Single-Hit and Multi-hit PIK3CA Short Variant Genomic Alterations in Clinically Advanced Prostate Cancer: A Genomic Landscape Study.

BACKGROUND: Tumors harboring two or more PIK3CA short variant (SV) ("multi-hit") mutations have been linked to improved outcomes with anti-PIK3CA-targeted therapies in breast cancer. The landscape and clinical implications of multi-hit PIK3CA alterations in clinically advanced prostate cancer (CAPC) remains elusive. OBJECTIVE: To evaluate the genomic landscape of single-hit and multi-hit PIK3CA genomic alterations in CAPC. PATIENTS AND METHODS: The Foundation Medicine FoundationCore database was used to identify 19,978 CAPC tumors that underwent hybrid capture-based comprehensive genomic profiling to evaluate all classes of genomic alterations (GA) and determine tumor mutational burden (TMB), microsatellite instability (MSI), genomic ancestry, single-base substitution mutational signatures, and homologous recombination deficiency signature (HRDsig). Tumor cell PD-L1 expression was determined by IHC (Dako 22C3). RESULTS: 18,741 (93.8%) tumors were PIK3CA wild type (WT), 1155 (5.8%) featured single PIK3CA SV, and 82 (0.4%) featured multi-hit PIK3CA SVs. Single-hit (6.6 versus 3.8; p < 0.0001) and multi-hit (12.8 versus 3.8; p < 0.0001) featured more driver GA per tumor than PIK3CA WT CAPC, as well as higher prevalence of MMR mutational signature, MSI high status, and TMB levels versus PIK3CA WT (p < 0.0001). Other differences in GA included higher frequencies of GA in BRCA2 in multi-hit versus WT (18.3% versus 8.5%; p = 0.0191), ATM in multi-hit versus WT (13.4% versus 5.6%; p = 0.02) and PTEN in single-hit versus WT (40.2% versus 30.1%; p < 0.0001). Homologous recombination deficiency signatures were higher in PIK3CA WT versus single-hit (11.2% versus 7.6%; p = 0.0002). There were no significant differences in PD-L1 expression among the three groups. CONCLUSIONS: Identification of multi-hit PIK3CA GA in CAPC highlights a potentially unique phenotype that may be associated with response to anti-PIK3CA targeted therapy and checkpoint inhibition, supporting relevant clinical trial designs.

Ancestry-, Sex-, and Age-Based Differences of Gene Alterations in NSCLC: From the Real-World Data of Cancer Genomic Profiling Tests.

BACKGROUND: Some genomic alterations in non-small cell lung cancer (NSCLC) are known to differ according to race, sex, or age. These studies have been limited in sample size and thus they cannot detect the differences precisely and comprehensively. METHODS: Tissue-based comprehensive genomic profiling was performed on 75,362 patients with NSCLC from the United States during routine clinical care. Additionally, we examined data of a Japanese NSCLC cohort with 1,019 patients. In the US cohort, 296 genes were examined for pathogenic alterations. Predominant genetic ancestry was inferred using a SNP-based approach, and patients were categorized into European (EUR), African (AFR), East Asian (EAS), Admixed American (AMR), and South Asian (SAS) ancestry groups. Patients were additionally stratified by histologic type, age (<40/≥40 years, <75/≥75 years), and sex. The prevalence of high tumor mutational burden (TMB-High) and microsatellite instability status was also calculated. RESULTS: Stratified by ancestry, EGFR alterations were significantly enriched in EAS versus other ancestry groups. The prevalence of ALK was significantly higher in the AMR, EAS, and SAS patients than in AFR and EUR patients. KRAS and STK11 were enriched in EUR and AFR patients versus other groups. TMB-High was significantly enriched in AFR patients versus all other groups. An analysis based on sex revealed differences in prevalence of alterations in 80 genes and TMB-High status. For example, EGFR, ALK, BRAF, and KRAS alterations were significantly enriched in females, whereas TP53, STK11, KEAP1, and TMB-High were significantly enriched in males. With respect to age, the prevalence of alterations in 41 genes, including ALK, RET, MET, EGFR, STK11, KEAP1, BRAF, and KRAS, as well as TMB-High, were significantly different between patients aged <40 years and those aged ≥40 years. CONCLUSIONS: Comprehensive analysis from a large real-world dataset revealed ancestry-associated differences in genomic alterations in NSCLC. Age- and sex-related differences in prevalence of genomic alterations and TMB-High status were also observed.

Ancestry-associated co-alteration landscape of KRAS and EGFR-altered non-squamous NSCLC.

Racial/ethnic disparities mar NSCLC care and treatment outcomes. While socioeconomic factors and access to healthcare are important drivers of NSCLC disparities, a deeper understanding of genetic ancestry-associated genomic landscapes can better inform the biology and the treatment actionability for these tumors. We present a comprehensive ancestry-based prevalence and co-alteration landscape of genomic alterations and immunotherapy-associated biomarkers in patients with KRAS and EGFR-altered non-squamous (non-Sq) NSCLC. KRAS was the most frequently altered oncogene in European (EUR) and African (AFR), while EGFR alterations predominated in East Asian (EAS), South Asian (SAS), and Admixed American (AMR) groups, consistent with prior studies. As expected, STK11 and KEAP1 alterations co-occurred with KRAS alterations while showing mutual exclusivity with EGFR alterations. EAS and AMR KRAS-altered non-Sq NSCLC showed lower rates of co-occurring STK11 and KEAP1 alterations relative to other ancestry groups. Ancestry-specific co-alterations included the co-occurrence of KRAS and GNAS alterations in AMR, KRAS, and ARID1A alterations in SAS, and the mutual exclusivity of KRAS and NF1 alterations in the EUR and AFR ancestries. Contrastingly, EGFR-altered tumors exhibited a more conserved co-alteration landscape across ancestries. AFR exhibited the highest tumor mutational burden, with potential therapeutic implications for these tumors.

Circulating Tumor DNA Dynamics Reveal KRAS G12C Mutation Heterogeneity and Response to Treatment with the KRAS G12C Inhibitor Divarasib in Solid Tumors.

PURPOSE: To inform prognosis, treatment response, disease biology, and KRAS G12C mutation heterogeneity, we conducted exploratory circulating tumor DNA (ctDNA) profiling on 134 patients with solid tumors harboring a KRAS G12C mutation treated with single-agent divarasib (GDC-6036) in a phase 1 study. EXPERIMENTAL DESIGN: Plasma samples were collected for serial ctDNA profiling at baseline (cycle 1 day 1 prior to treatment) and multiple on-treatment time points (cycle 1 day 15 and cycle 3 day 1). RESULTS: KRAS G12C ctDNA was detectable from plasma samples in 72.9% (43/59) and 92.6% (50/54) of patients with non-small cell lung cancer and colorectal cancer, respectively, the majority of whom were eligible for study participation based on a local test detecting the KRAS G12C mutation in tumor tissue. Baseline ctDNA tumor fraction was associated with tumor type, disease burden, and metastatic sites. A decline in ctDNA level was observed as early as cycle 1 day 15. Serial assessment showed a decline in ctDNA tumor fraction associated with response and progression-free survival. Except for a few cases of KRAS G12C sub-clonality, on-treatment changes in KRAS G12C variant allele frequency mirrored changes in the overall ctDNA tumor fraction. CONCLUSIONS: Across tumor types, the KRAS G12C mutation likely represents a truncal mutation in the majority of patients. Rapid and deep decline in ctDNA tumor fraction was observed in patients responding to divarasib treatment. Early on-treatment dynamics of ctDNA were associated with patient outcomes and tumor response to divarasib treatment.

Intratumoral Escherichia Is Associated With Improved Survival to Single-Agent Immune Checkpoint Inhibition in Patients With Advanced Non-Small-Cell Lung Cancer.

PURPOSEThe impact of the intratumoral microbiome on immune checkpoint inhibitor (ICI) efficacy in patients with non-small-cell lung cancer (NSCLC) is unknown. Preclinically, intratumoral Escherichia is associated with a proinflammatory tumor microenvironment and decreased metastases. We sought to determine whether intratumoral Escherichia is associated with outcome to ICI in patients with NSCLC.PATIENTS AND METHODSWe examined the intratumoral microbiome in 958 patients with advanced NSCLC treated with ICI by querying unmapped next-generation sequencing reads against a bacterial genome database. Putative environmental contaminants were filtered using no-template controls (n = 2,378). The impact of intratumoral Escherichia detection on overall survival (OS) was assessed using univariable and multivariable analyses. The findings were further validated in an external independent cohort of 772 patients. Escherichia fluorescence in situ hybridization (FISH) and transcriptomic profiling were performed.RESULTSIn the discovery cohort, read mapping to intratumoral Escherichia was associated with significantly longer OS (16 v 11 months; hazard ratio, 0.73 [95% CI, 0.59 to 0.92]; P = .0065) in patients treated with single-agent ICI, but not combination chemoimmunotherapy. The association with OS in the single-agent ICI cohort remained statistically significant in multivariable analysis adjusting for prognostic features including PD-L1 expression (P = .023). Analysis of an external validation cohort confirmed the association with improved OS in univariable and multivariable analyses of patients treated with single-agent ICI, and not in patients treated with chemoimmunotherapy. Escherichia localization within tumor cells was supported by coregistration of FISH staining and serial hematoxylin and eosin sections. Transcriptomic analysis correlated Escherichia-positive samples with expression signatures of immune cell infiltration.CONCLUSIONRead mapping to potential intratumoral Escherichia was associated with survival to single-agent ICI in two independent cohorts of patients with NSCLC.

Characterizing the Genomic Landscape of the Micropapillary Subtype of Urothelial Carcinoma of the Bladder Harboring Activating Extracellular Mutations of ERBB2.

The micropapillary subtype of urothelial carcinoma (MPUC) of the bladder is a very aggressive histological variant of urothelial bladder cancer (UBC). A high frequency of MPUC contains activating mutations in the extracellular domain (ECD) of ERBB2. We sought to further characterize ERBB2 ECD-mutated MPUC to identify additional genomic alterations that have been associated with tumor progression and therapeutic response. In total, 5,485 cases of archived formalin-fixed, paraffin-embedded UBC underwent comprehensive genomic profiling to identify ERBB2 ECD-mutated MPUC and evaluate the frequencies of genomic co-alterations. We identified 219 cases of UBC with ERBB2 ECD mutations (74% S310F and 26% S310Y), of which 63 (28.8%) were MPUC. Genomic analysis revealed that TERT, TP53, and ARID1A were the most common co-altered genes in ERBB2-mutant MPUC (82.5%, 58.7%, and 39.7%, respectively) and did not differ from ERBB2-mutant non-MPUC (86.5%, 51.9%, and 35.3%). The main differences between ERBB2 ECD-mutated MPUC compared with non-MPUC were KMT2D, RB1, and MTAP alterations. KMT2D and RB1 are tumor-suppressor genes. KMT2D frequency was significantly decreased in ERBB2 ECD-mutated MPUC (6.3%) in contrast to non-MPUC (27.6%; P < .001). RB1 mutations were more frequent in ERBB2 ECD-mutated MPUC (33.3%) than in non-MPUC (17.3%; P = .012). Finally, MTAP loss, an emerging biomarker for new synthetic lethality-based anticancer drugs, was less frequent in ERBB2 ECD-mutated MPUC (11.1%) than in non-MPUC (26.9%; P = .018). Characterizing the genomic landscape of MPUC may not only improve our fundamental knowledge about this aggressive morphological variant of UBC but also has the potential to identify possible prognostic and predictive biomarkers that may drive tumor progression and dictate treatment response to therapeutic approaches.

Increased KRAS G12C Prevalence, High Tumor Mutational Burden, and Specific Mutational Signatures Are Associated With MUTYH Mutations: A Pan-Cancer Analysis.

The aim of this study was to determine the pan-cancer landscape of MUTYH alterations and the relationship between MUTYH mutations and potentially actionable biomarkers such as specific genomic alterations, tumor mutational burden, and mutational signatures. We used a large pan-cancer comprehensive genomic dataset from patients profiled (tissue next generation sequencing) during routine clinical care. Overall, 2.8% of 229 120 solid tumors had MUTYH alterations, of which 55% were predicted germline. Thirty tumor types had a 2% or greater MUTYH mutation rate. MUTYH-altered versus -WT cancers had significantly higher tumor mutational burden and more frequent alterations in KRAS G12C, but not in KRAS in general; these observations were statistically significant, especially in colorectal cancers. Across cancers, PD-L1 expression levels (immunohistochemistry) were not associated with MUTYH alteration status. In silico computation demonstrated that MUTYH mutational signatures are associated with higher levels of hydrophobicity (which may reflect higher immunogenicity of neoantigens) relative to several other signature types such as microsatellite instability. Survival of patients with MUTYH-altered versus -WT tumors was similar. In conclusion, comprehensive genomic profiling suggests that several features of MUTYH-altered cancers may be pharmacologically targetable. Drugs such as sotorasib (targeting KRAS G12C) and immune checkpoint inhibitors, targeting the increased mutational load and higher neo-antigen hydrophobicity/immunogenicity merit investigation in MUTYH-mutated malignancies.

Prevalence of targetable genomic alterations in young women with advanced breast cancer: a cross-sectional study.

PURPOSE: Approximately 5% of breast cancers each year are diagnosed in young women < 40 years who tend to have worse clinical outcomes. We compared genomic alterations using comprehensive genomic profiling (CGP) of tumor tissue among very young women (< 30 years) and young women (30-39 years) compared to women ≥ 40 years at diagnosis. METHODS: 2049 advanced breast cancer cases were submitted to Foundation Medicine within a 22-month window for CGP. Hybrid-capture based CGP was performed to evaluate all classes of genomic alterations. Tumor mutational burden was determined on at least 0.8 Mbp of sequenced DNA and microsatellite instability was determined on at least 95 loci. Immunocyte PD-L1 expression was determined by immunohistochemistry. RESULTS: Of the total cases, 28 (1.37%) were < 30 years, 159 (7.76%) were 30-39 years, and 1862 (90.87%) were ≥ 40 at time of diagnosis. Breast tumors were less likely to be estrogen receptor positive in younger women (54% of < 30 years, p > 0.05; 60% of 30-39 years, p < 0.001; 69.4% of ≥ 40 years) and more likely to be triple negative (43%, p = 0.05; 33%, p = 0.05; 26.1% respectively). Young women had higher rates of BRCA1 mutations (17.9% <30 years, p < 0.001; 10.1% 30-39 years, p < 0.001; 2.6% ≥40 years), but lower rates of CDH1 (7.1% <30 years, p > 0.05; 5.0% 30-39 years, p < 0.001; 15.4% ≥40 years) and PIK3CA mutations (17.9% <30 years, p = 0.02; 17.6% 30-39 years, p < 0.001; 40.0% ≥40 years). CONCLUSION: Our findings contribute to the growing literature demonstrating unique genetic profiles among young women diagnosed with breast cancer, compared to older women.

Liquid biopsy comprehensive genomic profiling of lung cancer in the Italian population: A real-world experience.

OBJECTIVES: Liquid biopsy with next-generation sequencing (NGS) has emerged as a promising tool for tumor mutation profiling. In this study, we describe the genomic profile of Italian lung cancer patients tested with blood-based comprehensive genomic profiling (CGP) to assess the genomic landscape complexity and its impact on enhancing treatment options for patients. MATERIALS AND METHODS: Between January 2021 and December 2021, a total of 229 lung cancer patients were profiled by FoundationOne®Liquid CDx (F1LCDx®) assay on circulating tumor DNA (ctDNA). F1LCDx® reports alterations across 324 cancer-related genes and genomic signatures, including tumor fraction (TF) and blood-based tumor mutational burden (bTMB). Detected variants were classified according to the ESMO Scale of Clinical Actionability for molecular Targets (ESCAT). RESULTS: 90.4% of patients had at least one detectable alteration in plasma. The most frequently mutated genes were TP53 (47.6%), DNMT3A (33.2%), EGFR (20.1%), and KRAS (15.7%). Elevated TF was detected in 18.3% of patients, suggesting high reliability of test results. According to the ESCAT classification, potentially actionable alterations (Tier I-II) were identified in 27.1% of samples. An additional 5.2% harbored an alteration for which an approved drug is available in other cancer types (Tier III). Furthermore, 13.1% of tumors exhibited high bTMB, which may predict response to immunotherapy. Overall, 156 (68.1%) patients were eligible for enrolment in clinical trials. CONCLUSION: Liquid biopsy NGS is a viable and valuable approach to guide personalized therapy. The use of blood-based CGP may help identify a larger number of actionable mutations and increase chances of enrolment in clinical trials.

Unique Spectrum of Activating BRAF Alterations in Prostate Cancer.

PURPOSE: Alterations in BRAF have been reported in 3% to 5% of prostate cancer, although further characterization is lacking. Here, we describe the nature of BRAF alterations in prostate cancer using a large cohort from commercially available tissue and liquid biopsies subjected to comprehensive genomic profiling (CGP). EXPERIMENTAL DESIGN: Tissue and liquid biopsies from patients with prostate cancer were profiled using FoundationOne CDx and FoundationOne Liquid CDx CGP assays, respectively. Tissue biopsies from non-prostate cancer types were used for comparison (n = 275,151). Genetic ancestry was predicted using a single-nucleotide polymorphism (SNP) based approach. RESULTS: Among 15,864 tissue biopsies, BRAF-activating alterations were detected in 520 cases (3.3%). The majority (463 samples, 2.9%) harbored class II alterations, including BRAF rearrangements (243 samples, 1.5%), K601E (101 samples, 0.6%), and G469A (58 samples, 0.4%). BRAF-altered prostate cancers were enriched for CDK12 mutations (OR, 1.87; 9.2% vs. 5.2%; P = 0.018), but depleted in TMPRSS2 fusions (OR, 0.25; 11% vs. 32%; P < 0.0001), PTEN alterations (OR, 0.47; 17% vs. 31%; P < 0.0001), and APC alterations (OR, 0.48; 4.4% vs. 8.9%; P = 0.018) relative to BRAF wild-type (WT) disease. Compared with patients of European ancestry, BRAF alterations were more common in tumors from patients of African ancestry (5.1% vs. 2.9%, P < 0.0001) and Asian ancestry (6.0% vs. 2.9%, P < 0.001). CONCLUSIONS: Activating BRAF alterations were detected in approximately 3% of prostate cancers, and most were class II mutations and rearrangements; BRAF V600 mutations were exceedingly rare. These findings suggest that BRAF activation in prostate cancer is unique from other cancers and supports further clinical investigation of therapeutics targeting the mitogen-activated protein kinase (MAPK) pathway.

Comprehensive genomic profiling and treatment patterns across ancestries in advanced prostate cancer: a large-scale retrospective analysis.

BACKGROUND: Men of African ancestry experience the greatest burden of prostate cancer globally, but they are under-represented in genomic and precision medicine studies. Therefore, we sought to characterise the genomic landscape, comprehensive genomic profiling (CGP) utilisation patterns, and treatment patterns across ancestries in a large, diverse, advanced prostate cancer cohort, to determine the impact of genomics on ancestral disparities. METHODS: In this large-scale retrospective analysis, the CGP-based genomic landscape was evaluated in biopsy sections from 11 741 patients with prostate cancer, with ancestry inferred using a single nucleotide polymorphism-based approach. Admixture-derived ancestry fractions for each patient were also interrogated. Independently, clinical and treatment information was retrospectively reviewed for 1234 patients in a de-identified US-based clinicogenomic database. Prevalence of gene alterations, including actionable gene alterations, was assessed across ancestries (n=11 741). Furthermore, real-world treatment patterns and overall survival was assessed in the subset of patients with linked clincogenomic information (n=1234). FINDINGS: The CGP cohort included 1422 (12%) men of African ancestry and 9244 (79%) men of European ancestry; the clinicogenomic database cohort included 130 (11%) men of African ancestry and 1017 (82%) men of European ancestry. Men of African ancestry received more lines of therapy before CGP than men of European ancestry (median of two lines [IQR 0-8] vs one line [0-10], p=0·029). In genomic analyses, ancestry-specific mutational landscapes were observed, but the prevalence of alterations in AR, the DNA damage response pathway, and other actionable genes were similar across ancestries. Similar genomic landscapes were observed in analyses that accounted for admixture-derived ancestry fractions. After undergoing CGP, men of African ancestry were less likely to receive a clinical study drug compared with men of European ancestry (12 [10%] of 118 vs 246 [26%] of 938, p=0·0005). INTERPRETATION: Similar rates of gene alterations with therapy implications suggest that differences in actionable genes (including AR and DNA damage response pathway genes) might not be a main driver of disparities across ancestries in advanced prostate cancer. Later CGP utilisation and a lower rate of clinical trial enrolment observed in men of African ancestry could affect genomics, outcomes, and disparities. FUNDING: American Society for Radiation Oncology, Department of Defense, Flatiron Health, Foundation Medicine, Prostate Cancer Foundation, and Sylvester Comprehensive Cancer Center.

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

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.

Integrative Analysis of a Large Real-World Cohort of Small Cell Lung Cancer Identifies Distinct Genetic Subtypes and Insights into Histologic Transformation.

Small cell lung cancer (SCLC) is a recalcitrant neuroendocrine carcinoma with dismal survival outcomes. A major barrier in the field has been the relative paucity of human tumors studied. Here we provide an integrated analysis of 3,600 "real-world" SCLC cases. This large cohort allowed us to identify new recurrent alterations and genetic subtypes, including STK11-mutant tumors (1.7%) and TP53/RB1 wild-type tumors (5.5%), as well as rare cases that were human papillomavirus-positive. In our cohort, gene amplifications on 4q12 are associated with increased overall survival, whereas CCNE1 amplification is associated with decreased overall survival. We also identify more frequent alterations in the PTEN pathway in brain metastases. Finally, profiling cases of SCLC containing oncogenic drivers typically associated with NSCLC demonstrates that SCLC transformation may occur across multiple distinct molecular cohorts of NSCLC. These novel and unsuspected genetic features of SCLC may help personalize treatment approaches for this fatal form of cancer. SIGNIFICANCE: Minimal changes in therapy and survival outcomes have occurred in SCLC for the past four decades. The identification of new genetic subtypes and novel recurrent mutations as well as an improved understanding of the mechanisms of transformation to SCLC from NSCLC may guide the development of personalized therapies for subsets of patients with SCLC. This article is highlighted in the In This Issue feature, p. 1501.

Genetic Heterogeneity and Tissue-specific Patterns of Tumors with Multiple PIK3CA Mutations.

PURPOSE: To comprehensively characterize tissue-specific and molecular subclasses of multiple PIK3CA (multi-PIK3CA) mutations and assess their impact on potential therapeutic outcomes. EXPERIMENTAL DESIGN: We profiled a pan-cancer cohort comprised of 352,392 samples across 66 tumor types using a targeted hybrid capture-based next-generation sequencing panel covering at least 324 cancer-related genes. Molecularly defined subgroups, allelic configuration, clonality, and mutational signatures were identified and tested for association with PI3K inhibitor therapeutic response. RESULTS: Multi-PIK3CA mutations are found in 11% of all PIK3CA-mutant tumors, including 9% of low tumor mutational burden (TMB) PIK3CA-mutant tumors, and are enriched in breast and gynecologic cancers. Multi-PIK3CA mutations are frequently clonal and in cis on the same allele and occur at characteristic positions across tumor types. These mutations tend to be mutually exclusive of mutations in other driver genes, and of genes in the PI3K pathway. Among PIK3CA-mutant tumors with a high TMB, 18% are multi-PIK3CA mutant and often harbor an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC) mutational signature. Despite large differences in specific allele combinations comprising multi-PIK3CA mutant tumors, especially across cancer types, patients with different classes of multi-PIK3CA mutant estrogen receptor-positive, HER2-negative breast cancers respond similarly to PI3K inhibition. CONCLUSIONS: Our pan-tumor study provides biological insights into the genetic heterogeneity and tissue specificities of multi-PIK3CA mutations, with potential clinical utility to guide PI3K inhibition strategies.

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

PURPOSE: Alpelisib 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 DESIGN: A 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). RESULTS: PIK3CAm 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). CONCLUSIONS: CGP 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.

Tissue and liquid biopsy profiling reveal convergent tumor evolution and therapy evasion in breast cancer.

Pathological and genomic profiling have transformed breast cancer care by matching patients to targeted treatments. However, tumors evolve and evade therapeutic interventions often through the acquisition of genomic mutations. Here we examine patients profiled with tissue (TBx) and liquid biopsy (LBx) as part of routine clinical care, to characterize the tumor evolutionary landscape and identify potential vulnerabilities in the relapsed setting. Real-world evidence demonstrates that LBx is utilized later in care and identifies associations with intervening therapy. While driver events are frequently shared, acquired LBx alterations are detected in a majority of patients, with the highest frequency in ER+ disease and in patients with longer biopsy intervals. Acquired mutations are often polyclonal and present at lower allelic fractions, suggesting multi-clonal convergent evolution. In addition to well-characterized resistance mutations (e.g., ESR1, NF1, RB1, ERBB2), we observe a diversity of rarer but potentially targetable mutations (e.g., PIK3CA, HRAS/NRAS/KRAS, FGFR1/2/3, BRAF) and fusions (e.g., FGFR1/2, ERBB2, RET), as well as BRCA1/2 reversions through a variety of mechanisms, including splice alterations and structural deletions. This study provides insights on treatment and selection-driven tumor evolution and identifies potential combinatorial treatment options in advanced breast cancer.

Comprehensive pan-cancer genomic landscape of KRAS altered cancers and real-world outcomes in solid tumors.

Recent clinical development of KRAS inhibitors has heightened interest in the genomic landscape of KRAS-altered cancers. We performed a pan-cancer analysis of KRAS-altered samples from 426,706 adult patients with solid or hematologic malignancies using comprehensive genomic profiling; additional analyses included 62,369 liquid biopsy and 7241 pediatric samples. 23% of adult pan-cancer samples had KRAS alterations; 88% were mutations, most commonly G12D/G12V/G12C/G13D/G12R, and prevalence was similar in liquid biopsies. Co-alteration landscapes were largely similar across KRAS mutations but distinct from KRAS wild-type, though differences were observed in some tumor types for tumor mutational burden, PD-L1 expression, microsatellite instability, and other mutational signatures. Prognosis of KRAS-mutant versus other genomic cohorts of lung, pancreatic, and colorectal cancer were assessed using a real-world clinicogenomic database. As specific KRAS inhibitors and combination therapeutic strategies are being developed, genomic profiling to understand co-alterations and other biomarkers that may modulate response to targeted or immunotherapies will be imperative.

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.

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

BACKGROUND: Tumor 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. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: Overall, our results suggest that tissue TMB remains stable with time, though specific therapies such as immunotherapy may correlate with an increase in TMB. TRIAL REGISTRATION: NCT02478931 , registered June 23, 2015.

Concordance of DNA Repair Gene Mutations in Paired Primary Prostate Cancer Samples and Metastatic Tissue or Cell-Free DNA.

IMPORTANCE: DNA damage repair (DDR) gene mutations represent actionable alterations that can guide precision medicine strategies for advanced prostate cancer. However, acquisition of contemporary tissue samples for molecular testing can be a barrier to deploying precision medicine approaches. We hypothesized that most DDR alterations represent truncal events in prostate cancer and that primary tissue would faithfully reflect mutations found in cell-free circulating tumor DNA (ctDNA) and/or metastatic tissue. OBJECTIVE: To assess concordance in DDR gene alterations between primary prostate cancer and metastases or ctDNA specimens. DESIGN, SETTING, AND PARTICIPANTS: Patients were included if a DDR pathway mutation was detected in metastatic tissue or ctDNA and primary tissue sequencing was available for comparison. Sequencing data from 3 cohorts were analyzed: (1) FoundationOne, (2) University of Washington clinical cases (University of Washington-OncoPlex or Stand Up to Cancer-Prostate Cancer Foundation International Dream Team sequencing pipelines), and (3) University of Washington rapid autopsy series. Only pathogenic somatic mutations were included, and more than 30 days between primary tumor tissue and ctDNA and/or metastatic tissue acquisition was required. Clonal hematopoiesis of indeterminate potential (CHIP) and germline events were adjudicated by an expert molecular pathologist and excluded. MAIN OUTCOMES AND MEASURES: The DDR gene alterations detected in primary prostate tissue matched with metastatic tissue and/or ctDNA findings. RESULTS: A total of 72 men with known DDR alterations were included in the analysis, and primary samples with paired ctDNA and/or metastatic tissue were sequenced. After excluding patients with ctDNA where only CHIP and/or germline events (n = 21) were observed, 51 patients remained and were included in the final analysis. The median (range) time from acquisition of primary tissue to acquisition of ctDNA or tumor tissue was 55 (5-193) months. Concordance in DDR gene mutation status across samples was 84% (95% CI, 71%-92%). Rates of concordance between metastatic-primary and ctDNA-primary pairs were similar when patients with CHIP events were excluded. Multiclonal BRCA2 reversion mutations associated with resistance to PARP inhibitors and platinum chemotherapy were detected in ctDNA from 2 patients. CONCLUSIONS AND RELEVANCE: In this genetic association study of 3 patient cohorts, primary prostate tissue accurately reflected the mutational status of actionable DDR genes in metastatic tissue, consistent with DDR alterations being truncal in most patients. After excluding likely CHIP events, ctDNA profiling accurately captured these DDR mutations while also detecting reversion alterations that may suggest resistance mechanisms.