NFE2L2 Mutations Research Articles

Circulating tumour DNA in patients with hepatocellular carcinoma across tumour stages and treatments

ObjectiveCirculating tumour DNA (ctDNA) is a promising non-invasive biomarker in cancer. We aim to assess the dynamic of ctDNA in patients with hepatocellular carcinoma (HCC).DesignWe analysed 772 plasmas from 173...

Mutant Nrf2E79Q enhances the promotion and progression of a subset of oncogenic Ras keratinocytes and skin tumors

Squamous cell carcinomas (SCCs), including lung, head & neck, bladder, and skin SCCs often display constitutive activation of the KEAP1-NRF2 pathway. Constitutive activation is achieved through multiple mechanisms, including activating mutations in NFE2L2 (NRF2). To determine the functional consequences of Nrf2 activation on skin SCC development, we assessed the effects of mutant Nrf2E79Q expression, one of the most common activating mutations in human SCCs, on tumor promotion and progression in the mouse skin multistage carcinogenesis model using a DMBA-initiation/TPA-promotion protocol where the Hras A->T mutation (Q61L) is the canonical driver mutation. Nrf2E79Q expression was temporally and conditionally activated in the epidermis at two stages of tumor development: 1) after DMBA initiation in the epidermis but before cutaneous tumor development and 2) in pre-existing DMBA-initiated/TPA-promoted squamous papillomas. Expression of Nrf2E79Q in the epidermis after DMBA initiation but before tumor occurrence inhibited the development/promotion of 70% of squamous papillomas. However, the remaining papillomas often displayed non-canonical Hras and Kras mutations and enhanced progression to SCCs compared to control mice expressing wildtype Nrf2. Nrf2E79Q expression in pre-existing tumors caused rapid regression of 60% of papillomas. The remaining papillomas displayed the expected canonical Hras A->T mutation (Q61L) and enhanced progression to SCCs. These results demonstrate that mutant Nrf2E79Q enhances the promotion and progression of a subset of skin tumors and alters the frequency and diversity of oncogenic Ras mutations when expressed early after initiation.

Young adults (YA) with non-small cell lung cancer (NSCLC): Snapshot of the oncogenic drivers and immune landscapes.

8538 Background: Approximately 5% of NSCLC occurs in patients 50 years or younger (median age:71) representing distinct clinicopathological features. Reports characterizing genomic alterations are scarce. Using a large real-world (RW) dataset, we characterize oncogenic drivers, and immune landscapes to better understand YA with NSCLC. Methods: 42,326 NSCLC specimens were analyzed using next-generation sequencing of DNA (DNA-592 panel or whole exome) or RNA (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). YA (<=50 years) were categorized into three groups (years): 18-30 (A1, n=61), 31-40 (A2, n=277) & 41-50 (A3, n=1,549); vs >50 (A4, n=40,439). Composition of tumor microenvironment (TME) was estimated from bulk RNA sequencing using QuanTIseq method. RW survival on Osimertinib (Osi-OS) was obtained from insurance claims and calculated from initiation of Osi treatment to last contact and was compared between ages 18-50 and >50 years. Hazard ratio (HR) was calculated using the Cox proportional hazards model. Statistical significance was determined using Chi-square, Fisher’s Exact, Mann-Whitney U and log-rank tests and corrected for multiple comparisons where applicable (q<0.05). Results: Consistent with previous reports, NSCLC in YA was more prevalent in females, non-smokers and was associated with adenocarcinoma histology. Among driver alterations, ALK(IHC+), ROS1, RET and NTRK1 fusion were enriched and KRAS mutations were reduced in YA. Interestingly, while the prevalence of KRASG12D from transition (non-smoker related) and EGFRE746_A750 decreased with age, KRASG12C from transversion increased with age in YA. YA were also associated with improved Osi-OS (HR: 0.79, median Osi-OS=37.4 months vs 32 months in >50 years old, p=0.019). High tumor mutation burden (>=10 mut/Mb), the prevalence of mutations in TP53, KMT2D, NF1, RBM10, NFE2L2 and NOTCH1 increased while GNAS decreased with age. The expression of immune checkpoint genes such as PDCD1LG2(A1/A4: 0.63, A3/A4: 0.89), LAG3(A2/A4: 0.73, A3/A4: 0.89) and IFNG (A1/A4: 0.32, A3/A4: 0.75) were reduced, while M2 macrophage (A2/A4: 1.2) and neutrophil (A2/A4: 1.2) were increased in YA (all q<0.05). Conclusions: We report an increased prevalence of RET and NTRK1 fusions in YA and key differences in distributions of frequent KRAS and EGFR mutations in YA (vs A4) along with decreased expression of immune related genes in the tumor microenvironment. The implications are under active investigation. [Table: see text]

Dual mTOR1/2 Inhibitor Sapanisertib (FTH-003/TAK-228) in Combination With Weekly Paclitaxel in Patients With Previously Treated Metastatic Urothelial Carcinoma: A Phase II Open-Label Study

BackgroundThe PI3K/AKT/mTOR pathway is frequently altered at genomic level in metastatic urothelial carcinoma (mUC). Since mTOR is the last protein in the PI3K signaling cascade, it may have the largest impact on the pathway and has been a focus of targeted therapies. Sapanisertib (FTH-003/TAK-228) is an oral highly selective mTOR1 and mTOR2 inhibitor. NFE2L2 mutations have been described as predictive biomarkers of response in patients with advanced squamous cell lung cancer treated with sapanisertib. Patients and MethodsThis was an open-label, investigator-initiated phase II study evaluating safety and efficacy of sapanisertib plus paclitaxel in patients with mUC who had progressed to prior platinum therapy, and the correlation with NFE2L2 mutations in responders. Primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS), overall survival (OS) and safety. Patients were treated with weekly paclitaxel at dose of 80 mg/m2 on days 1, 8, and 15 in combination with sapanisertib 4 mg administered orally 3 days per week on days 2-4, 9-11, 16-18, and 23-25 of a 28-day cycle. NFE2L2 mutations were analyzed by Sanger sequencing in responders. Results22 patients were enrolled from May 2018 to April 2020; the trial was halted early due to slow accrual and the COVID-19 pandemic. ORR was 18.2% (n = 4). Disease control rate was 50% (7 SD and 4 PR). Median PFS was 3.4 months (95% CI: 1.8-6.1) and median OS was 6.1 months (95% CI: 1.8-13.4). Adverse events (AE) of grade 3-4 were seen in 86% of patients, but no patients discontinued treatment due to AEs. NFE2L2 mutations were not found in responders. ConclusionsAlthough the primary endpoint was no met, sapanisertib and paclitaxel combination demonstrated clinical activity in a heavily pretreated population of mUC. This trial generates insight for future combination of sapaniserib with immunotherapy and/or antibody drug conjugates.

Abstract 2028: The triterpenoid CDDO-Methyl ester requires Nrf2 to decrease lung tumor burden and to protect against the toxicity of chemotherapy in experimental lung cancer

Abstract The NRF2 cytoprotective pathway, a drug target for many inflammation-related diseases, regulates immune cell function. While the anti-inflammatory nature of NRF2 activation protects healthy cells from malignant transformation, cancer cells can utilize the pathway to promote resistance to anti-cancer drugs. Up to 30% of human lung adenocarcinomas acquire mutations in either NFE2L2 or its negative regulator KEAP1 which result in constitutive activation of the NRF2 pathway. However, despite our knowledge of the important immunomodulatory effects of NRF2, these effects are not well characterized in the context of cancer. With NRF2 activators now approved for clinical use, it is critical to understand the impact of these drugs in cancer. Triterpenoids including CDDO-methyl ester (CDDO-Me, also known as bardoxolone methyl) are potent pharmacological NRF2 activators with demonstrated anti-cancer activity in preclinical models. In an early-stage preclinical model of lung cancer, CDDO-Me significantly decreased tumor burden in a dose- and Nrf2-dependent manner and improved immune signatures within the tumor microenvironment. However, most human lung cancers are not diagnosed until more advanced stages. To test CDDO-Me in an established tumor intervention model, lung tumors were initiated with vinyl carbamate in A/J WT and Nrf2 knockout (KO) mice. Tumors were allowed to develop for 8 weeks post initiation, after which mice were fed either a vehicle control diet or CDDO-Me (50-100 mg/kg of diet) for an additional 8-12 weeks, alone or in combination with carboplatin and paclitaxel (C/P) at 50 mg/kg and 15 mg/kg, respectively, by IP injection every other week. CDDO-Me significantly (p < 0.05) decreased surface lung tumor counts 35-71% in a Nrf2-dependent manner, and C/P significantly (p < 0.001) reduced tumor burden 53-59% regardless of Nrf2 status. The combination of CDDO-Me + C/P reduced tumor burden in WT lungs by 84% (p < 0.05), more than either agent alone. Nrf2 KO mice had an approximately two-fold increase (p < 0.001) of surface tumors compared to WT mice in both studies, regardless of treatment. C/P reduced tumor burden similarly in Nrf2 WT and Nrf2 KO mice. Interestingly, the combination of CDDO-Me + C/P increased T cell infiltration into the lungs of WT mice, but this change was not observed in groups treated with either agent alone. Importantly, CDDO-Me did not decrease the efficacy of C/P but protected WT mice from mortality and weight loss and lowered white and red blood cell counts. These studies suggest that NRF2 activation in advanced lung cancers can still decrease tumor burden by favorably modulating the immune microenvironment and, importantly, complements the anti-tumor efficacy of conventional chemotherapy while decreasing drug toxicity. Citation Format: Jessica Ann Moerland, Karen T. Liby. The triterpenoid CDDO-Methyl ester requires Nrf2 to decrease lung tumor burden and to protect against the toxicity of chemotherapy in experimental lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2028.

Abstract 377: Saturation mutagenesis of KEAP1 to identify pathogenic mutations in lung cancer patients and gain insight into KEAP1 molecular function

Abstract Introduction: KEAP1 and NFE2L2 (NFR2) mutations occur in roughly 20% of non-small cell lung cancer (NSCLC) patients and are associated with resistance to a broad range of therapies including radiotherapy, chemotherapy, targeted therapies, and immunotherapy. In prior work, we demonstrated that not all KEAP1 mutations observed in NSCLC patients are pathogenic. These benign mutations have a wild-type phenotype that does not impart therapy resistance. However, only roughly 100 of ~13,000 (<1%) possible KEAP1 single amino acid substitution mutations have been characterized. Here, we performed saturation mutagenesis of KEAP1 to comprehensively evaluate all possible point mutation phenotypes in vitro. Methods: Saturation mutagenesis was accomplished using the High-throughput Mutagenesis by Integrated TilEs (MITE) method. In brief, the KEAP1 sequence was divided into 21 tiles each containing a 90 bp variable region flanked by a 30 bp constant region. At each codon position, mutations representing the 20 possible natural amino acids and a stop codon were included for a total of 13,076 KEAP1 variants. The tiles were assembled such that each full length KEAP1 construct contained a single desired mutation. The KEAP1 MITE lentivirus library was transduced into H1299-KEAP1NULL cells that were then treated with vehicle or H2O2 for 24 days. The KEAP1 ORF was amplified from gDNA and mutations were quantified by next generation sequencing. Mutation enrichment analysis was used to derive functional annotations. Results: Oxidative stress resulted in a bimodal distribution of KEAP1 mutations. Cells with KEAP1 nonsense mutations were enriched while cells with silent mutations were depleted. Using a threshold of 95% specificity for silent mutations to distinguish benign from pathogenic mutations, 94% of nonsense mutations and 88% of previously characterized pathogenic mutations were identified as pathogenic. While the majority of KEAP1 mutations were benign (64%), analysis of mutations found in TCGA NSCLC patients showed an enrichment of pathogenic mutations (72%). Overlaying mutation calls onto the KEAP1 structure showed enrichment of pathogenic mutations in the BTB homodimerization interface, hydrophobic core of the oxidative stress sensing IVR domain, and NRF2 binding pocket of the Kelch domain. Conclusions: Saturation mutagenesis of KEAP1 generated a comprehensive list of pathogenic KEAP1 single amino acid substitutions. Assignments strongly agreed with biologic function and prior literature. The assessment of KEAP1 mutation phenotypes is important as benign mutations occur in a significant subset of NSCLC patients and are not expected to impart therapy resistance. Our results will allow identification of benign and pathogenic KEAP1 mutations in clinical studies, which will enable testing of personalized therapeutic strategies for patients with KEAP1 mutations. Citation Format: Noah Kastelowitz, Shashank Shrishrimal, Soyeong Jun, Anni M. Zhang, Rui Wang, Ilayda Ilerten, Maximilian Diehn. Saturation mutagenesis of KEAP1 to identify pathogenic mutations in lung cancer patients and gain insight into KEAP1 molecular function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 377.

Evaluation of a real-world clinico-genomics database of patients with upper gastrointestinal (GI) malignancies in Japan.

400 Background: The genomic profile of gastrointestinal (GI) cancers provides essential information for precision medicine, however, there is limited availability of real-world data in Asian populations as they are not well-represented in most publicly accessible clinico-genomic databases. In this retrospective study, we evaluated the clinical and genomic characteristics of upper GI cancer patients in the database from an academia-industry collaborative nationwide cancer genome screening project in Japan. Methods: The Screening Projectfor Individualized Medicine in Japan (J-SCRUM) GI-SCREEN enrolled advanced GI cancer patients from 26 sites before planned or during systemic therapy from 2015 to 2019. Archived formalin-fixed paraffin-embedded tumor tissue was analyzed by next-generation sequencing (NGS) using Oncomine Cancer Research Panel and Oncomine Comprehensive Assay. Patients with gastric (GC), esophageal (EC) and pancreatic (PC) cancers were identified in the database. The frequency of key actionable genomic alterations in each cancer cohort was explored and compared to similar upper GI cohorts in The Cancer Genome Atlas (TCGA), a publicly available genomics database initiated in 2005 and concluded sample collection in 2013 with most samples from White cancer patients. Results: A total of 872 GC (mean age at registration [SD]: 64y [12]; percent males: 65%), 265 EC (64y [9]; 78%) and 495 PC (64y [10]; 60%) patients were identified. The majority (>70%) of samples were obtained from primary sites. Adenocarcinoma was the most common histological type for GC (85%) ad PC (76%), while squamous cell (76%) was the most common among EC patients. In the GC cohort, the prevalence of key actionable genomic alterations in J-SCRUM GI-SCREEN vs TCGA (N = 380) was, TP53 mutation (49% vs 49%), ERBB2 amplification (14% vs 13%), PIK3CA mutation (11% vs 14%), MYC amplification (9% vs 12%) and MET amplification (2% vs 3%). In the EC cohort, the prevalence in J-SCRUM vs TCGA (N = 182) was TP53 mutation (77% vs 87%), NFE2L2 mutation (21% vs 10%), PIK3CA mutation (9% vs 9%) and CDKN2A mutation (8% vs 8%). In the PC cohort, the prevalence in J-SCRUM vs TCGA (N = 150) was KRAS hotspot mutation (90% vs 93%), KRAS G12D (43% vs 41%), KRAS G12V (32% vs27%), TP53 mutation (58% vs 64%), BRCA1/2 deleterious mutation (5% vs 5%), ATM deleterious mutation (6% vs 5%) and MYC amplification (5% vs 5%). Conclusions: As part of a nationwide cancer genome screening project, J-SCRUM GI SCREEN database provides comprehensive genomic information for Asian population (Japan). The prevalence of key actionable gene alterations in each tumor type was highly comparable between J-SCRUM GI-SCREEN and TCGA data.

Pan-cancer analysis of NFE2L2 mutations identifies a subset of lung cancers with distinct genomic and improved immunotherapy outcomes

BackgroundMutations in the KEAP1-NFE2L2 signaling pathway were linked to increased tumorigenesis and aggressiveness. Interestingly, not all hotspot mutations on NFE2L2 were damaging; some even were activating. However, there was conflicting evidence about the association between NFE2L2 mutation and Nrf2-activating mutation and responsiveness to immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) and other multiple cancers.MethodsThe study with the largest sample size (n = 49,533) explored the landscape of NFE2L2 mutations and their impact response/resistance to ICIs using public cohorts. In addition, the in-house WXPH cohort was used to validate the efficacy of immunotherapy in the NFE2L2 mutated patients with NSCLC.ResultsIn two pan-cancer cohorts, Nrf2-activating mutation was associated with higher TMB value compared to wild-type. We identified a significant association between Nrf2-activating mutation and shorter overall survival in pan-cancer patients and NSCLC patients but not in those undergoing ICIs treatment. Similar findings were obtained in cancer patients carrying the NFE2L2 mutation. Furthermore, in NSCLC and other cancer cohorts, patients with NFE2L2 mutation demonstrated more objective responses to ICIs than patients with wild type. Our in-house WXPH cohort further confirmed the efficacy of immunotherapy in the NFE2L2 mutated patients with NSCLC. Lastly, decreased inflammatory signaling pathways and immune-depleted immunological microenvironments were enriched in Nrf2-activating mutation patients with NSCLC.ConclusionsOur study found that patients with Nrf2-activating mutation had improved immunotherapy outcomes than patients with wild type in NSCLC and other tumor cohorts, implying that Nrf2-activating mutation defined a distinct subset of pan-cancers and might have implications as a biomarker for guiding ICI treatment, especially NSCLC.

Comprehensive analyses of the clinicopathological features and genomic mutations of combined hepatocellular-cholangiocarcinoma.

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer that has two different tumor phenotypes in a single tumor nodule. The relationship between genetic mutations and clinicopathological features of cHCC-CCA remains to be elucidated. Whole-exome sequencing analyses were carried out in 13 primary and 2 recurrent cHCC-CCAs. The whole-exome analyses and clinicopathological information were integrated. TP53 was the most frequently mutated gene in this cohort, followed by BAP1, IDH1/2, and NFE2L2 mutations in multiple cases. All tumors with diameters <3cm had TP53 mutations. In contrast, six of seven tumors with diameters ≥3cm did not have TP53 mutations, but all seven tumors had mutations in genes associated with various pathways, including Wnt, RAS/PI3K, and epigenetic modulators. In the signature analysis, the pattern of mutations shown in the TP53 mutation group tended to be more similar to HCC than the TP53 nonmutation group. Mutations in recurrent cHCC-CCA tumors were frequently identical to those in the primary tumor, suggesting that those tumors originated from identical clones of the primary cHCC-CCA tumors. Recurrent and co-occurrent HCC tumors in the same patients with cHCC-CCA had either common or different mutation patterns from the primary cHCC-CCA tumors in each case. The study suggested that there were two subtypes of cHCC-CCA, one involving TP53 mutations in the early stage of the carcinogenic process and the other not involving such mutations. The comparison of the variants between primary and recurrent tumors suggested that cHCC-CCA was derived from an identical clone.

359. NFE2L2 MUTATIONS ARE PREDICTIVE AND PROGNOSTIC OF TREATMENT OUTCOME IN RESECTABLE ESOPHAGEAL SQUAMOUS CELL CARCINOMA PATIENTS

Abstract Background The poor prognosis of resectable esophageal squamous cell carcinoma (ESCC) poses an unmet need to identify genomic early predictive and prognostic biomarkers to improve treatment outcome and risk stratification. Methods Retrospective targeted sequencing was performed on 80 ESCC formalin-fixed, paraffin-embedded (FFPE) tumor specimens from 37 good responders and 43 poor responders receiving neoadjuvant treatment. Somatic mutations and copy number variations (CNVs) were detected in 130 cancer druggable genes with TOMA Os-seq. Kaplan Meier curve and COX regression analysis were used to estimate the risk of relapse and survival. Results Twelve patients (12/80, 15%) with hot spot NFE2L2 mutations occurred with significantly higher frequency in poor responders (p = 0.0003) and in patients with non-complete pathological response (p = 0.007) in resectable ESCC treated by chemo-radiotherapy (CRT). These patients also had poorer prognosis with higher risk of death (HR = 1.942, p = 0.039) and relapse (HR = 1.817, p = 0.063) by COX analysis. The independent prognostic role of hot spot NFE2L2 mutations was validated by the presence of recurrent plasma cell-free (ct)-DNA NFE2L2 mutations in an independent prospective cohort of resectable ESCC patients. Conclusions In summary, our study now demonstrates the clinical usefulness of hot spot NFE2L2 mutations, as a potential predictive and prognostic biomarker associated with pre-surgery CRT responses, and provides novel insights for future therapeutic options targeting the NRF signaling in resectable ESCC treated by neoadjuvant CRT. The novel prognostic role of hot spot NFE2L2 mutations may be useful markers to guide clinicians for treatment decisions in resectable ESCC patients following pre-surgery treatment.

Roles of NRF2 in DNA damage repair.

The transcription factor NF-E2-related factor 2 (NRF2) is a master regulator widely involved in essential cellular functions such as DNA repair. By clarifying the upstream and downstream links of NRF2 to DNA damage repair, we hope that attention will be drawn to the utilization of NRF2 as a target for cancer therapy. Query and summarize relevant literature on the role of NRF2 in direct repair, BER, NER, MMR, HR, and NHEJ in pubmed. Make pictures of Roles of NRF2 in DNA Damage Repair and tables of antioxidant response elements (AREs) of DNA repair genes. Analyze the mutation frequency of NFE2L2 in different types of cancer using cBioPortal online tools. By using TCGA, GTEx and GO databases, analyze the correlation between NFE2L2 mutations and DNA repair systems as well as the degree of changes in DNA repair systems as malignant tumors progress. NRF2 plays roles in maintaining the integrity of the genome by repairing DNA damage, regulating the cell cycle, and acting as an antioxidant. And, it possibly plays roles in double stranded break (DSB) pathway selection following ionizing radiation (IR) damage. Whether pathways such as RNA modification, ncRNA, and protein post-translational modification affect the regulation of NRF2 on DNA repair is still to be determined. The overall mutation frequency of the NFE2L2 gene in esophageal carcinoma, lung cancer, and penile cancer is the highest. Genes (50 of 58) that are negatively correlated with clinical staging are positively correlated with NFE2L2 mutations or NFE2L2 expression levels. NRF2 participates in a variety of DNA repair pathways and plays important roles in maintaining genome stability. NRF2 is a potential target for cancer treatment.

Molecular characterization of nectin-4 in non-small cell lung cancer subtypes.

e21119 Background: Nectin-4 (Nectin cell adhesion molecule-4) is a tumor associated antigen overexpressed in multiple solid tumor types. Studies show that over 60% of NSCLC are nectin-4 overexpressed. As enfortumab vedotin, an antibody-drug conjugate directed against nectin-4, has been FDA approved for advanced urothelial carcinoma, this holds treatment implications. Here, we investigated nectin-4 expression in NSCLC subtypes. Methods: 27,607 NSCLC tumors were tested for whole transcriptome sequencing (WTS, NovaSeq) and NextGen DNA sequencing (592 gene panel or whole exome sequencing, WES) at Caris Life Sciences (Phoenix, AZ). Tumors were analyzed overall in quartiles by nectin-4 expression (Q4-greatest, Q1-least) and further analyzed in the adenocarcinoma (AD) and squamous carcinoma (SQ) cohorts. PD-L1 expression was tested by IHC (Dako 22C3). Immune cell populations were estimated by QuanTISeq. Statistical significance was determined using Chi-square/Fisher-Exact/Mann-Whitney U tests and adjusted for multiple comparisons (q &lt; 0.05). Results: We saw significantly greater expression of nectin-4 in SQ (median TPM- 15.68) versus AD tumors (median TPM - 9.88) (p &lt; 0.05). We saw a greater prevalence of NFE2L2, STK11, ERBB2 mutations and genomic loss of heterozygosity (LOH) but a lower percentage of PD-L1+ tumors in Q4 vs Q1 tumors (all q-value &lt; 0.05, Table 1). mRNA expression of immune checkpoints PDL1, CTLA4, CD80, CD86, HAVCR2, and IDO1 was increased in Q4 tumors (1.1-1.3-fold, all q &lt; 0.05). Q4 tumors had reduced estimates of M1 macrophages, regulatory T cells (Tregs), and CD8+ T cells and increased estimates of neutrophils and NK cells. The association of nectin-4 expression with the immune landscapes of AD and SQ was similar. Q4 tumors in AD and SQ displayed increased expression of the above-mentioned immune checkpoints (q &lt; 0.05). In AD and SQ, estimates of M1 macrophages, Tregs, and CD8+ T cells were reduced, but neutrophils and NK cells were increased in Q4 tumors (q &lt; 0.05). The pattern of molecular alterations was different in AD and SQ. While KEAP1, STK11 and ERBB2 mutations were significantly enriched in AD Q4 tumors, NFE2L2 mutations were significantly enriched in SQ Q4 tumors (Table 1). Prevalence of LOH was higher and PD-L1+ tumors was lower in Q4 tumors in both AD and SQ (Table 1). Conclusions: NFE2L2 alterations in SQ and STK11, KEAP1 mutations in AD, which are associated with poor immunotherapy (IO) response, were enriched in Q4 tumors. This suggests that the immune landscape associated with high nectin-4 expression in NSCLC may be driven by the dysregulated KEAP1-NFE2L2 pathway. Targeted therapy towards nectin-4 may benefit these NSCLC subtypes that do not respond well to IO. [Table: see text]

The impact of single gene testing (SGT) on subsequent comprehensive genomic profiling (CGP) success in community oncology practice for advanced non-small cell lung cancer (NSCLC): Results from a prospective observational reference laboratory testing program.

6506 Background: CGP concurrently tests tumor tissue for guideline-recommended predictive biomarkers for optimal therapy selection and identification of clinical trials in NSCLC patients. A common practice in the community oncology setting is to first order SGT, then consider CGP when SGT results are negative or after treatment failure. Given the limited tissue available and the scope of alterations not tested by SGT, we sought to characterize the effects of prior SGT on subsequent CGP testing success, and therapeutic opportunities identified by CGP beyond SGT for NSCLC patients in the community setting. Methods: Reference laboratory information systems were used to prospectively contact clinicians across 80 community practices who ordered at least 1 SGT for guideline-recommended genomic variants in NSCLC. CGP was offered for their patients prior to SGT or upon receipt of negative SGT results. SGT included individual assays for BRAF, EGFR, KRAS, MET exon 14 skipping mutations; ALK, RET, and ROS1 rearrangements; and PD-L1 IHC . CGP included DNA-seq for mutations, copy number variants in 523 genes including guideline-recommended genomic variants, MSI, and tumor mutational burden; RNA-seq for rearrangements/fusions/splice variants; and PD-L1 IHC. Results: Among a total of 580 NSCLC patients with CGP ordered (May 2021-December 2022), 168 (29%) had ≥1 SGT ordered prior to CGP (median=5). No patients had all SGT performed, with untested cases ranging from 10% for ALK to 90% for MET exon 14. The same FFPE tissue block was used for CGP in 150/168 (89%) of cases with prior negative SGT. Compared to CGP-only cases, CGP for cases with prior negative SGT was canceled twice as often at tissue review (16% vs 7%; p=.001), had higher DNA extraction failures (13% vs 8%; p=.09), and lower DNA sequencing success (70% vs 83%, p&lt;.001). CGP detected guideline-recommended variants in 51% of all cases. Among prior negative SGT cases, frequencies were higher in genes where SGT was performed less often and is less sensitive than CGP RNA-seq ( RET fusions , MET exon 14 skipping). CGP also identified guideline-recommended variants in genes with no SGT offered during the study period ( ERBB2 mutations , NTRK2/3 fusions), as well as variants with emerging evidence for FDA expedited program-designated clinical trial therapies in 28% of patients, including NRG1 fusions and BRAF non-V600E , KEAP1, KRAS non-G12C, NFE2L2, STK11, and TP53 Y220C mutations. Conclusions: Prior negative SGT doubled subsequent CGP test cancellations for NSCLC due to tissue insufficiency and increased CGP DNA extraction failures. SGT practice in the community oncology setting does not meet practice guideline recommendations and negatively impacts the potential benefit of subsequent CGP for NSCLC patients.

Mutational analysis of microsatellite-stable gastrointestinal cancer with high tumour mutational burden: a retrospective cohort study.

Genomic signatures contributing to high tumour mutational burden (TMB-H) independent from mismatch-repair deficiency (dMMR) or microsatellite instability-high (MSI-H) status are not well studied. We aimed to characterise molecular features of microsatellite stable (MSS) TMB-H gastrointestinal tumours. Molecular alterations of 48 606 gastrointestinal tumours from Caris Life Sciences (CARIS) identified with next-generation sequencing were compared among MSS-TMB-H, dMMR/MSI-H, and MSS-TMB-low (L) tumours, using χ2 or Fisher's exact tests. Antitumour immune response within the tumour environment was predicted by analysing the infiltration of immune cells and immune signatures using The Cancer Genome Atlas database. The Kaplan-Meier method and the log-rank test were used to evaluate the impact of gene alterations on the efficacy of immune checkpoint inhibitors in MSS gastrointestinal cancers from the CARIS database, a Memorial Sloan Kettering Cancer Center cohort, and a Peking University Cancer Hospital cohort. MSS-TMB-H was observed in 1600 (3·29%) of 48 606 tumours, dMMR/MSI-H in 2272 (4·67%), and MSS-TMB-L in 44 734 (92·03%). Gene mutations in SMAD2, MTOR, NFE2L2, RB1, KEAP1, TERT, and RASA1 might impair antitumour immune response despite TMB-H, while mutations in 16 other genes (CDC73, CTNNA1, ERBB4, EZH2, JAK2, MAP2K1, MAP2K4, PIK3R1, POLE, PPP2R1A, PPP2R2A, PTPN11, RAF1, RUNX1, STAG2, and XPO1) were related to TMB-H with enhanced antitumour immune response independent of dMMR/MSI-H, constructing a predictive model (modified TMB [mTMB]) for immune checkpoint inhibitor efficacy. Patients with any mutation in the mTMB gene signature, in comparison with patients with mTMB wildtype tumours, showed a superior survival benefit from immune checkpoint inhibitors in MSS gastrointestinal cancers in the CARIS cohort (n=95, median overall survival 18·77 months [95% CI 17·30-20·23] vs 7·03 months [5·73-8·34]; hazard ratio 0·55 [95% CI 0·31-0·99], p=0·044). In addition, copy number amplification in chromosome 11q13 (eg, CCND1, FGF genes) was more prevalent in MSS-TMB-H tumours than in the dMMR/MSI-H or MSS-TMB-L subgroups. Not all mutations related to TMB-H can enhance antitumour immune response. More composite biomarkers should be investigated (eg, mTMB signature) to tailor treatment with immune checkpoint inhibitors. Our data also provide novel insights for the combination of immune checkpoint inhibitors and drugs targeting cyclin D1 or FGFs. US National Cancer Institute, Gloria Borges WunderGlo Foundation, Dhont Family Foundation, Gene Gregg Pancreas Research Fund, San Pedro Peninsula Cancer Guild, Daniel Butler Research Fund, Victoria and Philip Wilson Research Fund, Fong Research Project, Ming Hsieh Research Fund, Shanghai Sailing Program, China National Postdoctoral Program for Innovative Talents, China Postdoctoral Science Foundation, National Natural Science Foundation of China.

NFE2L2 Mutations Enhance Radioresistance in Head and Neck Cancer by Modulating Intratumoral Myeloid Cells.

NFE2L2 mutations are predictive biomarkers of radioresistance in head and neck cancer and confer sensitivity to glutaminase inhibitors to overcome radioresistance.

The higher body mass index is associated with a lower somatic mutation dependency in hepatocellular carcinoma

BackgroundReaching a certain somatic mutation threshold is a prerequisite of neoplastic initiation. High Body Mass Indexes (BMI) have been introduced as an independent risk factor for cancer development. However, there is evidence for lower mutational dependency in obese individuals in some types of cancers. In this study, we conducted a differential mutation analysis among patients with hepatocellular carcinoma (HCC) with different BMI. MethodsMutation annotation files of 158 normal-weights, 112 overweight and 76 obese HCC patients were retrieved from the TCGA-LIHC project using the TCGAbiolinks library [3]. The Ensembl Variant Effect Predictor (VEP) tool was used to further annotate the identified common and exclusive mutations in both BMI categories. The pathway enrichment of the characterized pathogenic mutations in different BMI categories was performed using the KEGG database. ResultHCC patients with normal BMIs showed a significantly higher frequency of pathogenic mutations compared with higher BMI groups. Pathogenic mutations in HCC-associated genes, namely TP53, CTTNB1, ARID2, ARID1A, AXIN1, and NFE2L2 were observed in the three BMI categories. However, they were more frequently targeted in normal-weights. Accordingly, a significantly larger number of mutated genes involved in cancer-associated signaling pathways, including Hepatocellular Carcinoma, PI3K-Akt, MAPK, and p53 were observed in patients with normal body weights compared with the higher BMI groups. In addition to the common targeted pathways among the three BMI groups, cAMP, Wnt, Hippo, and JAK-STAT signaling pathways were shared between normal-weight and overweight patients with HCC., Ras and mTOR signaling pathways were also exclusively targeted in the normal-weight group. ConclusionObese and overweight patients with HCC represent lower mutation dependency to develop HCC. More investigations considering central obesity measures will help to specify the molecular alterations caused by the presence of central fat that may promote tumor progression regardless of BMI.

Integrated analysis of racial disparities in genomic architecture identifies a trans-ancestry prognostic subtype in bladder cancer.

The incidence of bladder cancer and patient survival vary greatly among different populations, but the influence of the associated molecular features and evolutionary processes on its clinical treatment and prognostication remains unknown. Here, we analyze the genomic architectures of 505 bladder cancer patients from Asian/Black/White populations. We identify a previously unknown association between AHNAK mutations and activity of the APOBEC-a mutational signature, the activity of which varied substantially across populations. All significantly mutated genes but only half of arm-level somatic copy number alterations (SCNAs) are enriched with clonal events, indicating large-scale SCNAs as rich sources of bladder cancer clonal diversities. The prevalence of TP53 and ATM clonal mutations as well as the associated burden of SCNAs is significantly higher in Whites/Blacks than in Asians. We identify a trans-ancestry prognostic subtype of bladder cancer characterized by enrichment of non-muscle-invasive patients and muscle-invasive patients with good prognosis, increased CREBBP/FGFR3/HRAS/NFE2L2 mutations, decreased intra-tumor heterogeneity and genome instability, and an activated tumor microenvironment.

Omics-based identification of an NRF2-related auranofin resistance signature in cancer: Insights into drug repurposing

Auranofin is a thioredoxin reductase-1 inhibitor originally approved for the treatment of rheumatoid arthritis. Recently, auranofin has been repurposed as an anticancer drug, with pharmacological activity reported in multiple cancer types. In this study, we characterized transcriptional and genetic alterations associated with auranofin response in cancer. By integrating data from an auranofin cytotoxicity screen with transcriptome profiling of lung cancer cell lines, we identified an auranofin resistance signature comprising 29 genes, most of which are classical targets of the transcription factor NRF2, such as genes involved in glutathione metabolism (GCLC, GSR, SLC7A11) and thioredoxin system (TXN, TXNRD1). Pan-cancer analysis revealed that mutations in NRF2 pathway genes, namely KEAP1 and NFE2L2, are strongly associated with overexpression of the auranofin resistance gene set. By clustering cancer types based on auranofin resistance signature expression, hepatocellular carcinoma, and a subset of non-small cell lung cancer, head-neck squamous cell carcinoma, and esophageal cancer carrying NFE2L2/KEAP1 mutations were predicted resistant, whereas leukemia, lymphoma, and multiple myeloma were predicted sensitive to auranofin. Cell viability assays in a panel of 20 cancer cell lines confirmed the augmented sensitivity of hematological cancers to auranofin; an effect associated with dependence upon glutathione and decreased expression of NRF2 target genes involved in GSH synthesis and recycling (GCLC, GCLM and GSR) in these cancer types. In summary, the omics-based identification of sensitive/resistant cancers and genetic alterations associated with these phenotypes may guide an appropriate repurposing of auranofin in cancer therapy.

267. CURRENT STATUS OF COMPREHENSIVE GENOME PROFILING FOR ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Esophageal squamous cell carcinoma (ESCC) is the dominant histological subtype of esophageal cancer in Japan. However, there are few effective drugs for advanced ESCC patients (pts), and the prognosis of advanced ESCC pts remains poor. In 2019, comprehensive genome profiling (CGP) tests were approved for patients with advanced solid cancers who were refractory or intolerant to standard treatments. However, there were little data on CGP tests for advanced ESCC patients. The subjects were advanced ESCC patients received CGP tests between June 2019 and February 2022 and expected to be refractory or intolerant to standard treatments. This study evaluated characteristics of advanced ESCC patients who received CGP tests and the clinical utility of CGP tests to receive investigational new drugs. Characteristics of 12 patients were follows; median age:61(range: 48–72), male/female:10(83.3%)/2(16.7%)pts, performance status 0/1/2:2(16.7%)/9(75.0%)/1(8.3%)pts, treatment line 2/3/4:3(25.0%)/8(66.7%)/1(8.3%)pts CGP tests FoundationOne CDx/OncoGuideTM NCC Oncopanel System:8(66.7%)/4(33.3%)pts. All pts(100%) and 7pts(58.3%), 5pts(41.7%), 4pts(33.3%), 2pts(16.7%) showed TP53 mutations and CDKN2A/B loss, FGF amplification, NFE2L2 mutations, TMB-high. Seven pts(58.3%) were candidates for investigational new drugs based on CGP, 1 pt(8.3%) received investigational treatment based on CGP (ERBB2 amplification), 1 pt(8.3%) received investigational treatment not based on CGP test. 5pts(41.7%) were ineligible to trials according to deteriorated general condition or prior treatments. In 5pts(41.7%) without druggable gene alteration based on CGP tests, 2pts(16.7%) received investigational treatments not based on CGP. This study clarified the current status of CGP tests for advanced ESCC patients in our hospital. Due to the small population, further investigation was needed to evaluate the utility of CGP for ESCC pts.

APOBEC mutagenesis and selection for NFE2L2 contribute to the origin of lung squamous-cell carcinoma

Lung squamous-cell carcinoma originates as a consequence of oncogenic molecular variants arising from diverse mutagenic processes such as tobacco, defective homologous recombination, aging, and cytidine deamination by APOBEC proteins. Only some of the many variants generated by these processes actually contribute to tumorigenesis. Therefore, molecular investigation of mutagenic processes such as cytidine deamination by APOBEC should also determine whether the mutations produced by these processes contribute substantially to the growth and survival of cancer. Here, we determine the processes that gave rise to mutations of 681 lung squamous-cell carcinomas, and quantify the probability that each mutation was the product of each process. We then calculate the contribution of each mutation to increases in cellular proliferation and survival. We performed in vitro experiments to determine cytidine deamination activity of APOBEC3B against oligonucleotides corresponding with genomic sequences that give rise to variants of high cancer effect size. The largest APOBEC-related cancer effects are attributable to mutations in PIK3CA and NFE2L2. We demonstrate that APOBEC effectively deaminates NFE2L2 at the locations that confer high cancer effect. Overall, we demonstrate that APOBEC activity can lead to mutations in NFE2L2 that have large contributions to cancer cell growth and survival, and that NFE2L2 is an attractive potential target for therapeutic intervention.