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Background Tumor mutational burden (TMB) is a promising biomarker for stratifying patient subpopulation who would benefit from immune checkpoint blockade (ICB) therapies. Although great efforts have been made for standardizing TMB measurement, mutation calling and TMB quantification can be challenging in samples with low tumor content including liquid biopsies. The effect of varying tumor content on TMB estimation by different assay methods has never been systematically investigated. Method We established a series of reference standard DNA samples derived from 11 pairs of tumor–normal matched human cell lines across different cancer types. Each tumor cell line was mixed with its matched normal at 0% (control), 1%, 2%, 5%, and 10% mass‐to‐mass ratio to mimic the clinical samples with low tumor content. TMB of these reference standards was evaluated by both ∼1000× whole‐exome sequencing (wesTMB) and targeted panel sequencing (psTMB) at four different vendors. Both regression and classification analyses of TMB were performed for theoretical investigation and clinical practice purposes. Results Linear regression model was established that demonstrated in silico psTMB determined by regions of interest (ROI) as a great representative of wesTMB based on TCGA dataset. It was also true in our reference standard samples as the predicted psTMB interval based on the observed wesTMB captured the intended 90% of the in silico psTMB values. Although ∼1000× deep WES was applied, reference standard samples with less than 5% of tumor proportions are below the assay limit of detection (LoD) of wesTMB quantification. However, predicted wesTMB based on observed psTMB accurately classify (>0.97 AUC) for TMB high and low patient stratification even in samples with 2% of tumor content, which is more clinically relevant, as TMB determination should be a qualitative assay for TMB high and low patient classification. One targeted panel sequencing vendor using an optimized blood psTMB pipeline can further classify TMB status accurately (>0.82 AUC) in samples with only 1% of tumor content. Conclusions We developed a linear model to establish the quantitative correlation between wesTMB and psTMB. A set of DNA reference standards was produced in aid to standardize TMB measurements in samples with low tumor content across different targeted sequencing panels. This study is a significant contribution aiming to harmonize TMB estimation and extend its future application in clinical samples with low tumor content including liquid biopsy., Established 11 sets of reference standard samples with variable tumor proportions for evaluating TMB estimation. In silico and experimentally verified the linear regression model between the TMB analyzed by deep whole‐exome sequencing and targeted panel sequencing in reference standard samples. Targeted panel sequencing method might outperform WES for TMB estimation and categorization in samples with low tumor proportion.

Osteosarcoma is a primary malignant bone tumor that has a high potential to metastasize to lungs. Little is known about the mechanisms underlying the dissemination of OS cancer cells to lungs. We performed whole exome sequencing of 13 OS primary tumors, with matched lung metastases and normal tissues. Phylogenetic analyses revealed that lung metastatic tumors often harbor clones that are nonexistent or rare in the matched primary OS tumors. Spatially and temporally separated lung metastases were from parallel seeding events with a polyphyletic pattern. Loss of TP53 or RB1 is among the early events during OS tumorigenesis, while loss of PTEN is involved at the later stages associated with lung metastases. Finally, KEAP1 was identified as a novel biomarker for increased metastatic risk. Patients whose primary tumors harbored KEAP1 amplification have significantly poorer lung-metastasis free survival. This finding was validated in two independent datasets. Further, in vitro experiments exhibited that KEAP1 depletion suppressed the invasion of OS cells. Our findings uncover the patterns of clonal evolution during OS progression and highlight KEAP1 as a novel candidate associated with the risk of lung metastasis in OS patients.

// Juan Du 1, * , Xue Wu 2, * , Xiaoling Tong 2 , Xiaonan Wang 3 , Jia Wei 1 , Yang Yang 1 , Zhili Chang 3 , Yu Mao 3 , Yang W Shao 2 , Baorui Liu 1 1 The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing, Jiangsu, 210008, China 2 Geneseeq Technology Inc., Toronto, Ontario, M5G1L7, Canada 3 Nanjing Geneseeq Technology Inc., Sino-Danish Life Science Park, Nanjing, Jiangsu, 210032, China * These authors contributed equally to this work Correspondence to: Baorui Liu, email: baoruiliu@nju.edu.cn Yang W Shao, email: yang.shao@geneseeq.com Keywords: circulating tumor DNA, next generation sequencing, MET, crizotinib, drug resistance Received: April 19, 2016 Accepted: February 04, 2017 Published: February 17, 2017 ABSTRACT Crizotinib has been used to counter MET gene amplification in a number of different human malignancies. Transient response to crizotinib in MET -amplified gastric cancer has been reported, but the mechanisms of resistance are not well studied. Here, we reported a stage IV gastric cancer patient with high levels of MET amplification. The implementation of crizotinib treatment led to significant symptomatic improvement in the first 2 months, but was followed by rapid disease progression. Periodic mutation profiling of patient’s circulating tumor DNA (ctDNA) by next generation sequencing (NGS) revealed a number of genetic alterations including re-occurrence of MET amplification, multiple secondary MET mutations, a dramatic increase of FGFR2 gene relative copy number as well as mutations in other downstream and bypassing elements, which may collectively related to the patient’s cancer progression. Our results illustrate the complex and heterogeneous molecular mechanisms for crizotinib resistance in this patient, and demonstrate the great potential of ctDNA profiling for treatment decision-making and prognosis in clinical practice.

The global epidemic of drug-resistant tuberculosis is a catastrophic example of how antimicrobial resistance is undermining the public health gains made possible by combination drug therapy. Recent evidence points to unappreciated bacterial factors that accelerate the emergence of drug resistance. In a genome-wide association study of Mycobacterium tuberculosis isolates from China, we find mutations in the gene encoding the transcription factor prpR enriched in drug-resistant strains. prpR mutations confer conditional drug tolerance to three of the most effective classes of antibiotics by altering propionyl-CoA metabolism. prpR-mediated drug tolerance is carbon-source dependent, and while readily detectable during infection of human macrophages, is not captured by standard susceptibility testing. These data define a previously unrecognized and clinically prevalent class of M. tuberculosis variants that undermine antibiotic efficacy and drive drug resistance.

Cancer is a disease of complex genetic alterations, and comprehensive genetic diagnosis is beneficial to match each patient to appropriate therapy. However, acquisition of representative tumor samples is invasive and sometimes impossible. Circulating tumor DNA (ctDNA) is a promising tool to use as a non-invasive biomarker for cancer mutation profiling. Here we implemented targeted next generation sequencing (NGS) with a customized gene panel of 382 cancer-relevant genes on 605 ctDNA samples in multiple cancer types. Overall, tumor-specific mutations were identified in 87% of ctDNA samples, with mutation spectra highly concordant with their matched tumor tissues. 71% of patients had at least one clinically-actionable mutation, 76% of which have suggested drugs approved or in clinical trials. In particular, our study reveals a unique mutation spectrum in Chinese lung cancer patients which could be used to guide treatment decisions and monitor drug-resistant mutations. Taken together, our study demonstrated the feasibility of clinically-useful targeted NGS-based ctDNA mutation profiling to guide treatment decisions in cancer.

Cell-free DNA (cfDNA) is short, extracellular, fragmented double-stranded DNA found in plasma. Plasma of patients with solid tumor has been found to show significantly increased quantities of cfDNA. Although currently poorly understood, the mechanism of cfDNA generation is speculated to be a product of genomic DNA fragmentation during cellular apoptosis and necrosis. Sequencing of cfDNA with tumor origin has identified tumor biomarkers, elucidating molecular pathology and assisting in accurate diagnosis. In this study, we performed whole-genome sequencing ofcfDNA samples with matching tumor and whole blood samples from five patients diagnosed with stage IV gastric or lung cancer. We analyzed the coverage spectrum of the human genome in our cfDNA samples. cfDNA exhibited no large regions with significant under-coverage, although we observed unbalanced coverage depth in cfDNA at transcription start sites and exon boundaries as a consequence of biased fragmentation due to ordered nucleosome positioning. We also analyzed the copy number variant status based on the whole-genome sequencing results and found high similarity between copy number profile constructed from tumor samples and cfDNA samples. Overall, we conclude that cfDNA comprises a good representation of the tumor genome in late stage gastric and lung cancer.

2572 Background: Third generation epithelial growth factor receptor ( EGFR) tyrosine kinase inhibitor (TKI) osimertinib (AZD9291) has proven effective in Non-small cell lung cancer (NSCLC) patients who have developed EGFR T790M-mediated resistance to other EGFR TKIs. Unfortunately, a majority of patients still undergo progressed disease after receiving osimertinib treatment. Acquired EGFR C797S mutation has been identified as one major mechanism; however, resistance mechanisms of remaining cases are still largely unknown. Methods: Using next generation sequencing (NGS) targeting 416 cancer-relevant genes, we analyzed the mutation profiles of 99 NSCLC patients that were clinically resistant to osimertinib. Results: In addition to the notable EGFR C797 variants (22%), L792 mutations were identified in 10% of patients, and a further 7% cases carry L718 mutations. Further analysis of 14 patients with paired pre-treatment samples confirmed that these EGFR mutations were acquired during treatment. Interestingly, all L792 mutations are in cis with T790M and in trans with C797 mutations (when present in the same patient). 2 out of 10 L792-positive patients and 6 out of 7 L718-positive patients did not have co-existing C797 mutations, suggesting C797-, L792- and L718-mutated cells may represent different resistant clones. In vitro experiments demonstrated that L792 and L718 mutants also increase osimertinib IC50, and therefore confer their resistance. Besides secondary EGFR mutations, alterations in other key genes such as MET, KRAS, ERBB2 and PIK3CA may also contribute to osimertinib resistance. Notably, MET and KRAS amplifications are present only in patients without above EGFR secondary mutations. Conclusions: In this study, we identified secondary mutations on C797, L792 or L718 residues of EGFR in 29% of osimertinib-resistant patients. Combined with in vitro study, our data strongly suggest that L792 and L718 mutations are likely to alternatively cause osimertinib resistance. Furthermore, MET and KRAS amplification may serve as bypass resistance mechanisms in patients who are EGFR C797-, L792- and L718-wild type.