Tumor Mutation Burden Level Research Articles

Ductal (PDC), acinar (PAC) and neuroendocrine (PNC) carcinomas of the prostate: A comparative comprehensive genomic profiling (CGP) study.

5064 Background: PDC, PAC and PNC are histologic subtypes of prostate cancer (PC). We queried whether these subsets would share similar genomic alterations (GA) reflecting their disease biology and clinical features. Methods: CGP was performed using a hybrid capture-based assay on 61 PDC, 4,132 PAC and 217 PNC. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. Results: The age, GA per tumor and TP53 GA of PDC, PAC and PNC were similar (Table). RB1 GA were predominant in PNC. TMPRSS2:ERG fusions were most frequent in PNC, intermediate in PAC and lowest in PDC. AR GA were more often identified in PAC than PDC or PNC whereas PTEN GA were most frequent in PDC than PAC or PNC. Targetable GA were identified in all 3 groups when focused on BRCA2 (PARP inhibitors) and PIK3CA (MTOR inhibitors). ATM GA (PARP inhibitors) were more common in PAC than PDC or PNC. BRAF GA (BRAF/MEK inhibitors) were more frequent in PDC and PAC than PNC. CDK12 GA potentially associated with immunotherapy (IO) benefit were similar in PDC and PAC and low in PNC. Low frequencies of MSI-High and low median TMB levels were similar in all 3 groups. Conclusions: The pathologic features of PDC, PAC and PNC have been classically maintained as representative of 3 different tumor types with potentially contrasting histogenesis. In the current CGP based study, all 3 tumor types did not display significant differences in genomic signatures other than the high RB1 GA. CGP may reveal biomarkers that could direct patients to targeted (PARP, MTOR and BRAF/MEK inhibitors) or immunotherapies ( CDK12 GA, MSI-High or high TMB status) especially in PDC and PAC. [Table: see text]

Evaluation of TMB estimates for the prediction of response to immune checkpoint blockage.

2632 Background: Tumor mutational burden (TMB) is an emerging biomarker in immuno-oncology (IO). Panel sequencing (PS) is a promising approach for its implementation in clinical practice. Methods: We analyzed TMB as predictor of IO response in a multi-cancer cohort published by Miao et. al. The performance of three large panels (Illumina TSO500, Qiagen TMB [QIAseq] and Oncomine TMB [OTMB]) and two small panels (Illumina TST170 and Oncomine Comprehensive Assay [OCAv3]) was compared to WES by in silico simulations. Separation of responders (PR/CR) from non-responders (PD) was analyzed in the multi-cancer cohort (n = 193), in the lung cancer (n = 36) and in the melanoma (n = 125) subcohort. We also simulated PS in the TCGA pan-cancer cohort. Results: In lung cancer, TMB was strongly predictive for IO response (area under ROC curve [AUC] 0.78-0.94). WES performed (borderline)-significantly better than PS for all five panels (OCAv3: p = 0.011, TST170: p = 0.01, QIAseq: p = 0.048, OTMB: p = 0.063, TSO500: p = 0.11). For the cut-point of 199 mutations, misclassification rates compared to WES (16.7%) were borderline-significantly higher for the small panels OCAv3 (33.3%, p = 0.087) and TST170 (36.1%, p = 0.054), but not for the large panels. In melanoma, TMB was moderately predictive (AUC 0.58-0.63) and WES performed (borderline)-significantly better than the OCAv3, TST170, QIAseq and TSO500 panels. In the multi-cancer cohort, WES did not perform better than PS. TBM estimates from PS include an inherent fuzziness originating from restriction to a limited part of the coding sequence. Based on a random mutation model, we derived a mathematical formula for the coefficient of variation (CV) of TMB: The CV decreases inversely proportional with both the square root of the TMB level and with the square root of the panel size. We showed that the mathematical law is valid for real-word mutation data. Conclusions: Small panels (size < 1 Mpb) performed imprecise in diagnostic TMB estimation. Even using the largest commercially available panels it can be challenging to capture the full predictive information of TMB. The detrimental effect of small panel size can be addressed by using larger panels, but halving the CV of TMB necessitates quadruplication of the panel size.

Tumor mutation burden in biliary tract cancers with ERBB family or DNA damage repair gene mutations.

e15602 Background: Biliary tract cancers (BTCs) are aggressive malignancies associated with resistance to chemotherapy. Though immunotherapy presents a promising results based on several studies, the overall response rate is relative low. Tumor mutation burden (TMB) has been considered as a predictive biomarker of immunotherapy. However, the association between common mutations and TMB in BTCs are still unclear. Methods: We analyzed 76 BTC samples (32 cholangiocarcinoma and 44 gallbladder cancer patients) from the Cancer Genome Altas (TCGA) by whole-exome sequencing and 408 samples (276 cholangiocarcinoma and 132 gallbladder cancer patients) from Chinese clinical cohort by 381-gene targeted sequencing. The association between ERBB family members or DNA damage repair (DDR) genes and TMB was explored, respectively. TMB was defined as total number of somatic non-synonymous mutations in coding region. Results: The mutation frequency of ERBB genes was similar between the TCGA cohort and the clinical cohort (cholangiocarcinoma, 13.6% vs 10.9%; gallbladder cancer, 21.9% vs 17.4%). However, DDR mutation rate from the TCGA data was greater than that from the clinical data without consideration the pathological type (cholangiocarcinoma, 40.9% vs 21.0%; gallbladder cancer, 62.5% vs 29.5%). In cholangiocarcinoma, any ERBB gene mutation was associated with higher TMB level in TCGA cohort (P = 0.0013) and clinical cohort (P = 0.0008). Any DDR gene mutation also significantly contributed to higher TMB in two cohorts (TCGA cohort, P = 0.0001; clinical cohort, P = 0.0139). In gallbladder cancer, the same result was discovered on the significant association between ERBB genes statue and higher TMB in both two data (TCGA cohort, P = 0.0370; clinical cohort, P = 0.0008). On the contrary, higher TMB level was observed in patients carrying any DDR alterations (P = 0.0017) in TCGA cohort, but the association was not found in Chinese cohort (P = 0.2660). Besides, prognosis analysis was performed on patients in TCGA cohort. Cholangiocarcinoma patients harboring any ERBB gene mutation represent significantly poorer overall survival (median, 10.6 vs. 40.1 months; HR, 3.37; P = 0.0186). Conclusions: Chinese BTC patients may exhibit distinct gene mutations. These findings were the supplement of better comprehending BTC genomic characteristics. Further studies are needed to evaluate the efficacy of immunotherapy in BTC patients with ERBB or DDR mutation in Chinese clinical cohort.

Genomic Alterations of Renal Cell Carcinoma and Clinical Implications in the Chinese Population.

BackgroundThe aim of this study was to investigate the genomic alterations of renal cell carcinoma (RCC) in Chinese patients and to evaluate the correlations between significantly mutated genes and tumor mutation burden (TMB) levels in RCC.Material/MethodsTwo batch of specimens were collected from patients with RCC. Cohort 1 enrolled 17 RCC patients. Specimens and clinicopathological data were collected and the duration of disease-free survival were evaluated with a follow-up from 2 weeks to longer than 1 year. Cohort 2 collected 70 clear cell RCC (ccRCC) tissues and blood specimens. Next-generation sequencing were used to detect the genomic variations in those specimens in both cohorts and TMB in cohort 2. Clinicopathological features of the 2 cohorts were collected and the χ2 test or Fisher’s exact test was used for categorical variables stratified by TMB values.ResultsOur present study demonstrated that the top 3 most frequent aberrated genes in Chinese ccRCC patients were ABCB1, UGT1A1, and VHL, with percentages of 50.00%, 42.86%, and 34.52% respectively. And only 1 gene, which was ABCB1, showed statistically significant difference (P=0.047) stratified by TMB levels. In addition, 6 oncogenic pathways were involved in ccRCC cases in the 2 cohorts. Only 5 out of the 8 most common altered genes of RCC from COSMIC or TCGA databases were detected in our study.ConclusionsThe genomic alterations of Chinese RCC patients were different from that in TCGA and COSMIC. No significant genomic alterations were found correlating to TMB levels in ccRCC. Non-silent mutation of VHL may be a predictor for the outcome of ccRCC treated with axitinib.

Ductal and acinar carcinomas of the prostate: A comparative comprehensive genomic profiling study.

271 Background: Prostatic ductal carcinoma (PDC) is an uncommon centrally located prostate with a characteristic tubulopapillary histology. PDC shares some biomarker features with the more common prostatic acinar carcinoma (PAC) including PSA expression. Methods: In this comparative comprehensive genomic profiling (CGP) study, FFPE samples from 61 clinically advanced PDC and 4,132 PAC were profiled for all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. Results: The age, frequency of genomic alterations (GA) per tumor and TP53 GA of PDA and PAC were similar (Table). TMPRSS2:ERG fusions and AR GA were more often identified in PAC than PDC whereas PTEN GA were more common in PDC than PAC. Targetable GA were identified at similar frequencies in both groups focused on BRCA2 (PARP inhibitors), BRAF (BRAF/MEK inhibitors) and PIK3CA (MTOR inhibitors). ATM GA (PARP inhibitors) were more common in PAC than PDC. CDK12 GA potentially associated with immunotherapy (IO) benefit were similar in PDA and PAC as were the frequencies of MSI-High status, median TMB and high TMB levels. Conclusions: The pathologic features of PDC and PAC have been classically maintained as representative of 2 different tumor types with potentially contrasting histogenesis. In the current CGP-based study, PDA and PAC did not display significant differences in genomic signatures. CGP may reveal biomarkers that could direct patients to targeted (PARP, MTOR and BRAF/MEK inhibitors) or immunotherapies ( CDK12 GA, MSI-High or high TMB status) in both PDA and PAC. [Table: see text]

Genomic findings in adenocarcinoma of the urinary bladder.

132 Background: Prostatic ductal carcinoma (PDC) is an uncommon centrally located prostate with a characteristic tubulopapillary histology. PDC shares some biomarker features with the more common prostatic acinar carcinoma (PAC) including PSA expression. Methods: In this comparative comprehensive genomic profiling (CGP) study, FFPE samples from 61 clinically advanced PDC and 4,132 PAC were profiled for all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. Results: The age, frequency of genomic alterations (GA) per tumor and TP53 GA of PDA and PAC were similar (Table). TMPRSS2:ERG fusions and AR GA were more often identified in PAC than PDC whereas PTEN GA were more common in PDC than PAC. Targetable GA were identified at similar frequencies in both groups focused on BRCA2 (PARP inhibitors), BRAF (BRAF/MEK inhibitors) and PIK3CA (MTOR inhibitors). ATM GA (PARP inhibitors) were more common in PAC than PDC. CDK12 GA potentially associated with immunotherapy (IO) benefit were similar in PDA and PAC as were the frequencies of MSI-High status, median TMB and high TMB levels. Conclusions: The pathologic features of PDC and PAC have been classically maintained as representative of 2 different tumor types with potentially contrasting histogenesis. In the current CGP-based study, PDA and PAC did not display significant differences in genomic signatures. CGP may reveal biomarkers that could direct patients to targeted (PARP, MTOR and BRAF/MEK inhibitors) or immunotherapies ( CDK12 GA, MSI-High or high TMB status) in both PDA and PAC. [Table: see text]

T-Cell–Inflamed Gene-Expression Profile, Programmed Death Ligand 1 Expression, and Tumor Mutational Burden Predict Efficacy in Patients Treated With Pembrolizumab Across 20 Cancers: KEYNOTE-028

Biomarkers that can predict response to anti-programmed cell death 1 (PD-1) therapy across multiple tumor types include a T-cell-inflamed gene-expression profile (GEP), programmed death ligand 1 (PD-L1) expression, and tumor mutational burden (TMB). Associations between these biomarkers and the clinical efficacy of pembrolizumab were evaluated in a clinical trial that encompassed 20 cohorts of patients with advanced solid tumors. KEYNOTE-028 ( ClinicalTrials.gov identifier: NCT02054806) is a nonrandomized, phase Ib trial that enrolled 475 patients with PD-L1-positive advanced solid tumors who were treated with pembrolizumab 10 mg/kg every 2 weeks for 2 years or until confirmed disease progression or unacceptable toxicity occurred. The primary end point was objective response rate (ORR; by RECIST v1.1, investigator review). Secondary end points included safety, progression-free survival (PFS), and overall survival (OS). Relationships between T-cell-inflamed GEP, PD-L1 expression, and TMB and antitumor activity were exploratory end points. ORRs (with 95% CIs) ranged from 0% (0.0% to 14.2%) in pancreatic cancer to 33% (15.6% to 55.3%) in small-cell lung cancer. Across cohorts, median (95% CI) PFS ranged from 1.7 months (1.5 to 2.9 months) to 6.8 months (1.9 to 14.1 months) in pancreatic and thyroid cancers, respectively, and median OS from 3.9 months (2.8 to 5.5 months) to 21.1 months (9.1 to 22.4 months) in vulvar and carcinoid tumors, respectively. Higher response rates and longer PFS were demonstrated in tumors with higher T-cell-inflamed GEP, PD-L1 expression, and/or TMB. Correlations of TMB with GEP and PD-L1 were low. Response patterns indicate that patients with tumors that had high levels of both TMB and inflammatory markers (GEP or PD-L1) represent a population with the highest likelihood of response. Safety was similar and consistent with prior pembrolizumab reports. A T-cell--inflamed GEP, PD-L1 expression, and TMB predicted response to pembrolizumab in multiple tumor types. These biomarkers (alone/in combination) may help identify patients who have a higher likelihood of response to anti-PD-1 therapies across a broad spectrum of cancers.

Tumor Mutational Burden and Other Immunotherapy Markers in Histiocytic Neoplasms Using Next Generation Sequencing

IntroductionHistiocytic neoplasms are rare hematological malignancies that have protean clinical manifestations and can pose significant management challenges. Recently, vemurafenib was approved by the US-FDA for treatment of BRAF-V600-mutant Erdheim-Chester disease (ECD). However, there is a lack of FDA-approved therapies for other histiocytic neoplasms such as Langerhans cell histiocytosis (LCH) and Rosai-Dorfman disease (RDD). Over the last 5 years, immune checkpoint inhibitors such as programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors have shown significant improvement in outcomes among patients with several hematological and solid organ malignancies. In order to identify appropriate treatment candidates for these therapies, predictive biomarkers have been developed in various cancers. Evidence from solid tumors has suggested a favorable response to checkpoint inhibitor therapy with higher tumor mutational burden (TMB), defined as the number of mutations within a tumor genome. Next generation sequencing (NGS) of various tumors has shown an association between TMB of < 5 mutations/megabase (mut/Mb) to be associated with an absence of benefit from checkpoint inhibitors. In addition, high levels of PD-1/PD-L1 expression and microsatellite instability (MSI) are also correlated with response to therapy. The latter may be a result of somatic or germline alterations in DNA mismatch repair genes. In this study, we report the results for these biomarkers using NGS in patients with histiocytic neoplasms.MethodsWe utilized TempusTM NGS platform to analyze the tissue specimen of patients with histiocytic neoplasm. The Tempus xO Assay (Tempus Labs; Chicago, IL) combines a 1,711 gene targeted somatic and germline DNA sequencing panel with RNA-sequencing to detect both germline and somatic single nucleotide polymorphisms, indels, copy number variants, and gene rearrangements causing chimeric mRNA transcript expression in a wide array of solid tumor types. The assay utilizes formalin-fixed paraffin-embedded tumor samples and matched blood samples. TMB was calculated and reported as somatic mutations in tumor tissue per million base-pairs or mut/Mb. RNA sequencing was utilized to assess for PD-L1 and PD-1 gene expressions as compared to matched tumor and normal reference sets. Both PD-L1 and PD-1 gene expressions were reported as percentiles. DNA mismatch repair status was predicted by analysis of alterations in five common mismatch repair genes in somatic and germline DNA (MSH2, MSH6, MLH1, PMS2, and EPCAM). If there were no alterations identified in these genes, the mismatch repair status was predicted as microsatellite stable (MSS).ResultsA total of 13 patients with histiocytic neoplasms were included in the study. The distribution of individual histiocytic neoplasms was as follows: RDD (n=9), ECD (n=3), and LCH (n=1). The median TMB for RDD and ECD patients was 0.17 mut/Mb. For the one patient with LCH, the TMB level was 0.51 mut/Mb. Individual TMB levels are shown in figure 1. PD-L1 and PD-1 expression levels are depicted in table 1. Compared to normal reference sets, the PD-L1 expression was elevated in one patient each with RDD and ECD, and PD-1 expression was elevated in two patients each with RDD and ECD. For both ECD patients with higher PD-1 expression, NGS also showed presence of BRAF-V600E in the tumor tissue. The LCH patient had a low level of PD-L1 and PD-1 expression. For patients where evaluation of DNA mismatch repair was feasible on the tissue specimen (n=4), none showed related somatic or germline alterations.ConclusionsIn our series, the histiocytic neoplasms RDD, ECD, and LCH demonstrated low levels of TMB. None of the RDD and ECD patients were found to have alterations in DNA mismatch repair genes. Other markers of immunotherapy such as PD-L1/PD-1 expression appeared to be higher in ECD patients with BRAF-V600E, but in only a small subset of RDD patients. The low TMB seen in our study suggests that these histiocytic neoplasms may be less likely to respond to immune checkpoint inhibitors such as anti-PD-1 and anti-PD-L1 agents as compared to the tumors with high TMB. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MA06.07 Genetic and Epigenetic Alterations are Associated with Tumor Mutation Burden in Non-Small Cell Lung Cancer

Although several studies have indicated that tumor mutation burden (TMB) is associated with non-small cell lung cancer (NSCLC) development and clinical efficacy of immune checkpoint inhibitors (CPIs), identification of factors associated with TMB is still a major biological issue. It is well-known that DNA transcription can be regulated through methylation and demethylation, gene silencing caused by DNA hypermethylation is associated with cancer development. However, the relationship between DNA methylation and TMB in NSCLCs remains unclear. The landscape of DNA sequence in Chinese NSCLCs population were surveyed by using whole-exome sequencing (WES) by profiling 178 lung tissues (89 without any systemic anti-cancer therapy tumors and matched normal lung tissues). According to the 104 median-level of TMB in our cohort, high TMB (n=16, 252-465 range mutations per tumor) and low TMB (n=13, 57-79 range mutations per tumor) groups were divded. The NSCLC methylome between high and low TMB was characterized on a genome-wide scale using Illumina Infinium MethylationEPIC arrays combined with the WES data. The results show frequently aberrant DNA methylation, abundant chromosomal amplifications and deletions, and mutational signatures in high TMB lung cancer. Combining with clinical data, cigarette smoking associated with high TMB were observed in our cohort. Cancer-specific epigenetic alterations were observed in 294,141 CpG sites, comprising both tumor hyper- (769,38) and hypo- (217,203) methylation in high TMB lung cancer while none in low. These different methylations sites cover 1232 genes including 25 HOX genes. Global DNA hypomethylation and TP53 mutation, associated with increased chromosomal instability, were associated with TMB in NSCLCs.The high TMB NSCLCs are characterized by numerous copy number alterations and aberrantly methylated sites and display distinct mutational signatures. 25 hypermethylated HOX genes can be potentially useful as DNA methylation markers for prediction of TMB level. The results provide insights into the epigenetic impact of TMB, which may contribute to improve precison management of NSCLCs.

Abstract LB-339: Biomarkers predictive of response to pembrolizumab in head and neck cancer (HNSCC)

Abstract Background: Biomarkers predictive of response to anti-PD-1 therapy include tumor mutational burden (TMB) and inflammatory biomarkers such as PD-L1 expression and T-cell activated gene expression profile (GEP). This study evaluated relationships between TMB, PD-L1 expression, and GEP and response to pembrolizumab in HNSCC patients in the KEYNOTE (KN)-012 and KN-55 trials. Methods: Data were combined from 261 HNSCC patients in KN-012 (NCT01848834, subsets of B1 [PD-L1+, ≥1%, modified proportion score or interface pattern, QualTek IHC; n=34] and B2 [n=73] cohorts) and KN-055 (NCT02255097, platinum/cetuximab resistant, n=154) who had TMB data available by whole exome sequencing. Of these, 258 patients had PD-L1 data (PD-L1 IHC using 22C3 pharmDx, CPS readout) and 240 patients had GEP score (RNA analyzed on NanoString nCounter). HPV+/- status was assessed by p16 IHC and WES methods. Statistical testing of relationships between biomarkers and best overall response (BOR) by logistic regression and progression free survival (PFS) by Cox regression was prespecified and performed in a blinded manner. Results: TMB, PD-L1 CPS and GEP were each significantly associated with BOR in the overall population (p&amp;lt;0.01). WES identified 181 HPV- and 80 HPV+ patients. TMB and GEP were each highly associated with BOR regardless of HPV- (p=0.0034 and 0.0084) and HPV+ (p=0.0446 and 0.0008) status, respectively. PD-L1 CPS showed similar associations with BOR in HPV- (p=0.0649) and HPV+ (p=0.0003) patients. TMB showed no correlation with PD-L1 (r = -0.047) and GEP (r = -0.135); whereas PD-L1 CPS and GEP were moderately correlated (r = 0.469), consistent with an interferon-γ induced T-cell activated microenvironment including PD-L1 induction. TMB, PD-L1 CPS and GEP remained significantly predictive for BOR when assessed individually in joint models of TMB and PD-L1, and TMB and GEP (all p&amp;lt;0.001). Responses were higher in patients who had high levels of both TMB and inflammation as measured by PD-L1 CPS or GEP, than in patients with low TMB and inflammation. Similar results were observed for PFS. There was good concordance (81.5%) between p16 IHC and WES HPV detection methods, and findings were generally similar for HPV- (n=201) and HPV+ (n=58) patients identified by p16 IHC, indicating the feasibility of using the WES method. Conclusion: TMB, PD-L1 and T-cell inflamed GEP were independently predictive of response to pembrolizumab in HNSCC patients, in general regardless of HPV status. When used alone or jointly, these biomarkers may have utility in characterizing responses to anti PD-1 therapies and novel cancer regimens in HNSCC. Citation Format: Tanguy Y. Seiwert, Robert Haddad, Joshua Bauml, Jared Weiss, David G. Pfister, Shilpa Gupta, Ranee Mehra, Iris Gluck, Hyunseok Kang, Francis Worden, J. Paul Eder, Makoto Tahara, Barbara Burtness, Stephen V. Liu, Andrea Webber, Lingkang Huang, Robin Mogg, Razvan Cristescu, Jonathan Cheng, Laura Q. Chow. Biomarkers predictive of response to pembrolizumab in head and neck cancer (HNSCC) [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-339.

Abstract 3437: Genomic alterations, mutation burden, and microsatellite instability status of Chinese intrahepatic cholangiocarcinoma

Abstract Background: Intrahepatic cholangiocarcinoma (ICC) is often diagnosed at an advanced stage with limited therapeutic options and poor prognosis. Targeted and immune-therapy have yielded promising therapeutic effects in various solid tumor patients (pts). Thus the understanding of ICC genomic profiling, especially which of Chinese pts, seems to be necessary for the treatment strategy for ICC. Methods: Comprehensive genomic profiling of molecular alterations and predictive biomarkers to personalized treatment genes were performed on Formalin Fixed Paraffin Embedded (FFPE) samples and matched blood samples of 69 Chinese ICC pts. Genomic alterations (GAs) including substitutions, short and long insertion/deletions (indels), copy number variations and gene rearrangements were assessed. Tumor mutation burden (TMB) of total somatic substitutions and indels per megabase after filtering known driver mutations was calculated. Microsatellite instability (MSI) status was determined by identifying and scoring multiple mono-nucleotide repeats loci. Results: Median age of the 69 ICC pts was 58 years (range: 38-88), and male vs female was approximately 3:1. Average of 3.7 GAs /sample and 0.9 actionable GAs/sample were detected. The most widespread genomic alterations among the pts were revealed as TP53 (44.9%), ARIDIA (23.2%), KRAS (18.8%), BAP1 (14.5%), IDH1/2 (14.5%), PIK3CA (11.6%), FGFR2/3 (10.1%), MLC1 (8.7%), CDKN2A/B (8.7%), BRAF (7.2%), TERT (7.2%), PBRM1 (7.2%). The actionable mutations of the PI3K/mTOR pathway were found in 18.8% of the pts. In total, pts with one or more actionable GAs were 62.3%. Pts presented MSI-high were 7.2% of the total. Proportion was 10.1% for the pts with TMB value higher than 20 mut/Mb, and 32.8% for those higher than 10 mut/Mb. The TMB range of the 69 pts was 1.1 to 58.4 mut/Mb, and median was 7.4 mut/Mb. Conclusions: In our study, 62.3% of Chinese ICC pts were detected to have actionable GAs which could potentially guide and influence their personalized treatments. The identification of more frequent high TMB level than the published western populations indicated that Chinese ICC pts may more likely derive therapeutic benefits from immune checkpoint inhibitors. Citation Format: Jingyu Cao, Bo Jiang, Zimin Liu, Weibin Shu, Lei Li, Han Yang, Hua Li, Zhiyu Xiao, Maolin Yan, Jie Lin, Siqin Liu, Peng Zhang, Milind Javle, Ming Yao, Kai Wang, Haitao Zhao. Genomic alterations, mutation burden, and microsatellite instability status of Chinese intrahepatic cholangiocarcinoma [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 3437.

Refractory testicular pure seminoma (PS) and non-seminomatous(NS) germ cell tumors (GCT): A comprehensive genomic profiling (CGP) study.

565 Background: Although PSand NS testicular GCT have a favorable prognosis, on occasion these tumors can be refractory to conventional systemic treatments. Methods: FFPE tissues from 22 PS and 86 NSunderwent hybrid-capture based CGP to evaluate all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and reported as mutations (mut) per megabase (Mb) and microsatellite instability (MSI) was determined on 114 loci. Results: PS patients were older than NS (P=0.007). The primary tumor was sequenced in 41% of PS and 18% of NS with a metastasis sample in 59% of PS and 82% of NS.Four (18%) of PS had syncytial trophoblast cells identified. The mean GA frequency at 4.1 mut/case for NS was higher than that seen in PS and this difference reached near significance (P=0.08). The KRAS, TP53, CCND2 and FGF6/23 GA frequencies were similar in both tumor types (Table). GA in KIT, PIK3CA/ MTOR, PTEN and BRCA2 were more frequent in PS than NS whereas BRAF and ERBB2 GA were more frequent in NS (Table). MSI-High status was absent in in PS (0%) and identified in 2% of NS. Higher levels of TMB were not encountered in PS (0% TMB ≥10 mut/Mb), but higher TMB levels were more frequent in NS (5% ≥ 10 mut/B and 1% ≥ 20 mut/Mb). Conclusions: The GA found in refractory PS and NS differ significantly. PS features a lower GA frequency with slightly higher potential for targeted therapies in kinase (KIT) and MTOR pathways but, has very low TMB predicting limited opportunities for immunotherapy for these patients. For NS targeted therapy biomarkers appear even more uncommon than seen in PS with only extremely rare kinase inhibitor opportunities. However, based on rare high TMB and MSI-High status, immunotherapies may be of benefit in a small subset of NS patients. Further study of genomic findings in relapsed and clinically aggressive PS and NS appears warranted. [Table: see text]

Abstract P5-21-20: Integrating comprehensive genomic profiling with treatment decisions – Experience gained while treating 139 advanced breast carcinomas

Abstract Background: Comprehensive Genomic Profiling (CGP) using next-generation sequencing (NGS) technology can provide insight into potentially clinically relevant genomic alterations (CRGA) within a patient's breast cancer. For example, HER2 amplification status and targetable short variants (SV), acquired ESR1 or BRCA1/2 resistance mutations, and the presence of targetable alterations in the PI3K kinase. We retrospectively reviewed CGP results and subsequent outcomes at one cancer center to illustrate the experience of using molecular subtyping to inform treatment decisions. Methods: DNA extracted from FFPE tumor tissue or blood samples obtained during routine clinical care for patients (n=139) with predominantly relapsed, refractory or metastatic breast cancer was analyzed by hybrid-capture, NGS for all classes of GA: 1. base substitutions, 2. insertion and deletions, 3. rearrangements, and 4. copy number changes. Treatment decisions based on comprehensive genomic profiles were captured retrospectively. Tumor mutational burden (TMB), scored as mutations (mut)/Mb, was calculated on 0.8-1.2 Mb of sequenced DNA. Alterations affecting the ERBB family included amplification of or oncogenic mutations in ERBB2 (HER2), ERBB3, and EGFR. Results: From Jan 2013 to May 2017, FFPE tissue samples for 136 patients with advanced breast cancer were analyzed by CGP and 3 additional patients had circulating tumor DNA analyzed for alterations; 11 patients received profiling on multiple biopsies. Tumors analyzed were carcinomas (Ca) NOS (n=84), invasive ductal Ca (n=46), invasive lobular Ca (n=7), a neuroendocrine Ca, and a phyllodes tumor. In total, 118/139 (84.9%) samples harbored CRGA in a targetable pathway: PI3K/MTOR (n=67; 48.2%), CDK cell-cycle (n=40; 28.8%), ERBB family (n=24; 17.3%), FGFR (n=24; 17.3%), ESR1 (n=16; 11.5%), homologous repair (HRD)( n=14; 10.1%), and RAS/RAF/MEK (n=11; 7.9%). Targetable alterations in other cancer-related kinases were found in 10 (7.2%) samples and 10 (7.2%) samples were TMB high (≥20 mut/Mb) or had CD274 (PD-L1) amplification. There were 3 patients (2.1%) with HER2 short variants detected in the absence of ERBB2 amplification; these patients may respond to HER2-targeted therapies but would be HER2-negative by IHC. Many samples had alterations in ≥1 pathway, and overlap is particularly high for the CDK and FGFR pathways (12 samples). Alterations in pathways targeted by MTOR inhibitors, HER2-targeted therapies, or the CDK inhibitors were found in 93/136 (66.9%) tumors. Evaluation of outcomes for these 139 patients is ongoing and will be presented. Conclusions: Genomic profiling of breast carcinomas, using either tissue or liquid biopsies, provides potentially actionable information to guide treatment decisions. Overall, 84.9% of patient samples harbored oncogenic alterations in a targetable pathway, with two-thirds of tumors having alterations in pathways targeted by therapies with FDA approval for breast cancer and 7.2% of patients having high levels of TMB or amplification of PD-L1, suggesting that checkpoint inhibitors may be relevant options. Citation Format: Mahtani R, Gay LM, Chung J, Hartmaier R, Sokol E, Elvin JA, Daniel S, Ramkissoon S, Severson E, Suh J, Vergilio J-A, Stephens PJ, Ross JS. Integrating comprehensive genomic profiling with treatment decisions – Experience gained while treating 139 advanced breast carcinomas [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 P5-21-20.

The prognostic role of microsatellite status, tumor mutational burden, and protein expression in CRC.

572 Background: Comprehensive molecular profiling of CRC can inform treatment decisions by identifying patient subgroups at varying risks of death. Microsatellite instability (MSI) is prognostic in CRC and is used to select patients for immunotherapy. High tumor mutational burden (TMB) is associated with genomic instability and is prognostic in melanoma. Expression of p16 protein is prognostic in many tumor types. We used proteomic and genomic profiling to measure MSI, TMB and p16 in CRC tumors and to assess associations with patient survival. Methods: In archived clinical samples of CRC, 76 proteins were quantitated with mass spectrometry-based proteomics. MSI was measured by WGS and RNA-seq; unstable loci were quantified in tumor and normal samples. Cutoffs were derived via ROC analysis: high TMB was defined as &gt; 4.5 somatic mutations per megabase; p16 as &gt; 108 amol/ug. Patients were grouped by microsatellite status (MSI vs. microsatellite stable [MSS]), TMB (high vs. low), and p16 protein expression level. Survival curves were compared with the Mantel-Cox log-rank test. Results: Of 145 samples, 39 (27%) had high TMB and 29 (20%) had MSI. Patients with MSI tumors had longer OS than patients with MSS tumors (HR: 0.096; p = 0.003). Similarly, patients with high TMB had longer OS than those with low TMB (HR: 0.076; p &lt; 0.001). High p16 expression was prognostic of poor survival (HR: 2.874; p = 0.019). Among patients with MSS tumors or low TMB, those with low p16 levels had longer OS than patients with high p16 (HR: 0.257; p = 0.002 and HR: 0.249; p = 0.002, for MSS and low TMB, respectively). A combination of MSS, low TMB, and low p16 also differentiated between long and short survivors (HR: 0.249; p = 0.002). These associations remained after adjustment for tumor sidedness. Further analyses of clinical correlates will be presented. Conclusions: A combination of MSS, low TMB and low p16 expression characterized a subset of patients with longer survival. This is important because patients with MSS tumors have limited treatment options but may respond to CDK4/6 inhibitors due to low p16 expression. Molecular profiling of CRC may identify patient subgroups with a relatively poor prognosis who could benefit from personalized therapy.

The Tumor Mutational Burden of Chinese Advanced Cancer Patients Estimated by a 381-cancer-gene Panel.

Purpose: Tumor mutational burden (TMB) calculated by whole-exome sequencing (WES) is proved to be effective to predict the clinical benefit of immune checkpoint blockades. However, WES is not commonly used in China. We aimed to determine if a 381-caner-gene panel (CGP) could be used to estimate TMB, delineate the landscape of TMB of Chinese patients and identify mutated genes and pathways related to higher TMB.Methods: We first evaluated the correlation between the TMB estimated by a 381-cancer-gene panel MasterView and WES using the data from the melanoma sample cohort. 3023 formalin fixed, paraffin-embedded tumor specimens from 2932 Chinese patients with advanced solid tumor were profiled for 381 gene sequencing, the baits of which covered 4,557 exons of 365 cancer-related genes and 47 introns of 25 genes frequently rearranged in cancer (All performed in a lab who achieved full marks five times in the external quality assessment by College of American Pathologists [CAP]). Using the sequencing data, we estimated the TMB of Chinese advanced solid tumor and identified mutated genes and pathways related to higher TMB level.Results: 381-CGP-mutational burden was strongly associated with those calculated by WES (R2 = 0.978). The median TMB for each tumor type was 5.65 (colorectal cancer), 4.84 (lung cancer), 4.03 (hepatobiliary cancer), 4.03 (gastric carcinoma), 4.03 (breast cancer) mutations/mb respectively. No correlation was observed between TMB level and age (P = 0.577) or gender (P = 0.307). The TMB of patients with mismatch repair (MMR) or DNA repair response (DDR) pathway deficiency was significantly higher than that without MMR or DDR pathway deficiency (P < 0.001).Conclusion: The 381-cancer gene panel is a clinical practicable method to assess tumor mutational burden compared with whole exome sequencing. MMR and DDR deficiency are correlated with higher tumor mutational burden of Chinese patients with advanced solid tumors.

Abstract 665: Molecular and clinical analyses of patients with gynecologic malignancies treated with PD-1 directed immunotherapy

Abstract Long term responses are seldom seen in treatments of advanced or recurrent gynecologic cancers. In contrast, immunotherapeutic strategies targeting immune checkpoints such as programmed cell death-1 (PD-1) are currently in active development in platinum resistant gynecologic malignancies with promising durable responses observed in subset of patients. We analyzed all 14 patients with gynecology malignancies that were treated with PD-1 directed immunotherapy, nivolumab or pembrolizumab at the developmental therapeutics program (DTP) clinic in Northwestern University during 2015 till 2016. Seven patients had ovarian/fallopian/peritoneal cancer; six, endometrial cancer; 1, squamous cell carcinoma (SCC) of cervix that are refractory to standard-of-care chemotherapies. Comprehensive genomic profiling including microsatellite instability (MSI) status and tumor mutational burden (TMB) analyses was performed using next generation sequencing (NGS) (FoundationOne). Among 7 patients with ovarian/fallopian/peritoneal cancer (median age, 62 years), objective response rate (ORR) was 20%. One patient had partial response (PR), another one had stable disease (SD), and 3, progressive disease (PD). Two patients were uable to be evaluated. In a patient with PR, CA 125 level normalized from 426.2 to 10.8 U/mL. Her tumor had very low TMB (1/Mbp). In all patients, TMB level varied from very low to low (1-5 mut/Mbp) with MSI status being stable. Progression free survival (PFS) ranged from 14 to 196 days; overall survival (OS) varied from 60 to 199 days. Among 6 patients with endometrial cancer (median age, 68 years), ORR was 20%. One patient had PR while four experienced PD. One patient whose disease could not be assessed due to early death from an event unrelated to treatment had a remarkable chemical response with a decrease in CA125 level from 5,889 to 182.1 U/mL after one single dose. Her tumor had MSI-stable status and intermediate TMB (6 mut/Mbp). In a patient with PR, CA125 level decreased from 1,216 to 226.5 U/mL. Her tumor had MSI-high status with unknown TMB. Of note, one of four patients with PD had MSI-high status and intermediate level of TMB. Other patients had tumor with MSI-stable status and low level of TMB (3-4 mut/MB). PFS ranged from 23 to 258 days; OS varied from 73 to 265 days. One patient with HPV positive SCC of cervix (34 year old) experienced PD. Her tumor had MSI-stable status with unknown TMB. PFS was 88 days; OS, 132 days. In summary, patients with ovarian and endometrial cancer treated with PD-1 directed immunotherapy demonstrated ORR of 20%. One additional patient with endometrial cancer had an exceptional chemical response. None of the tumors had high TMB (&amp;gt;6/Mbp). No clear associations between genomic traits including TMB and MSI staus and responses were found. Further larger prospecitve studies are warranted to explore potential biomarkers for response with immunotherapy in gynecologic malignancies. Note: This abstract was not presented at the meeting. Citation Format: Young Kwang Chae, Sabina Murshudova, William H. Bae, Jonathan F. Anker, Mario J. Pineda, Wilberto Nieves-Neira, Daniela E. Matei, John R. Lurain, Shohreh Shahabi, Francis J. Giles. Molecular and clinical analyses of patients with gynecologic malignancies treated with PD-1 directed immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 665. doi:10.1158/1538-7445.AM2017-665

Mutational burden of tumors with primary site unknown.

3039 Background: Higher levels of tumor mutational burden (TMB) can predict sensitivity to immunotherapies (IO), which are FDA approved to treat NSCLC, melanoma, and urothelial carcinoma (Ca). TMB may be a biomarker for sensitivity to IO, irrespective of tumor type. TMB has not been explored widely for tumors of unknown primary site, but may reveal additional treatment options. Methods: Comprehensive genomic profiling of DNA from FFPE tissue samples was performed using hybrid-capture, next-generation sequencing. TMB was calculated by counting all coding short variant alterations (base substitutions and indels), including synonymous alterations, and subtracting from this functionally oncogenic or germline alterations (per ExAC, dbSNPT, or internal algorithmic analysis). To calculate the TMB per Mb, the total number of relevant mutations is divided by the coding region of the bait set (0.8 Mb, 1.1 Mb, or 1.2 Mb). High, intermediate, and low TMB were defined as ≥20 mut/Mb, ≥6 and &lt;20 mut/Mb, or &lt;6 mut/Mb, respectively. Tumor types with &gt;100 samples were analyzed. Results: From a database of 102,878 samples sequenced during routine clinical care, 6116 samples for which the primary tumor site was unclear at sequencing were identified. Table shows TMB metrics (mut/Mb) and median patient age for these cohorts. Conclusions: Significant numbers of patients with each tumor type have high TMB that may indicate benefit from IO, excepting GIST. As expected, urothelial tumors have higher than average TMB and more patients have high TMB. SCC tumors are commonly TMB high (23%), as are tumors difficult to define histologically (15%). For ACUP or CUP, the most common tumors, 8-11% have high TMB. Analysis of responses to treatment with IO are ongoing. [Table: see text]

Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden

BackgroundHigh tumor mutational burden (TMB) is an emerging biomarker of sensitivity to immune checkpoint inhibitors and has been shown to be more significantly associated with response to PD-1 and PD-L1 blockade immunotherapy than PD-1 or PD-L1 expression, as measured by immunohistochemistry (IHC). The distribution of TMB and the subset of patients with high TMB has not been well characterized in the majority of cancer types.MethodsIn this study, we compare TMB measured by a targeted comprehensive genomic profiling (CGP) assay to TMB measured by exome sequencing and simulate the expected variance in TMB when sequencing less than the whole exome. We then describe the distribution of TMB across a diverse cohort of 100,000 cancer cases and test for association between somatic alterations and TMB in over 100 tumor types.ResultsWe demonstrate that measurements of TMB from comprehensive genomic profiling are strongly reflective of measurements from whole exome sequencing and model that below 0.5 Mb the variance in measurement increases significantly. We find that a subset of patients exhibits high TMB across almost all types of cancer, including many rare tumor types, and characterize the relationship between high TMB and microsatellite instability status. We find that TMB increases significantly with age, showing a 2.4-fold difference between age 10 and age 90 years. Finally, we investigate the molecular basis of TMB and identify genes and mutations associated with TMB level. We identify a cluster of somatic mutations in the promoter of the gene PMS2, which occur in 10% of skin cancers and are highly associated with increased TMB.ConclusionsThese results show that a CGP assay targeting ~1.1 Mb of coding genome can accurately assess TMB compared with sequencing the whole exome. Using this method, we find that many disease types have a substantial portion of patients with high TMB who might benefit from immunotherapy. Finally, we identify novel, recurrent promoter mutations in PMS2, which may be another example of regulatory mutations contributing to tumorigenesis.

Abstract P1-05-08: Comprehensive genomic profiling of 8,654 breast carcinoma reveals therapeutically targetable molecular subtypes beyond those defined by hormone-receptor expression

Abstract Background: Breast carcinomas (BC) are commonly classified into 4 subtypes based on hormone receptor expression: basal, luminal A, luminal B, and HER2 overexpressed. Comprehensive genomic profiling (CGP) reveals targetable genomic alterations (GA) across all four mutation classes, as well measuring tumor mutational burden (TMB), and can redefine BC classification into therapeutically relevant subtypes. Testing with immunohistochemistry or hotspot testing can miss a substantial number of targetable alterations and cannot measure TMB. Methods: DNA was extracted from 40 µm of FFPE sections for 8654 consecutive BCs. CGP was performed on hybridization-captured, adaptor ligation-based libraries (mean coverage &amp;gt;500X) for up to 315 cancer-related genes and select introns from up to 28 genes frequently rearranged in cancer. Sequences were analyzed for substitutions, small insertions/deletions, copy number changes, and rearrangements. TMB was determined by counting non-driver, non-germline alterations across 1.1 Mbp of sequenced DNA. Clinically relevant GA (CRGA) are GA linked to therapies on the market or under evaluation in clinical trials. Immunotherapy (IO) sensitivity is defined as TMB &amp;gt;20 mut/Mbp or mutation of specific DNA repair pathways. Results: The table below outlines 7 distinct functional or signal transduction pathways commonly altered in BC. Several are targetable with therapies that are FDA approved for an oncology indication. Mutations can also be found in other targetable kinases such as RET, ROS1, and RAF. 6959 (80.4%) tumors harbor a GA in at least one pathway, and 2697 (31.2%) BC harbor alterations in just one pathway (unique cases). Only 9.8% of BC would be HER2-positive by IHC. Almost 4% (352/8654) of cases harbor rearrangements or gene fusions that may not be detectable with other assays. Mutations in ESR1 characterize an eighth category of tumors with acquired resistance to endocrine therapy; 796/8654 (9%) samples harbor ESR1 alterations. Conclusions: CGP can identify CRGA and TMB that can stratify tumors by predicted sensitivity to a variety of therapies, including HER2- or mTOR-targeted therapies, immunotherapies, and other kinase inhibitors. 80% of BC harbor targetable GA, and 30% of samples harbor mutations in only one pathway. CGP can provide crucial information for identifying which of several treatment modalities is most appropriate for these 30% of patients. High levels of TMB and most GA would not be identified by IHC or hotspot testing, but can be detected by next-generation sequencing. CGP is a powerful tool for guiding treatment across therapeutically distinct, but targetable, pathways. PI3K/AKT/mTOR pathwayFGFR pathwayCDK pathwayERBB pathwayHR deficientIO sensitiveOther kinasesTotal Cases43752650268512941266419424% Total Cases51%31%31%15%15%5%5%Unique Cases14422262312743094858% Unique Cases17%3%3%3%4%1%1%TherapiesEverolimus, TemsirolimusPazopanib, PonatinbPalbociclibTrastuzumab, Pertuzumab, Afatinib, Lapatinib, NeratinibOlaparibPembrolizumab, Nivolumab, Atezolizumab, IpilumumabSorafenib, Regorafenib, Dabrafenib, Vemurafenib, Crizotinib, Cabozantinib, Sunitinib Citation Format: Ross JS, Gay LM, Elvin JA, Suh J, Vergilio J-A, Ramkissoon S, Schrock A, Ali S, Miller VA, Stephens PJ. Comprehensive genomic profiling of 8,654 breast carcinoma reveals therapeutically targetable molecular subtypes beyond those defined by hormone-receptor expression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-08.

Agranulocytosis coincident with amodiaquine therapy.

<h3>Background</h3> Neoadjuvant chemoimmunotherapy for non-small cell lung cancer (NSCLC) has improved pathological responses and survival rates compared with chemotherapy alone, leading to Food and Drug Administration (FDA) approval of nivolumab plus chemotherapy for resectable stage IB-IIIA NSCLC (AJCC 7th edition) without ALK or EGFR alterations. Unfortunately, a considerable percentage of tumors do not completely respond to therapy, which has been associated with early disease progression. So far, it is impossible to predict these events due to lack of knowledge. In this study, we characterized the gene expression profile of tumor samples to identify new biomarkers and mechanisms behind tumor responses to neoadjuvant chemoimmunotherapy and disease recurrence after surgery. <h3>Methods</h3> Tumor bulk RNA sequencing was performed in 16 pretreatment and 36 post-treatment tissue samples from 41 patients with resectable stage IIIA NSCLC treated with neoadjuvant chemoimmunotherapy from NADIM trial. A panel targeting 395 genes related to immunological processes was used. Tumors were classified as complete pathological response (CPR) and non-CPR, based on the total absence of viable tumor cells in tumor bed and lymph nodes tested at surgery. Differential-expressed genes between groups and pathway enrichment analysis were assessed using DESeq2 and gene set enrichment analysis. CIBERSORTx was used to estimate the proportions of immune cell subtypes. <h3>Results</h3> CPR tumors had a stronger pre-established immune infiltrate at baseline than non-CPR, characterized by higher levels of <i>IFNG, GZMB, NKG7</i>, and M1 macrophages, all with a significant area under the receiver operating characteristic curve (ROC) &gt;0.9 for CPR prediction. A greater effect of neoadjuvant therapy was also seen in CPR tumors with a reduction of tumor markers and IFNγ signaling after treatment. Additionally, the higher expression of several genes, including <i>AKT1, BST2, OAS3,</i> or <i>CD8B</i>; or higher dendritic cells and neutrophils proportions in post-treatment non-CPR samples, were associated with relapse after surgery. Also, high pretreatment PD-L1 and tumor mutational burden levels influenced the post-treatment immune landscape with the downregulation of proliferation markers and type I interferon signaling molecules in surgery samples. <h3>Conclusions</h3> Our results reinforce the differences between CPR and non-CPR responses, describing possible response and relapse immune mechanisms, opening the possibility of therapy personalization of immunotherapy-based regimens in the neoadjuvant setting of NSCLC.