Next-generation Sequencing Of Tumor DNA Research Articles

Up-front mutation detection in circulating tumor DNA by droplet digital PCR has added diagnostic value in lung cancer

Identification of actionable mutations in advanced stage non-squamous non-small-cell lung cancer (NSCLC) patients is recommended by guidelines as it enables treatment with targeted therapies. In current practice, mutations are identified by next-generation sequencing of tumor DNA (tDNA-NGS), which requires tissue biopsies of sufficient quality. Alternatively, circulating tumor DNA (ctDNA) could be used for mutation analysis. This prospective, multicenter study establishes the diagnostic value of ctDNA analysis by droplet digital PCR (ctDNA-ddPCR) in patients with primary lung cancer.CtDNA from 458 primary lung cancer patients was analyzed using a panel of multiplex ddPCRs for EGFR (Ex19Del, G719S, L858R, L861Q and S768I), KRAS G12/G13 and BRAF V600 mutations. For 142 of 175 advanced stage non-squamous NSCLC patients tDNA-NGS results were available to compare to ctDNA-ddPCR. tDNA-NGS identified 98 mutations, of which ctDNA-ddPCR found 53 mutations (54%), including 32 of 45 (71%) targetable driver mutations. In 2 of these 142 patients, a mutation was found by ctDNA-ddPCR only. In 33 advanced stage patients lacking tDNA-NGS results, ctDNA-ddPCR detected 15 additional mutations, of which 7 targetable.Overall, ctDNA-ddPCR detected 70 mutations and tDNA-NGS 98 mutations in 175 advanced NSCLC patients. Using an up-front ctDNA-ddPCR strategy, followed by tDNA-NGS only if ctDNA-ddPCR analysis is negative, increases the number of mutations found from 98 to 115 (17%). At the same time, up-front ctDNA-ddPCR reduces tDNA-NGS analyses by 40%, decreasing the need to perform (additional) biopsies.

Somatic IDH1 Hotspot Variants in Chinese Patients With Pheochromocytomas and Paragangliomas.

IDH1 is a pheochromocytoma/paraganglioma (PPGL) susceptibility gene; however, its role, especially in the Chinese population, has not been characterized. To determine the prevalence of somatic IDH1 hotspot variants in a large cohort of Chinese patients with PPGLs and to summarize associated phenotypes. This retrospective cross-sectional study was based on a main cohort of 1141 patients with PPGLs from 2 tertiary-care centers in China. We included 50 cases with urinary bladder paragangliomas (UBPGLs), of whom 29 were part of the main cohort and 21 were from other centers. Two additional cases with IDH1 hotspot variants not part of the main cohort were also included for summarizing IDH1-associated phenotypes. Next-generation sequencing of tumor DNA was used to analyze a customized panel of genes. The overall prevalence of IDH1 hotspot variants in the main cohort was 0.5% (6/1141). Among those PPGLs without mutations in 15 common driver genes, the prevalence of IDH1 variants was 0.9% (4/455). When restricted to paraganglioma (PGL) without mutations, the prevalence reached 4.7% (4/86). Among UBPGLs, IDH1 hotspot variants accounted for 8% (4/50). Together, all 10 patients (9 PGLs and 1 pheochromocytoma) with IDH1 hotspot variants, including 3 females with concurrent EPAS1 hotspot variants, had apparently sporadic tumors, without metastasis or recurrence. There were 3 patients with biochemical data, all showing a non-adrenergic phenotype. The somatic IDH1 hotspot variants cause PPGL development in some Chinese patients, especially among those apparently sporadic PGLs with a non-adrenergic phenotype and without mutations in major PPGL driver genes.

Use of next-generation sequencing (NGS) to study the impact of demographic variables on prevalence of driver mutations in non-small cell lung cancer (NSCLC).

e21030 Background: The management of NSCLC is rapidly evolving with the discovery of new driver mutations and the introduction of targeted therapies (TT). The list of targets with FDA-approved TT is estimated to cover over half of the cases of non-squamous NSCLC. The prevalence of driver mutations varies significantly depending on several demographic variables such as smoking status, race, and sex. This abstract presents data obtained from a unique, racially diverse cohort of patients in the Washington, DC metropolitan area. Methods: Retrospective review of circulating tumor DNA NGS data from 2 commercial testing companies (Guardant and Tempus) and demographic data from our health records on 66 patients with NSCLC treated at the George Washington University Medical Faculty Associates between October 2017 and November 2021 was performed. Demographic information including age, sex, race, and smoking history, as well as histology, stage at diagnosis, and driver mutations were collected. Only patients with non-squamous NSCLC and identifiable mutations were included. Results: Mean age was 67. Most patients in this cohort were female (56.1%, male 43.9%), African-American (62.1%, Caucasian 25.8%, African 9.1%, Asian 3.0%), had smoking history (former smoker 48.5%, current smoker 25.8%, never smoker 25.8%), had adenocarcinoma (78.8%, other NSCLC (not squamous cell carcinoma or adenocarcinoma) 21.2%) with advanced stage (stage 4 62.1%, stage 3 28.8%, stage 1 3.0%, unknown 6.1%). Mutations are listed as follows: KRAS 28.8% (G12C accounted for 58% of all KRAS mutations), EGFR 19.7% (exons 19 and 21 mutations such as del19 and L858R accounted for 69%), MET 9.1% (MET amplification made up 83%), Her2 3.0% (2/66), ALK 3.0% (2/66), RET 1.5% (1/66), non-targetable mutations 34.8% (23/66). A table at the end of this abstract shows the frequency of three most prevalent mutations in this cohort distinguished by race, sex, and smoking status. Conclusions: KRAS was the most prevalent driver mutation in this cohort, followed by EGFR and MET. In concordance with the existing literature, EGFR was more prevalent in non-smokers and females, while KRAS was more common among smokers. Interestingly, a larger percentage of Africans had EGFR mutations compared to African-Americans. Given the small sample size, however, a larger scale study is needed to investigate this further. These findings show that demographic factors influence the prevalence of driver mutations which can have therapeutic implications in patients with NSCLC.[Table: see text]

TreeClone: Reconstruction of tumor subclone phylogeny based on mutation pairs using next generation sequencing data

We present TreeClone, a latent feature allocation model to reconstruct tumor subclones subject to phylogenetic evolution that mimics tumor evolution. Similar to most current methods, we consider data from next-generation sequencing of tumor DNA. Unlike most methods that use information in short reads mapped to single nucleotide variants (SNVs), we consider subclone phylogeny reconstruction using pairs of two proximal SNVs that can be mapped by the same short reads. As part of the Bayesian inference model, we construct a phylogenetic tree prior. The use of the tree structure in the prior greatly strengthens inference. Only subclones that can be explained by a phylogenetic tree are assigned non-negligible probabilities. The proposed Bayesian framework implies posterior distributions on the number of subclones, their genotypes, cellular proportions and the phylogenetic tree spanned by the inferred subclones. The proposed method is validated against different sets of simulated and real-world data using single and multiple tumor samples. An open source software package is available at http://www.compgenome.org/treeclone.

Precision oncology based on omics data: The NCT Heidelberg experience.

Precision oncology implies the ability to predict which patients will likely respond to specific cancer therapies based on increasingly accurate, high-resolution molecular diagnostics as well as the functional and mechanistic understanding of individual tumors. While molecular stratification of patients can be achieved through different means, a promising approach is next-generation sequencing of tumor DNA and RNA, which can reveal genomic alterations that have immediate clinical implications. Furthermore, certain genetic alterations are shared across multiple histologic entities, raising the fundamental question of whether tumors should be treated by molecular profile and not tissue of origin. We here describe MASTER (Molecularly Aided Stratification for Tumor Eradication Research), a clinically applicable platform for prospective, biology-driven stratification of younger adults with advanced-stage cancer across all histologies and patients with rare tumors. We illustrate how a standardized workflow for selection and consenting of patients, sample processing, whole-exome/genome and RNA sequencing, bioinformatic analysis, rigorous validation of potentially actionable findings, and data evaluation by a dedicated molecular tumor board enables categorization of patients into different intervention baskets and formulation of evidence-based recommendations for clinical management. Critical next steps will be to increase the number of patients that can be offered comprehensive molecular analysis through collaborations and partnering, to explore ways in which additional technologies can aid in patient stratification and individualization of treatment, to stimulate clinically guided exploratory research projects, and to gradually move away from assessing the therapeutic activity of targeted interventions on a case-by-case basis toward controlled clinical trials of genomics-guided treatments.

Abstract 2240: A case series of ERBB2 indel driver mutations in non-small cell lung cancer identified by cell-free circulating tumor DNA NGS

Abstract Background: In-frame insertions between codons 775 and 881 in exon 20 of the ERBB2 gene, of which a 12 base pair YVMA insertion is the most common, are activating mutations in 2%-4% of non-small cell lung cancers (NSCLC), and have also been reported in exon 19 and 20 in breast cancer. These driver mutations are not captured with IHC or FISH staining because in the majority of cases, the ERBB2 gene is not amplified and HER2 protein is not overexpressed. Next generation sequencing (NGS) of circulating cell-free DNA (cfDNA) provides a non-invasive means of identifying these potential driver mutations. Method: Guardant360TM is a targeted cfDNA NGS panel using hybrid capture and complete exon sequencing for single nucleotide variant detection in 70 genes, copy number amplifications (CNA) in 16 genes, and fusions in six genes and indels in EGFR, ERBB2 and MET exon 14 skipping. De-identified pathology and genotyping reports were reviewed for consecutive patients in which ERBB2 indels were identified in clinical practice. Results: Guardant360 identified ERBB2 indels in 27 of 2,093 (1.3%) of non-squamous NSCLC cases, with a single concomitant ERBB2 gene amplification. For this ERBB2 indel series, pathology reports revealed no patients with HER2 amplification via IHC or FISH, nor ERBB2 point mutation via NGS, but were only available in 25% of cases. Eight of the ERBB2 indels were confirmed by tissue NGS reports with zero false positives (100% PPV). 80% of ERBB2 indels were the common p.Tyr772_Ala775dup (YVMA insertion) in exon 20 followed by other insertions in codons 772 through 814. A single ERBB2 exon 20 p.Leu755_Glu757delinsProLys net deletion at 3.9% mutant allele fration (MAF) was noted in one patient, for whom outcome data was available. Initial tissue was IHC negative for HER2 overexpression at the referring hospital where the archival tissue biopsy was exhausted and so could not be sequenced. Based on the cfDNA finding of ERBB2 indel, the patient was switched from cytotoxic chemotherapy to trastuzumab with objective response on PET/CT and a repeat Guardant360 showed the ERBB2 indel MAF had dropped below the test limit of detection. After four months the patient progressed and it was decided to switch to ado-emtansine trastuzumab (T-DM1) in late November. Results: ERBB2 indels can be identified without tissue in NSCLC patients with 100% PPV in this modest cfDNA series. In a patient whose tissue was not available for sequencing, an objective response with trastuzumab was obtained. Citation Format: Nir Peled, Anna Belilovski-Rozenblum, Lior Soussan-Gutman, Christine Lee, AmirAli Talasaz, Richard B. Lanman. A case series of ERBB2 indel driver mutations in non-small cell lung cancer identified by cell-free circulating tumor DNA NGS. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2240.

Abstract OT1-1-14: Open-label, Phase II trial of afatinib, with or without vinorelbine, for the treatment of HER2-overexpressing inflammatory breast cancer (IBC)*

Abstract Background: IBC is a rare but aggressive form of BC accounting for 1–5% of all BC. Despite recent treatment advances, prognosis remains poor, with 3-year survival rate of 40% vs. 85% in non-IBC. IBC commonly lack ER/PR and more frequently harbor HER2 gene amplification (∼40%), and EGFR overexpression (∼30%) that are associated with poor prognosis. Recent data reported clinical efficacy of lapatinib, a reversible EGFR/HER2 inhibitor, in trastuzumab naïve/resistant HER2-positive IBC. Afatinib is an oral irreversible ErbB Family Blocker that blocks signaling through activated EGFR (ErbB1), HER2 (ErbB2), ErbB4 receptors, and transphosphorylation of ErbB3. Afatinib demonstrated preclinical activity in trastuzumab-resistant cell lines, HER2-positive tumor xenografts and HER2-negative SUM149 xenograft model derived from an IBC cell line. Its clinical efficacy was shown in heavily pretreated, HER2-positive metastatic BC patients (pts) who progressed after trastuzumab, with a partial response in 10% and a clinical benefit in 46% of pts. The combination of afatinib and vinorelbine has been shown to be more effective than either compound alone in reducing tumor volume in the SUM190 xenograft model. The purpose of this biomarker-driven trial is to investigate the efficacy and safety of afatinib alone and in combination with vinorelbine following progression on afatinib monotherapy and the genomic changes that occur during treatment in pts with trastuzumab pretreated or naïve HER2-positive IBC. Methods: Pts are recruited in two parallel cohorts of 20 pts each – trastuzumab-naïve and trastuzumab failure cohorts. Key eligibility criteria: Histologically-confirmed HER2-positive BC; investigator-confirmed IBC characterized by diffuse erythema and edema (peau d'orange) – dermal lymphatic emboli or palpable mass are not necessary for diagnosis; no prior anti-HER2 therapy except trastuzumab in the trastuzumab failure cohort; no prior vinorelbine; ECOG performance status 0–2; disease must be biopsiable. All pts start afatinib monotherapy (40 mg/day oral) in 4-week cycles (Part A). Following disease progression, pts may receive afatinib (40 mg/day) + vinorelbine (i.v. 25 mg/m2/week) (Part B). Primary endpoint: Clinical benefit defined as stable disease (for ≥6 months), partial response or complete response (RECIST 1.1). Secondary endpoints: Objective response, duration of objective response and progression-free survival. Other endpoints include overall survival and safety. Endpoints are assessed separately for Part A and Part B. In Part A (afatinib monotherapy) fresh tumor biopsies are taken pretreatment and following progressive disease, and blood samples are taken pretreatment to explore predictive markers of response/resistance to afatinib, to describe the IBC population that may benefit most and for tumor DNA next-generation sequencing and proteomic analysis. This is one of the first prospective clinical trials to integrate deep exome sequencing into trial design to assess proof of principle of a personalized cancer medicine approach to improve individualized pt treatment. The trial is planned to accrue 40 pts worldwide and recruitment is ongoing since August 2011. *Updated abstract from ASCO 2012. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT1-1-14.

MATH, a novel measure of intratumor genetic heterogeneity, is high in poor-outcome classes of head and neck squamous cell carcinoma

Differences among cancer cells within a tumor are important in tumorigenesis and treatment resistance, yet no measure of intratumor heterogeneity is suitable for routine application. We developed a quantitative measure of intratumor genetic heterogeneity, based on differences among mutated loci in the mutant-allele fractions determined by next-generation sequencing (NGS) of tumor DNA. We then evaluated the application of this measure to head and neck squamous cell carcinoma (HNSCC). We analyzed published electronically available NGS results for 74 HNSCC. For each tumor we calculated mutant-allele tumor heterogeneity (MATH) as the ratio of the width to the center of its distribution of mutant-allele fractions among tumor-specific mutated loci. Intratumor heterogeneity assessed by MATH was higher in three poor-outcome classes of HNSCC: tumors with disruptive mutations in the TP53 gene (versus wild-type TP53 or non-disruptive mutations), tumors negative versus positive for human papillomavirus (even when restricted to tumors having wild-type TP53), and HPV-negative tumors from smokers with more pack-years of cigarette exposure (with TP53 status taken into account). The relation of this type of intratumor heterogeneity to HNSCC outcome classes supports its further evaluation as a prognostic biomarker. As NGS of tumor DNA becomes widespread in clinical research and practice, MATH should provide a simple, quantitative, and clinically practical biomarker to help evaluate relations of intratumor genetic heterogeneity to outcome in any type of cancer.