EGFR Somatic Mutations Research Articles

Beyond tobacco: genomic disparities in lung cancer between smokers and never-smokers

BackgroundTobacco use is one of the main risk factors for Lung Cancer (LC) development. However, about 10–20% of those diagnosed with the disease are never-smokers. For Non-Small Cell Lung Cancer (NSCLC) there are clear differences in both the clinical presentation and the tumor genomic profiles between smokers and never-smokers. For example, the Lung Adenocarcinoma (LUAD) histological subtype in never-smokers is predominately found in young women of European, North American, and Asian descent. While the clinical presentation and tumor genomic profiles of smokers have been widely examined, never-smokers are usually underrepresented, especially those of a Latin American (LA) background. In this work, we characterize, for the first time, the difference in the genomic profiles between smokers and never-smokers LC patients from Chile.MethodsWe conduct a comparison by smoking status in the frequencies of genomic alterations (GAs) including somatic mutations and structural variants (fusions) in a total of 10 clinically relevant genes, including the eight most common actionable genes for LC (EGFR, KRAS, ALK, MET, BRAF, RET, ERBB2, and ROS1) and two established driver genes for malignancies other than LC (PIK3CA and MAP2K1). Study participants were grouped as either smokers (current and former, n = 473) or never-smokers (n = 200) according to self-report tobacco use at enrollment.ResultsOur findings indicate a higher overall GA frequency for never-smokers compared to smokers (58 vs. 45.7, p-value < 0.01) with the genes EGFR, KRAS, and PIK3CA displaying the highest prevalence while ERBB2, RET, and ROS1 the lowest. Never-smokers present higher frequencies in seven out of the 10 genes; however, smokers harbor a more complex genomic profile. The clearest differences between groups are seen for EGFR (15.6 vs. 21.5, p-value: < 0.01), PIK3CA (6.8 vs 9.5) and ALK (3.2 vs 7.5) in favor of never-smokers, and KRAS (16.3 vs. 11.5) and MAP2K1 (6.6 vs. 3.5) in favor of smokers. Alterations in these genes are comprised almost exclusively by somatic mutations in EGFR and mainly by fusions in ALK, and only by mutations in PIK3CA, KRAS and MAP2K1.ConclusionsWe found clear differences in the genomic landscape by smoking status in LUAD patients from Chile, with potential implications for clinical management in these limited-resource settings.

Mutational Characteristics and Clinical Outcomes for Lung Adenocarcinoma With EGFR Germline Mutations

IntroductionGermline mutations driving lung cancer have been infrequently reported in the literature, with EGFR T790M being a known germline mutation identified in 1% of NSCLCs. Typically, a somatic EGFR mutation is acquired to develop lung adenocarcinoma. Osimertinib has become a standard-of-care treatment for EGFR T790M-positive lung cancer. MethodsWe perform a retrospective analysis through the Lung Cancer Moon Shot GEMINI database at the University of Texas MD Anderson Cancer Center. Of the patients that underwent cell-free DNA analysis, germline mutations were identified by those with high variant allelic fraction approximating 50%, followed by further confirmation on genetic testing. ResultsWe identified 22 patients with germline EGFR mutations, with the majority harboring an EGFR T790M mutation (95.5%) and an EGFR L858R somatic mutation (50%). Notably, most patients were female (86.4%), non-smokers (81.8%), white (86.4%), had a family history of lung cancer (59.1%), and stage IV at diagnosis (72.7%). A distinct radiographic pattern of small multifocal ground-glass pulmonary nodules was observed in the majority of our cohort (72.7%).Among the 18 with advanced-stage NSCLC, 12 patients (66.7%) were treated with first-line osimertinib, demonstrating a median progression-free survival (PFS) of 16.9 months (95% confidence interval [CI]: 6.3–not reached [NR]). Others were treated with first-line afatinib (11.1%) or chemotherapy (22.2%). Among the 17 patients treated with osimertinib (in first or second-line), median PFS was 20.4 months (95% CI: 6.3–NR) and median overall survival was 82.0 months (95% CI: 28.4–NR). ConclusionsBased on our institutional cohort, NSCLC driven by EGFR germline mutations occurs more frequently in non-smoking, white females with multi-focal pulmonary nodules radiographically. Osimertinib for advanced germline EGFR-mutated NSCLC renders similar PFS compared to somatic T790M EGFR-mutated NSCLC.

Abstract 5547: Evaluation of two clinically focused targeted NGS systems for liquid biopsy testing shows a high level of concordance in resulting actionable mutations

Abstract Introduction: The use of blood-based molecular diagnostics is increasingly becoming routine in clinical oncology practice. The purpose of this study was to evaluate variant resulting concordance for the four major somatic variant classes using two clinical next-generation sequencing (NGS) systems for conducting highly sensitive blood-based analyses. All donor specimens were de-identified remnants from patients previously diagnosed with advanced NSCLC. We utilized two independent panels, systems and bioinformatic pipelines that are focused on clinical testing for key actionable variants. The fifty-gene panel Oncomine Precision Assay (OPA) GX was performed on the Genexus Integrated Sequencer and the 52 gene GeneStrat NGS (GSNGS) test was run as a reference on the Ion GeneStudio S5 PRIME system. The four major classes of mutations evaluated included Single Nucleotide Variants (SNV), Insertions and Deletions (INDEL), copy number amplifications (CNAs) and gene fusions. Methods: For this study, de-identified reference GSNGS cell-free nucleic acid (cfNA) remnant specimens (n=33 variants) that passed all validated QC bioinformatics thresholds were blinded and tested on the Genexus. DNA concentration was measured by fluorometry, and specimens were diluted to an input of 6.6 ng to 53.4 ng for library preparation. The Genexus system is pre-programmed to process specimens to result generation. Variant calls for all mutation classes were conducted using the on-board bioinformatics pipelines, and results were compared to those generated by the GeneStrat NGS Test. We included specimens harboring somatic variant mutations in KRAS, NRAS, BRAF, EGFR, ERBB2, and FGFR; CNAs; EML4-ALK fusions; and MET exon 14 skipping. Results: All samples passed the OPA (Genexus) bioinformatic quality control criteria and mapped reads were highly consistent, demonstrating accuracy of calls between both the Ion Torrent platforms, informatic pipelines and panels. We observed 100% concordance in variant calls for SNVs, INDELs and fusions between the two platforms. There was one CNA variant observed on the S5 that was not called by the Genexus workflow, yielding overall concordance of 97% (32 of 33). Of note, 44 additional somatic variants (SNV and INDELs) and 1 additional fusion were detected using the Genexus workflow. This was due to the lower threshold for variant calling on this system. Conclusion: The high concordance of the independent workflows for the detection of nucleic acid variants in circulation demonstrates the capability of both systems to be used for testing of clinical specimens. Specifically, actionable variants in the four major mutation classes were successfully detected in the reference and test specimen set. As a part of clinical validation, orthogonal testing of variants will need to be conducted. Citation Format: Sarah Kreston, Claire Gould, Kylie Blair, Leisa Jackson, Janice Riley, Gary A. Pestano. Evaluation of two clinically focused targeted NGS systems for liquid biopsy testing shows a high level of concordance in resulting actionable mutations. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5547.

Abstract 5675: Discovery and development of an asymmetric IgG-like bispecific antibody targeting EGFR and c-MET, engineered through H-H and H-L chain charge-based heterodimerization

Abstract Background: Lung cancer remains the leading cause of cancer mortality and is the most commonly diagnosed cancer with an estimated 2.2 million cases per year. EGFR, a tyrosine kinase receptor, plays a central role in cellular proliferation, survival, differentiation, and migration. Gain-of-function somatic mutations of EGFR significantly drives disease progression. Small molecule tyrosine kinase inhibitors (TKIs), designed to inhibit EGFR signaling specific to these mutations (e.g., L858R and E19del etc.), have achieved excellent clinical outcomes. However, patients carrying TKI-insensitive mutations (e.g., E20ins) and/or those with disease progression after TKI treatment, by the upregulation of complementary signaling pathways (HGF/c-MET, etc.), remain an unmet medical need. Here, we report the discovery of a fully-human afucosylated anti-EGFR x cMET IgG-like bispecific antibody (PM1080). Methods: PM1080 was generated by introducing unique mutations to the CH1-CL domains of each binding arm to promote correct HC-LC pairing. KIH mutations were also introduced to each CH3 domain to support HC-HC heterodimerization and generate an IgG-like bispecific with one arm binding to EGFR and the other to cMET. PM1080 was expressed via a four-chain expression system in CHO cells carrying a fut8−/− knockout to generate afucosylated molecules with enhanced ADCC activity. Results: Through monovalent affinity measurements, PM1080 interacted with EGFR at single digit nanomolar affinity and at sub-nanomolar affinity to c-MET. PM1080 preferentially bound to EGFR/c-MET double-positive cells rather than EGFR single-positive cells. PM1080 blocked both EGF/EGFR and HGF/c-MET signals via physical blockade of the interaction between EGF/HGF to their corresponding receptors and induced EGFR/c-MET internalization and degradation from the cell surface. Through intricate engineering, PM1080 was expressed via a four-chain system and produced as an afucosylated antibody with significantly improved ADCC function. PM1080 induced potent anti-tumor efficacy against EGFR/c-MET-positive tumor cells as a single agent. Importantly, PM1080 induced anti-tumor activity regardless of EGFR mutations at the intracellular signaling domain, and thus may serve as a promising combinational agent to TKI therapies. As expected, synergistic anti-tumor activity was observed when PM1080 was combined with either 1st or 3rd generation TKIs in both in vitro and in vivo models. Conclusion: PM1080, a bispecific antibody targeting EGFR &amp; c-MET was discovered for cancer treatment. PM1080 displayed potent anti-tumor efficacy either as a single agent or in combination with EGFR TKIs. Preclinical PK and toxicity studies have been conducted to support future first-in-human studies. Citation Format: Ping Wang, Weifeng Huang, Zhenting Zhao, Xiaoniu Miao, Shaogang Peng, Chao Wang, Yao Yan, Tiantian Dong, Andy Tsun, Yingda Xu, Xiaolin Liu, Luo Yi. Discovery and development of an asymmetric IgG-like bispecific antibody targeting EGFR and c-MET, engineered through H-H and H-L chain charge-based heterodimerization. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5675.

HPV Infection is Associated with FoxM1 Overexpression in Dysplastic Changes of Sinonasal Inverted Papilloma

This study aims to describe the factors associated with dysplastic changes in sinonasal inverted papilloma (SIP) including somatic EGFR mutation, FoxM1 expression, HPV status, and their association with dysplastic changes. A cross-sectional, analytical study was conducted comprising 34 samples of histologically-confirmed diagnosis of SIP. The samples were further grouped into 2 groups: 20 samples without associated dysplastic changes, and 14 samples with associated dysplastic changes. The numbers of FoxM1 positively-expressed cells, EGFR mutation, and HPV status were compared among two groups using appropriate comparative statistics. There was statistically-significant difference of FoxM1 expression between SIP and SIP with dysplasia (10% vs 100%; p<0.001). EGFR mutation was identified in 6 samples (30.0%) of the SIP and 5 samples (35.7%) of SIP with dysplasia. No difference of EGFR mutant proportion among two groups. HPV DNA was detected in 5 samples (25.0%) of SIP versus 9 samples (64.3%) of SIP with dysplasia. There was significant difference of HPV status among two groups (p=0.022). The high-risk subtypes were found in most HPV positive samples (57.1%), while low-risk subtypes and out panel subtypes were found 14.3% and 21.4%, respectively. FoxM1 was overexpressed in SIP with malignant transformation. FoxM1 along with HPV status is associated with dysplastic changes in the SIP. FoxM1 immunostaining is potential to be a biomarker of malignant transformation in SIP.

Abstract PL03-02: Somatic mutations, germline risk and ancestry in lung adenocarcinoma

Abstract Like most cancer types, lung cancer is caused predominantly by somatic genome alterations. In lung adenocarcinoma, the most common histological type of lung cancer, these alterations are known to occur in multiple pathways including the p53 pathway, the receptor tyrosine kinase (RTK)/Ras/Raf/MAP kinase (MAPK) pathway and PI3 kinase pathway, the Rb1/cell cycle pathway, the Swi/Snf pathway, the Myc pathway, and the telomerase pathway, among others. Genome-wide association studies comparing polymorphisms in germline DNA from patients with lung cancer and from unaffected control subjects have led to the identification of multiple germline risk alleles for lung adenocarcinoma, including the HLA locus, the telomerase catalytic subunit gene locus, and, notably, the nicotinic acetylcholine receptor locus, where polymorphisms appear to modulate individual propensity for smoking. The components of inherited lung cancer risk, particularly in nonsmokers, are not fully understood, but there appear to be variations in lung adenocarcinoma mutations in distinct ancestry groups, which may be related to inherited factors. In particular, the frequency of somatic EGFR and KRAS mutations in lung cancer varies by ethnicity. Somatic EGFR mutation rates are higher in lung cancers from patients with East Asian ancestry and lower in lung cancers from patients with European or African ancestry. Somatic KRAS and STK11 mutation rates show the opposite pattern, higher in lung cancers from patients with European or African ancestry and lower in lung cancers from patients with East Asian ancestry. In patients from Latin America, somatic lung cancer EGFR mutation frequencies vary by country, with higher frequencies in Peru, intermediate frequencies in Mexico, Colombia, Costa Rica and Brazil, and lower frequencies in Argentina. To explore the relationship between the somatic genome in lung cancer and ethnicity related germline risk, we performed cancer gene panel sequencing of DNA from over 1,000 lung cancers from Mexico and Colombia, countries with significant population admixture. We developed methods to infer ancestry from the tumor DNA sequence based on coverage of single nucleotide polymorphisms. This study revealed associations between ancestry and somatic lung cancer genomic alterations, including tumor mutational burden, and specific driver mutations in EGFR, KRAS, and STK11. A local ancestry score was more strongly correlated with EGFR mutation frequency compared with global ancestry correlation, suggesting that germline genetics, rather than environmental exposure, could underlie these disparities. Our study suggests that the variation in EGFR and KRAS somatic mutation frequency in lung cancer is associated with genetic ancestry in patients from Latin America, and suggests further studies to identify germline alleles that underpin this association. If we find a germline locus or loci that could impact the development of lung cancers with EGFR and/or KRAS mutation, this might help in improving lung cancer prevention and screening for populations of Latin American origin and others. Furthermore, multiple studies now highlight the importance of EGFR mutation screening and EGFR-directed targeted therapy for lung cancer patients in Latin America and with origins in Latin America. Citation Format: Matthew L. Meyerson. Somatic mutations, germline risk and ancestry in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr PL03-02.

Abstract 5754: The molecular characteristics of EGFR co-mutations in lung adenocarcinoma

Abstract Background: With the development of sensitive next-generation sequencing (NGS) techniques that go beyond the genotyping of specific mutations, detection rates for uncommon mutations have risen clinically. Given this rise in mutation identification, discoveries regarding isolation or coexistent mutations with the EGFR mutation (termed a “complex” mutation) are in “uncharted territory” in terms of their biological impacts on oncogenic pathways and their sensitivity or resistance, to EGFR TKIs. Methods: Targeted NGS for 450 genes was performed in OrigiMed (Shanghai, China), a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Results: A cohort of 262 Chinese lung adenocarcinoma patients were recruited. The most common mutated gene was EGFR (63%, 165/262), and followed by TP53 gene (59.2%, 155/262). Beyond single genetic lesions, we found that enriched co-mutations in lung adenocarcinoma were significantly different for TP53 (p &amp;lt; 0.001) and RTK-RAS (p = 0.0012) signaling pathways between the EGFR-positive and EGFR-negative groups. In all EGFR co-mutated patients, the frequency of co-mutation for EGFR/TP53 and the classical EGFR mutation was 106 (64.2%) and 144 (87.3%), respectively. EGFR-only mutations occurred in 50 (30.3%) patients, while co-mutation of EGFR and at least one tumor suppressor gene (TSG) occurred in 84 (50.9%) patients. Patients with EGFR-only mutation had a lower tumor mutational burden (p = 0.026) as compared to patients with only a TSG mutation. Patients with co-mutations of EGFR and at least one TSG also displayed a lower tumor mutational burden, although only a slight difference (p = 0.062) was determined. Conclusions: Profiles of EGFR co-mutational TSGs should be regarded as a unique subgroup for predictive insights in treatments for patients with co-occurrence of somatic EGFR and TSG mutations. To improve predictions related to drug sensitivity in targeted therapies for oncogenes with diverse mutations, our future studies will include publications dedicated to this topic. Citation Format: Yanhong Shang, Hao Zhang, Aiming Zang, Shaohua Yuan, Xiaofang Li, Wenpan Zhang, Ran Huo, Guotao Fang, Zhengyue Dou, Weiwei Liu, Xiao Han, Qi Zhao, Chenglin Xi. The molecular characteristics of EGFR co-mutations in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5754.

Abstract 527: A streamlined workflow for liquid biopsy extraction and highplex digital PCR analysis using the Maxwell® RSC system and 6-color Crystal Digital PCR™

Abstract Circulating cell free DNA (cfDNA) extracted from liquid biopsy samples have become established sample types for characterizing oncology targets. Currently, there are several extraction protocols and genomic platforms for researchers to select when interrogating genetic information. The focus of this work was to identify a flexible method for sample extraction that seamlessly integrates into a straightforward and sensitive genetic analysis workflow from plasma samples. Here, we present a workflow combining the Maxwell RSC system for automated cfDNA extraction and Crystal Digital PCR on the naica system for ultrasensitive high-plex detection from liquid biopsy samples of EGFR mutations in non-small cell lung cancer (NSCLC) and PIK3CA mutations and HER2 amplification in breast cancer. By bridging sample extraction with the Maxwell RSC system and high-plex digital PCR with the 6-color naica® system, we detected with high sensitivity and precision 32 common and rare somatic EGFR mutations in exons 18, 19, 20, and 21, representing more than 90% of EGFR mutations described in NSCLC. Furthermore, following the same extraction and sample testing workflow, we simultaneously detected a set of PIK3CA mutations and HER2 amplification from cfDNA samples. This proof-of-concept workflow creates the foundation for further development of streamlined sample-to-answer protocols that will better assist cancer researchers across the biomarker testing landscape. Citation Format: Cécile Jovelet, Myrtille Remy, Mylene Menanteau, Doug White, Douglas Horejsh, Allison Mallory. A streamlined workflow for liquid biopsy extraction and highplex digital PCR analysis using the Maxwell® RSC system and 6-color Crystal Digital PCR™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 527.

First-in-human phase 1/2 study of autologous T cells engineered using the Sleeping Beauty System transposon/transposase to express T-cell receptors (TCRs) reactive against cancer-specific mutations in patients with advanced solid tumors.

TPS2679 Background: In 2022 approximately 1.7 million Americans will die from solid cancers. Recently there have been significant advances in the genetic engineering of T lymphocytes to recognize neoantigens on tumors in vivo, resulting in remarkable cases of tumor regression and remission. Cancer cells frequently harbor KRAS, TP53, and EGFR somatic hotspot mutations that can be processed and presented by tumor HLA as neoantigens to T cells through their T-cell receptor (TCR). These neoantigens are not present in the normal tissues; thus, they are attractive targets for adoptive T cell therapy. Given the number and complexity of different neoantigen/HLA combinations on solid tumors, a TCR library approach is warranted. Therefore, we have developed a library of TCR-T cell therapies including those targeting shared KRAS, TP53 and EGFR mutations. Methods: Patients for whom a TCR matching the subject’s somatic mutation(s) and HLA type is available in our TCR library, and have progressive or recurrent disease following standard therapy are eligible for enrollment on this protocol. Patients with the following tumor types will be enrolled: ovarian, endometrial, colorectal, pancreatic, cholangiocarcinoma, and non-small cell lung cancer. This first-in-human study includes Screening, Pre-Treatment, Treatment and Follow-up Periods. During the Pre-Treatment Period, subjects will undergo apheresis for PBMCs isolation. The PBMCs will be transposed using the Sleeping Beauty system to express the subject’s mutation specific TCR. Bridging therapy after apheresis is allowed once the apheresis product has been accepted. During the Treatment Period, patients will undergo lymphodepletion with cyclophosphamide and fludarabine. After which, the TCR-T cell drug product will be administered to the subject by infusion at the assigned dose level. The starting dose level of Arm A (monotherapy) will be DL1 (5 x10 9 TCR+ Cells) administered on Day 0. Dose escalation will continue utilizing the accelerated BOIN design (planned escalation dose levels: 5 x10 9, 4 x10 10 and 1 x10 11 TCR+ Cells). In Arm B, if initiated by protocol, subjects will also receive aldesleukin (interleukin-2) infusion starting on Day 0 (within 24 hours of TCR-T cell product infusion) at 720K IU/kg, every eight hours for up to 4 days. The Follow-up Period will begin after the subject completes their Day 28 visit. Clinical and radiologic response will be assessed at 6 and 12 weeks after TCR-T cell drug product infusion and every 12 weeks thereafter until up to 2 years or study discontinuation (e.g., disease progression, initiation of new anti-cancer therapy, consent withdrawn), whichever occurs first. All subjects will continue to be followed in the Long-Term Follow-up Protocol for up to 15 years post-TCR-T cell drug product infusion. Clinical trial information: NCT05194735.

Accounting for EGFR Mutations in Epidemiologic Analyses of Non-Small Cell Lung Cancers: Examples Based on the International Lung Cancer Consortium Data.

Somatic EGFR mutations define a subset of non-small cell lung cancers (NSCLC) that have clinical impact on NSCLC risk and outcome. However, EGFR-mutation-status is often missing in epidemiologic datasets. We developed and tested pragmatic approaches to account for EGFR-mutation-status based on variables commonly included in epidemiologic datasets and evaluated the clinical utility of these approaches. Through analysis of the International Lung Cancer Consortium (ILCCO) epidemiologic datasets, we developed a regression model for EGFR-status; we then applied a clinical-restriction approach using the optimal cut-point, and a second epidemiologic, multiple imputation approach to ILCCO survival analyses that did and did not account for EGFR-status. Of 35,356 ILCCO patients with NSCLC, EGFR-mutation-status was available in 4,231 patients. A model regressing known EGFR-mutation-status on clinical and demographic variables achieved a concordance index of 0.75 (95% CI, 0.74-0.77) in the training and 0.77 (95% CI, 0.74-0.79) in the testing dataset. At an optimal cut-point of probability-score = 0.335, sensitivity = 69% and specificity = 72.5% for determining EGFR-wildtype status. In both restriction-based and imputation-based regression analyses of the individual roles of BMI on overall survival of patients with NSCLC, similar results were observed between overall and EGFR-mutation-negative cohort analyses of patients of all ancestries. However, our approach identified some differences: EGFR-mutated Asian patients did not incur a survival benefit from being obese, as observed in EGFR-wildtype Asian patients. We introduce a pragmatic method to evaluate the potential impact of EGFR-status on epidemiological analyses of NSCLC. The proposed method is generalizable in the common occurrence in which EGFR-status data are missing.

Abstract IA-07: Genetic ancestry and lung cancer somatic mutations in patients of Latin American descent: Etiology and treatment implications

Abstract Inherited lung cancer risk, particularly in nonsmokers, is poorly understood. In particular, the frequency of somatic EGFR and KRAS mutations in lung cancer varies by ethnicity. Somatic EGFR mutation rates are higher in lung cancers from patients with East Asian ancestry and lower in patients with European or African ancestry. Somatic KRAS mutation rates show the opposite pattern. In patients from Latin America, somatic lung cancer EGFR mutation rates vary by country, highest in countries with more Native American ancestry. To ask whether the somatic genome in lung cancer is affected by ethnicity related germline risk, we studied genomes from 1,153 lung cancers from Mexico and Colombia. This study revealed striking associations between ancestry and somatic lung cancer alterations, including tumor mutational burden, and specific driver mutations in EGFR, KRAS, and STK11. A local ancestry score was more strongly correlated with EGFR mutation frequency compared with global ancestry correlation, suggesting that germline genetics (rather than environmental exposure) underlie these disparities. Our study suggests that the variation in EGFR and KRAS mutation frequency in lung cancer is associated with genetic ancestry in patients from Latin America, and suggests further studies to identify germline alleles that underpin this association. If we find a germline locus, this might help in improving lung cancer prevention and screening for populations of Latin American origin and others. Furthermore, multiple studies now highlight the special importance of EGFR mutation screening and EGFR-directed targeted therapy for lung cancer patients in Latin America and with origins in Latin America. Citation Format: Jian Carrot-Zhang, Giovanny Soca-Chafre, Nick Patterson, Aaron Thorner, Anwesha Nag, Jacqueline Watson, Giulio Genovese, July Rodriguez, Maya Gelbard, Luis Corrales-Rodriguez, Yoichiro Mitsuishi, Gavin Ha, Joshua Campbell, Geoffrey Oxnard, Oscar Arrieta, Andres Cardona, Alexander Gusev, Matthew Meyerson. Genetic ancestry and lung cancer somatic mutations in patients of Latin American descent: Etiology and treatment implications [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA-07.

Multiplex SuperSelective PCR Assays for the Detection and Quantitation of Rare Somatic Mutations in Liquid Biopsies

SuperSelective primers, by virtue of their unique design, enable the simultaneous identification and quantitation of inherited reference genes and rare somatic mutations in routine multiplex PCR assays, while virtually eliminating signals from abundant wild-type sequences closely related to the target mutations. These assays are sensitive, specific, rapid, and low cost, and can be performed in widely available spectrofluorometric thermal cyclers. Herein, we provide examples of SuperSelective PCR assays that target eight different somatic EGFR mutations, irrespective of whether they occur in the same codon, occur at separate sites within the same exon, or involve deletions. In addition, we provide examples of SuperSelective PCR assays that detect specific EGFR mutations in circulating tumor DNA present in the plasma of liquid biopsies obtained from patients with non-small-cell lung cancer. The results suggest that multiplex SuperSelective PCR assays may enable the choice, and subsequent modification, of effective targeted therapies for the treatment of an individual's cancer, utilizing frequent noninvasive liquid biopsies.

Canonical NF-κB Promotes Lung Epithelial Cell Tumour Growth by Downregulating the Metastasis Suppressor CD82 and Enhancing Epithelial-to-Mesenchymal Cell Transition.

Simple SummaryCanonical NF-κB signalling pathway acts as a tumour promoter in several types of cancer including non-small cell lung cancer (NSCLC), but the mechanism(s) by which it contributes to NSCLC is still under investigation. We show here that NF-κB RelA/p65 is required for the tumour growth of human NSCLC cells grown in vivo as xenografts in immune-compromised mice. RNA-seq transcriptome profile analysis identified the metastasis suppressor CD82/KAI1/TSPAN27 as a canonical NF-κB target. Loss of CD82 correlated with malignancy. RelA/p65 stimulates cell migration and epithelial-to-mesenchymal cell transition (EMT), mediated, in part, by CD82/KAI1, through integrin-mediated signalling, thus, identifying a mechanism mediating NF-κB RelA/p65 lung tumour promoting function.Background: The development of non-small cell lung cancer (NSCLC) involves the progressive accumulation of genetic and epigenetic changes. These include somatic oncogenic KRAS and EGFR mutations and inactivating TP53 tumour suppressor mutations, leading to activation of canonical NF-κB. However, the mechanism(s) by which canonical NF-κB contributes to NSCLC is still under investigation. Methods: Human NSCLC cells were used to knock-down RelA/p65 (RelA/p65KD) and investigate its impact on cell growth, and its mechanism of action by employing RNA-seq analysis, qPCR, immunoblotting, immunohistochemistry, immunofluorescence and functional assays. Results: RelA/p65KD reduced the proliferation and tumour growth of human NSCLC cells grown in vivo as xenografts in immune-compromised mice. RNA-seq analysis identified canonical NF-κB targets mediating its tumour promoting function. RelA/p65KD resulted in the upregulation of the metastasis suppressor CD82/KAI1/TSPAN27 and downregulation of the proto-oncogene ROS1, and LGR6 involved in Wnt/β-catenin signalling. Immunohistochemical and bioinformatics analysis of human NSCLC samples showed that CD82 loss correlated with malignancy. RelA/p65KD suppressed cell migration and epithelial-to-mesenchymal cell transition (EMT), mediated, in part, by CD82/KAI1, through integrin-mediated signalling involving the mitogenic ERK, Akt1 and Rac1 proteins. Conclusions: Canonical NF-κB signalling promotes NSCLC, in part, by downregulating the metastasis suppressor CD82/KAI1 which inhibits cell migration, EMT and tumour growth.

Abstract 448: High prevalence of pathogenic germline variants in patients with oncogene-driven non-small cell lung cancer

Abstract BACKGROUND: Preliminary data has highlighted inherited predisposition to lung cancer related to certain genes. The frequency of pathogenic germline variants (PGV) PGV in patients (pts) with lung cancer according to the presence of an oncogenic driver is unknown. We studied the PGV of genes predisposing to cancer in pts with non-small cell lung cancer (NSCLC), and the somatic molecular profile of lung tumors. METHODS: Retrospective study of whole exome sequencing (WES) from tissue biopsies performed in pts with advanced NSCLC enrolled, after signature of the inform consent, in the MOSCATO/MATCH-R trials between 2012 and 2018. Variants were considered as PGVs in the cancer predisposing genes (PMID: 29625052) if they satisfied the following criteria: (i) they had a “PASS” flag in HaplotypeCaller, (ii) were annotated as “Pathogenic” or “Likely Pathogenic” in ClinVar (PMID: 29165669) or InterVar (PMID: 28132688), or (iii) were truncating variants. Somatic driver mutations and Loss of Heterozygocity (LOH) of PGV harboring genes were further evaluated. The overlap to loss of heterozygocity regions was reported only when the variant allele frequency of the PGV was significantly higher than in the normal tissue. Cancer history, clinical and molecular data were retrospectively collected. The somatic mutations (m) in EGFR/BRAF/MET/HER2/KRAS and fusions in ALK/ROS1/RET were also considered for analysis. RESULTS: Among 134 pts, 48% were women, median age was 61 (range 24-83), 45% were nonsmokers, 74% had adenocarcinoma. The most common somatic oncogenic driver alterations were: EGFRm in 44 pts (33%), KRASm in 19 pts (14%), BRAFm in 12 pts (9%) and ALK in 12 pts (9%).PGV were found in 22 out of 152 (15%) cancer-predisposing genes; 4 pts had additional somatic mutations (2) or LOHs (2) in the same genes. 77% of PGVs were in genes which are part of DNA repair pathways including 3.6% nucleotide excision repair (ERCC1/2/3, XPA), 6.5% homologous recombination/Fanconi Anemia: (FANC/A/C/M/D2, BRCA1, RECQL), 2.1% base excision repair (MUTYH, NTHL1), while the others were represented by genes related to cell signaling and metabolism (NF1, MET, ELANE, PRDM9, TRIM37).In the 22 PGV-carriers, 68% had a somatic oncogene-driven alteration (15/22) : EGFRm (n=7; 5 ex19del, 2 ex21(L858R)), KRASm (n=3; 2 G12D, 1 G12V), METm (n=2), HER2m (n=1), ROS1 (n=1) and RET (n=1). PGV were observed in 16% of EGFRm (7/44), 67% of METm (2/3), 15% in KRASm (3/19), 33% of HER2m (1/3), 25% of ROS1 (1/4), 50% of RET (1/2); but no PGV was identified in pts with BRAFVm (12) or ALK (12). CONCLUSION: In our cohort, 15% of pts with NSCLC were PGV-carriers; 68% of PGV-carriers had oncogene-driven tumors, particularly with somatic EGFR mutations. PGV and oncogene-driven lung carcinogenesis need further evaluation. Citation Format: Laura Mezquita, Andrei Iurchenko, Jose Carlos Benitez, Maria Baz, Sergey Nikolaev, David Planchard, Felix Blanc-Durand, Mihaela Aldea, Patricia Martín-Romano, Yohann Loriot, Claudio Nicotra, Maud Ngocamus, Jean-Yves Scoazec, Stefan Michiels, Sophie Postel-Vinay, Julien Viot, Luc Friboulet, Antoine Italiano, Fabrice Andre, Christophe Massard, Jean-Charles Soria, Etienne Rouleau, Daniel Gautheret, Benjamin Besse. High prevalence of pathogenic germline variants in patients with oncogene-driven non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 448.

Increased ROS1 expression in nsclc associated with somatic EGFR mutations

Increased ROS1 expression in nsclc associated with somatic EGFR mutations

Characteristic of genetic and t cell receptor repertoire of Chinese multiple primary lung cancer patients.

e20510 Background: As the incidence of lung cancer soars, the number of patients diagnosed with multiple primary lung cancers (MPLC) was also rising. It was already known that genetic and immune microenvironment were involved in oncogenesis and treatment of tumors. The aim of this study was to investigate genetic and T Cell Receptor (TCR) Repertoire characteristic in patients with multiple primary lung cancers. Methods: We queried 84 lesions analyzed by 1021 gene panel and TCRβ repertoire for 32 patients with MPLC profiled by NGS. Shannon index was calculated on the clonal abundance of all productive TCR sequences. T cell clonality was defined as 1 − (Shannon index)/ln(# of productive unique sequences). MOI was a measure of the similarity in the T cell repertoire between tissue and blood taking into account the specific rearrangements and their respective frequencies. Results: 98.7% (77/78) of lesions from 30 patients were detected somatic mutations and EGFR mutations were detected in 44.2% (34/77) lesions; BRAF were detected in 22.1% (17/77) lesions; and KRAS were detected in 14.3% (11/77) lesions. We found different combinations of three driving genes in 13 patients. Almost all lesions (97.4%) had mutations in the RAS-RTK pathway. Shared mutations were detected between different lesions from 8 patients, and two patients were intrapulmonary metastases; The others were more meet to convergent evolution. The overall immune characteristics of 76 lesions from 30 patients were further studied. The shannon index showed heterogeneity in difference lesions of each patients and the comparing the most and least shannon index lesions variations ranged between 0.066 to 3.344. The lesions with shared mutations showed higher Shannon index than the non-share lesions (7.31 vs 6.96; p = 0.011). The heterogeneity also identified in MOI and 55% MOI of lesions was less than 0.5. The lesions with MOI higher than 0.5 had some pathologically identical and some have shared mutations. Furthermore, the shared TCR between lesions in same patient were analyzed. The more than 78% of T cell clones were restricted to individual lesions, which indicated the presence of heterogeneity between lesions due to T cell clones distribution heterogeneity. Meanwhile, the shared TCR was significantly higher in identical pathology group than difference pathology group (17.18% vs 6.60%; p = 0.0006); Besides, we found that shared TCR was comparable in both shared mutations and non-shared mutations groups (13.1% vs 15.33%, p = 0.537). These results suggested that the pathology between lesions in patients was affected to 100% shared TCR. Conclusions: In conclusion, the multiple lesions of MPLC showed heterogeneity and this heterogeneity is due to the distribution of T cell clones between lesions. Besides, the MOI and shared TCR was higher in MPLC with same pathology, which may increase the understanding of MPLC and influence the treatment choice of MPLC patients.

Cutaneous Lymphadenoma Is a Distinct Trichoblastoma-like Lymphoepithelial Tumor With Diffuse Androgen Receptor Immunoreactivity, Notch1 Ligand in Reed-Sternberg-like Cells, and Common EGFR Somatic Mutations.

The term “cutaneous lymphadenoma” was coined in this journal for an unusual lymphoepithelial cutaneous adnexal neoplasm, possibly with immature pilosebaceous differentiation. Some authors further proposed that cutaneous lymphadenoma was an adamantinoid trichoblastoma. However, although a hair follicle differentiation is widely accepted, the fact that this is a lymphoepithelial tumor is not appropriately explained by the trichoblastoma hypothesis. Our goal was to further clarify the phenotypic and genotypic features of cutaneous lymphadenoma in a series of 11 cases. Histologically, a lobular architecture surrounded by a dense fibrous stroma was present in all cases. The lobules were composed of epithelial cells admixtured with small lymphocytes and isolated or clustered large Reed-Sternberg–like (RS-L) cells. The epithelial cells were diffusely positive for the hair follicle stem cell markers CK15, PHLDA1, and for androgen receptor. No immunostaining for markers of sebaceous differentiation was found. Intraepithelial lymphocytes were predominantly CD3+, CD4+, FoxP3+ T cells. RS-L cells showed both strong Jagged-1 and Notch1 cytoplasmic immunostaining. Androgen-regulated NKX3.1 nuclear immunostaining was present in a subset of large intralobular cells in all cases. Double immunostaining showed coexpression of NKX3.1 and CD30 in a subset of RS-L cells. No immunostaining for lymphocytic or epithelial markers was present in RS-L cells. EGFR, PIK3CA, and FGFR3 somatic mutations were found by next-generation sequencing in 56% of the cases. We consider that cutaneous lymphadenoma is a distinct benign lymphoepithelial tumor with androgen receptor and hair follicle bulge stem cell marker expression, RS-L cell-derived Notch1 ligand, and common EGFR gene mutations.

Genetic Ancestry Contributes to Somatic Mutations in Lung Cancers from Admixed Latin American Populations.

Inherited lung cancer risk, particularly in nonsmokers, is poorly understood. Genomic and ancestry analysis of 1,153 lung cancers from Latin America revealed striking associations between Native American ancestry and their somatic landscape, including tumor mutational burden, and specific driver mutations in EGFR, KRAS, and STK11. A local Native American ancestry risk score was more strongly correlated with EGFR mutation frequency compared with global ancestry correlation, suggesting that germline genetics (rather than environmental exposure) underlie these disparities. SIGNIFICANCE: The frequency of somatic EGFR and KRAS mutations in lung cancer varies by ethnicity, but we do not understand why. Our study suggests that the variation in EGFR and KRAS mutation frequency is associated with genetic ancestry and suggests further studies to identify germline alleles that underpin this association.See related commentary by Gomez et al., p. 534.This article is highlighted in the In This Issue feature, p. 521.

Abstract PS18-07: Association between pathological response and tumor genomic profiling in triple negative breast cancer patients treated with neoadjuvant chemotherapy

Abstract Background: Triple negative breast cancer (TNBC) has a marked molecular diversity that promotes clinical heterogeneity. Less than 40% of TNBC patients will achieve a pathological complete response (pCR) to standard neoadjuvant chemotherapy. Patients who do not achieve pCR have a high risk of disease recurrence and subsequent death from breast cancer. Molecular characterization may identify TNBC patients unlikely to achieve pCR and subsequently develop recurrent disease. Methods: We are conducting a multicenter prospective study of clinical stages II and III TNBC patients treated with neoadjuvant docetaxel and carboplatin. We performed tumor whole exome sequencing on 56 patients pre-treatment samples to identify somatic mutation associated with pCR. Thirteen matching samples from cycle 1 day 3 (C1D3) were also analyzed to assess changes in somatic mutation profiles. Results: In this biomarker study, thirty-seven (66.1%) patients are Caucasians, 17 (30.4%) African American. Nineteen (33.9%) achieved pCR following six cycles of neoadjuvant docetaxel and carboplatin. 9063 variants were detected in 5386 unique genes. The overall mutation burden for patients who achieved pCR was not significantly different from non-pCR patients (median of 80 variants, IQR 51-135 in pCR, vs median 72, IQR 44-102 in non-pCR, Wilcoxon rank sum test p=0.78). As expected, TP53 is the most frequently mutated gene observed in 48 of all 56 patients (85.7%). There was a non-significant trend with lower TP53 mutations occurring in 78.9% of patients with pCR, versus 89.2% of non-pCR patients (OR 0.46, 95% CI 0.07 - 2.83; p value 0.42). BRCA2 somatic mutations were observed in 5.4% and 5.3% of pCR and non-pCR samples, respectively. No BRCA1 somatic mutations were identified. EGFR, RAD51AP2, SDK2, L1CAM, KPRP, CACNA1S, CFAP58, COL22A1, and COL4A5 were differentially mutated and almost exclusively found in pCR samples. PCDHA1 and TRMT9B were observed in 18.9% and 16.2%, respectively, of non-pCR samples only. There was a trend of higher variant counts in the thirteen matched samples at C1D3 (median of 82, IQR 49-157) versus corresponding pre-treatment samples (median of 72, IQR 42-92), Wilcoxon rank sum test p=0.29, suggesting clone emergence under treatment pressure. Using the Molecular Signatures Database v7.1, several gene families involved in immune related pathways showed differences between pCR and non-pCR samples. Additionally, borderline differences in hedgehog signaling pathway were identified between pCR and non-pCR samples. There were no differences in apoptosis, DNA repair, EMT, inflammatory response, NOTCH signaling pathways. Conclusion: Across TNBC tumors analyzed, TP53 mutation frequency does not differ in pCR versus non-pCR patients. Somatic mutations in EGFR, RAD51AP2, immune pathways genes, and hedgehog signaling pathway genes may predict pCR to docetaxel and carboplatin chemotherapy. Citation Format: Foluso O Ademuyiwa, Jingqin Luo, Bryan Fisk, Gejae Jeffers, Tracy Summa, Isabella Grigsby, Mothaffar Rimawi, Meenakshi Anurag, Matthew Ellis, Obi Griffith, Malachi Griffith. Association between pathological response and tumor genomic profiling in triple negative breast cancer patients treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-07.

Prevalence of the Brazilian TP53 Founder c.1010G>A (p.Arg337His) in Lung Adenocarcinoma: Is Genotyping Warranted in All Brazilian Patients?

In Southern and Southeastern Brazil, there is a germline pathogenic variant with incomplete penetrance located in the oligomerization domain of TP53, c.1010G>A (p.Arg337His). Due to a founder effect, the variant is present in 0.3% of the general population of the region. Recently, this variant was identified in 4.4 and 8.9% of two apparently unselected, single center case series of Brazilian lung adenocarcinoma (LUAD) patients from the Southeastern and Central regions of the country, respectively. In the present study, our aim was to examine TP53 c.1010G>A allele and genotype frequencies in LUAD samples obtained from patients diagnosed in Southern Brazil. A total of 586 LUAD samples (tumor DNA) recruited from multiple centers in the region were tested, and the mutant allele was identified using TaqMan® assays in seven cases (7/586, 1.2%) which were submitted to next generation sequencing analyses for confirmation. Somatic EGFR mutations were more frequent in TP53 c.1010G>A carriers than in non-carriers (57.1 vs. 17.6%, respectively). Further studies are needed to confirm if TP53 c.1010G>A is a driver in LUAD carcinogenesis and to verify if there is a combined effect of EGFR and germline TP53 c.1010G>A. Although variant frequency was higher than observed in the general population, it is less than previously reported in LUAD patients from other Brazilian regions. Additional data, producing regional allele frequency information in larger series of patients and including cost-effectiveness analyses, are necessary to determine if TP53 c.1010G>A screening in all Brazilian LUAD patients is justified.