COMPUTATIONAL GENOMIC MODELS FOR SPATIO-TEMPORAL INVESTIGATION OF EARLY LUNG CANCER PATHOLOGY

Lung cancer, of which non-small cell lung cancer (NSCLC) is the most common form, is the second most prevalent cancer and the leading cause of cancer-related deaths. NSCLCs primarily comprise adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC). Advances in early detection and prevention have been limited by the lack of early-stage biomarkers and targets. A comprehensive molecular characterization of premalignant lesions and tumor-adjacent normal tissue can aid in better understanding NSCLC pathogenesis. However, these investigations are further challenged by limited tissue availability and low cellular fractions of detectable somatic mutations. Therefore, there is a dearth of knowledge about the pathogenesis of premalignant lung lesions, especially for atypical adenomatous hyperplasia (AAH), the only known precursor to LUADs. We performed a cross-platform integrative analysis comprising targeted DNA sequencing, genotype array profiling and transcriptome sequencing of matched AAHs, LUADs and normal tissues from 23 early-stage patients. The study revealed potentially divergent pathways based on the mutation status of AAH (BRAF vs KRAS), recurrent chromosomal aberrations (17p loss) and the presence of immune deregulation early in the pathogenesis of AAHs. Molecular changes, characteristic of NSCLCs, might also occur in normal tissues, preceding identifiable premalignancy-associated morphological changes. We sought to comprehensively survey the somatic mutational architecture of the normal airway in early-stage NSCLCs. Targeted DNA sequencing allowed us to capture driver mutations at low cellular fractions, typical of these non-malignant tissues. Additionally, genotype array profiling helped characterize subtle chromosomal aberrations in these tissues. This multi-region study included tumor-adjacent and -distant airways, nasal epithelia and uninvolved normal lung (collectively cancerized field) along with matched multi-region NSCLCs and blood cells from 48 patients. Integrative computational analysis revealed genomic airway field carcinogenesis in 52% of cases. The airway field exhibited mutations in known drivers, that were present at lower frequencies compared to NSCLCs, suggestive of selection-driven clonal expansion. These driver events also comprised somatic “two-hit” alterations in matched airway field and NSCLCs. Our study design offers spatiotemporal insights into NSCLC development and suggests potential targets for early detection and treatment, in possibly less hostile environments of premalignancy. To validate and enhance the utility of the bioinformatic techniques devised and implemented for these investigations, I also provide methods to expand such analyses across multiple tumor sites

ROS1 genomic rearrangements are rare actionable drivers in microsatellite stable colorectal cancer.

c-Ros oncogene 1, receptor tyrosine kinase (ROS1) genomic rearrangements have been reported previously in rare cases of colorectal cancer (CRC), yet little is known about the frequency, molecular characteristics, and therapeutic vulnerabilities of ROS1-driven CRC. We analyzed a clinical dataset of 40 589 patients with CRC for ROS1 genomic rearrangements and their associated genomic characteristics (Foundation Medicine, Inc [FMI]). We moreover report the disease course and treatment response of an index patient with ROS1-rearranged metastatic CRC. ROS1 genomic rearrangements were identified in 34 (0.08%) CRC samples. GOPC-ROS1 was the most common ROS1 fusion identified (11 samples), followed by TTC28-ROS1 (3 samples). Four novel 5' gene partners of ROS1 were identified (MCM9, SRPK1, EPHA6, P4HA1). Contrary to previous reports on fusion-positive CRC, ROS1-rearrangements were found exclusively in microsatellite stable (MSS) CRCs. KRAS mutations were significantly less abundant in ROS1-rearranged vs ROS1 wild type cases. The index patient presented with chemotherapy-refractory metastatic right-sided colon cancer harboring GOPC-ROS1. Molecularly targeted treatment with crizotinib induced a rapid and sustained partial response. After 15 months on crizotinib disseminated tumor progression occurred and KRAS Q61H emerged in tissue and liquid biopsies. ROS1 rearrangements define a small, yet therapeutically actionable molecular subgroup of MSS CRC. In summary, the high prevalence of GOPC-ROS1 and noncanonical ROS1 fusions pose diagnostic challenges. We advocate NGS-based comprehensive molecular profiling of MSS CRCs that are wild type for RAS and BRAF and patient enrollment in precision trials

ROS1 genomic rearrangements are rare actionable drivers in microsatellite stable colorectal cancer

c-Ros oncogene 1, receptor tyrosine kinase (ROS1) genomic rearrangements have been reported previously in rare cases of colorectal cancer (CRC), yet little is known about the frequency, molecular characteristics, and therapeutic vulnerabilities of ROS1-driven CRC. We analyzed a clinical dataset of 40 589 patients with CRC for ROS1 genomic rearrangements and their associated genomic characteristics (Foundation Medicine, Inc [FMI]). We moreover report the disease course and treatment response of an index patient with ROS1-rearranged metastatic CRC. ROS1 genomic rearrangements were identified in 34 (0.08%) CRC samples. GOPC-ROS1 was the most common ROS1 fusion identified (11 samples), followed by TTC28-ROS1 (3 samples). Four novel 5' gene partners of ROS1 were identified (MCM9, SRPK1, EPHA6, P4HA1). Contrary to previous reports on fusion-positive CRC, ROS1-rearrangements were found exclusively in microsatellite stable (MSS) CRCs. KRAS mutations were significantly less abundant in ROS1-rearranged vs ROS1 wild type cases. The index patient presented with chemotherapy-refractory metastatic right-sided colon cancer harboring GOPC-ROS1. Molecularly targeted treatment with crizotinib induced a rapid and sustained partial response. After 15 months on crizotinib disseminated tumor progression occurred and KRAS Q61H emerged in tissue and liquid biopsies. ROS1 rearrangements define a small, yet therapeutically actionable molecular subgroup of MSS CRC. In summary, the high prevalence of GOPC-ROS1 and noncanonical ROS1 fusions pose diagnostic challenges. We advocate NGS-based comprehensive molecular profiling of MSS CRCs that are wild type for RAS and BRAF and patient enrollment in precision trials. Keywords: ROS1 rearrangement; acquired resistance; colorectal cancer; crizotinib; molecular subgroups; precision treatmen

Intra-patient stability of tumor mutational burden from tissue biopsies at different time points in advanced cancers

BackgroundTumor mutational burden (TMB) may be a predictive biomarker of immune checkpoint inhibitor (ICI) responsiveness. Genomic landscape heterogeneity is a well-established cancer feature. Molecular characteristics may differ even within the same tumor specimen and undoubtedly evolve with time. However, the degree to which TMB differs between tumor biopsies within the same patient has not been established.MethodsWe curated data on 202 patients enrolled in the PREDICT study (NCT02478931), seen at the University of California San Diego (UCSD), who had 404 tissue biopsies for TMB (two per patient, mean of 722 days between biopsies) to assess difference in TMB before and after treatment in a pan-cancer cohort. We also performed an orthogonal analysis of 2872 paired pan-solid tumor biopsies in the Foundation Medicine database to examine difference in TMB between first and last biopsies.ResultsThe mean (95% CI) TMB difference between samples was 0.583 [- 0.900-2.064] (p = 0.15). Pearson correlation showed a flat line for time elapsed between biopsies versus TMB change indicating no correlation (R2 = 0.0001; p = 0.8778). However, in 55 patients who received ICIs, there was an increase in TMB (before versus after mean mutations/megabase [range] 12.50 [range, 0.00-98.31] versus 14.14 [range, 0.00-100.0], p = 0.025). Analysis of 2872 paired pan-solid tumor biopsies in the Foundation Medicine database also indicated largely stable TMB patterns; TMB increases were only observed in specific tumors (e.g., breast, colorectal, glioma) within certain time intervals.ConclusionsOverall, our results suggest that tissue TMB remains stable with time, though specific therapies such as immunotherapy may correlate with an increase in TMB.Trial registrationNCT02478931 , registered June 23, 2015

Strategies for identification of somatic variants using the Ion Torrent deep targeted sequencing platform

Abstract Background ‘Next-generation’ (NGS) sequencing has wide application in medical genetics, including the detection of somatic variation in cancer. The Ion Torrent-based (IONT) platform is among NGS technologies employed in clinical, research and diagnostic settings. However, identifying mutations from IONT deep sequencing with high confidence has remained a challenge. We compared various computational variant-calling methods to derive a variant identification pipeline that may improve the molecular diagnostic and research utility of IONT. Results Using IONT, we surveyed variants from the 409-gene Comprehensive Cancer Panel in whole-section tumors, intra-tumoral biopsies and matched normal samples obtained from frozen tissues and blood from four early-stage non-small cell lung cancer (NSCLC) patients. We used MuTect, Varscan2, IONT’s proprietary Ion Reporter, and a simple subtraction we called “Poor Man’s Caller.” Together these produced calls at 637 loci across all samples. Visual validation of 434 called variants was performed, and performance of the methods assessed individually and in combination. Of the subset of inspected putative variant calls (n=223) in genomic regions that were not intronic or intergenic, 68 variants (30%) were deemed valid after visual inspection. Among the individual methods, the Ion Reporter method offered perhaps the most reasonable tradeoffs. Ion Reporter captured 83% of all discovered variants; 50% of its variants were visually validated. Aggregating results from multiple packages offered varied improvements in performance. Conclusions Overall, Ion Reporter offered the most attractive performance among the individual callers. This study suggests combined strategies to maximize sensitivity and positive predictive value in variant calling using IONT deep sequencing

Biology and Targetability of the Extended Spectrum of PIK3CA Mutations Detected in Breast Carcinoma

PurposeAlpelisib is a PI3K alpha (PI3Kα)-selective inhibitor approved for the treatment of hormone receptor-positive/HER2-negative (HR+/HER2-) PIK3CA-mutated advanced breast cancer (ABC) based on the SOLAR-1 trial, which defined 11 substitutions in exons 7, 9, and 20 in PIK3CA (SOLAR1m). We report alpelisib effectiveness for ABC harboring SOLAR1m, as well as other pathogenic PIK3CA mutations (OTHERm) using comprehensive genomic profiling (CGP).Experimental designA total of 33,977 tissue and 1,587 liquid biopsies were analyzed using hybrid capture-based CGP covering the entire coding sequence of PIK3CA. Clinical characteristics and treatment history were available for 10,750 patients with ABC in the deidentified Flatiron Health-Foundation Medicine clinico-genomic database (FH-FMI CGDB).ResultsPIK3CAm were detected in 11,767/33,977 (35%) of tissue biopsies, including 2,300 (7%) samples with OTHERm and no SOLAR1m. Liquid biopsy had 77% sensitivity detecting PIK3CAm, increasing to 95% with circulating tumor DNA fraction ≥2%. In patients with HR+/HER2- ABC and PIK3CAm receiving alpelisib/fulvestrant (ALP+FUL; n = 182) or fulvestrant alone (FUL; n = 119), median real-world progression-free survival (rwPFS) was 5.9 months on ALP+FUL [95% confidence interval (CI): 5.1-7.4] versus 3.1 months on FUL (95% CI: 2.7-3.7; P < 0.0001). In patients with OTHERm, median rwPFS was 4.0 months on ALP+FUL (95% CI: 2.8-10.1) versus 2.5 months on FUL (95% CI: 2.2-3.7; P = 0.0054).ConclusionsCGP detects diverse PIK3CAm in a greater number of patients with ABC than PCR hotspot testing; 20% of patients with PIK3CAm do not have SOLAR1m. These patients may derive benefit from alpelisib. See related commentary by Tau and Miller, p. 989

Ancestral characterization of the genomic landscape, comprehensive genomic profiling utilization, and treatment patterns may inform disparities in advanced prostate cancer: A large-scale analysis

5003 Background: Prostate cancer (PCa) incidence, mortality, and outcomes vary widely across race/ethnicity. The underlying drivers of these differences are multifactorial, including systemic barriers that lead to wide variation in access to care including genomic and precision medicine. Men of African ancestry (AFR) are particularly underrepresented in genomic and precision medicine studies. Therefore, we sought to comprehensively assess patterns of gene alterations, comprehensive genomic profiling (CGP) utilization, and treatment patterns in a large, diverse advanced PCa cohort. Methods: 11,741 PCa patients with CGP, as part of routine clinical care (Foundation Medicine Inc., FMI) were evaluated for their genomic landscape. Predominant ancestry was inferred using a SNP-based approach (Connelly et al, AACR 2018). Independently, the US-based de-identified Flatiron Health (FH)-FMI clinico-genomic database (CGDB) of 897 evaluable PCa patients was also queried. Clinical characteristics and treatment selections were described for patients who received metastatic or castrate-resistant diagnosis between 1/2011 and 6/2020. Results: The FMI cohort included 1,422 (12%) men of AFR and 9,244 (79%) men of European ancestry (EUR). Median age was lower in AFR compared with EUR men (64 vs. 67, p < 0.001). TP53 and PTEN alterations and TMPRSS2-ERG rearrangements occurred less frequently in AFR than EUR men (35% vs. 43%, 21% vs. 33%, 15% vs. 33% respectively, p < 0.05). In contrast, alterations in SPOP (11.9% vs. 7.3%), CDK12 (10.0% vs. 5.2%), CCND1 (6.0% vs. 3.8%), KMT2D (7.7% vs. 5.1%), HGF (4.1% vs. 2.5%), and MYC (13.4% vs. 10.6%) were enriched in the AFR cohort (p < 0.05). Alteration frequency in BRCA1/2, AR, DNA damage response pathway genes, and actionable genes with therapy implications, were similar across ancestry. Of note, BRAF alterations were slightly enriched in AFR (5.0% vs. 3.2%, p < 0.05). In the CGDB cohort (79 AFR, 762 EUR), AFR men received a median of 2 lines of therapy prior to CGP, compared to 1 line for EUR men. Notably, the proportion of patients receiving immunotherapy and PARPi was similar across ancestry, however AFR men were less likely to receive clinical study drug compared with EUR men (11% vs 30%, p < 0.001), even among men with actionable alterations (1% vs 6%, p < 0.001). Conclusions: To our knowledge, this study encompasses the largest cohort, particularly of AFR men in a genomic study, that defines CGP utilization, the genomic landscape and therapeutic implications of CGP in PCa across ancestry. Overall, there were largely similar rates of actionable gene alterations across ancestry. Notably, AFR men were less likely to receive CGP earlier in their treatment course, and less likely to be treated on clinical trials, which could impact the genomic landscape, outcomes, and ultimately disparities

Additional file 1 of Strategies for identification of somatic variants using the Ion Torrent deep targeted sequencing platform

Supplementary Materials. (PDF 1208 kb

Multiple PIK3CA mutation clonality correlates with outcomes in taselisib + fulvestrant-treated ER+/HER2–, PIK3CA-mutated breast cancers

Abstract Background Mutations in the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), encoded by the PIK3CA gene, cause dysregulation of the PI3K pathway in 35–40% of patients with HR+/HER2– breast cancer. Preclinically, cancer cells harboring double or multiple PIK3CA mutations (mut) elicit hyperactivation of the PI3K pathway leading to enhanced sensitivity to p110α inhibitors. Methods To understand the role of multiple PIK3CAmut in predicting response to p110α inhibition, we estimated the clonality of multiple PIK3CAmut in circulating tumor DNA (ctDNA) from patients with HR+/HER2– metastatic breast cancer enrolled to a prospectively registered clinical trial of fulvestrant ± taselisib, and analyzed the subgroups against co-altered genes, pathways, and outcomes. Results ctDNA samples with clonal multiple PIK3CAmut had fewer co-alterations in receptor tyrosine kinase (RTK) or non-PIK3CA PI3K pathway genes compared to samples with subclonal multiple PIK3CAmut indicating a strong reliance on the PI3K pathway. This was validated in an independent cohort of breast cancer tumor specimens that underwent comprehensive genomic profiling. Furthermore, patients whose ctDNA harbored clonal multiple PIK3CAmut exhibited a significantly higher response rate and longer progression-free survival vs subclonal multiple PIK3CAmut. Conclusions Our study establishes clonal multiple PIK3CAmut as an important molecular determinant of response to p110α inhibition and provides rationale for further clinical investigation of p110α inhibitors alone or with rationally-selected therapies in breast cancer and potentially other solid tumor types