1 wild-type were more likely to show no activity on CAT (P < 0.05). SSC is a robust paradigm for localizing Wernicke’s area, making it an important clinical tool for function-preserving surgeries. We also found a correlation between tumor genomics and functional activation, which deserves more comprehensive study.

Phase I study of trametinib with neoadjuvant chemoradiation (CRT) in patients with locally advanced rectal cancers (LARC).

3612 Background: The RAS/RAF/MEK signal transduction pathway is critical to the development of colorectal cancer, and tumors harboring KRAS, NRAS, and BRAF mutations were shown to be resistant to radiation. Thus, we conducted a phase I study of trametinib, a potent MEK1/2 inhibitor, in combination with CRT in patients with LARC. Methods: Phase I trial for patients with Stage II/III rectal cancers with a 3+3 design, and an expansion cohort of 12 patients at the maximum tolerated dose (MTD). Trametinib is given orally at 3 dose levels: 0.5mg, 1mg, and 2mg (Mon-Fri). CRT regimen is infusional 5-flourouracil (5FU) 225mg/m2/day (Mon-Fri) and daily fractions of 1.8 Gy (total 50.4Gy). There is a 5-day trametinib lead-in followed by trametinib and CRT. Six to 10 weeks after completion of CRT, patients then proceed to their surgery and adjuvant therapy. The primary endpoint is to identify the MTD and recommended phase 2 dose of trametinib with CRT. Immunohistochemistry staining for phosphorylated –ERK (pERK) and genomic profiling is performed on the tumor samples. Results: Enrollment is complete at all dose levels with18 patients, and 15 patients evaluable for toxicity and response as of Feb 6. One dose-limiting toxicity of diarrhea was observed at the 2mg dose. Grade 3 and most common toxicities are shown in Table 1. No grade 4/5 toxicities have been observed. At 2mg dose level, 3/9 (33%) patients had pathological complete response (pCR) and 2/9 (22%) had a near pCR. Correlative studies confirm decrease in pERK levels with increasing doses of trametinib. Correlation of genomic mutational status with toxicity, response, and outcomes is being analyzed. Conclusions: The combination of trametinib with 5FU CRT is tolerable with promising preliminary activity. Final results will be presented at the meeting. Toxicities. Clinical trial information: NCT01740648. [Table: see text]

Gene Mutations and Treatment Outcome in the Context of Chlorambucil (Clb) without or with the Addition of Rituximab (R) or Obinutuzumab (GA-101, G) - Results of an Extensive Analysis of the Phase III Study CLL11 of the German CLL Study Group

BACKGROUND The CLL11 trial evaluated front line treatment with Clb vs. R-Clb vs. G-Clb in 781 physically unfit patients. Genomic aberrations, IGHV and TP53 mutation status are established prognostic factors in CLL, while NOTCH1, SF3B1, ATM and othersare recurrently mutated genes of potential prognostic and/or predictive value. METHODS We performed amplicon-based targeted next generation sequencing of all coding exons (TP53, ATM, MYD88, FBXW7, BIRC3, XPO1, POT1) or hotspots (NOTCH1 exons 33/34, SF3B1 exons 14-16/18) via Illumina TSCA™ in a representative subset of 689 (88.2%) patients of the CLL11 trial at baseline. Mean read depth was 1363x (range 636-1879) and a variant allele frequency >5% was determined as mutation. RESULTS Incidences of gene mutations were: ATM 19.6%, NOTCH1 18.6%, SF3B1 12.9%, TP53 10.7%, POT1 7.3%, XPO1 6.8%, BIRC3 5.2%, FBXW7 3.8%, and MYD88 3.3%. We found several significant associations with clinical and genetic baseline characteristics including most prominently IGHVunmut with ATMmut (p=0.013), NOTCH1mut (p Regarding response to treatment, TP53mut was associated with reduced ORR for R-Clb (p At a median observation time of 41.9 months, there were 529 (76.8%) events for PFS and 195 (28.3%) events for OS in the cohort studied. G-Clb prolonged PFS as compared to R-Clb in the overall cohort (median 27.1 vs. 15.7 months, HR 0.49, p We performed multivariable Cox regression analysis including clinical and genetic prognostic parameters that were significant in univariate analyses to evaluate their independent prognostic value for PFS and OS. For PFS we identified G-Clb vs. Clb (HR 0.21, p 10 U/l (HR 1.22, p=0.037) as independent prognostic factors. Regarding OS, 17p- (HR 2.57, p 80 years (HR 1.68, p=0.006), Binet stage C (HR 2.1, p 10 U/l (HR 1.59, p=0.004) were identified as independent adverse prognostic factors, while G-Clb vs. Clb (HR 0.61, p=0.022) and G-Clb vs. R-Clb (HR 0.69, p=0.029) correlate with a better OS. CONCLUSION The advent of targeted next generation sequencing has led to a more comprehensive molecular characterization of CLL patients and can provide the basis for genotype specific treatment concepts. G-Clb improves treatment outcome as compared to R-Clb for most subgroups defined by gene mutations and overcomes NOTCH1mut-associated Rituximab resistance. In particular, addition of G achieved more MRD negativity in NOTCH1mut, ATMmut, BIRC3mut and FBXW7mut patients. Both ATMmut and TP53mut remain strong prognostic factors. New treatment regimens with novel agents should be considered for patients with adverse independent prognostic factors for PFS and OS, most notably 17p-, 11q-, IGHVunmut and TP53mut. Disclosures Tausch:Gilead: Other: Travel support, Speakers Bureau; Amgen: Other: Travel support; Celgene: Other: Travel support. Bahlo:F. Hoffman-La Roche: Honoraria, Other: Travel grant. Goede:Glaxo Smith Kline: Consultancy, Honoraria; F- Hoffmann-LaRoche: Consultancy, Honoraria, Other: Travel grants; Janssen: Consultancy, Other: Travel grants; Gilead: Consultancy; Mundipharma: Consultancy, Honoraria; Bristol Myer Squibb: Honoraria. Ritgen:Roche: Membership on an entity9s Board of Directors or advisory committees, Research Funding. Langerak:InVivoScribe Technologies: Patents & Royalties: Royalties are provided to European Network (EuroClonality); F. Hofmann-LaRoche, Genentech: Research Funding. Fingerle-Rowson:F. Hoffmann-LaRoche: Employment. Kneba:Amgen: Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants, Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants; Janssen-Cilag: Consultancy, Honoraria, Other: Travel grants; Glaxo-SmithKline: Other: Travel grants; Roche: Consultancy, Honoraria, Other: Travel grants, Research Funding. Fischer:Roche: Other: travel grants. Hallek:F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau. Stilgenbauer:Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding.

Gene Mutations and Treatment Outcome in CLL Patients Treated with Chlorambucil (Chl) or Ofatumumab-Chl (O-Chl): Results from the Phase III Study COMPLEMENT1 (OMB110911)

BACKGROUND: Genomic aberrations and IGHV mutation status are established prognostic factors in CLL. With TP53, NOTCH1, SF3B1, ATM, MYD88, FBXW7, BIRC3 and POT1 recurrently mutated genes were found in CLL and were discussed to associate with disease characteristics and to affect therapy efficacy and outcome.METHODS: We assessed the incidence and impact of gene mutations in the COMPLEMENT1 trial (1st line Chl vs. O-Chl). Pretreatment samples were available from 376 patients (84.1%) and this cohort was representative of the full trial population. Mutations were analyzed by amplicon-based targeted NGS using Illumina Miseq for all coding exons (TP53, ATM, MYD88, FBXW7, BIRC3 and POT1) or hotspot exons (NOTCH1, SF3B1). Additionally, the exact variant frequency was determined.RESULTS: The incidences of gene mutations were: TP53 8.2%, NOTCH1 14.9%, SF3B1 14.1%, ATM 10.9%, MYD88 2.7%, FBXW7 3.5%, POT1 7.7%, and BIRC3 2.7%.Regarding baseline characteristics, we found significant associations: TP53mut with high ß2MG (p=0.01), 17p- (p<0.01), and unmutated IGHV (p=0.01); ATMmut with high WBC (p=0.02), and 11q- (p<0.01); MYD88mut with mutated IGHV (p=0.02); FBXW7mut with 17p- (p=0.02), and +12q (p<0.01). BIRC3mut was only present in IGHV unmutated cases (p<0.01), was more frequent in 11q- (p<0.01), +12q (p=0.05), and in cases with NOTCH1mut (p=0.05). POT1mut was more frequent in NOTCH1mut cases (p=0.02) without associations with any other baseline parameter.Regarding response to treatment, TP53mut was significantly associated with reduced ORR rate (p<0.01). CR rate was not correlated with mutations in the covered genes.At a median follow-up of 31.7 months, there were 249 (66%) events for PFS and 63 (16.8%) events for OS. O-Chl as compared to Chl resulted in significantly improved PFS (median 22.4 vs. 13.1 months, HR 0.54, p<0.01). In univariate analyses, TP53mut (HR 2.07, p<0.01), NOTCH1mut (HR 1.50, p=0.01) and SF3B1mut (HR 1.66, p=0.01) were associated with shorter PFS, whereas ATM and other candidate genes showed no association (ATMmut: HR 1.40, p=0.07).Analyzing both treatment arms separately, TP53mut had an impact on PFS with Chl and O-Chl treatment (HR 1.92, p=0.04 and HR 2.49, p<0.01). Notably, NOTCH1mut was associated with outcome in O-Chl only (HR 2.01, p<0.01 vs. HR 1.14, p=0.59) resulting in a reduced beneficial effect from the addition of Ofatumumab to Chlorambucil treatment. ATMmut and BIRC3mut mutations were only adverse prognostic factors with Chl monotherapy (ATMmut: HR 1.69, p=0.05 vs. HR 1.35, p=0.27; BIRC3mut: HR 2.84, p=0.04 vs. HR 0.99, p=0.99). OS was reduced significantly only in TP53mut cases (HR 3.69, p<0.01). Of note, none of the MYD88mut cases (n=10) had died within the follow-up period.To identify genomic factors of independent prognostic impact, we performed multivariable Cox regression analyses for PFS and OS including treatment arms, 11q-, +12q, 17p-, IGHV and all candidate gene mutations. For PFS, the following independent prognostic factors were identified: O-Chl (HR 0.46, p<0.01), 17p- (HR 3.14, p<0.01), 11q- (HR 1.57, p=0.01), unmutated IGHV (HR 1.43, p=0.02), TP53mut (HR 1.81, p=0.03), NOTCH1mut (HR 1.63, p<0.01) and SF3B1mut (HR 1.54, p=0.02). Regarding OS, only 17p- (HR 4.07, p<0.01), and unmutated IGHV (HR 1.81, p=0.05) were identified as independent adverse prognostic factors with TP53mut showing a trend (HR 2.14, p=0.10).CONCLUSION: We performed mutational analyses for the 8 most frequent mutated genes in CLL in the COMPLEMENT1 trial evaluating 1st line O-Chl against Chl. An independent prognostic impact was identified for TP53mut, NOTCH1mutand SF3B1mut regarding PFS. Notably, NOTCH1mut affected outcome mainly with O-Chl treatment, whereas ATMmut and BIRC3mut were associated with outcome with Chl monotherapy. In multivariate analysis for OS, none of the gene mutations, but the established parameters IGHV and 17p- had independent prognostic impact. DisclosuresTausch:GlaxoSmithKline: Research Funding, Travel support Other. Hillmen:GSK: Honoraria, Research Funding. Offner:GlaxoSmithKline: Honoraria, Research Funding. Janssens:GSK: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Roche: Speakers Bureau; Mundipharma: Speakers Bureau. Mayer:Glaxo: Research Funding; Roche: Research Funding. Panagiotidis:GlaxoSmithKline: Consultancy, Honoraria. McKeown:GlaxoSmithKline: Employment. Gupta:GlaxoSmithKline: Employment. Stilgenbauer:GlaxoSmithKline: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Platform comparison for evaluation of ALK protein immunohistochemical expression, genomic copy number and hotspot mutation status in neuroblastomas.

ALK is an established causative oncogenic driver in neuroblastoma, and is likely to emerge as a routine biomarker in neuroblastoma diagnostics. At present, the optimal strategy for clinical diagnostic evaluation of ALK protein, genomic and hotspot mutation status is not well-studied. We evaluated ALK immunohistochemical (IHC) protein expression using three different antibodies (ALK1, 5A4 and D5F3 clones), ALK genomic status using single-color chromogenic in situ hybridization (CISH), and ALK hotspot mutation status using conventional Sanger sequencing and a next-generation sequencing platform (Ion Torrent Personal Genome Machine (IT-PGM)), in archival formalin-fixed, paraffin-embedded neuroblastoma samples. We found a significant difference in IHC results using the three different antibodies, with the highest percentage of positive cases seen on D5F3 immunohistochemistry. Correlation with ALK genomic and hotspot mutational status revealed that the majority of D5F3 ALK-positive cases did not possess either ALK genomic amplification or hotspot mutations. Comparison of sequencing platforms showed a perfect correlation between conventional Sanger and IT-PGM sequencing. Our findings suggest that D5F3 immunohistochemistry, single-color CISH and IT-PGM sequencing are suitable assays for evaluation of ALK status in future neuroblastoma clinical trials.

Identification of driver gene alterations in Chinese female patients with squamous cell carcinomas of the lung.

e19087 Background: Smoking remains the leading risk factor of NSCLC. Activating mutations in EGFR gene have been reported more frequently in never smokers than smokers with NSCLC. Female pts with squamous cell carcinomas of the lung (SQCLC) differ from males in that most females are life-long never/light smokers. However, molecular abnormalities in female pts with SQCLC have not been well-characterized. Methods: We retrospectively screened 38 female and 40 male pts with SQCLC in China from 2009 to 2012 to determine the prevalence of alterations in EGFR, KRAS, ALK, PI3K, PTEN and FGFR1. Mutational analysis of EGFR, PIK3CA, KRAS and PTEN was performed using PCR-based DNA sequencing. FGFR1 amplification and ALKrearrangements were detected by FISH. A Cox regression model was used to assess the association between genomic mutation status and clinical features as well as OS. Results: 10.3% (8/78) pts were detected with EGFR mutation, 6.4% (5/78) with KRAS mutation, 7.7% (6/78) with PI3K mutation and 6.4% (5/78)...

Somatic copy number abnormalities (SCNAs) and mutations in PI3K/AKT pathway as prognostic factors for overall survival (OS) in platinum-treated locally advanced or metastatic urothelial tumors.

4511 Background: An integrative analysis was conducted to identify genomic alterations and mutational status on the gene pathway level that could predict OS in pts with urothelial cancers (UC) trea...

Somatic copy number abnormalities (SCNAs) and mutations in PI3K/AKT pathway and prognostic significance for overall survival (OS) in platinum-treated locally advanced or metastatic urothelial cancer (UC).

358 Background: An integrative analysis was conducted to identify genomic alterations and mutational status on the gene pathway level that could predict OS in patients (pt) with UC treated with platinum chemotherapy. Methods: DNA was extracted from 94 FFPE samples from invasive UC and screened for mutations and CNAs. Of those, complete clinical data was available from 85 cases. Mutations in the cancer genome were analyzed by a mass-spectrometry based genotyping platform (Oncomap version 3) and genomic imbalances were detected by comparative genomic hybridization (CGH) analysis. Regions with threshold of log2 ratio ≥0.4, or ≤ -0.6 were defined as either having copy number gain or loss and significantly recurrent CNAs across the set of samples was determined using a GISTIC analysis. To better define the co-occurrence pattern of mutations and CNAs, we grouped genes into 3 core signal transduction pathways: (1) TP53 pathway; (2) Abnormalities of in the RTK-RAS-RAF pathway including either ERBB2, FGFR3, MET, FGFR1, KRAS BRAF, RAF1 and NF1; (3) abnormalities on PI3K/AKT pathway including PTEN, PIK3CA, AKT1, TSC1, and MTOR. OS was measured from treatment to time of death or censored. Cox regression was used to assess pathways abnormalities and mutations with outcome. Results: Thirty five samples (41%) harbored mutations on at least one gene, mainly TP53 (16%), PIK3CA (9%), FGFR3 (2%), HRAS/KRAS (5%), and CTNNB1 (1%). Sixty-six per cent of patients had some sort of copy number variation. PI3K/AKT was the most affected pathway and was associated with shorter overall survival (hazard ratio (HR) =2.3 with 95% CI: 1.3 to 4.2). There was no significant association between mutational status and OS. Combined analysis of SNCA and mutations in the same pathway will be presented. Conclusions: Patients with advanced urothelial cancer receiving platinum chemotherapy have a significantly poor survival which correlated with copy number abnormalities in the PI3K/AKT pathway. This can help to identify patients that can benefit from targeted pathway specific agents.