Single Circulating-Tumor-Cell-Targeted Sequencing to Identify Somatic Variants in Liquid Biopsies in Non-Small-Cell Lung Cancer Patients

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths worldwide. Liquid biopsy by a blood draw to detect circulating tumor cells (CTCs) is a tool for molecular profiling of cancer using single-cell and next-generation sequencing (NGS) technologies. The aim of the study was to identify somatic variants in single CTCs isolated from NSCLC patients by targeted NGS. Thirty-one subjects (20 NSCLC patients, 11 smokers without cancer) were enrolled for blood draws (7.5 mL). CTCs were identified by immunofluorescence, individually retrieved, and DNA-extracted. Targeted NGS was performed to detect somatic variants (single-nucleotide variants (SNVs) and insertions/deletions (Indels)) across 65 oncogenes and tumor suppressor genes. Cancer-associated variants were classified using OncoKB database. NSCLC patients had significantly higher CTC counts than control smokers (p = 0.0132; Mann–Whitney test). Analyzing 23 CTCs and 13 white blood cells across seven patients revealed a total of 644 somatic variants that occurred in all CTCs within the same subject, ranging from 1 to 137 per patient. The highest number of variants detected in ≥1 CTC within a patient was 441. A total of 18/65 (27.7%) genes were highly mutated. Mutations with oncogenic impact were identified in functional domains of seven oncogenes/tumor suppressor genes (NF1, PTCH1, TP53, SMARCB1, SMAD4, KRAS, and ERBB2). Single CTC-targeted NGS detects heterogeneous and shared mutational signatures within and between NSCLC patients. CTC single-cell genomics have potential for integration in NSCLC precision oncology.     

An Integrated Approach for Analyzing Clinical Genomic Variant Data from Next-Generation Sequencing

Next-generation sequencing (NGS) technologies provide the potential for developing high-throughput and low-cost platforms for clinical diagnostics. A limiting factor to clinical applications of genomic NGS is downstream bioinformatics analysis for data interpretation. We have developed an integrated approach for end-to-end clinical NGS data analysis from variant detection to functional profiling. Robust bioinformatics pipelines were implemented for genome alignment, single nucleotide polymorphism (SNP), small insertion/deletion (InDel), and copy number variation (CNV) detection of whole exome sequencing (WES) data from the Illumina platform. Quality-control metrics were analyzed at each step of the pipeline by use of a validated training dataset to ensure data integrity for clinical applications. We annotate the variants with data regarding the disease population and variant impact. Custom algorithms were developed to filter variants based on criteria, such as quality of variant, inheritance pattern, and impact of variant on protein function. The developed clinical variant pipeline links the identified rare variants to Integrated Genome Viewer for visualization in a genomic context and to the Protein Information Resource’s iProXpress for rich protein and disease information. With the application of our system of annotations, prioritizations, inheritance filters, and functional profiling and analysis, we have created a unique methodology for downstream variant filtering that empowers clinicians and researchers to interpret more effectively the relevance of genomic alterations within a rare genetic disease.     

Overview of the role of liquid biopsy in cancer management

With the emergence of novel targeted therapeutic options in early-stage and advanced-stage malignancies, researchers have shifted their focus on developing personalized treatment plans through molecular profiling. Circulating tumor DNA (ctDNA) is a cell-free DNA (ctDNA) fragment, originating from tumor cells, and circulating in the bloodstream as well as biological fluids. Over the past decade, many techniques were developed for liquid biopsies through next-generation sequencing. This alternative non-invasive biopsy offers several advantages in various types of tumors over traditional tissue biopsy. The process of liquid biopsy is considered minimally invasive and therefore easily repeatable when needed, providing a more dynamic analysis of the tumor cells. Moreover, it has an advantage in patients with tumors that are not candidates for tissue sampling. Besides, it offers a deeper understanding of tumor burden as well as treatment response, thereby enhancing the detection of minimal residual disease and therapeutic guidance for personalized medicine. Despite its many advantages, ctDNA and liquid biopsy do have some limitations.This paper discusses the basis of ctDNA and the current data available on the subject, as well as its clinical utility. We also reflect on the limitations of using ctDNA in addition to its future perspectives in clinical oncology and precision medicine.     

Complex Mutations & Subpopulations of Deletions at Exon 19 of EGFR in NSCLC Revealed by Next Generation Sequencing: Potential Clinical Implications

Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.     

Managing expectational language: translational genetic professionals consider the clinical potential of next-generation sequencing technologies

Clinical genetic professionals are used to being flooded by claims about the seemingly endless potential and promise of next-generation sequencing (NGS) in medicine today. This paper is about managing expectations in translational medicine. From 2009 to 2011, we conducted focus groups with genetic and allied professionals concerned with genomics in the clinic to examine their attitudes and perspectives of genetic and genomic tools in this environment. In this paper, we examine and explore some of their discussions, specifically related to NGS and whole genome sequencing tests and their introduction as normal clinical tools. Informed by sociology of expectations (SE), we discuss expectational language in the arena of translational medicine. Through SE, illuminated are some barriers and strategies used by professionals to manage expectations. Further, our work suggests the importance of SE and more nuanced study to understand the discursive realm of translational genomic medicine.     

Pilot Study of a Next-Generation Sequencing-Based Targeted Anticancer Therapy in Refractory Solid Tumors at a Korean Institution

We evaluated the preliminary efficacy and feasibility of a next-generation sequencing (NGS)-based targeted anticancer therapy in refractory solid tumors at a Korean institution. Thirty-six patients with advanced cancer underwent molecular profiling with NGS with the intent of clinical application of available matched targeted agents. Formalin-fixed paraffin-embedded (FFPE) tumors were sequenced using the Comprehensive Cancer Panel (CCP) or FoundationOne in the Clinical Laboratory Improvement Amendments-certified laboratory in the USA. Response evaluations were performed according to RECIST v1.1. Four specimens did not pass the DNA quality test and 32 specimens were successfully sequenced with CCP (n = 31) and FoundationOne (n = 1). Of the 32 sequenced patients, 10 (31.3%) were ≤40 years. Twelve patients (37.5%) had received ≥3 types of prior systemic therapies. Of 24 patients with actionable mutations, five were given genotype-matched drugs corresponding to actionable mutations: everolimus to PIK3CA mutation in parotid carcinosarcoma (partial response) and tracheal squamous cell carcinoma (stable disease; 21% reduction), sorafenib to PDGFRA mutation in auditory canal adenocarcinoma (partial response), sorafenib to BRAF mutation in microcytic adnexal carcinoma (progressive disease), and afatinib to ERBB2 mutation in esophageal adenocarcinoma (progressive disease). Nineteen of 24 patients with actionable mutations could not undergo targeted therapy based on genomic testing because of declining performance status (10/24, 41.7%), stable disease with previous treatment (5/24, 20.8%), and lack of access to targeted medication (4/24, 16.7%). NGS-based targeted therapy may be a good option in selected patients with refractory solid tumors. To pursue this strategy in Korea, lack of access to clinical-grade NGS assays and a limited number of genotype-matched targeted medications needs to be addressed and resolved.     

Gene Alterations in Paired Supernatants and Precipitates from Malignant Pleural Effusions of Non-Squamous Non-Small Cell Lung Cancer

OBJECTIVE: This study investigated the feasibility of using malignant pleural effusion (MPE) supernatant and paired cell blocks (precipitate) for gene profiling in patients with non-small cell lung cancer (NSCLC) using next-generation sequencing (NGS) technique. METHODS: Stage IV non-squamous NSCLC patients with MPE were eligible in this prospective study and recruited from Zhejiang Cancer Hospital between May 2014 and October 2015. MPE supernatant and paired precipitate sample gene alterations were determined with NGS containing 14 cancer-related genes. Progression free survival (PFS) was evaluated using Kaplan–Meier method and compared using log-rank test. RESULTS: A total of 102 patients were enrolled in the present study. All pleural effusions were confirmed as malignant with cytological smears. A total of 77 paired MPE supernatant and precipitate samples were acquired from the 102 patients. The results revealed that there were no statistically significant differences in the detection rate and maximum allelic fraction between supernatant and precipitate samples (P = 1.0 and P = .6). Collectively, 172 and 158 genomic alterations with 112 shared mutations were identified in supernatant and precipitate samples, respectively. Comparable PFS was found in EGFR mutation patients according to the supernatant and precipitate sample results (14.0 vs.13.9 months, P = .90). CONCLUSIONS: These results demonstrated that MPE supernatants were comparable to precipitate samples for detection of genetic alterations. However, gene mutation heterogeneity was found between these two media types.     

On being “actionable”: clinical sequencing and the emerging contours of a regime of genomic medicine in oncology

This article explores commercial, academic, and national initiatives aimed at using sequencing technologies to generate “actionable” genomic results that can be applied to the clinical management of oncology patients. We argue that the term “actionable” is not merely a buzzword, but signals the emergence of a distinctive sociotechnical regime of genomic medicine in oncology. Unlike other regimes of genomic medicine that are organized around assessing and managing inherited risk for developing cancer (e.g. BRCA testing), actionable regimes aim to generate predictive relationships between genetic information and drug therapies, thereby generating new kinds of clinical actions. We explore how these genomic results are made actionable by articulating them with existing clinical routines, clinical trials, regulatory regimes, and health care systems; and in turn, how clinical sequencing programs have begun to reconfigure knowledge and practices in oncology. Actionability regimes confirm the emergence of bio-clinical decision-making in oncology, whereby the articulation of molecular hypotheses and experimental therapeutics become central to patient care.     

Mixed responses to first-line alectinib in non-small cell lung cancer patients with rare ALK gene fusions: A case series and literature review

Anaplastic lymphoma kinase (ALK) fusion is a well-defined biomarker for ALK tyrosine kinase inhibitors (TKIs) treatment in non-small cell lung cancer (NSCLC). Alectinib, a second-generation ALK-TKI, has been shown to have significantly longer progression-free survival (PFS) than first-generation ALK inhibitors in untreated ALK-rearranged NSCLC patients. However, its clinical efficacy on rare ALK fusions remains unclear. Herein, two advanced NSCLC patients received first-line alectinib treatment, given their positive ALK fusion status as determined by immunohistochemistry (IHC) testing results. Patients showed limited clinical response (PFS: 4 months) and primary resistance to alectinib respectively. Molecular profiling using next-generation sequencing (NGS) further revealed a striatin (STRN)-ALK fusion in the first patient accompanied by MET amplification, and a LIM domain only protein 7 (LMO7)-ALK fusion in another patient without any other known oncogenic alterations. Both patients demonstrated improved survival after they switched to second-line crizotinib (PFS: 11 months) and ensartinib (PFS: 18 months), respectively, up till the last follow-up assessment. In conclusion, the clinical efficacy of ALK-TKIs including alectinib for lung cancer with uncommon ALK gene fusions is still under evaluation. This study and literature review results showed mixed responses to alectinib in NSCLC patients who harboured rare ALK fusions. Comprehensive molecular profiling of tumour is thus strongly warranted for precise treatment strategies.     

Current and emerging applications of liquid biopsy in pan-cancer

Cancer morbidity and mortality are growing rapidly worldwide and it is urgent to develop a convenient and effective method that can identify cancer patients at an early stage and predict treatment outcomes. As a minimally invasive and reproducible tool, liquid biopsy (LB) offers the opportunity to detect, analyze and monitor cancer in any body fluids including blood, complementing the limitations of tissue biopsy. In liquid biopsy, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are the two most common biomarkers, displaying great potential in the clinical application of pan-cancer. In this review, we expound the samples, targets, and newest techniques in liquid biopsy and summarize current clinical applications in several specific cancers. Besides, we put forward a bright prospect for further exploring the emerging application of liquid biopsy in the field of pan-cancer precision medicine.     

Comparison of Hepatocellular Carcinoma miRNA Expression Profiling as Evaluated by Next Generation Sequencing and Microarray

MicroRNA (miRNA) expression profiling has proven useful in diagnosing and understanding the development and progression of several diseases. Microarray is the standard method for analyzing miRNA expression profiles; however, it has several disadvantages, including its limited detection of miRNAs. In recent years, advances in genome sequencing have led to the development of next-generation sequencing (NGS) technologies, which significantly advance genome sequencing speed and discovery. In this study, we compared the expression profiles obtained by next generation sequencing (NGS) with the profiles created using microarray to assess if NGS could produce a more accurate and complete miRNA profile. Total RNA from 14 hepatocellular carcinoma tumors (HCC) and 6 matched non-tumor control tissues were sequenced with Illumina MiSeq 50-bp single-end reads. Micro RNA expression profiles were estimated using miRDeep2 software. As a comparison, miRNA expression profiles for 11 out of 14 HCCs were also established by microarray (Agilent human microRNA microarray). The average total sequencing exceeded 2.2 million reads per sample and of those reads, approximately 57% mapped to the human genome. The average correlation for miRNA expression between microarray and NGS and subtraction were 0.613 and 0.587, respectively, while miRNA expression between technical replicates was 0.976. The diagnostic accuracy of HCC, p-value, and AUC were 90.0%, 7.22×10⁻⁴, and 0.92, respectively. In summary, NGS created an miRNA expression profile that was reproducible and comparable to that produced by microarray. Moreover, NGS discovered novel miRNAs that were otherwise undetectable by microarray. We believe that miRNA expression profiling by NGS can be a useful diagnostic tool applicable to multiple fields of medicine.     

Identification of canine circulating miRNAs as tumor biospecific markers using Next-Generation Sequencing and Q-RT-PCR

Delay in cancer diagnosis often results in metastasis and an inability to successfully treat the tumor. The use of broadly cancer-specific biomarkers at an early stage may improve cancer treatment and staging. This study has explored circulatory exosomal miRNAs as potential diagnostic biomarkers to identify cancer patients. Secretory exosomal miRNAs were isolated from 13 canine cancer cell lines (lymphoma, mast cell tumor, histiocytic cell line, osteosarcoma, melanoma, and breast tumor) and were sequenced by Next-Generation sequencing (NGS). We have identified 6 miRNAs (cfa-miR-9, -1841, ?1306, ?345, ?132, and ?26b) by NGS that were elevated in all cancer cell types. The miRNAs identified by NGS were then examined by Q-RT-PCR. The PCR data demonstrated similar expression patterns to those seen with NGS but provided fold differences that were much lower than those seen for NGS. Cfa-miR-9 was found to be the most consistently elevated miRNA in NGS and PCR, making it the most likely miRNA to prove diagnostic. In this study, we have demonstrated that it is possible to identify exosomal miRNAs with elevated secretion across multiple tumor types that could be used as circulatory diagnostic biomarkers for liquid biopsy in the future.     

Management of Incidental Findings in the Era of Next-generation Sequencing

Next-generation sequencing (NGS) technologies allow for the generation of whole exome or whole genome sequencing data, which can be used to identify novel genetic alterations associated with defined phenotypes or to expedite discovery of functional variants for improved patient care. Because this robust technology has the ability to identify all mutations within a genome, incidental findings (IF)- genetic alterations associated with conditions or diseases unrelated to the patient’s present condition for which current tests are being performed- may have important clinical ramifications. The current debate among genetic scientists and clinicians focuses on the following questions: 1) should any IF be disclosed to patients, and 2) which IF should be disclosed – actionable mutations, variants of unknown significance, or all IF? Policies for disclosure of IF are being developed for when and how to convey these findings and whether adults, minors, or individuals unable to provide consent have the right to refuse receipt of IF. In this review, we detail current NGS technology platforms, discuss pressing issues regarding disclosure of IF, and how IF are currently being handled in prenatal, pediatric, and adult patients.     

Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome.In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient’s resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.     

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next‐Generation Sequencing Technologies for Cell Line Development

Early analytical clone screening is important during Chinese hamster ovary (CHO) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. Sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. Next‐generation sequencing (NGS) technologies emerged as alternatives for CHO clone analytics. We report an efficient NGS workflow applying the targeted locus amplification (TLA) strategy for genomic screening of antibody expressing CHO clones. In contrast to previously reported RNA sequencing approaches, TLA allows for targeted sequencing of genomic integrated transgenic DNA without prior locus information, robust detection of single‐nucleotide variants (SNVs) and transgenic rearrangements. During clone selection, TLA/NGS revealed CHO clones with high‐level SNVs within the antibody gene and we report in another case the utility of TLA/NGS to identify rearrangements at transgenic DNA level. We also determined detection limits for SNVs calling and the potential to identify clone contaminations by TLA/NGS. TLA/NGS also allows to identify genetically identical clones. In summary, we demonstrate that TLA/NGS is a robust screening method useful for routine clone analytics during cell line development with the potential to process up to 24 CHO clones in less than 7 workdays.     

Detecting and targetting oncogenic fusion proteins in the genomic era

The advent of widespread cancer genome sequencing has accelerated our understanding of the molecular aberrations underlying malignant disease at an unprecedented rate. Coupling the large number of bioinformatic methods developed to locate genomic breakpoints with increased sequence read length and a deeper understanding of coding region function has enabled rapid identification of novel actionable oncogenic fusion genes. Using examples of kinase fusions found in liquid and solid tumours, this review highlights major concepts that have arisen in our understanding of cancer pathogenesis through the study of fusion proteins. We provide an overview of recently developed methods to identify potential fusion proteins from next‐generation sequencing data, describe the validation of their oncogenic potential and discuss the role of targetted therapies in treating cancers driven by fusion oncoproteins.     

Exome sequencing reveals comprehensive genomic alterations across eight cancer cell lines

It is well established that genomic alterations play an essential role in oncogenesis, disease progression, and response of tumors to therapeutic intervention. The advances of next-generation sequencing technologies (NGS) provide unprecedented capabilities to scan genomes for changes such as mutations, deletions, and alterations of chromosomal copy number. However, the cost of full-genome sequencing still prevents the routine application of NGS in many areas. Capturing and sequencing the coding exons of genes (the "exome") can be a cost-effective approach for identifying changes that result in alteration of protein sequences. We applied an exome-sequencing technology (Roche Nimblegen capture paired with 454 sequencing) to identify sequence variation and mutations in eight commonly used cancer cell lines from a variety of tissue origins (A2780, A549, Colo205, GTL16, NCI-H661, MDA-MB468, PC3, and RD). We showed that this technology can accurately identify sequence variation, providing ～95% concordance with Affymetrix SNP Array 6.0 performed on the same cell lines. Furthermore, we detected 19 of the 21 mutations reported in Sanger COSMIC database for these cell lines. We identified an average of 2,779 potential novel sequence variations/mutations per cell line, of which 1,904 were non-synonymous. Many non-synonymous changes were identified in kinases and known cancer-related genes. In addition we confirmed that the read-depth of exome sequence data can be used to estimate high-level gene amplifications and identify homologous deletions. In summary, we demonstrate that exome sequencing can be a reliable and cost-effective way for identifying alterations in cancer genomes, and we have generated a comprehensive catalogue of genomic alterations in coding regions of eight cancer cell lines. These findings could provide important insights into cancer pathways and mechanisms of resistance to anti-cancer therapies.     

Evaluation of personalized cancer therapies based on comprehensive genomic profiling in a middle-sized oncologic center in Austria,the University Clinic Krems

Background and AimTo successfully apply personalized cancer therapies, thorough understanding of the patient's tumor is needed. In-depth, comprehensive genomic profiling systems allow gathering this knowledge by testing hundreds of cancer-related genes. Several large institutions have established precision oncology programs in recent years with promising results for patients. However, especially middle-sized oncologic institutions face challenges to implement such programs.This study aims to retrospectively analyze the effects of comprehensive genomic tumor profiling with respect to feasibility and effectiveness in a middle-sized oncologic center in Austria.MethodsFrom May 1st, 2016 to December 31st, 2019 patients at the University Clinic Krems, who suffered from CUP-syndromes plus patients, who were resistant to conventional therapy or have progressed after all available therapy lines, were offered to get their tumors analyzed by comprehensive genomic profiling in order to establish a customized therapy.ResultsOf 69 considered patients, 64 patients’ samples could be profiled. The median number of detected alterations was 4 (minimum 0; maximum 23). Most frequent alterations were reported for TP53, KRAS and CDKN2A/B.In 13 patients (20% of 64 successful profiles), personalized therapies could be initiated. 22 patients were treated with another line of chemotherapy as no actionable alteration could be detected. Effectiveness, determined by a PFS of the newly initiated therapy longer than 130% of the last conventional therapy line, could be seen in 8 of 13 patients (61,5%) of the precision oncology cohort compared to only 3 of 22 patients (13,5%) in the chemotherapy group. Additionally, Kaplan-Meier curves of PFS demonstrate a significant benefit for personalized treated patients (p = 0,0165 with a median PFS of 151 days, compared to 83 days in the chemotherapy group).ConclusionIn summary, personalized cancer therapy based on comprehensive genomic profiling is effective and feasible also in the setting of a middle-sized oncologic center.     

Potential predictive value of comutant LRP1B and FAT for immune response in non-small cell lung cancer: LRP1B and FAT comutation enhance immune response

BackgroundPreliminary investigation revealed that Low-density lipoprotein receptor-related protein 1b (LRP1B) and FAT atypical cadherin (FAT) family mutation might serve as immune regulators under certain tumor microenvironment.Experimental designWe curated a total of 70 non-small cell lung cancer (NSCLC) patients who harbored alterations in LRP1B and/or FAT family (FAT1/2/3/4) based on next-generation sequencing (NGS) to analyze multiple-dimensional data types, including comutant status, tumor mutation burden (TMB), programmed death receptor ligand 1 (PD-L1) expression, T cell-inflamed gene expression profiling (GEP) and therapy response.Results20 patients with co-occurring mutations in LRP1B and FAT1/2/3/4 revealed a relatively higher TMB level of 17.05 mut/Mb compared with 7.60 mut/Mb and 8.80 mut/Mb in single LRP1B and FAT mutation groups, respectively. LRP1B and FAT members showed specifically enriched T cell-inflamed genes and the co-occurring mutant TP53 status in NSCLC patients who harbor LRP1B/FAT comutations.ConclusionsThis work provides evidence that co-occurring mutations of LRP1B and FAT in NSCLC may serve as a group of potential predictive factors in guiding immunotherapy on the basis of their association with TMB status.