KEAP1 Mutations Research Articles

Abstract 871: KYNU expression is a prognostic factor in KEAP1/STK11 co-mutated lung adenocarcinoma

Abstract We searched for gene expression prognostic markers with bimodal distribution in lung adenocarcinoma (LUAD) that might help specify molecular subtypes and identified a highly consistent biomarker KYNU, an enzyme in the pathway that converts tryptophan to nicotinamide adenine dinucleotide (NAD+). KYNU performed better in predicting LUAD outcomes than genes in related pathways. High KYNU expression is particularly observed in tumors with mutations in the tumor suppressor genes KEAP1 and STK11, but not in the oncogene KRAS. From clinical data of LUAD, we found more frequent co-mutation of KEAP1 and STK11 than KRAS with either KEAP1 or STK11. Tumors with mutations in both KEAP1 and STK11 had poorer outcomes than tumors with mutations in neither or only one of these genes, regardless of KRAS status. Importantly, KYNU expression associated with poor outcomes regardless of KEAP1/STK11 status and other covariates. We also assessed clinical features associated with KEAP1/STK11 mutations with AACR GENIE data. By examining co-expression of KYNU with other genes, we determined that high KYNU expression had low expression of gene sets related to macrophages. Citation Format: Ling Cai, Thomas J. Rogers, Huiyu Li, Jiyeon Kim, Yang Xie, Guanghua Xiao, John Minna, Ralph J. DeBerardinis. KYNU expression is a prognostic factor in KEAP1/STK11 co-mutated lung adenocarcinoma [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 871.

Abstract 1908: Artificial intelligence-powered spatial analysis of tumor-infiltrating lymphocytes reveals distinct genomic profile of immune excluded phenotype in pan-carcinoma

Abstract Introduction: Immune-excluded phenotype, defined by the existence of tumor-infiltrating lymphocytes (TIL) exclusively confined to cancer-associated stroma (CS) without protruding to tumor nest, has been suggested to be an intrinsic resistance mechanism of immune checkpoint inhibitor. However, little is known about the genomic landscape of immune-excluded phenotype across cancer types. In the current study, we analyzed genomic correlates of immune excluded phenotype in pan-carcinoma, using Lunit SCOPE IO, an artificial intelligence (AI)-powered whole slide image (WSI) software analyzer. Methods: Lunit SCOPE IO has been trained for > 100,000 WSI across > 30 cancer types to detect lymphocyte, cancer epithelium (CE), and CS with > 95% accuracy. WSI was divided into 1 mm2 sized patch and lymphocyte density in CE or its density in CS were calculated to classify immune phenotype as follows: lymphocyte infiltration into CE was considered as inflamed, lymphocyte exclusively enriched in CS as immune-excluded, and sparse lymphocyte infiltration as immune-desert. We analyzed 7,128 The Cancer Genome Atlas (TCGA) pan-carcinoma samples across 20 cancer types excluding those of mesenchymal origin. Results: Lunit SCOPE IO classifies WSI of TCGA pan-carcinoma into three immune phenotypes: inflamed (17.9%), immune-excluded (27.7%), immune-desert (34.2%) and remaining 20.2% of mixed phenotype. Immune-excluded phenotype is highly enriched in lung squamous cell carcinoma (61.5%), lung adenocarcinoma (53.6%), colorectal cancer (51.7%), and pancreatic cancer (42.2%) whereas inflamed phenotype is less dominant in colorectal cancer (3.4%) and pancreatic cancer (3.3%). Tumor mutational burden (TMB) is increased in both inflamed (mean 7.30/Mb) and immune-excluded (7.18/Mb) compared to that in immune-desert (3.28/Mb), however, genomic instability assessed by fraction altered loss-of-heterozygosity (LOH) is only increased in immune-excluded (mean 0.208) compared to that in inflamed (0.143) and immune-desert (0.148, P < 10-16). Consistent with this result, tumors with high TMB (> 10/Mb) and LOH of HLA genes (N = 124) is predominantly enriched by immune-excluded (45.2%), compared to inflamed (23.4%) or immune-desert (8.9%). Interestingly, a majority of oncogenic drivers such as mutations of TP53, KRAS, and KEAP1 are significantly enriched in immune-excluded (fold change > 1.5, P < 10-10), and gene sets associated with epithelial-mesenchymal transition, apical junction, and TGF-beta signaling are enriched in immune-excluded. Conclusion: AI-powered spatial analysis of WSI can classify immune phenotype in pan-carcinoma and it reveals that plausible immune intrinsic resistance pathways including genomic instability, LOH of HLA genes, and alteration of major oncogenic drivers are highly enriched in immune-excluded phenotype. Citation Format: Chan-Young Ock, Changhee Park, Kyunghyun Paeng, Donggeun Yoo, Seokhwi Kim, Sehhoon Park, Se-Hoon Lee, Tony S. Mok, Yung-Jue Bang. Artificial intelligence-powered spatial analysis of tumor-infiltrating lymphocytes reveals distinct genomic profile of immune excluded phenotype in pan-carcinoma [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 1908.

Abstract 102: Clinicopathological and molecular characteristics, treatment patterns, and outcomes in patients with KRAS p.G12C-mutated metastatic non-small cell lung cancer (NSCLC) in the AACR Project GENIE database

Abstract Introduction: Mutations in the RAS family of proto-oncogenes are frequently found in NSCLC, with KRAS being the most prevalent mutated isoform. Of KRAS mutations, the most common is KRAS G12C, representing ~40% of KRAS mutations and occurring in ~13% of all lung adenocarcinoma cases. The objective of this study is to describe clinicopathological and molecular characteristics, treatment patterns, and outcomes in patients with KRAS G12C-mutated metastatic NSCLC using the AACR Project GENIE database. Methods: An observational retrospective study of adult patients (>18 years of age) with KRAS G12C-mutated metastatic NSCLC from three US academic comprehensive cancer centers in the AACR Project GENIE database was undertaken using de-identified data. Patients were eligible if their tumor tested positive for KRAS G12C mutation by next-generation sequencing at the participating institutions. Event-time distributions were estimated using the Kaplan-Meier method, and multivariable models were fitted to estimate adjusted hazard ratios (aHRs) by Cox proportional models. Patients with large panel sequencing performed up to Dec 31, 2018 were included. All available data until Aug 1, 2019 were collected. Results: Of the 416 eligible patients included in this analysis, 91.6% were white, 64.4% were female, median age at metastatic diagnosis was 68.2 years (range: 37.6-86.2), and 97.1% were former or current smokers. 65.1% of patients had stage 4 NSCLC at diagnosis, and most tumors were adenocarcinomas (85.8%). The KRAS G12C mutation was nearly mutually exclusive (≤ 1.0%) with other actionable driver mutations in EGFR, ALK, ROS1, and BRAF but was co-occurred with STK11 (23.6%) and KEAP1 (9.9%) mutations. Among patients who received PD-(L)1 monotherapy (n=110), median progression-free (mPFS) and overall survival (mOS) were 4.6 and 11.9 months, respectively, while among those who received chemotherapy only (n=133) mPFS and mOS were 4.1 and 12.1 months, respectively. When clinical outcomes were analyzed according to co-occurring genomic alterations, we found that KRAS/STK11 co-mutations were associated with significantly worse outcomes with immunotherapy (PFS: aHR 0.44, P=0.002; OS: aHR 0.42, P=0.003) but not with chemotherapy (PFS: aHR 0.82, P=0.44; OS: aHR 0.81, P=0.43). There was no significant impact of co-occurring KRAS mutations with KEAP1, SMARCA4, ATM, RBM10 and TP53 on clinical outcomes to either PD-(L)1 inhibition or chemotherapy. Conclusions: KRAS G12C mutant NSCLC represents a heterogeneous group of lung cancers and its genomic diversity is reflected in differential responses to therapy. These findings highlight the urgent clinical need for molecular biomarker-driven therapies in this subset of patients. Citation Format: Biagio Ricciuti, Chenguang Wang, Hira Rizvi, Jacklynn Egger, Michele LeNoue-Newton, Marilyn Holt, Shivani Aggarwal, Huakang Tu, Hil Hsu, Xuena Wang, Gataree Ngarmchamnanrith, Victoria Chia, Jocelyn Lee, Robert Scharpf, Valsamo Anagnostou, Christine Lovly, Greg Riely, Mark Awad. Clinicopathological and molecular characteristics, treatment patterns, and outcomes in patients with KRAS p.G12C-mutated metastatic non-small cell lung cancer (NSCLC) in the AACR Project GENIE database [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 102.

Abstract 2507: Does a threshold exist for NRF2 hyperactivation to block tumor progression in KRAS mutant, TP53-deficient NSCLC

Abstract Lung cancer is responsible for the most cancer-related deaths worldwide. Within the most prominent histological subtype, non-small cell lung cancer (NSCLC), there is an unmet clinical need: lung adenocarcinomas (ADCs) driven by mutant KRAS. Within this subset of tumors, KRAS mutations co-occur with mutations in tumor suppressor genes including TP53 and the redox regulator KEAP1. KEAP1 is the negative regulator of transcription factor NRF2, which directs the antioxidant response and multiple facets of metabolism. In NSCLC, alterations in the KEAP1-NRF2 circuit result in constitutive NRF2 activation and are often associated with resistance to therapy and poor outcomes in patients. While NRF2 hyperactivation has been associated with tumor progression, our lab's recent findings suggest that this may be context-dependent, and that too much NRF2 activation may be detrimental. To study the role of NRF2 hyperactivation on tumor progression, we have utilized KRAS mutant genetically engineered mouse models of NSCLC harboring TP53 deletion. These studies are based on our lab's finding that the homozygous KEAP1R554Q loss-of-function mutation decreases tumor size in a Kras mutant, Trp53-deficient (KP) lung ADC model (Kang et al. 2019 eLife). In parallel to these studies, we have also developed a conditional murine allele of the NRF2D29H mutation found in human NSCLC to serve as a secondary model of NRF2 hyperactivation in the KP mouse (KPN). Consistent with our homozygous KEAP1 mutant model (KPKK), we found that KPN mice demonstrated constitutive NRF2 activation, as observed by increased immunohistochemical staining of canonical NRF2 target, NQO1. This degree of NRF2 activation in KPN mice was slightly lower than that of KPKK mice, suggesting that the KPN mouse is an intermediate model of NRF2 activation. Supportingly, we also found that KPN mice had decreased tumor burden, although not to the same extent as KPKK mice. Interestingly, our heterozygous KEAP1 mutant model (KPK) demonstrates only modest NRF2 activation but did not exhibit decreased tumor burden. Importantly, analyses of tumor number suggested that KPKK and KPN tumors are impaired in tumor progression, rather than initiation. KPKK and KPN tumors also exhibited lower proliferative indices when compared to KP mice, in correspondence with their reduced tumor burden. Collectively, these results suggest that there may be a threshold for NRF2 activation to block tumor progression in the KP model. Current studies are focused on determining whether this impediment to tumor burden is NRF2-dependent, and what NRF2-dependent mechanisms may impair tumor progression. Importantly, these studies may help identify whether a threshold for NRF2 hyperactivation to promote or block tumor progression exists, and if this can be therapeutically exploited in patients with KRAS mutant, TP53-deficient lung tumors. Citation Format: Janine M. DeBlasi, Aimee Falzone, Gina M. DeNicola. Does a threshold exist for NRF2 hyperactivation to block tumor progression in KRAS mutant, TP53-deficient NSCLC [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 2507.

A Novel Nrf2 Pathway Inhibitor Sensitizes Keap1-Mutant Lung Cancer Cells to Chemotherapy.

The nuclear factor erythroid-2-related factor 2 (Nrf2)-Keap1-ARE pathway, a master regulator of oxidative stress, has emerged as a promising target for cancer therapy. Mutations in NFE2L2, KEAP1, and related genes have been found in many human cancers, especially lung cancer. These mutations lead to constitutive activation of the Nrf2 pathway, which promotes proliferation of cancer cells and their resistance to chemotherapies. Small molecules that inhibit the Nrf2 pathway are needed to arrest tumor growth and overcome chemoresistance in Nrf2-addicted cancers. Here, we identified a novel small molecule, MSU38225, which can suppress Nrf2 pathway activity. MSU38225 downregulates Nrf2 transcriptional activity and decreases the expression of Nrf2 downstream targets, including NQO1, GCLC, GCLM, AKR1C2, and UGT1A6. MSU38225 strikingly decreases the protein level of Nrf2, which can be blocked by the proteasome inhibitor MG132. Ubiquitination of Nrf2 is enhanced following treatment with MSU38225. By inhibiting production of antioxidants, MSU38225 increases the level of reactive oxygen species (ROS) when cells are stimulated with tert-butyl hydroperoxide (tBHP). MSU38225 also inhibits the growth of human lung cancer cells in both two-dimensional cell culture and soft agar. Cancer cells addicted to Nrf2 are more susceptible to MSU38225 for suppression of cell proliferation. MSU38225 also sensitizes human lung cancer cells to chemotherapies both in vitro and in vivo Our results suggest that MSU38225 is a novel Nrf2 pathway inhibitor that could potentially serve as an adjuvant therapy to enhance the response to chemotherapies in patients with lung cancer.

The prognostic impact of KRAS, TP53, STK11 and KEAP1 mutations and the influence of the NLR in NSCLC patients treated with immunotherapy.

e21010 Background: ICIs changed the way NSCLC is treated, but not all patients benefit from it. PD-L1 level is used to predict response to therapy, but its performance is sub-optimal. KRAS is important in NSCLC tumorigenesis, but the impact of its mutations in patients treated with ICIs is unclear. Similarly, studies evaluating co-mutations in TP53, STK11 and KEAP1 as well as the NLR showed that they may predict the benefit of ICIs. Methods: We conducted a retrospective study including all consenting patients with NSCLC treated with ICIs at the CHUM between July 2015 and June 2020. OS and PFS were compared in co-mutation subgroups using Kaplan-Meier and logrank methods. Co-mutations in TP53, STK11 and KEAP1 as well as the NLR were accounted for. Overall response rate (ORR) and safety data was also compared in subgroups and will be detailed at the meeting. Results: We included 100 patients with known KRAS status. From these, 50 were wild-type ( KRASWT) and 50 were mutated ( KRASMut). The most frequent mutation was G12C (54%). Co-mutation status for TP53, STK11 and KEAP1 were known for, respectively, 40, 39 and 38 patients. Co-mutations for these genes were present in respectively 19 (47.5%), 8 (20.5%) and 4 (10.5%). Data comparing KRASMut and KRASWT showed non-significant differences in survival (median OS of respectively 21.1 vs. 17.7 months, p = 0.27). The presence of STK11 and/or KEAP1 mutations was associated with a negative impact on survival when compared with wild-type (median OS 7.4 vs 20.4 months, p = 0.001). When the presence of a KRAS mutation was compounded with STK11 and KEAP1, KRASMut (vs KRASWT) trended to a better prognosis in STK11+KEAP1WT tumors (median OS of 21.1 for KRASMut vs 15.8 for KRASWT, p = 0.15), but not in STK11+/-KEAP1Mut tumors (7.4 for KRASMut vs 7.0 for KRASWT). No influence on survival was seen in relationship to the TP53 co-mutation. Interestingly, the NLR was significantly higher with STK11 mutations (6.66Mut vs 3.59WT, p = 00012), slightly lower with TP53 mutations (3.23Mut vs 4.82WT, p = 0.047) but not impacted by KEAP1 (3.72Mut vs 4.20WT, p = 0.72) or KRAS mutations (4.32Mut vs 5.21WT, p = 0.34). Conclusions: The STK11 and KEAP1 mutations are significant adverse predictors of ICI therapy benefit. The NLR is strongly impacted by STK11 mutations but not by KEAP1 mutations suggesting marked differences in the resistance mechanism for both mutations. In STK11-KEAP1WT tumors, KRAS mutations seems to be associated with improved survival in NSCLC patient treated with ICIs.

KEAP1 mutations in squamous cell lung cancer.

e21098 Background: KEAP1 mutations have been shown to decrease the efficacy of both chemotherapy (CTX) and immune-checkpoint inhibition (ICI) in lung adenocarcinoma. However, few is known about their impact on systemic treatment of squamous cell lung cancer (SqCC). The aim of this study was to assess the impact of KEAP1 mutations on systemic treatment outcome in SqCC. Methods: Tumor biopsies of SqCC patients were analyzed within the German Network Genomic Medicine (NGM) using a next-generation DNA sequencing (NGS) panel comprising 17 genes. In subsets, PD-L1 expression was tested with immunohistochemistry (IHC). MET amplification and FGFR1 amplification was tested with fluorescence in situ hybridization (FISH). Overall survival was estimated using Kaplan Meier statistics. For comparisons, we used log rank. A cohort with KEAP1 wild-type patients from the same panel served as control group. Results: Out of 1399 SqCC patients analyzed, 151 had a KEAP1 mutation (11%). The most common co-occurring mutations besides TP53 were PTEN, KRAS and NFE2L2. The median overall survival (OS) of stage IV KEAP1 mutated patients (n = 82) compared to stage IV control group patients (n = 82) was 7.3 vs. 11.4 months (hazard ratio (HR) 0.87 [95% confidence interval (CI) 0.62-1.23], p = 0.43). The addition of a second treatment line with ICI led to marked OS improvements in both KEAP1 mutant patient group (18.7 vs. 6.6 months, HR 0.11, [95% CI 0.04-0.25], p < 0.0001) and control group (20.3 vs. 5.0 months, HR 0.12 [95% CI 0.06-0.24], p < 0.0001). PD-L1 expression did not differ significantly in both groups. Conclusions: KEAP1 mutations occur commonly in SqCC patients and do not impact the efficacy of ICI in terms of OS. To identify prognostic markers for response to ICI further research is needed.

An investigation on biomarkers of hyperprogressive disease after PD-1/PDL-1 therapies in Chinese non-small cell lung cancer patients.

e21173 Background: Immune checkpoint inhibitors (ICIs), such as PD-1/PD-L1 antibodies, are becoming standard of care for lung cancer treatment. However, low response rates to ICI antibodies and even hyperprogressive disease (HPD) have limited the clinical application of ICIs. Considering HPD for patients means shorter survival times and worse prognoses, valid biomarkers are in urgent demand to predict the occurrence of HPD and the efficacy of ICI. Here, we explored the genomic biomarkers of HPD in Chinese non-small cell lung cancer (NSCLC) patients. Methods: 749 NSCLC patients with both genomic information and PD-L1 expression data were screened from HapLab database. HaploX 605-gene panel sequencing, covering 1.31 MB genome, was performed to analyze the genomic data of patients. PDL-1 expression was detected by immunochemistry. Results: 15 genes related to HPD were detected altered in NSCLC patients. Collectively, 511 out of 749 patients (68.22%) had at least one alteration of HPD related genes. The frequent alterations were EGFR mutation (46.86%), ALK fusion (7.49%), MDM2 amplification (6.54%), KEAP1 mutation (5.34%), STK11 mutation (4.27%), FGF3/4 amplification (4.14%), DNMT3A mutation (2.14%) and MDM4 amplification (2.00%). While in EGFR wild-type and ALK-negative patients, the top 5 frequent alterations were KEAP1 mutation (8.96%), FGF3/4 amplification (7.00%), STK11 mutation (6.16%), PTEN mutation (5.32%) and MDM2 amplification (4.20%). Notably, EGFR mutation (49.27%, 68/138), ALK fusion (9.42%, 13/138) and MDM2 amplification (4.35%, 6/138) were also observed in PDL-1 positive (TPS≥1%) patients. Conclusions: It is urgent to identify specific biomarkers that could predict HPD and to develop effective methods to prevent HPD. Our study investigated the genomic alterations associated with HPD in Chinese NSCLC patients.

Chemo-immunotherapy outcomes of KRAS-G12C mutant lung cancer compared to other molecular subtypes of KRAS-mutant lung cancer.

9088 Background: KRAS mutations are identified in approximately 30% of NSCLC, with G12C mutations being the most common subtype and representing 12% of all non-small cell lung cancer cases. Novel direct inhibitors are in clinical development and have shown promising activity, although the efficacy of these agents compared to other standard therapies for lung cancer is not yet known. We hypothesized that patients with KRAS-G12C mutations may have distinct responses to chemo-immunotherapy regimens both with respect to STK11 and KEAP1 co-mutation status and compared to patients with non-G12C subtypes. Methods: Patients with KRAS-mutant lung cancers at Memorial Sloan Kettering Cancer Center and Dana Farber Cancer Institute treated with chemo-immunotherapy regimens as first line therapy for advanced/metastatic disease were identified. Subset with KRAS G12C mutations non-G12C subtypes were compared and response to therapy was assessed by investigator. Baseline characteristics were compared with the Chi-square and Fisher’s exact test for categorical data and Wilcoxon rank-rum test for continuous data. Response evaluations where performed by investigators and compared between groups with the Fisher’s exact test. Progression free survival and overall survival was calculated from start of therapy to date of progression or death/last follow up, respectively and compared between groups using the Cox proportional-hazards model. Results: We identified 137 patients with KRAS -mutant NSCLC treated with chemo-immunotherapy: 45% (62/137) had mutations in KRAS-G12C and 55% harbored non-G12C mutations (17% G12V, 15% G12D, 4% G12A, 4% G12S, 3% G13D). The median OS was 21 and 14 months for G12C and non-G12C patients, respectively (p = 0.24). ORR to chemo-immunotherapy for patients harboring a KRAS-G12C mutation was 40% (25/62) compared to 31% (23/75) in non-G12C subtypes (p = 0.3). Median PFS was similar for both G12C and non-G12C subtypes (7.3 vs 6.1 months, respectively, p = 0.12). Concurrent STK11 mutation was identified in 40% of patients with KRAS-G12C and KEAP1 alterations were observed in 32% of patients. In patients with KRAS-G12C, co-mutation in STK11 and/or KEAP1 was associated with shorter PFS (15.8 vs 5.1 months, p = 0.01). Conclusions: KRAS-G12C mutations are present in 12% of patients with NSCLC and represent a relevant subtype of NSCLC given KRAS G12C inhibitors now in clinical development. Treatment outcomes to chemo-immunotherapy are similar in patients with G12C and non-G12C subtypes. Outcomes are poor for patients with concurrent STK11 and/or KEAP1 mutations representing a significant unmet need.

Genomic profiling identifies somatic mutations predicting thromboembolic risk in patients with solid tumors

Venous thromboembolism (VTE) associated with cancer (CAT) is a well-described complication of cancer and a leading cause of death in patients with cancer. The purpose of this study was to assess potential associations of molecular signatures with CAT, including tumor-specific mutations and the presence of clonal hematopoiesis. We analyzed deep-coverage targeted DNA-sequencing data of >14 000 solid tumor samples using the Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets platform to identify somatic alterations associated with VTE. End point was defined as the first instance of cancer-associated pulmonary embolism and/or proximal/distal lower extremity deep vein thrombosis. Cause-specific Cox proportional hazards regression was used, adjusting for pertinent clinical covariates. Of 11 695 evaluable individuals, 72% had metastatic disease at time of analysis. Tumor-specific mutations in KRAS (hazard ratio [HR], 1.34; 95% confidence interval (CI), 1.09-1.64; adjusted P = .08), STK11 (HR, 2.12; 95% CI, 1.55-2.89; adjusted P < .001), KEAP1 (HR, 1.84; 95% CI, 1.21-2.79; adjusted P = .07), CTNNB1 (HR, 1.73; 95% CI, 1.15-2.60; adjusted P = .09), CDKN2B (HR, 1.45; 95% CI, 1.13-1.85; adjusted P = .07), and MET (HR, 1.83; 95% CI, 1.15-2.92; adjusted P = .09) were associated with a significantly increased risk of CAT independent of tumor type. Mutations in SETD2 were associated with a decreased risk of CAT (HR, 0.35; 95% CI, 0.16-0.79; adjusted P = .09). The presence of clonal hematopoiesis was not associated with an increased VTE rate. This is the first large-scale analysis to elucidate tumor-specific genomic events associated with CAT. Somatic tumor mutations of STK11, KRAS, CTNNB1, KEAP1, CDKN2B, and MET were associated with an increased risk of VTE in patients with solid tumors. Further analysis is needed to validate these findings and identify additional molecular signatures unique to individual tumor types.

Abstract PO-059: Investigating gene expression profiles associated with clinical radiation resistance in KEAP1/NFE2L2 wildtype lung cancer

Abstract Background: We previously reported that approximately half of local recurrences (LR) after radiotherapy for localized NSCLC harbor mutations in KEAP1 or NFE2L2. Here we sought to explore factors associated with LR after radiotherapy in KEAP1/NFE2L2 wildtype NSCLC. Methods: We identified consecutive patients with stage IA1-IIIC NSCLC treated at our institution with chemoradiotherapy (CRT) or stereotactic ablative radiotherapy (SABR) from 2009-2018 and who had genotyping performed on tumor tissue with full coverage of common recurrent lung cancer driver genes including TP53, KRAS, KEAP1, and NFE2L2. We defined LR as tumor regrowth within the prescription dose radiotherapy volume. Our primary objective was to identify factors associated with LR in KEAP1/NFE2L2WT tumors. We performed RNA-seq on a subset of cases with available tissue using the SMARTer Stranded total RNA-seq Kit v2 (Takara Bio USA, Inc.). Statistical analysis was performed using R (version 3.6) with differential gene expression performed using ‘DESeq2’. All P-values were two-sided and considered significant at P&amp;lt;0.05 with adjustment for multiple hypothesis testing when appropriate. Results: We identified 139 consecutive patients with localized NSCLC who received tumor genotyping and were treated with CRT for stage IIB-IIIC NSCLC (n=58) or SABR for stage I-IIB NSCLC (n=81). 26 (18.7%) of these patients harbored KEAP1/NFE2L2WT tumors. Clinical factors such as tumor volume (P=0.18), histology (P=0.87), and radiation dose (P=0.3) were not associated with LR in this subset. Similarly, somatic mutation analysis did not reveal association of any recurrent driver mutations with LR in these KEAP1/NFE2L2WT tumors, including in TP53 (n=19; P=0.73) or KRAS (n=9; P=0.98). Tissue was available for RNA-seq analysis of 38 KEAP1/NFE2L2WT tumors, of which the majority were adenocarcinomas (n=25, 65.8%) and approximately half each received CRT (n=20, 52.6%) and SABR (n=18, 47.4%). Gene set enrichment analysis revealed a trend for association of LR with expression of hypoxia genes (P=0.07, Q=0.28). Similarly, a previously reported 10-gene radiation sensitivity index (RSI) was not associated with LR (P=0.34). Individual gene analysis identified KRT14 as being significantly less expressed in cases with LR (adjusted P=2.2e-9). In a validation cohort of 24 stage I-IIA KEAP1/NFE2L2WT patients from the TCGA who were treated with radiotherapy, those who died had lower expression of KRT14 than those who did not (P=0.0003). Conclusions: In summary, we identify low expression of KRT14, a squamous cell carcinoma differentiation gene, as a potential biomarker for increased risk of LR after definitive radiotherapy of KEAP1/NFE2L2WT NSCLC. Validation in larger cohorts and biological characterization will be required to determine if this biomarker could be useful for guiding precision radiotherapy approaches. Citation Format: Michael S. Binkley, Young-Jun Jeon, Monica Nesselbush, Everett J. Moding, Barzin Nabet, Diego Almanza, Christopher Yoo, David M. Kurtz, Susie Grant Owen, Leah M. Backhus, Mark F. Berry, Joseph B. Shrager, Kavitha J. Ramchandran, Sukhmani K. Padda, Millie Das, Joel W. Neal, Heather A. Wakelee, Ash A. Alizadeh, Billy W. Loo, Maximilian Diehn. Investigating gene expression profiles associated with clinical radiation resistance in KEAP1/NFE2L2 wildtype lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-059.

The acylfulvene alkylating agent, LP-184, retains nanomolar potency in non-small cell lung cancer carrying otherwise therapy-refractory mutations.

More than 40% of non-small cell lung cancer (NSCLC) patients lack actionable targets and require non-targeted chemotherapeutics. Many become refractory to drugs due to underlying resistance-associated mutations. KEAP1 mutant NSCLCs further activate NRF2 and upregulate its client PTGR1. LP-184, a novel alkylating agent belonging to the acylfulvene class is a prodrug dependent upon PTGR1. We hypothesized that NSCLC with KEAP1 mutations would continue to remain sensitive to LP-184. LP-184 demonstrated highly potent anticancer activity both in primary NSCLC cell lines and in those originating from brain metastases of primary lung cancers. LP-184 activity correlated with PTGR1 transcript levels but was independent of mutations in key oncogenes (KRAS and KEAP1) and tumor suppressors (TP53 and STK11). LP-184 was orders of magnitude more potent in vitro than cisplatin and pemetrexed. Correlative analyses of sensitivity with cell line gene expression patterns indicated that alterations in NRF2, MET, EGFR and BRAF consistently modulated LP-184 sensitivity. These correlations were then extended to TCGA analysis of 517 lung adenocarcinoma patients, out of which 35% showed elevated PTGR1, and 40% of those further displayed statistically significant co-occurrence of KEAP1 mutations. The gene correlates of LP-184 sensitivity allow additional personalization of therapeutic options for future treatment of NSCLC.

Abstract PO-069: Circulating tumor DNA kinetics to identify genomic predictors of rapid response to chemoradiation in non-small cell lung cancer

Abstract Introduction: Despite evidence that a subset of patients with locoregionally advanced non-small cell lung cancer (NSCLC) can be cured with radiation doses less than 60 Gy, there are currently no validated approaches to identify patients that could benefit from radiation dose de-escalation. Normal tissue changes including inflammation and fibrosis can be difficult to distinguish from residual disease on standard imaging during and following chemoradiation therapy (CRT), making assessment of treatment response and identification of favorable responders challenging. We hypothesized that circulating tumor DNA (ctDNA) kinetics during CRT could be used as a surrogate of response to identify genomic predictors of rapid response to treatment. Methods: We applied cancer personalized profiling by deep sequencing (CAPP-Seq) ctDNA analysis to 61 patients treated with CRT for Stage II-III NSCLC. We quantified ctDNA concentrations pre-CRT and a median of 21 days into CRT (mid-CRT) to determine the log-fold change in ctDNA concentration and identify “rapid responders.” The association between ctDNA log-fold change as a continuous variable with progression-free survival (PFS) was analyzed using univariable and multivariable regression, including gender, age, and stage as co-variables. The prevalence of driver gene single nucleotide variants in rapid responders versus slow responders was compared for each gene using Fisher’s exact tests with P-values adjusted using the Benjamini-Hochberg procedure. Results: Mid-CRT ctDNA log-fold change was significantly associated with progression-free survival as a continuous variable on both univariable (P=0.02) and multivariable analysis (P=0.03). Among patients whose ctDNA log-fold change was more negative than -2.15, 10/11 (91%) did not recur within the radiation field. We defined ctDNA rapid responders as the 10 patients with the largest decrease in ctDNA concentration mid-CRT without local progression. Compared with slow responders, ctDNA rapid responders had a trend towards more TP53 mutations (P=0.12), but no driver mutations were significantly enriched in rapid responders. Notably, mutations in common driver genes KEAP1, NFE2L2, KRAS, and EGFR were observed in 36% of slow responders and 0% of rapid responders (P=0.03). Conclusions: Our results suggest that ctDNA kinetics during CRT can identify patients responding favorably to treatment. Additional molecular characterization of ctDNA rapid responders may enable identification of patients who could benefit from treatment de-escalation. Citation Format: Everett J. Moding, Yufei Liu, Angela B. Hui, Jianzhong He, Yawei Qiao, Ting Xu, Luyang Yao, Saumil Gandhi, Zhongxing Liao, Millie Das, Kavitha J. Ramchandran, Sukhmani K. Padda, Joel W. Neal, Heather A. Wakelee, Billy W. Loo, Steven H. Lin, Ash A. Alizadeh, Maximilian Diehn. Circulating tumor DNA kinetics to identify genomic predictors of rapid response to chemoradiation in non-small cell lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-069.

122P STK11 and/or KEAP1 mutations and outcomes in non-small cell lung cancer patients treated with immune checkpoint inhibitors: A systematic literature review

Background: Immune checkpoint inhibitors (ICIs) have recently become a major strategy in treatment of non-small cell lung cancer (NSCLC), but somatic mutations involving the STK11 and KEAP1 genes may elicit resistance to anti-PD(L)1 agents. We conducted a systematic review aiming to determine if these mutations are significantly related to poorer outcomes in NSCLC patients (pts) treated with ICIs.

Mutations Associated with No Durable Clinical Benefit to Immune Checkpoint Blockade in Non-S-Cell Lung Cancer.

Simple SummaryWith the application of immunotherapy in patients with non-small-cell lung cancer (NSCLC), we found that immunotherapy for some patients cannot achieve long-term effects. Therefore, the purpose of this study is to explore the characteristics of these patients and make a model that can effectively predict the prognosis of immunotherapy patients. The results of this study will make it easier for clinicians to screen out NSCLC patients for immunotherapy.(1) Background: The immune checkpoint blockade (ICB) has shown promising efficacy in non-small-cell lung cancer (NSCLC) patients with significant clinical benefits and durable responses, but the overall response rate to ICBs is only 20%. The lack of responsiveness to ICBs is currently a central problem in cancer immunotherapy. (2) Methods: Four public cohorts comprising 2986 patients with NSCLC were included in the study. We screened 158 patients with NSCLC with no durable clinical benefit (NDB) to ICBs in the Rizvi cohort and identified NDB-related gene mutations in these patients using univariate and multivariate Cox regression analyses. Programmed death-ligand 1 (PD-L1) expression, tumor mutation burden (TMB), neoantigen load, tumor-infiltrating lymphocytes, and immune-related gene expression were analyzed for identifying gene mutations. A comprehensive predictive classifier model was also built to evaluate the efficacy of ICB therapy. (3) Results: Mutations in FAT1 and KEAP1 were found to correlate with NDB in patients with NSCLC to ICBs; however, the analysis suggested that only mutation in FAT1 was valuable in predicting the efficacy of ICB therapy, and that mutation in KEAP1 acted as a prognostic but not a predictive biomarker for NSCLC. Mutations in FAT1 were associated with a higher TMB and lower multiple lymphocyte infiltration, including CD8 (T-Cell Surface Glycoprotein CD8)+ T cells. We established a prognostic model according to PD-L1 expression, TMB, smoking status, treatment regimen, treatment type, and FAT1 mutation, which indicated good accuracy by receiver operating characteristic (ROC) analysis (area under the curve (AUC) for 6-months survival: 0.763; AUC for 12-months survival: 0.871). (4) Conclusions: Mutation in FAT1 may be a predictive biomarker in patients with NSCLC who exhibit NDB to ICBs. We proposed an FAT1 mutation-based model for screening more suitable NSCLC patients to receive ICBs that may contribute to individualized immunotherapy.

Efficacy and safety of anti-PD-1-based therapy in combination with PARP inhibitors for patients with advanced solid tumors in a real-world setting

BackgroundRationale exists for combining immune checkpoint inhibitors and PARP inhibitors (PARPi), and results of clinical trials in ovarian cancer are promising, but data in other cancers are limited.MethodEfficacy and safety of PARPi/anti-PD-1 in advanced solid tumors were retrospectively analyzed. The efficacy measures included objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS).ResultsThis retrospective study included data from 40 patients. The ORR was 27.5% (95% CI, 13.0–42.0%), with a DCR of 85.0% (95% CI, 73.4–96.6%). Except four patients in first-line treatment (three with PR and one with SD), the ORR of ≥second-line treatment, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) was 22.2%, 23.1% and 28.6%, and the DCR was 83.3%, 84.6% and 71.4%, separately. The median PFS of all patients, ≥second-line treatment, NSCLC and SCLC was 4.6 m, 4.2 m, 4.5 m and 3.7 m. The median OS was 9.4 m, 11.4 m, 12.7 m and 5.4 m, respectively. Multivariable analysis revealed that BRCA1/2 mutation was positively correlated with ORR (P = 0.008), and LDH≥250U/L was negatively correlated with lowered DCR (P = 0.018), while lymphocyte number, ECOG and LDH significantly influenced both PFS and OS. We found that the possible resistant mechanisms were sarcomatous degeneration and secondary mutation, including BRCA2 truncation mutation, A2M, JAK1,T790M, KEAP1 and mTOR mutation. 37.5% patients had ≥grade 3 adverse events.ConclusionPARPi/anti-PD-1 is an effective and tolerable method for patients with advanced solid tumors, and BRCA1/2 is a potential biomarker.

Landscape of Biomarkers in Non-small Cell Lung Cancer Using Comprehensive Genomic Profiling and PD-L1 Immunohistochemistry.

Comprehensive genomic profiling (CGP) and immunohistochemistry (IHC) are important biomarker tools used for patients with non-small cell lung cancer (NSCLC) given the expanding number of standard-of-care therapies that require companion diagnostic testing. We examined 9450 NSCLC real-world patient samples that underwent both CGP and programmed death-ligand 1 (PD-L1) IHC to understand the biomarker landscape in this patient cohort. By assessing National Comprehensive Cancer Network (NCCN)-recommended biomarkers including genomic alterations, tumor mutational burden (≥10 mutations/Mb cut-off), and PD-L1 expression (Tumor Proportion Score (TPS) ≥ 50% cut-off), we show that CGP + PD-L1 IHC yielded potentially actionable results for 70.5% of the 9,450 patients with NSCLC. Among the remaining 29.5% (2,789/9,450) of patients, 86.7% (2,419/2,789) were potentially eligible for another biomarker-associated therapy and/or clinical trial based on their genomic profile. In addition, in the PD-L1TPS≥50% disease subset, BRAF mutations, MET mutations, MET amplifications, and KRAS mutations were significantly enriched; and in the PD-L1TPS<50%, EGFR mutations, ERBB2 mutations, STK11 mutations, and KEAP1 mutations were enriched. These findings highlight the improved clinical utility of combining CGP with IHC to expand the biomarker-guided therapeutic options available for patients with NSCLC, relative to single biomarker testing alone.

P75.02 Non-Squamous NSCLC Patients Harbored STK11 or KEAP1 Mutation Showed Insensitivity to any Treatment Including Immunotherapy

The objective of this study was mainly to analyze the prognostic effect for patients with non-squamous non-small cell lung cancer (NSCLC) harbored STK11 or KEAP1 mutation received chemotherapy and PD1/PD-L1 inhibitors. The data from OAK and POPLAR clinical trials was firstly applied to analyze the relationship between STK11 or KEAP1 mutation and PD-L1 expression and blood tumor mutation burden (bTMB) for patients with non-squamous NSCLC; Then, the overall survival (OS) difference was compared between the patients who received atezolizumab or docetaxel harbored STK11 or KEAP1 mutation and none. Importantly, the OS difference of patients between STK11 or KEAP1 mutation and none was further confirmed using public database from the cBioPortal platform. Most STK11 (7.33% vs.1.57%) or KEAP1 (10.76% vs. 8.46%) mutation was found in non-squamous NSCLC patients comparing with squamous NSCLC. Interestingly, only 0.96% STK11 mutation and 1.92% KEAP1 mutation occurred in EGFR mutated non-squamous NSCLC patients. Compared with wild-type, higher bTMB were found in patients harbored STK11 or KEAP1 mutation (both, P < 0.001), while PD-L1 expression was higher (25% VS. 14.54%) in KEAP1 mutated patients and lower (7.89% VS. 15.9%) in STK11 mutated patients. Importantly, we found that non-squamous NSCLC patients harbored STK11 or KEAP1 mutations had worse OS no matter treated with atezolizumab monotherapy or docetaxel (all, P < 0.001). Moreover, these mutated patients could not benefit from atezolizumab compared with docetaxel (all, P > 0.05). Similarly, in further analysis of cBioPortal data, we also found that STK11 or KEAP1 mutated non-squamous NSCLC patients who received PD1/PD-L1 inhibitors or other treatment had worse survival than wild-type (all, P < 0.05). Non-squamous NSCLC patients harbored STK11 or KEAP1 mutation were not sensitive to any treatment, suggesting they may be a special subtype.

P83.01 Updated Survival and Biomarker Analysis of Camrelizumab and Apatinib in Previously Treated pts of Advanced Non-Squamous NSCLC

Our previous report showed that camrelizumab combination with apatinib have showed promising results in previously chemotherapy-treated patients with advanced non-squamous NSCLC. We further report the updated survival data and biomarkers analysis here. We conduct a multi-center single-arm phase 1b/II study investigating the safety and efficacy of camrelizumab and apatinib in previously treated patients with advanced NSCLC. This study included phase 1b apatinib dose escalation phase and phase II population expansion cohort. The primary endpoints were safety and ORR respectively. Patients of non-squamous NSCLC who received apatinib 250 mg orally once daily in combination with camrelizumab 200 mg intravenously on day 1 every 2 weeks were included into this analysis (NCT03083041). 22C3 array was used for PD-L1 immunohistochemistry and OseqTM-pan cancer panel (including 636 genes and 1.95Mb) was used for the genomic alternation testing. Between March 21, 2017 and October 11, 2018, 105 patients were enrolled, 91patients had PD-L1 expression testing and 46 had sufficient tissue for NGS. As the cutoff of Aug 15, 2019, one had a confirmed complete response, 28 had confirmed partial response, and 48 had stable disease, ORR was 30.9% (29/94, 95% CI, 21.7-41.2%) in the efficacy-evaluable population (n=94). Median progression-free survival was 5.7 months (95% CI, 4.5–8.8) and median overall survival was 19.2 months (95% CI, 11.2-24.5) in all patients. PD-L1 expression was positive in 25 (27.4%) patients, median TMB is 9 mutations/Mb, while STK11 and KEAP1 mutation were found in 7 and 10 patients respectively. Patients with PD-L1 TPS>1% and high TMB could not predict higher ORR (36.0% vs 22.7%, P = 0.20; 29.2% vs 36.4%, p=0.564, respectively) or longer PFS (median 6.8 vs 5.1 months, P = 0.61; 7.8 vs 8.0 months, P = 0.98). Notably, patients with STK11/KEAP1 mutation had a numerically higher ORR (42.9% vs 28.1%, P =0.327), longer PFS (median 9.4 vs 5.3, P = 0. 592) and statistically significantly longer OS (median NR vs NR, P = 0. 047) than those of wild type. The most common treatment-related adverse events of grade 3 or higher were hypertension (18 [17.1%]), palmar-plantar erythrodysesthesia syndrome (14 [13.3%]), and increased gamma-glutamyltransferase (10 [9.5%]). Combined camrelizumab and apatinib had promising antitumor activity and acceptable safety in previously treated patients with advanced non-squamous NSCLC, especially in these with STK11 or KEAP1 mutation, phase III trial is ongoing for further validation (NCT04203485).

P33.20 Evaluation of Combined Biomarker of Response to Immunotherapy in Patients with Advanced Non-Small Cell Lung Cancer

Immunotherapy revolutionized NSCLC treatment. At present, the only established predictive biomarker for I/O-therapy stratification is PD-L1 expression. However, the expression of PD-L1 is limited by heterogeneous expression and even low expressors harbor patients that respond to I/O therapy. The aim of the study is to evaluate the value of combinations of positive (Tumor Mutational Burden, PD-L1) and negative (a.o. CD73 and VISTA expression, inactivating STK11 and KEAP1 mutations) predictive markers in patients (pts) with advanced NSCLC on I/O-therapy.