Circulating tumor DNA (ctDNA) represents a promising biomarker for noninvasive assessment of cancer burden, but existing methods have insufficient sensitivity or patient coverage for broad clinical applicability. Here we introduce CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), an economical and ultrasensitive method for quantifying ctDNA. We implemented CAPP-Seq for non-small cell lung cancer (NSCLC) with a design covering multiple classes of somatic alterations that identified mutations in >95% of tumors. We detected ctDNA in 100% of stage II–IV and 50% of stage I NSCLC patients, with 96% specificity for mutant allele fractions down to ~0.02%. Levels of ctDNA significantly correlated with tumor volume, distinguished between residual disease and treatment-related imaging changes, and provided earlier response assessment than radiographic approaches. Finally, we explored biopsy-free tumor screening and genotyping with CAPP-Seq. We envision that CAPP-Seq could be routinely applied clinically to detect and monitor diverse malignancies, thus facilitating personalized cancer therapy.

High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by ~3 fold, and synergize when combined to yield ~15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to clinical non-small cell lung cancer (NSCLC) samples, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and 96% specificity and detection of ctDNA down to 4 in 105 cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings.

Background Early reports on patients with cancer and COVID-19 have suggested a high mortality rate compared with the general population. Patients with thoracic malignancies are thought to be particularly susceptible to COVID-19 given their older age, smoking habits, and pre-existing cardiopulmonary comorbidities, in addition to cancer treatments. We aimed to study the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on patients with thoracic malignancies. MethodsThe Thoracic Cancers International COVID-19 Collaboration (TERAVOLT) registry is a multicentre observational study composed of a cross-sectional component and a longitudinal cohort component. Eligibility criteria were the presence of any thoracic cancer (non-small-cell lung cancer [NSCLC], small-cell lung cancer, mesothelioma, thymic epithelial tumours, and other pulmonary neuroendocrine neoplasms) and a COVID-19 diagnosis, either laboratory confirmed with RT-PCR, suspected with symptoms and contacts, or radiologically suspected cases with lung imaging features consistent with COVID-19 pneumonia and symptoms. Patients of any age, sex, histology, or stage were considered eligible, including those in active treatment and clinical follow-up. Clinical data were extracted from medical records of consecutive patients from Jan 1, 2020, and will be collected until the end of pandemic declared by WHO. Data on demographics, oncological history and comorbidities, COVID-19 diagnosis, and course of illness and clinical outcomes were collected. Associations between demographic or clinical characteristics and outcomes were measured with odds ratios (ORs) with 95% CIs using univariable and multivariable logistic regression, with sex, age, smoking status, hypertension, and chronic obstructive pulmonary disease included in multivariable analysis. This is a preliminary analysis of the first 200 patients. The registry continues to accept new sites and patient data. Findings Between March 26 and April 12, 2020, 200 patients with COVID-19 and thoracic cancers from eight countries were identified and included in the TERAVOLT registry; median age was 68•0 years (61•8-75•0) and the majority had an Eastern Cooperative Oncology Group performance status of 0-1 (142 [72%] of 196 patients), were current or former smokers (159 [81%] of 196), had non-small-cell lung cancer (151 [76%] of 200), and were on therapy at the time of COVID-19 diagnosis (147 [74%] of 199), with 112 (57%) of 197 on first-line treatment. 152 (76%) patients were hospitalised and 66 (33%) died. 13 (10%) of 134 patients who met criteria for ICU admission were admitted to ICU; the remaining 121 were hospitalised, but were not admitted to ICU. Univariable analyses revealed that being older than 65 years (OR 1•88, 95% 1•00-3•62), being a current or former smoker (4•24, 1•70-12•95), receiving treatment with chemotherapy alone (2•54, 1•09-6•11), and the presence of any comorbidities (2•65, 1•09-7•46) were associated with increased risk of death. However, in multivariable analysis, only smo...

Background Anti-PD1/PD-L1 directed immune checkpoint inhibitors (ICI) are widely used to treat patients with advanced non-small-cell lung cancer (NSCLC). The activity of ICI across NSCLC harboring oncogenic alterations is poorly characterized. The aim of our study was to address the efficacy of ICI in the context of oncogenic addiction. Patients and methods We conducted a retrospective study for patients receiving ICI monotherapy for advanced NSCLC with at least one oncogenic driver alteration. Anonymized data were evaluated for clinicopathologic characteristics and outcomes for ICI therapy: best response (RECIST 1.1), progression-free survival (PFS), and overall survival (OS) from ICI initiation. The primary end point was PFS under ICI. Secondary end points were best response (RECIST 1.1) and OS from ICI initiation. Results We studied 551 patients treated in 24 centers from 10 countries. The molecular alterations involved KRAS ( n = 271), EGFR ( n = 125), BRAF ( n = 43), MET ( n = 36), HER2 ( n = 29), ALK ( n = 23), RET ( n = 16), ROS1 ( n = 7), and multiple drivers ( n = 1). Median age was 60 years, gender ratio was 1 : 1, never/former/current smokers were 28%/51%/21%, respectively, and the majority of tumors were adenocarcinoma. The objective response rate by driver alteration was: KRAS = 26%, BRAF = 24%, ROS1 = 17%, MET = 16%, EGFR = 12%, HER2 = 7%, RET = 6%, and ALK = 0%. In the entire cohort, median PFS was 2.8 months, OS 13.3 months, and the best response rate 19%. In a subgroup analysis, median PFS (in months) was 2.1 for EGFR , 3.2 for KRAS , 2.5 for ALK , 3.1 for BRAF , 2.5 for HER2 , 2.1 for RET , and 3.4 for MET . In certain subgroups, PFS was positively associated with PD-L1 expression ( KRAS , EGFR ) and with smoking status ( BRAF , HER2 ). Conclusions : ICI induced regression in some tumors with actionable driver alterations, but clinical activity was lower compared with the KRAS group and the lack of response in th...

Identifying molecular residual disease (MRD) after treatment of localized lung cancer could facilitate early intervention and personalization of adjuvant therapies. Here, we apply cancer personalized profi ling by deep sequencing (CAPP-seq) circulating tumor DNA (ctDNA) analysis to 255 samples from 40 patients treated with curative intent for stage I–III lung cancer and 54 healthy adults. In 94% of evaluable patients experiencing recurrence, ctDNA was detectable in the fi rst posttreatment blood sample, indicating reliable identifi cation of MRD. Posttreatment ctDNA detection preceded radiographic progression in 72% of patients by a median of 5.2 months, and 53% of patients harbored ctDNA mutation profi les associated with favorable responses to tyrosine kinase inhibitors or immune checkpoint blockade. Collectively, these results indicate that ctDNA MRD in patients with lung cancer can be accurately detected using CAPP-seq and may allow personalized adjuvant treatment while disease burden is lowest.

Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment.

The isolation and analysis of circulating cell-free tumor DNA in plasma is a powerful tool with considerable potential to improve clinical outcomes across multiple cancer types, including NSCLC. Assays of this nature that use blood as opposed to tumor samples are frequently referred to as liquid biopsies. An increasing number of innovative platforms have been recently developed that improve not only the fidelity of the molecular analysis but also the number of tests performed on a single specimen. Circulating tumor DNA assays for detection of both EGFR sensitizing and resistance mutations have already entered clinical practice and many other molecular tests - such as detection of resistance mutations for Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase rearrangements - are likely to do so in the near future. Due to an abundance of new evidence, an appraisal was warranted to review strengths and weaknesses, to describe what is already in clinical practice and what has yet to be implemented, and to highlight areas in need of further investigation. A multidisciplinary panel of experts in the field of thoracic oncology with interest and expertise in liquid biopsy and molecular pathology was convened by the International Association for the Study of Lung Cancer to evaluate current available evidence with the aim of producing a set of recommendations for the use of liquid biopsy for molecular analysis in guiding the clinical management of advanced NSCLC patients as well as identifying unmet needs. In summary, the panel concluded that liquid biopsy approaches have significant potential to improve patient care, and immediate implementation in the clinic is justified in a number of therapeutic settings relevant to NSCLC.

Rociletinib was active in patients with EGFR-mutated NSCLC associated with the T790M resistance mutation. (Funded by Clovis Oncology; ClinicalTrials.gov number, NCT01526928.).