Liquid biopsy assay for lung carcinoma using centrifuged supernatants from fine-needle aspiration specimens

IntroductionTumor mutation profiling is standard-of-care in lung carcinoma patients. However, comprehensive molecular profiling of small specimens, including core needle biopsy (CNB) and fine-needle aspiration (FNA) specimens, may often be inadequate due to limited tissue. Centrifuged FNA supernatants, which are typically discarded, have emerged recently as a novel liquid-based biopsy for molecular testing. In this study, we evaluate the use of lung carcinoma FNA supernatants for detecting clinically relevant mutations.MethodsSupernatants from lung carcinoma FNA samples (n = 150) were evaluated. Samples were further analyzed using next-generation sequencing (NGS) and ultrasensitive droplet digital PCR (ddPCR). Mutation profiles in a subset of samples were compared with results derived from paired tissue samples from the same patient (n = 67) and available plasma liquid biopsy assay (n = 45).ResultsAll 150 samples yielded adequate DNA and NGS were carried out successfully on 104 (90%) of 116 selected samples. Somatic mutations were detected in 82% of the samples and in 50% of these patients a clinically relevant mutation was identified that would qualify them for targeted therapy or a clinical trial. There was high overall concordance between the mutation profiles of supernatants and the corresponding tissue samples, with 100% concordance with concurrent FNA and 96% with concurrent CNB samples. Comparison of actionable driver mutations detected in supernatant versus plasma samples showed 84% concordance.ConclusionsFNA supernatants can provide a valuable specimen source for genotyping lung carcinoma especially in patients with insufficient tumor tissue, thereby reducing multigene mutation profiling failure rates, improving turnaround times, and avoiding repeat biopsies.     

Plasma Circulating Tumor DNA and Clonal Hematopoiesis in Metastatic Renal Cell Carcinoma

BackgroundThere is a lack of molecularly-informed biomarkers for patients with metastatic renal cell carcinoma (RCC). Plasma cell-free DNA (cfDNA) sequencing is a minimally-invasive alternative to tissue for profiling the genome in other cancers but relevance in metastatic RCC remains unclear.Materials and MethodsWhole blood was collected from 55 patients with metastatic RCC. Plasma cfDNA and leukocyte DNA were subjected to targeted sequencing across 981 cancer genes. Matched tumor tissue from 14 patients was analyzed.ResultsThirty-three percent of patients had evidence for RCC-derived circulating tumor DNA (ctDNA), significantly lower than patients with metastatic prostate or bladder cancer analyzed using the same approach. Among ctDNA-positive patients, ctDNA fraction averaged only 3.9% and showed no strong association with clinical variables. In these patients, the most commonly mutated genes were VHL, BAP1, and PBRM1, and matched tissue concordance was 77%. Evidence of somatic expansions unrelated to RCC, such as clonal hematopoiesis of indeterminate potential, were detected in 43% of patients. Pathogenic germline mutations in DNA repair genes were detected in 11% of patients. CtDNA-positive patients had shorter overall survival and progression-free survival on first-line therapy. Patients with evidence of clonal hematopoiesis of indeterminate potential had an intermediate prognosis compared with ctDNA-positive and -negative patients.ConclusionsCfDNA sequencing enables straightforward characterization of the somatic RCC genome in a minority of patients with metastatic RCC. Owing to low ctDNA abundance, and the presence of non-RCC derived somatic clones in circulation, cfDNA sequencing may not be a simple pan-patient alternative to tissue biopsy in metastatic RCC.     

Feasibility of real time next generation sequencing of cancer genes linked to drug response: Results from a clinical trial

The successes of targeted drugs with companion predictive biomarkers and the technological advances in gene sequencing have generated enthusiasm for evaluating personalized cancer medicine strategies using genomic profiling. We assessed the feasibility of incorporating real‐time analysis of somatic mutations within exons of 19 genes into patient management. Blood, tumor biopsy and archived tumor samples were collected from 50 patients recruited from four cancer centers. Samples were analyzed using three technologies: targeted exon sequencing using Pacific Biosciences PacBio RS, multiplex somatic mutation genotyping using Sequenom MassARRAY and Sanger sequencing. An expert panel reviewed results prior to reporting to clinicians. A clinical laboratory verified actionable mutations. Fifty patients were recruited. Nineteen actionable mutations were identified in 16 (32%) patients. Across technologies, results were in agreement in 100% of biopsy specimens and 95% of archival specimens. Profiling results from paired archival/biopsy specimens were concordant in 30/34 (88%) patients. We demonstrated that the use of next generation sequencing for real‐time genomic profiling in advanced cancer patients is feasible. Additionally, actionable mutations identified in this study were relatively stable between archival and biopsy samples, implying that cancer mutations that are good predictors of drug response may remain constant across clinical stages.     

Ultra-deep next-generation sequencing of plasma cell-free DNA in patients with advanced lung cancers: results from the Actionable Genome Consortium

BackgroundNoninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients while also elucidating disease heterogeneity. We sought to develop an ultra-deep plasma next-generation sequencing (NGS) assay for patients with non-small-cell lung cancers (NSCLC) that could detect targetable oncogenic drivers and resistance mutations in patients where tissue biopsy failed to identify an actionable alteration.Patients and methodsPlasma was prospectively collected from patients with advanced, progressive NSCLC. We carried out ultra-deep NGS using cfDNA extracted from plasma and matched white blood cells using a hybrid capture panel covering 37 lung cancer-related genes sequenced to 50 000× raw target coverage filtering somatic mutations attributable to clonal hematopoiesis. Clinical sensitivity and specificity for plasma detection of known oncogenic drivers were calculated and compared with tissue genotyping results. Orthogonal ddPCR validation was carried out in a subset of cases.ResultsIn 127 assessable patients, plasma NGS detected driver mutations with variant allele fractions ranging from 0.14% to 52%. Plasma ddPCR for EGFR or KRAS mutations revealed findings nearly identical to those of plasma NGS in 21 of 22 patients, with high concordance of variant allele fraction (r = 0.98). Blinded to tissue genotype, plasma NGS sensitivity for de novo plasma detection of known oncogenic drivers was 75% (68/91). Specificity of plasma NGS in those who were driver-negative by tissue NGS was 100% (19/19). In 17 patients with tumor tissue deemed insufficient for genotyping, plasma NGS identified four KRAS mutations. In 23 EGFR mutant cases with acquired resistance to targeted therapy, plasma NGS detected potential resistance mechanisms, including EGFR T790M and C797S mutations and ERBB2 amplification.ConclusionsUltra-deep plasma NGS with clonal hematopoiesis filtering resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsy was inadequate for genotyping.     

Clinical Application of the FoundationOne CDx Assay to Therapeutic Decision-Making for Patients with Advanced Solid Tumors

BackgroundImplementation of personalized medicine requires the accessibility of tumor molecular profiling in order to allow prioritization of appropriate targeted therapies for individual patients. Our aim was to study the role of comprehensive genomic profiling assays that may inform treatment recommendations for patients with solid tumors.Materials and MethodsWe performed a prospective study to evaluate the feasibility of application of the FoundationOne CDx panel—which detects substitutions, insertions and deletions, and copy number alterations in 324 genes, select gene rearrangements, and genomic signatures including microsatellite instability and tumor mutation burden (TMB)—to patients with advanced or recurrent solid tumors before its approval in Japan.ResultsA total of 181 samples were processed for genomic testing between September 2018 and June 2019, with data being successfully obtained for 175 of these samples, yielding a success rate of 96.7%. The median turnaround time was 41 days (range, 21–126 days). The most common known or likely pathogenic variants were TP53 mutations (n = 113), PIK3CA mutations (n = 33), APC mutations (n = 32), and KRAS mutations (n = 29). Among the 153 patients assessed for TMB, the median TMB was 4 mutations/Mb, and tumors with a high TMB (≥10 mutations/Mb) were more prevalent for lung cancer (11/32) than for other solid tumor types (9/121, Fisher''s exact test p < .01). No clear trend toward increased efficacy for immune checkpoint inhibitor (ICI) monotherapy or ICI combination chemotherapy in patients with a high programmed cell death–ligand 1 tumor proportion score or a high TMB was apparent. Among the 174 patients found to harbor known or likely pathogenic actionable alterations, 24 individuals (14%) received matched targeted therapy.ConclusionThe FoundationOne CDx assay was performed with formalin‐fixed, paraffin‐embedded tumor specimens with a success rate of >95%. Such testing may inform the matching of patients with cancer with investigational or approved targeted drugs.Implications for PracticeThis prospective cohort study was initiated to investigate the feasibility and utility of clinical application of FoundationOne CDx. A total of 181 samples were processed for genomic testing between September 2018 and June 2019, with data being successfully obtained for 175 of these samples, yielding a success rate of 96.7%, and 24 individuals (14%) received matched targeted therapy.     

Clinical next generation sequencing to identify actionable aberrations in a phase I program

Purpose

We determined the frequency of recurrent hotspot mutations in 46 cancer-related genes across tumor histologies in patients with advanced cancer.

Methods

We reviewed data from 500 consecutive patients who underwent genomic profiling on an IRB-approved prospective clinical protocol in the Phase I program at the MD Anderson Cancer Center. Archival tumor DNA was tested for 740 hotspot mutations in 46 genes (Ampli-Seq Cancer Panel; Life Technologies, CA).

Results

Of the 500 patients, 362 had at least one reported mutation/variant. The most common likely somatic mutations were within TP53 (36%), KRAS (11%), and PIK3CA (9%) genes. Sarcoma (20%) and kidney (30%) had the lowest proportion of likely somatic mutations detected, while pancreas (100%), colorectal (89%), melanoma (86%), and endometrial (75%) had the highest. There was high concordance in 62 patients with paired primary tumors and metastases analyzed. 151 (30%) patients had alterations in potentially actionable genes. 37 tumor types were enrolled; both rare actionable mutations in common tumor types and actionable mutations in rare tumor types were identified.

Conclusion

Multiplex testing in the CLIA environment facilitates genomic characterization across multiple tumor lineages and identification of novel opportunities for genotype-driven trials.     

Benefit of Targeted DNA Sequencing in Advanced Non–Small-Cell Lung Cancer Patients Without EGFR and ALK Alterations on Conventional Tests

BackgroundGenetic sequencing testing has become widely used to inform treatment decisions for advanced non–small-cell lung cancer (NSCLC) patients. We analyzed benefits of genetic sequencing testing in real practice.Patients and MethodsWe retrospectively reviewed 209 NSCLC patients who had no EGFR and ALK alterations on routine molecular tests and underwent next-generation targeted DNA sequencing of 380 cancer-related genes between November 2013 and October 2016. Median patient age was 59 years. A total of 96 patients (46%) were never smokers, and 195 patients (93%) had adenocarcinoma.ResultsAmong 209 total patients, 64 (31%) demonstrated actionable genetic alterations; 20 had EGFR mutations (6 L858R, 8 exon 19 deletions, 1 L861Q, 1 G719S, 4 exon 20 duplications), 4 ALK fusions, 9 ROS1 fusions, 6 BRAF V600E mutations, 15 RET fusions, 1 MET high-level amplification, 6 MET exon 14 skipping mutations, and 3 ERBB2 exon 20 insertion mutations. Of the 64 patients harboring actionable alterations, 28 patients received therapy targeted to their own actionable alterations (15 EGFR, 3 ALK, 1 ROS1, 8 RET, 1 BRAF). There were significant differences in overall survival between individuals with no actionable alterations, those with actionable alterations but no targeted therapy, and those with actionable alterations and targeted therapy (20.1 vs. 17.1 vs. 66.2 months, P < .001).ConclusionThe results of targeted DNA sequencing testing could provide improved treatment options for some NSCLC patients and result in a survival benefit to NSCLC patients with no EGFR and ALK alterations on routine tests who are treated with targeted therapy.     

Capture-Based Targeted Ultradeep Sequencing in Paired Tissue and Plasma Samples Demonstrates Differential Subclonal ctDNA-Releasing Capability in Advanced Lung Cancer

IntroductionCirculating tumor DNA (ctDNA), which represents an unbiased way to assess tumor genetic profile noninvasively, facilitates studying intratumor heterogeneity. Although intratumor heterogeneity has been elucidated substantially in a few cancer types, including NSCLC, how it influences the ability of tumor cells harboring different genetic abnormalities in releasing their DNA remains elusive. We designed a capture-based panel targeting NSCLC to detect and quantify genetic alterations from plasma samples by using deep sequencing. By applying the panel to paired biopsy and plasma samples, we imputed and compared the ctDNA-releasing efficiency in subclones harboring distinct genetic variants.MethodsWe collected 40 pairs of matched biopsy and plasma samples from patients with advanced lung cancer and applied capture-based sequencing using our LungPlasma panel, which consists of critical exons and introns of 168 genes. We derived a normalized relative allelic fraction score (NRAFS) to reflect ctDNA-releasing efficiency.ResultsBy using mutations detected in biopsy samples as a reference, we achieved 87.2% by-variant sensitivity, including for single-nucleotide variants, insertions or deletions, and gene fusions. Furthermore, the by-variant sensitivity for the seven most critical and actionable genes was 96.2%. The average NRAFS for subclones carrying mutations from seven actionable genes was 0.877; in contrast, the average NRAFS for other mutations was 0.658. Mutations from four genes involved in cell cycle pathways had a particularly low NRAFS (0.480) compared with the other two groups (p = 0.07).ConclusionsWe have demonstrated that subclones carrying driver mutations are more prone to release DNA. We have also demonstrated the quantitative ability of capture-based sequencing, paving its way for routine utilization in clinical settings.     

Drug-sensitiveFGFR3 mutations in lung adenocarcinoma

BackgroundLung cancer is the leading cause of cancer-related deaths across the world. In this study, we present therapeutically relevant genetic alterations in lung adenocarcinoma of Indian origin.Materials and methodsForty-five primary lung adenocarcinoma tumors were sequenced for 676 amplicons using RainDance cancer panel at an average coverage of 1500 × (reads per million mapped reads). To validate the findings, 49 mutations across 23 genes were genotyped in an additional set of 363 primary lung adenocarcinoma tumors using mass spectrometry. NIH/3T3 cells over expressing mutant and wild-typeFGFR3 constructs were characterized for anchorage independent growth, constitutive activation, tumor formation and sensitivity to FGFR inhibitors usingin vitro and xenograft mouse models.ResultsWe present the first spectrum of actionable alterations in lung adenocarcinoma tumors of Indian origin, and shows that mutations ofFGFR3 are present in 20 of 363 (5.5%) patients. TheseFGFR3 mutations are constitutively active and oncogenic when ectopically expressed in NIH/3T3 cells and using a xenograft model in NOD/SCID mice. Inhibition ofFGFR3 kinase activity inhibits transformation of NIH/3T3 overexpressingFGFR3 constructs and growth of tumors driven byFGFR3 in the xenograft models. The reduction in tumor size in the mouse is paralleled by a reduction in the amounts of phospho-ERK, validating thein vitro findings. Interestingly, theFGFR3 mutations are significantly higher in a proportion of younger patients and show a trend toward better overall survival, compared with patients lacking actionable alterations or those harboringKRAS mutations.ConclusionWe present the first actionable mutation spectrum in Indian lung cancer genome. These findings implicateFGFR3 as a novel therapeutic in lung adenocarcinoma.     

Treatment of Non–Small-Cell Lung Cancer Based on Circulating Cell-Free DNA and Impact of Variation Allele Frequency

BackgroundNext-generation sequencing of circulating cell-free DNA (cfDNA) can identify sensitizing and resistance mutations in non–small-cell lung cancer (NSCLC). cfDNA is helpful when tissue is insufficient for genomic testing or repeat biopsy is not feasible or poses unacceptable risk. Here we report the experience of cfDNA testing at the time of diagnosis and how this intervention can help avoid further invasive interventions, how it can be used to determine initiation of therapy, and how variation allele frequency of the somatic alteration affects response to subsequent treatment.Patients and MethodsThis is a single-institution retrospective study of patients with advanced NSCLC who had cfDNA from plasma tested using the Guardant360 panel, which identifies somatic genomic alterations by massive parallel sequencing of target genes. An institutional Clinical Laboratory Improvement Amendments tissue panel using fluorescence in situ hybridization (for MET, RET, ROS1, and ALK) and next-generation sequencing for selected genes was used for tissue analysis. Actionable mutations are those with US Food and Drug Administration–approved targeted therapies (EGFR, ALK, ROS, BRAF, NTRK fusions) or therapies soon to be approved (RET fusions and MET amplifications, or MET exon 14 skipping mutation).ResultsA total of 163 blood samples from 143 patients were evaluated, 82 at diagnosis and 81 at disease progression. A total of 94 cases had tissue and cfDNA testing performed within 12 weeks of each other. Seventy-six (81%) of 94 cases were concordant, of which 22 cases were concordantly positive and 54 concordantly negative. Eighteen (19%) of 94 cases were discordant, of which 11 had negative blood and positive tissue results, and 7 had positive blood and negative tissue results. cfDNA testing had a sensitivity of 67% (95% confidence interval [CI], 51%, 83%), specificity of 89% (95% CI, 81%, 97%), negative predictive value of 83% (95% CI, 74%, 92%), and positive predictive value of 76% (95% CI, 60%, 91%). Nineteen (21%) of 82 cfDNA samples analyzed at diagnosis had actionable mutations identified (4 EGFR exon 19 deletion, 2 EGFR exon 21 L858R, 2 EGFR L861Q, 1 L861R, 4 EML4-ALK fusion, 2 CD74-ROS1 fusion, 2 MET exon 14 skipping mutation, 2 KIF5B-RET fusion). Of the 82 patients with cfDNA testing performed at the time of diagnosis, 8 patients (10%) initiated targeted therapy on the basis of cfDNA results only, with 6 patients experiencing partial response, 1 patient complete response, and 1 patient stable disease. The response rate for patients who initiated targeted therapies on the basis of cfDNA only at diagnosis was 88%. Variant allele frequency had no impact on response.ConclusionsInitiation of targeted therapy for advanced NSCLC was feasible based only on identification of actionable mutations by cfDNA testing in 9% of the cases for which tissue diagnosis could not be obtained. Actionable targets were identified by cfDNA in 20% of the samples sent at diagnosis. A substantial number of patients benefited from cfDNA testing at initial diagnosis because it identified actionable mutations that led to appropriate targeted treatments.     

Investigating the feasibility of tumour molecular profiling in gastrointestinal malignancies in routine clinical practice

BackgroundTargeted capture sequencing can potentially facilitate precision medicine, but the feasibility of this approach in gastrointestinal (GI) malignancies is unknown.Patients and methodsThe FOrMAT (Feasibility of a Molecular Characterisation Approach to Treatment) study was a feasibility study enrolling patients with advanced GI malignancies from February 2014 to November 2015. Targeted capture sequencing (mainly using archival formalin-fixed paraffin-embedded diagnostic/resection samples) was carried out to detect mutations, copy number variations and translocations in up to 46 genes which had prognostic/predictive significance or were targets in current/upcoming clinical trials.ResultsOf the 222 patients recruited, 215 patients (96.8%) had available tissue samples, 125 patients (56.3%) had ≥16 genes successfully sequenced and 136 patients (61.2%) had ≥1 genes successfully sequenced. Sample characteristics influenced the proportion of successfully sequenced samples, e.g. tumour type (colorectal 70.9%, biliary 52.6%, oesophagogastric 50.7%, pancreas 27.3%, P = 0.002), tumour cellularity (high versus low: 78.3% versus 13.3%, P ≤ 0.001), tumour content (high versus low: 78.6% versus 27.3%, P = 0.001) and type of sample (resection versus biopsy: 82.4% versus 47.6%, P ≤ 0.001). Currently, actionable alterations were detected in 90 (40.5%) of the 222 patients recruited (66% of the 136 patients sequenced) and 2 patients subsequently received a targeted therapy. The most frequently detected currently actionable alterations were mutations in KRAS, BRAF, TP53 and PIK3CA. For the 205 patients with archival samples, the median time to obtain sequencing results was 18.9 weeks, including a median of 4.9 weeks for sample retrieval and 5.1 weeks for sequencing.ConclusionsTargeted sequencing detected actionable alterations in formalin-fixed paraffin-embedded samples, but tissue characteristics are of critical importance in determining sequencing success. Routine molecular profiling of GI tumours outside of clinical trials is not an effective use of healthcare resources unless more targeted drugs become available.ClinicalTrials.gov identifierNCT02112357     

Top advances of the year: Precision oncology

The advent of precision medicine has changed the landscape of oncologic biomarkers, drug discovery, drug development, and, more importantly, outcomes for patients with cancer. Precision oncology entails the genomic profiling of tumors to detect actionable aberrations. The advances in clinical next-generation sequencing from both tumor tissue and liquid biopsy and availability of targeted therapies has rapidly entered mainstream clinical practice. In this review, recent major developments in precision oncology that have affected outcomes for patients with cancer are discussed. Rapid clinical development was seen of targeted agents across various mutational profiles such as KRASG12C (which was considered “undruggable” for almost 4 decades), Exon 20 insertions, and RET mutations. Approaches to precision chemotherapy delivery by the introduction of antibody drug conjugates in the armamentarium against lung cancer has been appreciated.     

Clinical utility of plasma-based digital next-generation sequencing in patients with advance-stage lung adenocarcinomas with insufficient tumor samples for tissue genotyping

BackgroundApproximately 30% of tumor biopsies from patients with advanced-stage lung adenocarcinomas yield insufficient tissue for successful molecular subtyping. We have analyzed the clinical utility of next-generation sequencing (NGS) of cell-free circulating tumor DNA (ctDNA) in patients with inadequate tumor samples for tissue genotyping.Patients and methodsWe conducted the study in a multi-institutional prospective cohort of clinically unselected patients with advanced-stage lung adenocarcinomas with insufficient tissue for EGFR, ALK or ROS1 genotyping across 12 Spanish institutions (n = 93). ctDNA NGS was carried out by Guardant Health (Guardant360, Redwood City, CA), using a hybrid-capture-based 73-gene panel. Variants were deemed actionable if they were part of the OncoKB precision oncology knowledge database and classified in four levels of actionability based on their clinical or preclinical evidence for drug response.ResultsEighty-three out of 93 patients (89%) had detectable levels of ctDNA. Potentially actionable level 1–4 genomic alterations were detected in 53 cases (57%), of which 13 (14%) had level 1–2A alterations (Food and Drug Administration-approved and standard-care biomarkers according to lung cancer guidelines). Frequencies of each genomic alteration in ctDNA were consistent with those observed in unselected pulmonary adenocarcinomas. The majority of the patients (62%), particularly those with actionable alterations (87%), had more than one pathogenic variant in ctDNA. The median turnaround time to genomic results was 13 days. Twelve patients (13%) received genotype-matched therapies based on ctDNA results, deriving the expected clinical benefit. Patients with co-occurring pathogenic alterations had a significantly shorter median overall survival as compared with patients without co-occurring pathogenic alteration (multivariate hazard ratio = 5.35, P = 0.01).ConclusionDigital NGS of ctDNA in lung cancers with insufficient tumor samples for tissue sequencing detects actionable variants that frequently co-occur with other potentially clinically relevant genomic alterations, allowing timely initiation of genotype-matched therapies.     

Comparison of Clinically Relevant Mutation Profiles Between Preoperative Biopsy and Corresponding Surgically Resected Specimens in Japanese Patients With Non–Small-cell Lung Cancer by Amplicon-based Massively Parallel Sequencing

Background

Amplicon-based massively parallel sequencing (MPS) is an effective platform for identifying clinically actionable mutations across many genes in limited amounts of tissue. Most lung cancers are diagnosed and staged using small tissue samples obtained by transbronchial biopsy (TBB). To determine whether the mutations in TBB specimens detected by amplicon-based MPS reflect those present in the tumors, we compared the mutational profiles of preoperative TBB specimens and corresponding surgically resected specimens.

Hybrid capture-based genomic profiling of circulating tumor DNA from patients with estrogen receptor-positive metastatic breast cancer

BackgroundGenomic changes that occur in breast cancer during the course of disease have been informed by sequencing of primary and metastatic tumor tissue. For patients with relapsed and metastatic disease, evolution of the breast cancer genome highlights the importance of using a recent sample for genomic profiling to guide clinical decision-making. Obtaining a metastatic tissue biopsy can be challenging, and analysis of circulating tumor DNA (ctDNA) from blood may provide a minimally invasive alternative.Patients and methodsHybrid capture-based genomic profiling was carried out on ctDNA from 254 female patients with estrogen receptor-positive breast cancer. Peripheral blood samples were submitted by clinicians in the course of routine clinical care between May 2016 and March 2017. Sequencing of 62 genes was carried out to a median unique coverage depth of 7503×. Genomic alterations (GAs) in ctDNA were evaluated and compared with matched tissue samples and genomic datasets of tissue from breast cancer.ResultsAt least 1 GA was reported in 78% of samples. Frequently altered genes were TP53 (38%), ESR1 (31%) and PIK3CA (31%). Temporally matched ctDNA and tissue samples were available for 14 patients; 89% of mutations detected in tissue were also detected in ctDNA. Diverse ESR1 GAs including mutation, rearrangement and amplification, were observed. Multiple concurrent ESR1 GAs were observed in 40% of ESR1-altered cases, suggesting polyclonal origin; ESR1 compound mutations were also observed in two cases. ESR1-altered cases harbored co-occurring GAs in PIK3CA (35%), FGFR1 (16%), ERBB2 (8%), BRCA1/2 (5%), and AKT1 (4%).ConclusionsGAs relevant to relapsed/metastatic breast cancer management were identified, including diverse ESR1 GAs. Genomic profiling of ctDNA demonstrated sensitive detection of mutations found in tissue. Detection of amplifications was associated with ctDNA fraction. Genomic profiling of ctDNA may provide a complementary and possibly alternative approach to tissue-based genomic testing for patients with estrogen receptor-positive metastatic breast cancer.     

Feasibility of preemptive biomarker profiling for personalised early clinical drug development at a Comprehensive Cancer Center

PurposeMultiple investigational drugs are currently explored in cancer patient populations defined by specific biomarkers. This demands a new process of patient selection for clinical trials.Patients and methodsStarting January 1, 2012, preemptive biomarker profiling was offered at the West German Cancer Center to all patients with advanced non-small-cell lung (NSCLC) or colorectal cancer (CRC), who met generic study inclusion criteria. Tumour specimens were subjected to prespecified profiling algorithms to detect ‘actionable biomarkers’ by amplicon sequencing, in situ hybridisation and immunohistochemistry. The clinical course was closely monitored to offer trial participation whenever applicable.ResultsWithin 12 months, 267 patients (188 NSCLC, 79 CRC) were profiled. Estimated additional cost for biomarker profiling was 219615.51 EUR excluding histopathology workup and administration. The most prevalent biomarkers in pulmonary adenocarcinoma were KRAS mutations (29%), loss of PTEN expression (18%), EGFR mutations (9%), HER2 amplification (5%) and BRAF mutations (3%), while the prevalence of ALK translocations and PIK3CA mutations was extremely low. In pulmonary squamous cell carcinoma FGFR1 amplifications were found in 15%, PTEN expression was lost in 20% and DDR2 was mutated in a single case. KRAS mutations (41%) predominated in CRC, followed by loss of PTEN expression (16%), PIK3CA (5%) and BRAF (5%) mutations. So far 13 patients (5%) have entered biomarker-stratified clinical trials. Therapeutic decisions for approved drugs were guided in another 45 patients (17%).ConclusionPreemptive biomarker profiling can be implemented into the diagnostic algorithm of a large Comprehensive Cancer Center. Substantial investments in diagnostics and administration are required.     

Concordance between Comprehensive Cancer Genome Profiling in Plasma and Tumor Specimens

IntroductionDetection of somatic genomic alterations in the plasma of patients with cancer (“liquid biopsy”) are increasingly being used in the clinic. However, the concordance of alterations identified in liquid biopsies with those detected in cancer specimens is not routinely being determined.MethodsWe sought to systematically compare alterations found by a massively parallel sequencing liquid biopsy assay covering 39 genes (NEOliquid [NEO New Oncology GmbH, Köln, Germany]) with those identified through routine diagnostic testing in a certified central pathology laboratory in a cohort of patients with nonsquamous NSCLC. NEOliquid is based on enrichment of the genomic territory of interest by hybrid capture and is thus capable of detecting point mutations, small insertions and deletions, copy number alterations, and gene rearrangements/fusions in a single assay.ResultsIn a cohort of 82 patients with matched blood/tissue samples, the concordance between NEOliquid and tissue-based routine testing was 98%, the sensitivity of NEOliquid was higher than 70%, and the specificity was 100%. Discordant cases included those with insufficient amounts of circulaating tumor DNA in plasma and cases in which known driver mutations (e.g., isocitrate dehydrogenase (NADP(+)), 1 systolic gene [IDH1] R132H, kinesin family member 5B gene [KIF5b–ret proto-oncogene [RET], or MNNG HOS Transforming gene [MET] exon 14) were found in the plasma but were not interrogated by routine tissue analyses.ConclusionsIn summary, NEOliquid offers accurate and reliable detection of clinically relevant driver alterations in plasma of patients with cancer.     

Expert Consensus Recommendations on Biomarker Testing in Metastatic and Nonmetastatic NSCLC in Asia

IntroductionMost published guidelines for genomic biomarker testing in NSCLC reflect the disease epidemiology and treatments readily available in Europe and North America. Nevertheless, 60% of annual global NSCLC cases occur in Asia, where patient characteristics, tumor molecular profiles, and treatments vary greatly from the Western world. For example, mutations in the EGFR occur at a higher prevalence in Asia than in other world regions. Although medical associations such as the International Association for the Study of Lung Cancer, European Society for Medical Oncology, and American Society of Clinical Oncology have described principles for tumor genomic biomarker testing in NSCLC, there is a need for recommendations specific for Asia.MethodsThis report provides consensus recommendations for NSCLC biomarker testing from Asian lung cancer experts for clinicians working in Asia to improve patient care. Biomarker testing approaches for actionable genetic alterations in EGFR, ALK, ROS1, and others are discussed.ResultsThese recommendations are divided into nonmetastatic and metastatic forms of adenocarcinoma and squamous cell carcinoma. Owing to the higher prevalence of EGFR mutations in Asia, the experts emphasized the need for EGFR testing to include not just common mutations (exon 19 deletions and L858R substitutions) but also other uncommon EGFR mutations. In addition to the assessment of biomarkers in the tumor tissue, the role of assessing tumor biomarkers by liquid biopsy is discussed.ConclusionThis consensus provides practical recommendations for biomarker testing in nonmetastatic and metastatic Asian NSCLC patients.