Subtype-specific KRAS mutations in advanced lung adenocarcinoma: A retrospective study of patients treated with platinum-based chemotherapy

BackgroundPlatinum-based chemotherapy is the most common treatment in advanced-stage lung adenocarcinoma. Because the clinical significance of KRAS mutational status in this setting has not yet been clearly determined, a mutation subtype-specific analysis was performed in the so far largest cohort of Caucasian patients with KRAS mutant advanced-stage lung adenocarcinoma treated with platinum-based chemotherapy.Methods505 Caucasian stage III–IV lung adenocarcinoma patients with known amino acid substitution-specific KRAS mutational status and treated with platinum-based chemotherapy were included. The correlations of subtype-specific KRAS mutations with smoking status, progression-free and overall survival (PFS and OS, respectively) and therapeutic response were analysed.ResultsAmong 338 KRAS wild-type, 147 codon 12 mutant and 20 codon 13 mutant patients, there were no mutation-related significant differences in PFS or OS (P values were 0.534 and 0.917, respectively). Eastern Cooperative Oncology Group (ECOG) status and clinical stage were significant independent prognostic factors. KRAS mutation showed a significant correlation with smoking status (P = 0.018). Importantly, however, G12V KRAS mutant patients were significantly more frequent among never-smokers than all other codon 12 KRAS mutant (G12x) subtypes (P = 0.016). Furthermore, this subgroup tended to have a higher response rate (66% versus 47%; P = 0.077). A modestly longer median PFS was also found in the G12V mutant cohort (233 days; versus 175 days in the G12x group; P = 0.145).ConclusionsWhile KRAS mutation status per se is neither prognostic nor predictive in stage III–IV lung adenocarcinoma, subtype-specific analysis may indeed identify clinically relevant subgroups of patients that may ultimately influence treatment decisions.     

First-line pemetrexed plus cisplatin followed by gefitinib maintenance therapy versus gefitinib monotherapy in East Asian patients with locally advanced or metastatic non-squamous non-small cell lung cancer: A randomised,phase 3 trial

BackgroundIn the Iressa Pan-ASia Study (IPASS), gefitinib claimed improved progression-free survival (PFS) versus carboplatin-paclitaxel in clinically selected lung cancer patients. The primary objective of this study was to assess the PFS of pemetrexed-cisplatin (PC) followed by gefitinib maintenance versus gefitinib monotherapy in an IPASS-like population.MethodsIn this open-label, randomised, phase 3 trial, eligible patients were ⩾18 years, chemonaïve, East Asian, light ex-smokers/never-smokers with advanced non-squamous non-small cell lung cancer, an Eastern Cooperative Oncology Group (ECOG) performance status 0–1 and unknown epidermal growth factor receptor (EGFR) mutation status who enrolled at 12 sites in Asia. Patients randomly received (1:1) pemetrexed (500 mg/m²) plus cisplatin (75 mg/m²) for six 21-day cycles, followed by gefitinib maintenance or gefitinib monotherapy (250 mg/day). Patient tissue was retrospectively analysed for EGFR mutations. This study is registered with ClinicalTrials.gov, NCT01017874.FindingsBetween 23rd November 2009 and 27th April 2012, 253 patients entered, and 236 patients were randomly assigned to and treated with PC therapy (N = 114) and gefitinib monotherapy (N = 118). Between-arm baseline characteristics were balanced. PFS was not significantly different between treatment arms (p = 0.217). The unadjusted hazard ratio (HR) was 0.85 (95% confidence interval (CI) 0.63–1.13). The HR should be cautiously interpreted as it was not constant. EGFR mutation status was determined for 74 tissue samples; 50 (67.6%) had mutations. In a pre-specified subgroup analysis, only the treatment-by-EGFR mutation interaction was significant (p = 0.008) for PFS. For the entire treatment period, a higher proportion of patients in the PC/gefitinib arm versus gefitinib experienced possibly drug-related grade 3–4 treatment-emergent adverse events (39 of 114 [34%] versus 19 of 118 [16%]; p = 0.002).InterpretationIn the intention-to-treat (ITT) population, PFS was not significantly different. In the biomarker-assessable population, front-line EGFR tyrosine kinase inhibitor monotherapy was not efficacious in patients with wild-type EGFR. Identification of EGFR mutation status is key in the management of advanced non-squamous non-small cell lung cancer.FundingEli Lilly and Company.     

Additional prognostic role of EGFR activating mutations in lung adenocarcinoma patients with brain metastasis: Integrating with lung specific GPA score

ObjectiveWhile several prognostic models have been presented in NSCLC patients with brain metastasis, none of these models have included molecular markers as an index. The aim of our study was to evaluate the prognostic value of EGFR mutations and to integrate these EGFR mutations into the prognostic index in NSCLC patients with brain metastasis.Materials and methodsWe analyzed retrospectively 292 lung adenocarcinoma patients with brain metastasis. Clinico-pathological features and overall survival (OS) were compared between patients with EGFR mutations and patients with EGFR wild type. EGFR mutation status was integrated with lung specific graded prognostic assessment (GPA) score.ResultsAmong 292 patients, EGFR mutation status was tested in 183 patients. One hundred and five patients (57.4%) had EGFR activating mutations, 14 (7.7%) had EGFR non-activating mutations and 64 (35.0%) had EGFR wild type. OS was significantly longer in patients with EGFR activating mutations than in those with EGFR wild type patients (20.4 vs. 10.1 months, p = 0.002). However, patients with EGFR non-activating mutations did not show superior OS compared with EGFR wild type patients (14.6 vs. 10.1 months, p = 0.83). Multivariate analysis revealed that the presence of EGFR activating mutation is an independent positive prognostic factor for OS (adjusted hazard ratio 0.56, p = 0.002).ConclusionsEGFR activating mutations have a prognostic role in lung adenocarcinoma patients with brain metastasis that is independent of other known prognostic factors. The frequency of EGFR mutation was higher than expected. The presence of EGFR activating mutations should be included as an index in the prognostic models for lung adenocarcinoma patients with brain metastasis.     

Radiation-enhancing effect of sodium glycididazole in patients suffering from non-small cell lung cancer with multiple brain metastases: A randomized,placebo-controlled study

PurposeMedian survival of patients with brain metastases from non-small cell lung cancer is poor. This study was to investigate the radiation-enhancing effect of sodium glycididazole combined with whole-brain radiotherapy of multiple brain metastases from non-small cell lung cancer.Patients and methodsSixty-four patients with multiple brain metastases from non-small cell lung cancer were included: the study group (n = 32) received whole-brain radiotherapy combined with sodium glycididazole at a dose of 700 mg/m² intravenous infusion 30 minutes before radiotherapy, three times a week; the control group (n = 32) only received whole-brain radiotherapy. The primary end point was central nervous system (CNS) progression-free survival and overall survival. The treatment-related toxicity was also recorded.ResultsThe CNS disease control rate was better (90.6% vs 65.6%, P = 0.016) in the study group than in the control group at 3 month of follow-up. The median CNS progression-free survival time was longer in the study group than in the control group (7.0 months vs 4.0 months, P = 0.038). There was no significant difference of the median overall survival time between the study group and the control group (11.0 months vs 9.0 months, P = 0.418). On the other hand, the treatment-related toxicity showed no statistically significant difference between these two groups (P > 0.05).ConclusionsThe study indicated that sodium glycididazole was an effective, promising radiation-enhancing agent that improved CNS disease control rate, extended the median CNS progression-free survival time and was well tolerated in patients suffering from non-small cell lung cancer with multiple brain metastases.     

Single French centre retrospective analysis of local control after high dose radiotherapy with or without chemotherapy and local control for Pancoast tumours

PurposeSuperior sulcus non-small cell lung cancer represents less than 5% of all lung cancers and is a challenge for the physicians because of clinical presentation, treatments related toxicities and poor prognosis. The aim of this preliminary retrospective report is to present outcomes of patients affected by a superior sulcus non-small cell lung cancer, treated by high dose radiotherapy (> 60 Gy) with or withour chemotherapy.Patients and methodsAll adult inoperable or unresectable patients (≥ 18 years) with a clinical and radiological diagnosis of superior sulcus non-small cell lung cancer treated in our department by radiotherapy with or without chemotherapy were retrospectively analysed. Primary endpoint was the local control. Overall survival, metastasis free survival and toxicity rates were also analysed and reported.ResultsFrom January 1999 to June 2009, 12 patients were treated by exclusive high-dose radiochemotherapy. Median age was 53 years (range: 33–64 years); mean follow-up time was 20 months (range: 2–75 months). Mean local control, overall survival and metastasis free survival were 20.2, 22 and 20 months, respectively. At the time of this analysis, seven patients died of cancer and three of them presented only a metastatic disease progression. One patient died of acute cardiac failure 36 months after the end of radiochemotherapy and was disease free. Treatment was well tolerated and any acute and/or late G3-4 toxicity was recorded (NCI-CTC v 3.0 score).ConclusionThis analysis confirms the interest of exclusive high-dose radiochemotherapy in treating inoperable superior sulcus non-small cell lung cancer patients, in achieving good local control and overall survival rates.     

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.     

Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma

BackgroundA major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients.Patients and methodsMatched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing.ResultsFor exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection.ConclusionsCombining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone.Clinical TrialsNCT01526928     

Clinical and molecular characteristics of non-small-cell lung cancer (NSCLC) harboring EGFR mutation: results of the nationwide French Cooperative Thoracic Intergroup (IFCT) program

BackgroundEGFR mutations cause inconsistent response to EGFR tyrosine-kinase inhibitors (TKI). To better understand these features, we reviewed all cases of EGFR-mutated non-small-cell lung cancer collected in the Biomarkers France database.Patients and methodsOf 17 664 patients, 1837 (11%) with EGFR-mutated non-small-cell lung cancer were retrospectively analyzed for clinical and molecular characteristics. Results were correlated with survival and treatment response for the 848 stage IV patients.ResultsEGFR exon 18, 19, 20 and 21 mutations were found in 102 (5.5%), 931 (51%), 102 (5.5%) and 702 (38%) patients, respectively. Over 50% of exon 18 and 20 mutated patients were smokers. The median follow-up was 51.7 months. EGFR mutation type was prognostic of overall survival (OS) versus wild-type {exon 19: hazard ratio (HR)=0.51 [95% confidence interval (CI): 0.41–0.64], P < 0.0001; exon 21: HR = 0.76 (95% CI: 0.61–0.95), P = 0.002; exon 20: HR = 1.56 (95% CI: 1.02–2.38), P = 0.004}. EGFR mutation type was prognostic of progression-free survival versus wild-type [exon 19: HR = 0.62 (95% CI: 0.49–0.78), P < 0.0001; exon 20: HR = 1.46 (95% CI: 0.96–2.21), P = 0.07]. First-line treatment choice did not influence OS in multivariate analysis. First-line TKI predicted improved progression-free survival versus chemotherapy [HR = 0.67 (95% CI: 0.53–0.85), P = 0.001]. OS was longer for del19 versus L858R, which was associated with better OS compared with other exon 21 mutations, including L861Q. TKI improved survival in patients with exon 18 mutations, while chemotherapy was more beneficial for exon 20-mutated patients.ConclusionEGFR mutation type can inform the most appropriate treatment. Therapeutic schedule had no impact on OS in our study, although TKI should be prescribed in first-line considering the risk of missing the opportunity to use this treatment.     

Intensity-modulated radiotherapy prolongs the survival of patients with nasopharyngeal carcinoma compared with conventional two-dimensional radiotherapy: A 10-year experience with a large cohort and long follow-up

BackgroundTo evaluate the survival benefit of intensity-modulated radiotherapy (IMRT) compared with conventional two-dimensional radiotherapy (2D-CRT) in nasopharyngeal carcinoma (NPC) using a large cohort with long follow-up.MethodsWe retrospectively analysed 7081 non-metastatic NPC patients who received curative IMRT or 2D-CRT from February 2002 to December 2011.ResultsOf the 7081 patients, 2245 (31.7%) were administered IMRT, while 4836 (68.3%) were administered 2D-CRT. At 5 years, the patients administered IMRT had significantly higher local relapse-free survival (LRFS), loco-regional relapse-free survival (LRRFS), progression-free survival (PFS) and overall survival (OS) (95.6%, 92.5%, 82.1% and 87.4%, respectively) than those administered 2D-CRT (90.8%, 88.5%, 76.7% and 84.5%, respectively; p < 0.001). The distant metastasis-free survival (DMFS) was higher for IMRT than 2D-CRT, with borderline significance (87.6% and 85.7%, respectively; p = 0.056). However, no difference was observed between IMRT and 2D-CRT in nodal relapse-free survival (NRFS; 96.3% and 97.4%, respectively; p = 0.217). Multivariate analyses showed that IMRT was an independent protective prognostic factor for LRFS, LRRFS and PFS, but not NRFS, DMFS or OS.ConclusionsIMRT provided an improved LRFS, LRRFS and PFS in both the early and advanced T classifications and overall stage for non-disseminated NPC compared with 2D-CRT. However, no significant advantage was observed in NRFS, DMFS or OS when IMRT was used.     

BRAF mutation status is an independent prognostic factor for resected stage IIIB and IIIC melanoma: Implications for melanoma staging and adjuvant therapy

Background5-year survival for melanoma metastasis to regional lymph nodes (American Joint Committee on Cancer stage III) is <50%. Knowledge of outcomes following therapeutic lymphadenectomy for stage III melanoma related to BRAF status may guide adjuvant use of BRAF/MEK inhibitors along with established and future therapies.AimsTo determine patterns of melanoma recurrence and survival following therapeutic lymph node dissection (TLND) associated with oncogenic mutations.MethodsDNA was obtained from patients who underwent TLND and had ⩾2 positive nodes, largest node >3 cm or extracapsular invasion. Mutations were detected using an extended Sequenom MelaCARTA panel.ResultsMutations were most commonly detected in BRAF (57/124 [46%] patients) and NRAS (26/124 [21%] patients). Patients with BRAF mutations had higher 3-year recurrence rate (77%) versus 54% for BRAF wild-type patients (hazard ratio (HR) 1.8, p = 0.008). The only prognostically significant mutations occurred in BRAF: median recurrence-free (RFS) and disease-specific survival (DSS) for BRAF mutation patients was 7 months and 16 months, versus 19 months and not reached for BRAF wild-type patients, respectively. Multivariate analysis identified BRAF mutant status and number of positive lymph nodes as the only independent prognostic factors for RFS and DSS.ConclusionsPatients with BRAF mutations experienced rapid progression of metastatic disease with locoregional recurrence rarely seen in isolation, supporting incorporation of BRAF status into melanoma staging and use of BRAF/MEK inhibitors post-TLND.     

Associations between RAD51D germline mutations and breast cancer risk and survival in BRCA1/2-negative breast cancers

BackgroundRAD51D is involved in DNA double-strand break repair by homologous recombination and plays an important role in the maintenance of genomic stability. The associations between RAD51D germline mutations and breast cancer risk and survival are not fully elucidated.Patients and methodsRAD51D germline mutations were determined using a multigene panel in 7657 unselected breast cancer patients who were negative for BRCA1/2 germline mutations. The RAD51D recurrent mutation p.K91fs was screened in 7947 healthy controls by Sanger sequencing.ResultsA total of 29 cases (0.38%) carried deleterious RAD51D germline mutations among this cohort of 7657 unselected breast cancer patients. The RAD51D recurrent mutation p.K91fs was identified in 18 cases (0.24%) of these 7657 patients. In contrast, the p.K91fs mutation was found in 8 of 7947 healthy controls with a frequency of 0.10%. The RAD51D p.K91fs mutation was significantly associated with increased breast cancer risk in unselected breast cancer [odds ratio = 2.34, 95% confidence interval (CI) 1.02–5.38; P = 0.040]. RAD51D mutation carriers were diagnosed at a younger age (P = 0.006) and were more likely to be triple-negative breast cancer (P = 0.003), estrogen receptor negative (P = 0.005) and high-grade cancers (P = 0.023) than noncarriers. Furthermore, RAD51D mutation carriers had a significantly worse recurrence-free survival [unadjusted hazard ratio (HR) = 3.00, 95% CI 1.56–5.80; P = 0.001] and distant recurrence-free survival (unadjusted HR = 2.54, 95% CI 1.14–5.67; P = 0.023) than noncarriers.ConclusionThe RAD51D recurrent mutation, p.K91fs, confers a moderately increased breast cancer risk, and RAD51D mutation carriers have an unfavorable survival compared with noncarriers.     

Évolution des cancers de l’œsophage : impact de la stratégie thérapeutique

PurposeTo assess the outcome of esophageal cancer according to therapeutic strategy.Patients and methodsOne-hundred and twenty patients with esophageal cancer treated by an association of radiotherapy and chemotherapy and possibly surgery, between 2004 and 2010, were retrospectively studied. The first site of relapse was classified as follows: local (tumour), locoregional (tumour and/or nodal: celiac, mediastinal, sus-clavicular) or metastatic.ResultsWith a 15.7-months (1.4–62) median follow-up, there were 89 deaths and 79 recurrences. Three types of treatments were performed: 50 Gy exclusive chemoradiotherapy (47 patients) or 50 to 65 Gy exclusive chemoradiotherapy (44 patients) or chemoradiotherapy followed by surgery (27 patients). The local first relapse was as much frequent as distant relapse (50 patients). With a-5 cm margin up and down to the tumour, there was only one nodal relapse. Two-year survival was 39.5% (95% confidence interval [IC]: 30.5–40.8) and relapse-free survival was 26.5% (CI: 18.6–35). Multivariate analysis revealed that treatment type and disease stage had a significant impact on survival, relapse-free survival and locoregional control. Compared to exclusive chemoradiotherapy, surgery improved locoregional control (40.2 versus 8.7 months, P = 0.0004) but in a younger population. Despite postoperative mortality, the gain was maintained for distance relapse-free survival (40.2 versus 10 months, P = 0.0147) and overall survival (29.3 versus 14.2 months, P = 0.0088). Compared to 50 Gy chemoradiotherapy, local control was improved if high dose chemoradiotherapy was performed (13.8 versus 7.5 months, P = 0.05) but not overall survival (14.0 versus 15.4 months, P = 0.24).ConclusionMore than one-third relapse is local. Locoregional control is better with high dose chemoradiotherapy. In this study, surgery performed in selected patients only, improved locoregional control, relapse-free disease and overall survival.     

Spectrum of gene mutations detected by next generation exome sequencing in brain metastases of lung adenocarcinoma

BackgroundBrain metastases (BM) are a life-threatening complication. We aimed to analyse gene mutations in lung adenocarcinoma BM.MethodsWe performed next generation sequencing (NGS) of a pre-defined set of 48 cancer-related genes in a cohort of 76 neurosurgical lung adenocarcinoma BM specimens using a cancer specific gene panel on the MiSeq platform (Illumina, San Diego, CA). NGS results were statistically correlated to patient characteristics. Data on ALK, ROS1, MET and FGFR1 gene status assessed by FISH were available from previous studies in the majority of patients.ResultsTwenty-nine (60.4%) of the 48 investigated cancer-related genes were mutated in at least one BM sample and 64 (84.2%) of the 76 BM samples carried at least one mutated gene. The number of mutated genes per sample ranged from 0 to 9 (median 2). The most commonly mutated genes were TP53, KRAS and CDKN2A, which were affected in 35/76 (46.1%), 29/76 (38.2%) and 17/76 (22.4%) samples, respectively. Other potentially druggable alterations included EGFR mutations (3/76, 3.9% of samples), PIK3CA mutation (2/76, 2.6%), BRAF mutation (1/76, 1.3%) and SMO mutation (1/76, 1.3%). Presence of KRAS mutations was associated with positive smoking history (p = 0.015, Chi square test) and presence of EGFR mutation correlated with unfavourable overall survival time from BM diagnosis (p = 0.019, log rank test).ConclusionsDeleterious gene mutations, some of them with potential therapeutic implications, are found in a high fraction of lung adenocarcinoma BM.     

Prevalence of germline MUTYH mutations among Lynch-like syndrome patients

Background and aimsIndividuals with tumours showing mismatch repair (MMR) deficiency not linked to germline mutations or somatic methylation of MMR genes have been recently referred as having ‘Lynch-like syndrome’ (LLS). The genetic basis of these LLS cases is unknown. MUTYH-associated polyposis patients show some phenotypic similarities to Lynch syndrome patients. The aim of this study was to investigate the prevalence of germline MUTYH mutations in a large series of LLS patients.MethodsTwo hundred and twenty-five probands fulfilling LLS criteria were included in this study. Screening of MUTYH recurrent mutations, whole coding sequencing and a large rearrangement analysis were undertaken. Age, sex, clinical, pathological and molecular characteristics of tumours including KRAS mutations were assessed.ResultsWe found a prevalence of 3.1% of MAP syndrome in the whole series of LLS (7/225) and 3.9% when only cases fulfilling clinical criteria were considered (7/178). Patients with MUTYH biallelic mutations had more adenomas than monoallelic (P = 0.02) and wildtype patients (P < 0.0001). Six out of nine analysed tumours from six biallelic MUTYH carriers harboured KRAS-p.G12C mutation. This mutation was found to be associated with biallelic MUTYH germline mutation when compared with reported series of unselected colorectal cancer cohorts (P < 0.0001).ConclusionsA proportion of unexplained LLS cases is caused by biallelic MUTYH mutations. The obtained results further justify the inclusion of MUTYH in the diagnostic strategy for Lynch syndrome-suspected patients.     

Amplicon-based next-generation sequencing of plasma cell-free DNA for detection of driver and resistance mutations in advanced non-small cell lung cancer

BackgroundGenomic analysis of plasma cell-free DNA is transforming lung cancer care; however, available assays are limited by cost, turnaround time, and imperfect accuracy. Here, we study amplicon-based plasma next-generation sequencing (NGS), rather than hybrid-capture-based plasma NGS, hypothesizing this would allow sensitive detection and monitoring of driver and resistance mutations in advanced non-small cell lung cancer (NSCLC).Patients and methodsPlasma samples from patients with NSCLC and a known targetable genotype (EGFR, ALK/ROS1, and other rare genotypes) were collected while on therapy and analyzed blinded to tumor genotype. Plasma NGS was carried out using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes, as well as intronic coverage for detection of ALK/ROS1 fusions. Diagnostic accuracy was compared with plasma droplet digital PCR (ddPCR) and tumor genotype.ResultsA total of 168 specimens from 46 patients were studied. Matched plasma NGS and ddPCR across 120 variants from 80 samples revealed high concordance of allelic fraction (R² = 0.95). Pretreatment, sensitivity of plasma NGS for the detection of EGFR driver mutations was 100% (30/30), compared with 87% for ddPCR (26/30). A full spectrum of rare driver oncogenic mutations could be detected including sensitive detection of ALK/ROS1 fusions (8/9 detected, 89%). Studying 25 patients positive for EGFR T790M that developed resistance to osimertinib, 15 resistance mechanisms could be detected including tertiary EGFR mutations (C797S, Q791P) and mutations or amplifications of non-EGFR genes, some of which could be detected pretreatment or months before progression.ConclusionsThis blinded analysis demonstrates the ability of amplicon-based plasma NGS to detect a full range of targetable genotypes in NSCLC, including fusion genes, with high accuracy. The ability of plasma NGS to detect a range of preexisting and acquired resistance mechanisms highlights its potential value as an alternative to single mutation digital PCR-based plasma assays for personalizing treatment of TKI resistance in lung cancer.     

Prediction and monitoring of relapse in stage III melanoma using circulating tumor DNA

BackgroundThe advent of effective adjuvant therapies for patients with resected melanoma has highlighted the need to stratify patients based on risk of relapse given the cost and toxicities associated with treatment. Here we assessed circulating tumor DNA (ctDNA) to predict and monitor relapse in resected stage III melanoma.Patients and methodsSomatic mutations were identified in 99/133 (74%) patients through tumor tissue sequencing. Personalized droplet digital PCR (ddPCR) assays were used to detect known mutations in 315 prospectively collected plasma samples from mutation-positive patients. External validation was performed in a prospective independent cohort (n = 29).ResultsctDNA was detected in 37 of 99 (37%) individuals. In 81 patients who did not receive adjuvant therapy, 90% of patients with ctDNA detected at baseline and 100% of patients with ctDNA detected at the postoperative timepoint relapsed at a median follow up of 20 months. ctDNA detection predicted patients at high risk of relapse at baseline [relapse-free survival (RFS) hazard ratio (HR) 2.9; 95% confidence interval (CI) 1.5–5.6; P = 0.002] and postoperatively (HR 10; 95% CI 4.3–24; P < 0.001). ctDNA detection at baseline [HR 2.9; 95% CI 1.3–5.7; P = 0.003 and postoperatively (HR 11; 95% CI 4.3–27; P < 0.001] was also associated with inferior distant metastasis-free survival (DMFS). These findings were validated in the independent cohort. ctDNA detection remained an independent predictor of RFS and DMFS in multivariate analyses after adjustment for disease stage and BRAF mutation status.ConclusionBaseline and postoperative ctDNA detection in two independent prospective cohorts identified stage III melanoma patients at highest risk of relapse and has potential to inform adjuvant therapy decisions.     

High plasma D-dimer level is associated with decreased survival in patients with lung cancer

AimsAn elevated plasma d-dimer level indicates the activation of coagulation and fibrinolysis. In the present study, we investigated the association of pre-treatment haemostatic parameters (d-dimer, fibrinogen and prothrombin fragment 1+2) with clinicopathological parameters and outcome in patients with lung cancer.Materials and methodsPlasma levels of d-dimer and other parameters were measured in 78 evaluable patients with lung cancer (60 non-small cell lung cancers, 18 small cell lung cancers). At diagnosis, 35 patients (44.9%) were locally advanced stage (IIIA/B) and 43 patients (55.1%) had metastatic disease (IV). Multivariate statistical analysis was carried out using Cox's proportional hazards model. The receiver operating characteristic curve was used to determine the cut-off values for d-dimer, fibrinogen and prothrombin fragment 1+2.ResultsThe median survival for all patients was 264 days (95% confidence interval 200–328 days). A significant association between the plasma levels of d-dimer and the response to chemotherapy was observed (P = 0.03). With the univariate analysis, tumour stage, pre-treatment plasma levels of d-dimer, fibrinogen, platelet count, lactate dehydrogenase concentration and Karnofsky performance status were predictive for survival. With the multivariate analysis (P ≤ 0.1), the plasma level of d-dimer (P < 0.001), tumour stage (P = 0.01) and Karnofsky performance status (P = 0.02) were identified as independent predictive factors. The median survival times were 405 days (95% confidence interval 165–644 days) and 207 days (95% confidence interval 146–267 days, P < 0.001), respectively, for patients with a low d-dimer level (≤0.65 μg/ml) and a high d-dimer level (>0.65 μg/ml).ConclusionsElevated plasma levels of d-dimer in patients with lung cancer are associated with decreased survival and a poor response to treatment. Pre-treatment for the d-dimer level may be useful in the prediction of survival and the response to treatment.     

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.