TP53 mutations in circulating tumor DNA in advanced epidermal growth factor receptor-mutant lung adenocarcinoma patients treated with gefitinib.

Tumor protein p53 (TP53) is a tumor suppressor gene and TP53 mutations are associated with poor prognosis in non-small cell lung cancer. However, the in-depth classification of TP53 and its relationship with treatment response and prognosis in epidermal growth factor receptor (EGFR)-mutant tumors treated with EGFR tyrosine kinase inhibitors are unclear. Circulating tumor DNA was prospectively collected at baseline in advanced treatment-naïve EGFR-mutant lung adenocarcinoma patients treated with gefitinib in an open-label, single-arm, prospective, multicenter, phase 2 clinical trial (BENEFIT trial) and analyzed using next-generation sequencing. Survival was estimated using the Kaplan-Meier method. Of the 180 enrolled patients, 115 (63.9%) harbored TP53 mutations. The median progression-free survival (PFS) and overall survival (OS) of patients with TP53-wild type tumors were significantly longer than those of patients with TP53-mutant tumors. Mutations in exons 5-8 accounted for 80.9% of TP53 mutations. Mutations in TP53 exons 6 and 7 were significantly associated with inferior PFS and OS compared to wild-type TP53. TP53 mutation also influenced the prognosis of patients with different EGFR mutations. Patients with TP53 and EGFR exon 19 mutations had significantly longer PFS and OS than patients with TP53 and EGFR L858R mutations, and both groups had worse survival than patients with only EGFR mutations. Patients with TP53 mutations, especially in exons 6 and 7, had a lower response rate and shorter PFS and OS when treated with gefitinib. Moreover, TP53 exon 5 mutation divided TP53 mutations in disruptive and non-disruptive types.

Titin mutation in circulatory tumor DNA is associated with efficacy to immune checkpoint blockade in advanced non-small cell lung cancer.

BACKGROUND: Only a fraction of patients with advanced non-small cell lung cancer (NSCLC) respond well to immune checkpoint blockade (ICB) therapy. Here, we investigated whether Titin (TTN) mutation, which has been demonstrated to be a predictive biomarker in tissue-based analysis, can identify patients with a greater likelihood in response to ICB based on circulatory tumor DNA (ctDNA) sequencing. METHODS: In this retrospective analysis, 92 patients with advanced NSCLC from two independent cohorts who received ICB treatment were included. A probe panel covering all exons of TTN was developed and validated to detect TTN mutation in ctDNA. Baseline plasma samples were collected and subjected to ctDNA sequencing with the TTN probe panel. RESULTS: Of the 92 patients, 28.3% harbored TTN mutation in their baseline ctDNA. Progression-free survival was significantly improved in patients with the mutated TTN (212 days and 334.5 days for cohort 1 and 2) compared to those without the mutation (113 days and 147 days for cohort 1 and 2). Objective response to ICB treatment (40% for TTNmut and 15.8% for TTNwt in cohort 1; 50% for TTNmut and 23.4% for TTNwt in cohort 2) was common in patients with mutated TTN. Stratified analysis showed a generally predictive potential of TTN mutation in patients with advanced NSCLC. CONCLUSIONS: The presence of mutated TTN in pre-treatment peripheral blood was associated with favorable objective response and survival with ICB administration. Therefore, circulatory TTN mutation may be applicable for guiding ICB immunotherapy in patients with NSCLC.

Circulating Tumour DNA Sequencing Identifies a Genetic Resistance-Gap in Colorectal Cancers with Acquired Resistance to EGFR-Antibodies and Chemotherapy.

Epidermal growth factor receptor antibodies (EGFR-Abs) confer a survival benefit in patients with RAS wild-type metastatic colorectal cancer (mCRC), but resistance invariably occurs. Previous data showed that only a minority of cancer cells harboured known genetic resistance drivers when clinical resistance to single-agent EGFR-Abs had evolved, supporting the activity of non-genetic resistance mechanisms. Here, we used error-corrected ctDNA-sequencing (ctDNA-Seq) of 40 cancer genes to identify drivers of resistance and whether a genetic resistance-gap (a lack of detectable genetic resistance mechanisms in a large fraction of the cancer cell population) also occurs in RAS wild-type mCRCs treated with a combination of EGFR-Abs and chemotherapy. We detected one MAP2K1/MEK1 mutation and one ERBB2 amplification in 2/3 patients with primary resistance and KRAS, NRAS, MAP2K1/MEK1 mutations and ERBB2 aberrations in 6/7 patients with acquired resistance. In vitro testing identified MAP2K1/MEK1 P124S as a novel driver of EGFR-Ab resistance. Mutation subclonality analyses confirmed a genetic resistance-gap in mCRCs treated with EGFR-Abs and chemotherapy, with only 13.42% of cancer cells harboring identifiable resistance drivers. Our results support the utility of ctDNA-Seq to guide treatment allocation for patients with resistance and the importance of investigating further non-canonical EGFR-Ab resistance mechanisms, such as microenvironmentally-mediated resistance. The detection of MAP2K1 mutations could inform trials of MEK-inhibitors in these tumours.

Tracking Neoantigens by Personalized Circulating Tumor DNA Sequencing during Checkpoint Blockade Immunotherapy in Non-Small Cell Lung Cancer.

The evolutionary dynamics of tumor-associated neoantigens carry information about drug sensitivity and resistance to the immune checkpoint blockade (ICB). However, the spectrum of somatic mutations is highly heterogeneous among patients, making it difficult to track neoantigens by circulating tumor DNA (ctDNA) sequencing using "one size fits all" commercial gene panels. Thus, individually customized panels (ICPs) are needed to track neoantigen evolution comprehensively during ICB treatment. Dominant neoantigens are predicted from whole exome sequencing data for treatment-naïve tumor tissues. Panels targeting predicted neoantigens are used for personalized ctDNA sequencing. Analyzing ten patients with non-small cell lung cancer, ICPs are effective for tracking most predicted dominant neoantigens (80-100%) in serial peripheral blood samples, and to detect substantially more genes (18-30) than the capacity of current commercial gene panels. A more than 50% decrease in ctDNA concentration after eight weeks of ICB administration is associated with favorable progression-free survival. Furthermore, at the individual level, the magnitude of the early ctDNA response is correlated with the subsequent change in tumor burden. The application of ICP-based ctDNA sequencing is expected to improve the understanding of ICB-driven tumor evolution and to provide personalized management strategies that optimize the clinical benefits of immunotherapies.

Genomic landscapes by multiregion sequencing combined with circulation tumor DNA detection contribute to molecular diagnosis in glioblastomas.

Glioblastoma is a highly aggressive brain malignancy with a poor prognosis. Its high intratumor heterogeneity contributes to therapeutic resistance, tumor progression and recurrence. We sequenced 31 loci in 11 patients with glioblastoma (including one patient with samples available from the primary and recurrent tumors) to determine the genetic basis and intratumor heterogeneity of glioblastoma. By analyzing the somatic mutations, known driver genes were identified, including EGFR, PTEN and TP53, and the MUC16 gene exhibited the highest mutation rate in the samples examined. Through an evolutionary analysis of the sequencing results, the EGFR p.L861Q mutation was determined to play a role in the progression from the primary tumor to a relapsing tumor in one patient. We analyzed 1403 genes in blood-derived ctDNA that were previously revealed to play a role in tumorigenesis and the progression of cancer. Somatic mutations identified through ctDNA sequencing that match the results of multipoint exon sequencing in tumor tissues were detected, such as EGFR p.L861Q. These findings provide new insights into the intratumor heterogeneity and evolution of glioblastoma. In addition, ctDNA detection in blood samples represents a convenient method to dynamically identify the genetic changes and new therapeutic targets during the treatment of glioblastoma.

Cell-Free DNA Next-Generation Sequencing Prediction of Response and Resistance to Third-Generation EGFR Inhibitor.

INTRODUCTION: The genomic alterations driving resistance to third-generation EGFR tyrosine kinase inhibitors (TKIs) are not well established, and collecting tissue biopsy samples poses potential complications from invasive procedures. Cell-free circulating DNA (cfDNA) testing provides a noninvasive approach to identify potentially targetable mechanisms of resistance. Here we utilized a 70-gene cfDNA next-generation sequencing test to interrogate pretreatment and progression samples from 77 EGFR-mutated non-small cell lung cancer (NSCLC) patients treated with a third-generation EGFR TKI. PATIENTS AND METHODS: Rociletinib was evaluated in advanced or metastatic (second line or higher) disease with EGFR T790M-positive NSCLC in the TIGER-X (NCT01526928) and TIGER-2 (NCT02147990) studies. Plasma samples were collected at baseline and at the time of systemic progression while receiving rociletinib. The critical exons in 70 genes were sequenced in cfDNA isolated from plasma samples to elucidate a comprehensive genomic profile of alterations for each patient. RESULTS: Plasma-based cfDNA analysis identified 93% of the initial EGFR activating and 85% of the EGFR T790M resistance mutations in pretreatment samples with detectable tumor DNA. Profiling of progression samples revealed significant heterogeneity, with different variant types (eg, mutations, amplifications, and fusions) detected in multiple genes (EGFR, MET, RB1) that may be driving resistance in patients. Novel alterations not previously described in association with resistance to third-generation TKIs were also detected, such as an NTRK1 fusion. CONCLUSION: cfDNA next-generation sequencing identified initial EGFR activating and secondary T790M resistance mutations in NSCLC patients with high sensitivity, predicted treatment response equivalent to tissue analysis, and identified multiple novel and established resistance alterations.