Xiaoling Tong

Circulating Tumor DNA Mutation Profiling by Targeted Next Generation Sequencing Provides Guidance for Personalized Treatments in Multiple Cancer Types

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.

Novel Mutations on EGFR Leu792 Potentially Correlate to Acquired Resistance to Osimertinib in Advanced NSCLC

Abstract Osimertinib is an irreversible third generation EGFR tyrosine kinase inhibitor (TKI) and has shown outstanding performances in treating EGFR T790M-positive advanced non-small cell lung cancer (NSCLC) patients, but acquired resistance is inevitable. EGFR C797S is the most notable resistance mechanism to this drug, but other EGFR mutations may also exist. In three lung adenocarcinoma patients resistant to osimertinib, we identified recurrent novel mutations at EGFR Leu792 codon by targeted next generation sequencing (NGS) of cell free DNA (cfDNA) from plasma or pleural effusion. In two cases with pre-treatment tumor samples tested, mutations at Leu792 were absent, indicating that they were newly acquired during the therapy. Interestingly, all Leu792 mutations are localized at the same gene allele as T790M, but different from C797S. Structural prediction suggests that Leu792 mutations could interrupt the binding of osimerbinib to EGFR and potentially cause drug resistance.

Mutational profiling of non-small-cell lung cancer patients resistant to first-generation EGFR tyrosine kinase inhibitors using next generation sequencing

Patients with advanced non-small-cell lung cancer (NSCLC) harboring sensitive epithelial growth factor receptor (EGFR) mutations invariably develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Identification of actionable genetic alterations conferring drug-resistance can be helpful for guiding the subsequent treatment decision. One of the major resistant mechanisms is secondary EGFR-T790M mutation. Other mechanisms, such as HER2 and MET amplifications, and PIK3CA mutations, were also reported. However, the mechanisms in the remaining patients are still unknown. In this study, we performed mutational profiling in a cohort of 83 NSCLC patients with TKI-sensitizing EGFR mutations at diagnosis and acquired resistance to three different first-generation EGFR TKIs using targeted next generation sequencing (NGS) of 416 cancer-related genes. In total, we identified 322 genetic alterations with a median of 3 mutations per patient. 61% of patients still exhibit TKI-sensitizing EGFR mutations, and 36% of patients acquired EGFR-T790M. Besides other known resistance mechanisms, we identified TET2 mutations in 12% of patients. Interestingly, we also observed SOX2 amplification in EGFR-T790M negative patients, which are restricted to Icotinib treatment resistance, a drug widely used in Chinese NSCLC patients. Our study uncovered mutational profiles of NSCLC patients with first-generation EGFR TKIs resistance with potential therapeutic implications.

Circulating tumor DNA profiling by next generation sequencing reveals heterogeneity of crizotinib resistance mechanisms in a gastric cancer patient with MET amplification

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 patients 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 patients 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.

Short-Term Responders of Non-Small Cell Lung Cancer Patients to EGFR Tyrosine Kinase Inhibitors Display High Prevalence of TP53 Mutations and Primary Resistance Mechanisms.

Non–small cell lung cancer (NSCLC) with activating EGFR mutations in exon 19 and 21 typically responds to EGFR tyrosine kinase inhibitors (TKI); however, for some patients, responses last only a few months. The underlying mechanisms of such short responses have not been fully elucidated. Here, we sequenced the genomes of 16 short-term responders (SR) that had progression-free survival (PFS) of less than 6 months on the first-generation EGFR TKI and compared them to 12 long-term responders (LR) that had more than 24 months of PFS. All patients were diagnosed with advanced lung adenocarcinoma and harbored EGFR 19del or L858R mutations before treatment. Paired tumor samples collected before treatment and after relapse (or at the last follow-up) were subjected to targeted next-generation sequencing of 416 cancer-related genes. SR patients were significantly younger than LR patients (P < .001). Collectively, 88% of SR patients had TP53 variations compared to 13% of LR patients (P < .001). Additionally, 37.5% of SR patients carried EGFR amplifications compared to 8% of LR patients. Other potential primary resistance factors were also identified in the pretreatment samples of 12 SR patients (75%), including PTEN loss; BIM deletion polymorphism; and amplifications of EGFR, ERBB2, MET, HRAS, and AKT2. Comparatively, only three LR patients (25%) were detected with EGFR or AKT1 amplifications that could possibly exert resistance. The diverse preexisting resistance mechanisms in SR patients revealed the complexity of defining treatment strategies even for EGFR-sensitive mutations.

GCC2-ALK as a targetable fusion in lung adenocarcinoma and its enduring clinical responses to ALK inhibitors

OBJECTIVES ALK, RET and ROS1 fusions have been identified as treatable targets in 5%-15% of non-small-cell lung cancers, and thanks to the advanced sequencing technologies, their new partner genes have been steadily detected. Here we identified a rare fusion of ALK (GCC2-ALK) in a patient with advanced lung adenocarcinoma and monitored the treatment efficacy of ALK inhibitors on this patient. We further performed in vitro functional studies of this fusion protein for evaluating its oncogenic potential. MATERIALS AND METHODS The GCC2-ALK fusion gene was identified by targeted next generation sequencing (NGS) from the tumor DNA samples, and its fusion product was confirmed by Sanger sequencing the cDNA product. The functional study of GCC2-ALK was performed in Ba/F3 cells with cell proliferation and viability assays. The activation of downstream signaling pathways of ALK and their responses to crizotinib inhibition were studied in HEK-293 and 293T cells with ectopic expression of GCC2-ALK. In parallel, disease progression in the patient was monitored by computed tomography scanning and targeted NGS of either liquid or tissue biopsy samples throughout and after crizotinib treatment. RESULTS Similarly to EML4-ALK, the GCC2-ALK fusion protein promotes IL-3-independent growth of Ba/F3 cells. Ectopic expression of GCC2-ALK leads to hyper-activation of ALK downstream signaling that can be inhibited by crizotinib. Crizotinib treatment of the patient resulted in 18 months of progression free survival without any trace of GCC2-ALK fusion in the liquid biopsies. Re-biopsy of a lung lesion at progression identified the re-occurrence of GCC2-ALK. The patient was then administrated with a second-generation ALK inhibitor, ceritinib, and received partial response until the last follow-up. CONCLUSION We identified and functionally validated GCC2-ALK as a constitutively activated fusion in NSCLC. The patient was benefited from crizotinib treatment initially and then ceritinib after progression, suggesting GCC2-ALK as a novel therapeutic target for ALK inhibitors.

Case Report: Osimertinib achieved remarkable and sustained disease control in an advanced non-small-cell lung cancer harboring EGFR H773L/V774M mutation complex

Missense mutations in EGFR exon 20 are rare in non-small-cell lung cancer (NSCLC), and mostly insensitive to the first generation tyrosine kinase inhibitors (TKIs) of EGFR. However, their responses to the third generation TKI are unclear. Here, we reported a patient with advanced NSCLC harboring a rare EGFR H773L/V774M mutation complex. Although he was irresponsive to the first generation TKI gefitinib, he demonstrated sustained disease control to osimertinib, suggesting that this complex is an activating mutation of EGFR and can be suppressed by osimertinib. The follow-up genetic profiling revealed multiple acquired new mutations that might be related to his resistance to osimertinib. This finding would provide valuable experience for future treatment of the same mutations.

Targeted next generation sequencing identified clinically actionable mutations in patients with esophageal sarcomatoid carcinoma

BackgroundEsophageal sarcomatoid carcinoma (ESC) is a rare disease with a mixture of both carcinomatous and sarcomatous components in the tumor. Its genetic background and mechanisms of oncogenesis remain largely unknown.MethodsHere we performed targeted next generation sequencing (NGS) on a pan-cancer gene panel in 15 ESC tumors to explore their genetic alterations, and aimed to identify clinically actionable mutations for future treatment instructions.ResultsTP53 alterations were identified in all patients. Alterations in receptor tyrosine kinases (RTK) were identified in 10 out of 15 patients. Members of downstream RAS and PI3-kinase pathways are also mutated in 10 patients, and PIK3CA is the top mutated gene in these pathways. In addition, we identified mutations on histone modification genes in 5 patients, including histone acetyltransferase gene EP300 and its homologue CREBBP, lysine methyltransferase genes KMT2A and KMT2B, and lysine demethylase gene KDM5A. Finally, mismatch repair (MMR) genes and proofreading gene POLE all together were mutated in one third of the ESC patients.ConclusionsThis is the first study to unravel the mutational profile of ESC tumors. Our findings could match 9 patients to the targeted therapies currently available in clinical practice or in active clinical trials, suggesting the potential utility of targeted therapies for this rare disease in the future.