EGFR-mutant Tumors Research Articles

Impact of HER2 expression on EGFR-TKI treatment outcomes in lung tumors harboring EGFR mutations: A HER2-CS study subset analysis

ObjectivesEpidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are standard treatment for EGFR-mutated non-small-cell lung carcinoma (NSCLC); however, a biomarker to predict their efficacy has not been established. Although human epidermal growth factor receptor-2 (HER2) aberrations constitute a potential mechanism for acquired resistance to EGFR-TKIs, the impact of HER2 on EGFR-TKI treatment outcomes has not been systematically evaluated. In this post-hoc subgroup study, we examined the impact of HER2 on the effect of EGFR-TKIs in patients with NSCLC harboring EGFR mutations. Materials and MethodsOf 1126 patients with NSCLC enrolled into a prospective cohort study (HER2-CS study), we analyzed data of 356 (32 %) patients with EGFR-mutant tumors. HER2 protein expression levels were determined by immunohistochemistry (IHC) with the gastric cancer criteria. Patients were divided either to an HER2-P group (HER2-IHC2+/3+) or an HER2-N group (HER2-IHC0/1+). We primarily assessed differences in the time-to-treatment failure (TTF) of EGFR-TKI between the groups. ResultsThe HER2 scoring was as follows: IHC0 (n = 76, 21 %), IHC1+ (n = 199, 56 %), IHC2+ (n = 72, 20 %), and IHC3+ (n = 9, 3 %). The patients’ demographics were similar in the HER2-P and HER2-N groups. The HER2-P group showed a significantly shorter EGFR-TKI TTF than the HER2-N group (hazard ratio [HR]: 1.657, 95 % confidence interval [CI]: 1.076–2.552; median: 13.3 vs. 19.1 months). The magnitude of the negative impact of TTF was especially dependent on performance status (PS). HER2 expression significantly deteriorated the TTF in the subgroup with PS 2 (HR: 5.497, 95 % CI: 1.510–20.02), but not in that with better PS (HR: 1.437, 95 % CI: 0.899–2.298) (pinteraction = 0.015). ConclusionIn the current cohort, HER2 protein expression in EGFR-mutant NSCLC may have a negative impact on the effect of EGFR-TKIs, the effect of which was PS dependent.

Endothelin-1-Mediated Drug Resistance in EGFR-Mutant Non-Small Cell Lung Carcinoma.

Progression on therapy in non-small cell lung carcinoma (NSCLC) is often evaluated radiographically, however, image-based evaluation of said therapies may not distinguish disease progression due to intrinsic tumor drug resistance or inefficient tumor penetration of the drugs. Here we report that the inhibition of mutated EGFR promotes the secretion of a potent vasoconstrictor, endothelin-1 (EDN1), which continues to increase as the cells become resistant with a mesenchymal phenotype. As EDN1 and its receptor (EDNR) is linked to cancer progression, EDNR-antagonists have been evaluated in several clinical trials with disappointing results. These trials were based on a hypothesis that the EDN1-EDNR axis activates the MAPK-ERK signaling pathway that is vital to the cancer cell survival; the trials were not designed to evaluate the impact of tumor-derived EDN1 in modifying tumor microenvironment or contributing to drug resistance. Ectopic overexpression of EDN1 in cells with mutated EGFR resulted in poor drug delivery and retarded growth in vivo but not in vitro. Intratumoral injection of recombinant EDN significantly reduced blood flow and subsequent gefitinib accumulation in xenografted EGFR-mutant tumors. Furthermore, depletion of EDN1 or the use of endothelin receptor inhibitors bosentan and ambrisentan improved drug penetration into tumors and restored blood flow in tumor-associated vasculature. Correlatively, these results describe a simplistic endogenous yet previously unrealized resistance mechanism inherent to a subset of EGFR-mutant NSCLC to attenuate tyrosine kinase inhibitor delivery to the tumors by limiting drug-carrying blood flow and the drug concentration in tumors. SIGNIFICANCE: EDNR antagonists can be repurposed to improve drug delivery in VEGFA-secreting tumors, which normally respond to TKI treatment by secreting EDN1, promoting vasoconstriction, and limiting blood and drug delivery.

Marked synergy by vertical inhibition of EGFR signaling in NSCLC spheroids shows SOS1 is a therapeutic target in EGFR-mutated cancer.

Drug treatment of 3D cancer spheroids more accurately reflects in vivo therapeutic responses compared to adherent culture studies. In EGFR-mutated lung adenocarcinoma, EGFR-TKIs show enhanced efficacy in spheroid cultures. Simultaneous inhibition of multiple parallel RTKs further enhances EGFR-TKI effectiveness. We show that the common RTK signaling intermediate SOS1 was required for 3D spheroid growth of EGFR-mutated NSCLC cells. Using two distinct measures of pharmacologic synergy, we demonstrated that SOS1 inhibition strongly synergized with EGFR-TKI treatment only in 3D spheroid cultures. Combined EGFR- and SOS1-inhibition markedly inhibited Raf/MEK/ERK and PI3K/AKT signaling. Finally, broad assessment of the pharmacologic landscape of drug-drug interactions downstream of mutated EGFR revealed synergy when combining an EGFR-TKI with inhibitors of proximal signaling intermediates SOS1 and SHP2, but not inhibitors of downstream RAS effector pathways. These data indicate that vertical inhibition of proximal EGFR signaling should be pursued as a potential therapy to treat EGFR-mutated tumors.

Circulating Proteoglycan Endocan Mediates EGFR-Driven Progression of Non-Small Cell Lung Cancer.

Although new generations of EGFR-tyrosine kinase inhibitors (EGFR-TKI) have been developed for the treatment of patients with non-small cell lung cancer (NSCLC) with EGFR-mutant tumors, TKI resistance often returns as a result of additional EGFR mutations. In addition to seeking for next-generation EGFR-TKI, developing novel EGFR-targeting strategies may hold the key to overcome the vicious cycle of TKI resistance. Endocan is known as a receptor tyrosine kinase ligand enhancer in tumorigenesis, but the impact of endocan on EGFR-driven NSCLC progression remains unknown. In this study, higher endocan levels were found in lung tumors compared with cancer-free tissues and correlated with poor prognosis in patients with NSCLC harboring mutant EGFR; circulating endocan levels were also significantly higher in patients with mutant EGFR. Endocan facilitated EGFR signaling via direct binding and enhancing of the EGF-EGFR interaction and supported the growth of tumors driven by mutated EGFR. Activated EGFR in turn upregulated expression of endocan via JAK/STAT3 and ERK/ELK cascades, thus forming a positive regulatory loop of endocan-EGFR signaling. On the basis of the binding region between endocan and EGFR, we designed therapeutic peptides and demonstrated promising therapeutic effects in xenografts harboring EGFR mutations including TKI-resistant T790M. Together, our findings highlight the novel interaction between endocan and EGFR and new opportunities to effectively target endocan-EGFR regulatory axis in patients with TKI-resistant NSCLC. SIGNIFICANCE: Endocan is a novel and critical regulator of EGF/EGFR signaling and serves as an alternative target of EGFR-TKI resistance in NSCLC.

Abstract 4981: Comprehensive analysis of immune phenotypes identifies CD73+ CD163high macrophages as a prognostic biomarker in lung adenocarcinoma

Abstract To better understand the immune profile within the tumor microenvironment (TME) and the potential immune-evasion capacity that may arise from administration of immune checkpoint inhibitors, we investigated immunological phenotypes and their correlations with genomic alterations in order to provide predictive/prognostic implications in lung adenocarcinoma (LAC).Surgical sections with corresponding peripheral blood mononuclear cells were collected from 76 patients with pathologically confirmed Ia-IIIa. The histological contents expressing immune checkpoint molecules in the tumor nest and stromal area were imaged and quantified using a multi-spectral imaging system. Distribution of tumor mutation burden (TMB) and non-synonymous somatic mutations were analyzed in whole exome sequencing (WES) data.Total 76 patients consisted of EGFR-mutant (n=37) and EGFR-wild type (n=39) tumors. As is well known, the number of tumor-infiltrating cytotoxic T cells (174.7 ± 24.4 cells/mm2) and TMB (3.8 ± 0.4) in EGFR-mutant tumors was significantly lower than those of EGFR wild type tumors (394.2 ± 82.3 cells/mm2 and 6.7 ± 1.2) (p=0.0146 and p=0.0254, respectively), elucidating EGFR mutation-mediated immune evasion. The subjects carrying EGFR wild-type were classified into two subgroups by the density of tumor-infiltrating cytotoxic T cells, defined as the high TIL (n=20) and low TIL (n=19). There was great difference of mean density of cytotoxic T cells between low TIL group (129.6 ± 16.8 cells/mm2) and high TIL group (672.8±143.5 cells/mm2). Tumor-infiltrating T cells were granzyme B-positive, suggesting most cells were tumor-specific. Interestingly, no significant difference in TMB was found between the two subgroups (7.5 ± 1.9 in low TIL group vs. 5.8±1.3 in high TIL group; p=0.4642). Tumor proportion score (TPS) of PD-L1 was remarkably higher in high TIL group than low TIL group (29.7 ± 6.5% vs. 13.9 ± 2.8%, p=0.0008, respectively), but no significant differences of tumoral CD73 and IDO between the two groups (p=0.643 and p=0.923, respectively). Even there were no significant differences in counts of tumor-infiltrating macrophages in all types of macrophage, significantly higher counts of PD-L1+ M1-type macrophages (CD68+ CD163low) in the stromal area were discovered in the high TIL group compared to those of the low TIL group (396.2 ± 93.6 vs. 139.8 ± 30.1, p=0.0114, respectively), indicating that most of macrophages resided in the stromal area. Interestingly, the low TIL group exhibited higher counts of CD73+ M2-type macrophages (CD68+ CD163high) in the stromal area than the high TIL group (644.5 ± 110.9 vs. 387.9 ± 73.2, p=0.0639, respectively). Cell count ratio (M1/M2) of macrophages was significantly higher in the high TIL group (p=0.0042), suggesting that the TME with a smaller number of TILs tends to contain more M2-type macrophages. The tumor recurrence was more frequent in the low TIL group compared to the high TIL group with no statistical significance (p=0.127). In surgically resected lung adenocarcinoma patients, the subgroup with a low density of tumor-infiltrating cytotoxic T cells had a large number of CD73+ M2-type macrophages, which may contribute to poor prognosis. Acknowledgement: This study was sponsored by Ono Pharma Co., Ltd Citation Format: Jae-Hwan Kim, Tae-Min Kim, Sung-Gu Her, Kyoung-Ho Pyo, Jeong- Oh Kim, Beung-Chul Ahn, Byoung Chul Cho, Jin Hyoung Kang. Comprehensive analysis of immune phenotypes identifies CD73+ CD163high macrophages as a prognostic biomarker in lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4981.

Abstract 911: Anti-PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR mutant NSCLC

Abstract It has been reported that NSCLC patients with EGFR mutation do not respond to anti-PD-1(L1) therapies as well as patients without the mutation. We have previously shown that EGFR mutant tumors express more CD73 compared to EGFR wildtype tumors and we hypothesized that this may contribute to the resistance to anti-tumor immunity and anti-PD-1(L1) therapies. To understand how CD73 expression in EGFR mutant tumors may confer immunosuppression, we compared EGFR mutant cell lines (PC9, HCC2935) and wildtype cell lines (H1666, H1355) in in vitro T cell killing assays. Furthermore, we investigated the effect of anti-CD73 antibody, either as a monotherapy or in combination with anti-PD-L1 antibody, in promoting tumor growth inhibition (TGI) using a xenograft mouse model. In this model, NSG mice were implanted with MART1-expressing PC9 cells and treated with MART1-specific CD8+ T cells together with anti-PD-L1, anti-CD73 or combination. We showed that CD73 expression is regulated by EGFR signaling in NSCLC, possibly through activation of Protein Kinase C (PKC). EGFR mutant cell lines were significantly more resistant to T cells killing compared to wildtype cell lines in vitro, by suppressing T cell proliferation and function as indicated by Ki-67 and granzyme B expression. In the xenograft mouse model using MART1-expressing PC9 cells, anti-PD-L1 did not promote TGI compared to isotype control, which closely mimicked the limited therapeutic effect of anti-PD-1(L1) antibodies on EGFR mutant NSCLC. Although anti-CD73 alone did not reduce tumor size, mice treated with anti-CD73 and anti-PD-L1 combination therapy had significantly smaller tumors compared to mice treated with isotype control (~38%, p<0.0001), anti-PD-L1 alone (~32%, p<0.0001) or anti-CD73 alone (~32%, p<0.01). The combination therapy increased the number of MART1-specific CD8+ T cells in the tumor compared to isotype control (~280%, p<0.01), anti-PD-L1 alone (~556%, p<0.001) and anti-CD73 alone (~270%, p<0.01). The combination therapy also increased the frequency of CD62L+CD45RO+CCR7+ phenotype in CD8+ T cells in the spleen compared to isotype control (~120%, p<0.001), but not in the tumor, as well as enhancing IFNγ production by tumor antigen-specific CD8+ T cells compared to isotype control (~224%, p<0.001). Together, these results further support the ongoing clinical trials evaluating combination of anti-CD73 and anti-PD-L1 in treating EGFR mutant NSCLC while suggesting the increased central memory T cells may play a role in response to therapy. Citation Format: Eric Tu, Kelly McGlinchey, Yelena Lazdun, James Kurasawa, Susan Wilson, Leslie Wetzel, Karen Coffman, Zachary Cooper, Katie Streicher. Anti-PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR mutant NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 911.

Abstract 2333: Mutational landscape of multiple primary lung cancers and its correlation with environmental factors

Abstract Background: Multiple primary lung cancers (MPLC) are reported to occur in 0.2-20 % of all primary lung cancer cases. MPLC are considered to harbor various genetic profiling patterns among the tumors even within the same individuals, though they developed from the same occupational and environmental exposure. In this study, we performed paired mutational analyses to clarify genetic mutations in MPLC and its relationship with environmental factors. Materials and methods: We obtained 38 surgical specimens from 17 patients who were pathologically diagnosed as MPLC and underwent surgery at Osaka City University Hospital between October 2007 and March 2019. In addition to the clinicopathological features, the patients' age, sex, smoking history, body mass index, radiologic features and surgical procedures were reviewed. Four of the 17 MPLC patients had metachronous lesions, whereas 13 patients only had synchronous lesions. For genetic profiling of MPLC patients, the extracted DNAs were deep-sequenced on Ion S5 sequencer (Thermo Fisher) for somatic mutations in 409 cancer-associated genes (Ion AmpliSeq Comprehensive Cancer Panel). The study was approved by institutional review board of Osaka City University Hospital and written informed consent was obtained from all of the patients. For statistically analyses, we assumed genetic mutations occurred by chance and calculated the frequency of concomitant mutations in multiple tumors within the same individuals based on a previous study (JME study; Kawaguchi T et al, J Clin Oncol 2016). Comparing the assumed frequency of mutations with the actual data, statistically analyses whether mutations would occur concomitantly or randomly were performed using an exact test. Results: Deep sequencing was successfully performed in all of the specimens with the mean coverage of 823. Results from comprehensive genetic analyses suggested that the mutation profiles differed among the tumors within the same individuals, whereas EGFR, KRAS, TP53, and PARP1 mutations were concomitantly detected in multiple tumors within the same individuals. In this study, the frequency of EGFR mutations was significantly higher in never or light -smokers and females. The occurrence of concomitant EGFR or KRAS mutations in multiple tumors within the same individuals was significantly more frequent than expected by chance (EGFR, P = .0023; KRAS, P = .0049), suggesting environmental factors play more role in EGFR and KRAS mutations than the other mutations including TP53 which occurred more randomly in individual tumors. Concomitant EGFR or KRAS mutations were particularly prominent in never or light-smokers and males. Conclusions: Results from our MPLC study confirmed that the development of EGFR or KRAS-mutated tumors were strongly related to environmental factors such as sex and smoking history, and suggested that the other mutations may occur randomly. Citation Format: Motohiro Izumi, Jun Oyanagi, Kenji Sawa, Mitsuru Fukui, Koichi Ogawa, Yoshiya Matsumoto, Yoko Tani, Tomohiro Suzumura, Tetsuya Watanabe, Hiroyasu Kaneda, Shigeki Mitsuoka, Kazuhisa Asai, Tatsuo Kimura, Nobuyuki Yamamoto, Tomoya Kawaguchi, Yasuhiro Koh. Mutational landscape of multiple primary lung cancers and its correlation with environmental factors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2333.

Abstract CT216: IMpower150 final analysis: Efficacy of atezolizumab (atezo) + bevacizumab (bev) and chemotherapy in first-line (1L) metastatic nonsquamous (nsq) non-small cell lung cancer (NSCLC) across key subgroups

Abstract Background: The Ph III IMpower150 study (NCT02366143) showed PFS and OS benefit with atezo + bev + carboplatin (carbo) + paclitaxel (pac; ABCP; Arm B) vs bev + carbo + pac (BCP; Arm C) as 1L treatment (tx) for patients (pts) with metastatic nsq NSCLC. Clinical benefit was also seen in pts with EGFR-mutant or ALK-positive (EGFR/ALK+) tumors. We report the final efficacy analyses from IMpower150, including those of the experimental tx arm evaluating atezo + carbo + pac (ACP; Arm A) vs Arm C. Methods: The randomized, open-label, Ph III IMpower150 study evaluated ACP (Arm A) or ABCP (Arm B) vs BCP (Arm C) in pts with metastatic nsq NSCLC (N = 1202). Key eligibility criteria included no prior chemotherapy, measurable disease per RECIST 1.1 and ECOG PS 0-1. Pts were stratified by sex, baseline liver metastases (mets) and PD-L1 expression. Coprimary endpoints were investigator-assessed PFS and OS in ITT wild-type (WT; no EGFR or ALK genetic alterations) pts. Secondary endpoints included PFS and OS in the ITT and PD-L1 expression subgroups. Results: With a minimum follow-up of 32.4 mo (data cutoff: Sep 13, 2019), median OS in ITT-WT pts was 19.0 mo in Arm A vs 14.7 mo in Arm C (HR, 0.84; 95% CI: 0.71, 1.00; Table). OS improvement in the TC1/2/3 or IC1/2/3-WT PD-L1 subgroup was seen in Arm A vs C; no improvement was seen in the TC0 and IC0-WT subgroup. In pts with EGFR/ALK+ tumors and pts with baseline liver mets, OS was comparable in Arms A and C; continued OS benefit in these subgroups was seen in Arm B vs C. The safety profile of each regimen was consistent with previously reported data at the second interim OS analysis (data cutoff: Jan 22, 2018). Arm A vs Arm CArm B vs Arm CaOS HR (95% CI)Median OS, moOS HR (95% CI)Median OS, moITTb (n = 402 [Arm A]; 400 [Arm B]; 400 [Arm C])0.86 (0.73, 1.01)19.0 vs 15.00.80 (0.68, 0.95)19.8 vs 15.0ITT-WTb (n = 350 [Arm A]; 359 [Arm B]; 338 [Arm C])0.84 (0.71, 1.00)19.0 vs 14.70.80 (0.67, 0.95)19.5 vs 14.7TC1/2/3 or IC1/2/3-WT (n = 185 [Arm A]; 192 [Arm B]; 165 [Arm C])0.71 (0.55, 0.91)24.4 vs 16.00.73 (0.57, 0.94)22.5 vs 16.0TC0 and IC0-WT (n = 164 [Arm A]; 167 [Arm B]; 173 [Arm C])0.96 (0.76, 1.22)14.8 vs 14.10.90 (0.71, 1.14)16.9 vs 14.1EGFR/ALK+ (n = 52 [Arm A]; 41 [Arm B]; 62 [Arm C])1.03 (0.67, 1.58)21.2 vs 17.50.77 (0.48, 1.26)25.3 vs 17.5EGFR sensitizing (n = 33 [Arm A]; 26 [Arm B]; 32 [Arm C])1.00 (0.57, 1.74)19.0 vs 18.10.60 (0.31, 1.14)29.4 vs 18.1Liver mets ITT (n = 52 [Arm A]; 52 [Arm B]; 57 [Arm C])1.01 (0.68, 1.51)7.7 vs 9.10.67 (0.45, 1.02)13.2 vs 9.1IC, tumor-infiltrating immune cells; TC, tumor cells. TC1/2/3 or IC1/2/3 = TC ≥ 1% or IC ≥ 1% PD-L1. TC0 and IC0 = TC < 1% and IC < 1% PD-L1.aAnalysis considered final at second OS interim analysis; data shown are for descriptive purposes only.bStratified analysis. Conclusions: At the final OS analysis, IMpower150 showed numerically but not statistically significant OS improvement in Arm A vs C. Final analysis data from IMpower150 continue to show OS benefit with 1L ABCP in nsq NSCLC, including in key subgroups (sensitizing EGFR mutations, liver mets). Citation Format: Mark A. Socinski, Tony S. Mok, Makoto Nishio, Robert M. Jotte, Federico Cappuzzo, Francisco Orlandi, Daniil Stroyakovskiy, Naoyuki Nogami, Delvys Rodriguez-Abreu, Denis Moro-Sibilot, Christian A. Thomas, Fabrice Barlesi, Gene Finley, Shengchun Kong, Xiaoyan Liu, Anthony Lee, Shelley Coleman, Geetha Shankar, Martin Reck. IMpower150 final analysis: Efficacy of atezolizumab (atezo) + bevacizumab (bev) and chemotherapy in first-line (1L) metastatic nonsquamous (nsq) non-small cell lung cancer (NSCLC) across key subgroups [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT216.

Abstract 3276: Molecular heterogeneity of anti-PD-1/PD-L1 immunotherapy efficacy is correlated with tumor immune microenvironment in non-small cell lung cancer from East-Asian patients

Abstract Background: Anti-PD-1/PD-L1 immunotherapy is widely used to treat patients with advanced non-small-cell lung cancer (NSCLC). Although PD-1/PD-L1 inhibitor efficacy across NSCLC is now better understood, it is largely unknown how the tumor immune microenvironment differs regarding tumor genomics and its impact on prognosis and response to immunotherapy in East-Asian NSCLC patients. Methods: We performed an integrated analysis on publicly available data to explore associations between anti-PD-1/PD-L1 immunotherapy efficacy and classic driver oncogene mutations, including EGFR, KRAS, and ALK fusion mutations, in East-Asian NSCLC patients. Four pooled and clinical cohort analyses were used to correlate driver oncogene mutation status and tumor microenvironment based on PD-L1 and CD8+ tumor-infiltrating lymphocytes (TILs). Immune infiltrating patterns were also established for genomic NSCLC subgroups by CIBERSORT algorithm. Results: Based on East-Asian NSCLC patients, TIDE analysis revealed that for anti-PD-1/PD-L1 immunotherapy, EGFR-mutant and ALK-rearranged tumors may yield inferior responses; however, KRAS-mutant tumors may respond better. Pooled and clinical cohort analyses demonstrated tumor immune microenvironment heterogeneity with oncogenic addiction. Data showed remarkably higher PD-L1- and TIL-positive KRAS-mutant tumors, suggesting KRAS mutations may drive an inflammatory phenotype with adaptive immune resistance. However, the EGFR-mutant or ALK-rearranged groups showed a remarkably higher proportion of PD-L1−/TIL− tumors, suggesting an uninflamed phenotype with immunological ignorance. Notably, similar to triple wild-type NSCLC, EGFR L858R-mutant tumors positively correlated with an inflammatory phenotype, suggesting responsiveness to anti-PD-1/PD-L1 immunotherapy. Furthermore, CIBERSORT algorithm results revealed that apart from enriched CD8+ T cells, KRAS-mutant NSCLC showed a similar intratumoral immune profile to triple wild-type NSCLC. Strikingly, EGFR-mutant and ALK-rearranged tumors were characterized by enriched resting memory CD4+ T cells, as well as a lack of CD8+ and activated memory CD4+ T cells. Conclusion: Our study highlights the complex relationship between immune heterogeneity and immunotherapeutic response in East-Asian NSCLC patients regarding oncogenic addiction. Importantly, our findings provide support for individualized treatments according to NSCLC molecular subtype. Keywords: NSCLC, East-Asian, oncogene mutations, PD-1/PD-L1 inhibitors, immune landscape Citation Format: Chengming Liu, Runsen Jin, Sufei Zheng, Xinfeng Wang, Nan Sun, Jie He. Molecular heterogeneity of anti-PD-1/PD-L1 immunotherapy efficacy is correlated with tumor immune microenvironment in non-small cell lung cancer from East-Asian patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3276.

Abstract 1943: SHP2 inhibition as the backbone of targeted therapy combinations for the treatment of cancers driven by oncogenic mutations in the RAS pathway

Abstract Direct targeting of oncogenic mutations in the RAS pathway, such as mutant EGFR, BRAFV600E, and most recently KRASG12C, has emerged as a beneficial therapeutic strategy for patients with cancers bearing these mutations. Mutant-selective inhibitors offer the advantage of a wide therapeutic window but are ultimately limited by the emergence of drug resistance. While a variety of resistance mechanisms have been described, escape from mutant-selective inhibitors frequently involves activation of wild type signaling nodes, including hyperactivation of receptor tyrosine kinases (RTKs), that lead to robust re-activation of the RAS pathway. SHP2 (PTPN11) is a phosphatase that functions as a convergent node downstream of multiple RTKs to regulate RAS activation. In the context of adaptive resistance to mutant-selective inhibitors, SHP2 inhibition has the potential to suppress oncoprotein-mediated signaling as well as adaptive signaling driving escape from therapy. Here we show, using in vitro and in vivo systems, that SHP2 inhibitors have the potential to become the backbone of targeted therapy combinations for RAS-dependent tumors. RMC-4630, a potent, selective, orally bioavailable allosteric inhibitor of SHP2, enhanced the anti-tumor activity of mutant-selective inhibitors, as exemplified by preclinical combination studies with EGFR mutant or KRASG12C inhibitors. RMC-4630 accelerated the time to, and increased the magnitude of, tumor regressions in osimertinib-sensitive EGFR-mutant tumors and delayed and/or reduced tumor regrowth in mice upon cessation of treatment. RMC-4630 was also effective at inhibiting tumor growth in a PDX model that had become resistant to osimertinib via amplification of c-MET. Lastly, SHP2 inhibition blocked EGFRL858R/T790M/C797S signaling in vitro indicative of activity against on-target osimertinib resistance mechanisms. Combination benefits were also observed preclinically with KRASG12C mutant-selective inhibitors, such as AMG-510. For other oncogenic drivers in the RAS pathway, including KRASG12D and KRASG12V, NF1LOF, KRAS amplifications or BRAFClass3, mutant-selective inhibitors are not currently available. In these contexts, a combination strategy simultaneously targeting nodes both up- and down-stream of the oncoprotein, a strategy we refer to as “oncoprotein clamping,” was able to drive regressions of tumors. In xenograft models bearing these mutations, RMC-4630 or cobimetinib alone dosed sub-MTD inhibited growth but induced few tumor regressions in mice. However, the corresponding combinations markedly increased the number and depth of tumor regressions. Translation of these preclinical findings into clinical benefit could position RMC-4630, an investigational therapeutic agent, as a backbone of targeted therapy combinations for patients bearing cancers with diverse oncogenic mutations in the RAS pathway. Citation Format: Jacqueline A. Smith, Mallika Singh, Robert J. Nichols, Elena S. Koltun, Yu C. Yang, David P. Wildes, Carlos Stahlhut, Dong Lee, Chris J. Schulze, Denise Reyes, Abby Marquez, Grace J. Lee, Shaoling Li, Christophe Marcireau, Laurent Debussche, Mark A. Goldsmith, Zhengping Wang, Adrian L. Gill, Steve M. Kelsey. SHP2 inhibition as the backbone of targeted therapy combinations for the treatment of cancers driven by oncogenic mutations in the RAS pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1943.

Targeting Immunometabolism Mediated by CD73 Pathway in EGFR-Mutated Non-small Cell Lung Cancer: A New Hope for Overcoming Immune Resistance.

Despite the relevant antitumor efficacy of immunotherapy in advanced non-small cell lung cancer (NSCLC), the results in patients whose cancer harbors activating epidermal growth factor receptor (EGFR) mutations are disappointing. The biological mechanisms underlying immune escape and both unresponsiveness and resistance to immunotherapy in EGFR-mutant NSCLC patients have been partially investigated. To this regard, lung cancer immune escape largely involves high amounts of adenosine within the tumor milieu with broad immunosuppressive effects. Indeed, besides immune checkpoint receptors and their ligands, other mechanisms inducing immunosuppression and including adenosine produced by ecto-nucleotidases CD39 and CD73 contribute to lung tumorigenesis and progression. Here, we review the clinical results of immune checkpoint inhibitors in EGFR-mutant NSCLC, focusing on the dynamic immune composition of EGFR-mutant tumor microenvironment. The adenosine pathway-mediated dysregulation of energy metabolism in tumor microenvironment is suggested as a potential mechanism involved in the immune escape process. Finally, we report the strong rationale for planning strategies of combination therapy with immune checkpoints blockade and adenosine signaling inhibition to overcome immune escape and immunotherapy resistance in EGFR-mutated NSCLC.

Diagnostic and Predictive Values of 18F-FDG PET/CT Metabolic Parameters in EGFR-Mutated Advanced Lung Adenocarcinoma.

PurposeThe clinical implications of the metabolic parameters of 18F-fluorodeoxyglucose positron emission tomography-computed tomography (18F-FDG PET/CT) in epidermal growth factor receptor (EGFR)-mutated lung cancer are not fully understood. The aim of this study was to evaluate the diagnostic and prognostic utility of the parameters in EGFR-mutated lung cancer patients.Patients and MethodsWe retrospectively enrolled 134 patients with advanced lung adenocarcinoma (72 EGFR-negative and 62 EGFR-positive). We evaluated the correlation between EGFR mutational status and the maximum standardized uptake value (SUVmax), as well as the associations between treatment outcomes in EGFR-mutated patients and various metabolic parameters of primary tumors. For the best predictive parameters, we calculated the metabolic tumor volume (MTV) and total lesion glycolysis (TLG) using two SUV cutoffs: 1.5 (MTV1.5, TLG1.5) and 2.5 (MTV2.5, TLG2.5).ResultsMean SUVmax was lower for EGFR-mutated tumors compared with EGFR wild-type (6.11 vs 10.41, p < 0.001) tumors. Low SUVmax was significantly associated with positive EGFR mutation (odds ratio = 1.74). Multivariate analysis for survival demonstrated that high MTV1.5, TLG1.5, MTV2.5, and TLG2.5 were independently associated with shorter progression-free survival (PFS) and overall survival (OS), and the highest hazard ratios were found in TLG1.5 (3.26 for PFS and 4.62 for OS).ConclusionSUVmax may be predictive for EGFR mutational status, and MTV and TLG of primary tumors may be promising prognostic parameters; 18F-FDG PET/CT has potential utility for the risk stratification of EGFR-mutated patients treated with targeted therapy.

Compared to plasma, bronchial washing fluid shows higher diagnostic yields for detecting EGFR-TKI sensitizing mutations by ddPCR in lung cancer

BackgroundThe rate of diagnosis of advanced lung adenocarcinoma must be improved. In this study, we compared the detection rates of EGFR-tyrosine kinase inhibitor-sensitizing mutations (mEGFRs) in bronchial washing fluid (BWF) and the plasma of patients with lung adenocarcinoma using the tissue genotype as the standard reference.MethodsPaired blood and BWF specimens were collected from 73 patients with lung cancer. The tumor EGFR mutation status was determined by genotyping of the plasma and BWF samples using droplet digital PCR (ddPCR).ResultsThe study cohort included 26, 10, 10, and 27 patients with stage I, II, III, and IV disease. Of the 73 cases, 35 had a wild-type EGFR, and 19 had the L858R substitution and exon 19 deletion mutations. The areas under the receiver operator characteristic curves for sensitivity vs. specificity of ddPCR were 0.895 [95% confidence interval (CI): 0.822–0.969] for BWF and 0.686 (95% CI: 0.592–0.780) for plasma (p < 0.001). The fractional abundance was higher in BWF of the mEGFR-positive cases than in the plasma (p = 0.004), facilitating easy threshold setting and discrimination between mEGFR-positive and negative cases. When genotyping results obtained using plasma and BWF were compared for early lung cancer (stages I–IIIA), the diagnostic yields were significantly higher for BWF ddPCR, and the same tendency was observed for the advanced stages, suggesting that the BWF data may reflect the genotype status in early-stage patients.ConclusionsThe mEGFR genotyping results obtained using BWF showed a higher diagnostic efficacy than did those obtained using the plasma. Thus, BWF-based genotyping may be a useful substitute for that using plasma in lung cancer.

Abstract B10: Feasibility of bronchial washing fluid-based approach to early-stage lung cancer diagnosis

Abstract Circulating tumor DNA has few applications for detecting early-stage lung cancers. Bronchial washing (BW) is minimally invasive, and BW fluids can contain tumor DNA. Thus, analysis of BW fluids could be used for precision medicine. Forty-eight paired specimens (primary tumor tissue, normal tissue, BW supernatant, and BW precipitate) from twelve patients with early-stage non-small cell lung cancer were analyzed using ultradeep next-generation sequencing with a custom panel containing 113 genes. In primary tumors, 130 missense mutations/indels (5–16 per patient) and 20 driver mutations (0–3) were found; 105 (80.8%) and 97 (74.6%) of these missense mutations/indels were identified in BW supernatants and precipitates, respectively, and 19 (95.0%) of the driver mutations in both BW supernatants and precipitates. Concordance between EGFR mutations in primary tumors, BW supernatants, and BW precipitates was 100%. The median allele frequencies (AFs) for missense mutations/indels in primary tumors, BW supernatants, and BW precipitates were 13.0%, 0.3%, and 0.1%, respectively. The AFs of missense mutations/indels in BW supernatants correlated with those in primary tumors and in BW precipitates (P&amp;lt;0.001). In conclusion, this study provides evidence that the BW fluids, especially supernatants, can be an alternative to overcome the limitation that clinical utility of ctDNA is often not reliable in early-stage NSCLC. Citation Format: Jeong Seon Ryu, Myoung Kyu Lee, Seung Jae Lee. Feasibility of bronchial washing fluid-based approach to early-stage lung cancer diagnosis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B10.

PD-L1 Recruits Phospholipase C and Enhances EGFR Signaling: Relevance to Resistance of EGFR-Mutated Lung Tumors to Immunotherapy

Cancer immunotherapy has focused on inhibitors of checkpoint proteins, such as Programmed Death Ligand 1 (PD-L1). Unlike RAS-mutated lung cancers, EGFR mutant tumors have generally low response to immunotherapy. Because treatment outcomes vary by EGFR allele, we assumed that intrinsic and microenvironmental factors are involved. Among all non-immunological signaling pathways we surveyed in patients’ datasets, EGFR signaling best associated with high PD-L1. Correspondingly, active EGFRs stabilized PD-L1’s transcripts and depleting PD-L1 severely inhibited EGFR-driven tumorigenicity and metastasis in mice. The underlying mechanisms involve physical recruitment of an auto-inhibited phospholipase C-g1 (PLC-g1) to PD-L1, which enhances trans-phosphorylation by EGFR. Once activated, pPLC-g1 stimulates calcium flux, RHO GTPases and protein kinase C, thereby establishing an aggressive mesenchymal phenotype. We show that approved anti-PD-L1 antibodies can inhibit these intrinsic functions of PD-L1. Our results portray PD-L1 as a molecular amplifier of EGFR signaling and lay the foundation for understanding resistance of EGFR mutant tumors to immunotherapy.

Phase II randomized trial of carboplatin + pemetrexed + bevacizumab, +/- atezolizumab in stage IV non-squamous non-small lung cancer (NSCLC) patients who harbor a sensitizing EGFR mutation or have never smoked.

TPS9629 Background: Stage IV NSCLC patients who are never-smokers or with EGFR-mutated tumors generally do not benefit from single-agent immunotherapy. Retrospective subgroup analyses from recent phase III trials suggest that immunotherapy-chemotherapy +/- VEGF inhibition may overcome resistance to PD-L1 inhibitors in these patients, however prospective research on this is needed. This trial will examine a patient population with stage IV non-squamous disease who either have tumors that possess an EGFR exon 19 or 21 mutation or who are never-smoker wild-types, to determine whether the PD-L1 inhibitor atezolizumab in combination with pemetrexed, carboplatin, and bevacizumab can improve outcomes. Methods: This is a randomized, phase II, multi-center, open-label trial to assess pemetrexed/carboplatin and bevacizumab +/- atezolizumab in 117 subjects with stage IV non-squamous NSCLC. Randomization will be 2:1 favoring the + atezolizumab arm. Patients are stratified by EGFR mutation status (i.e. EGFR exon 19 or 21 vs. never-smoker wild-type). Never-smoker wild-type is defined as smoking &lt; 100 cigarettes in a lifetime and without any EGFR mutation or ALK or ROS1 rearrangement. Patients with EGFR exon 19 or 21 mutated tumors must have progression of disease or intolerance of treatment with one or more prior TKIs. Primary endpoint is progression-free survival (PFS). Secondary endpoints include overall survival (OS), overall response rate, duration of response, and time to response. Primary objective is to compare PFS between arms. Secondary objectives include a safety analysis in all treated subjects, and comparisons of PFS and OS between arms for the patient subset with EGFR-mutated tumors. Correlative studies include interrogating flow cytometry-based peripheral blood biomarkers, examining the role of desmoplasia in local tumor immunosuppression, and assessing the contribution of estrogen metabolites to tumorigenesis. This study opened in August 2019 with 2 patients enrolled at the time of submission. Twenty U.S. sites through the NCCN are participating. This study was approved and funded by the National Comprehensive Cancer Network (NCCN) Oncology Research Program from general research support provided by Genentech, Inc. Clinical trial information: NCT03786692 .

First-iGAP: A Randomized Placebo-Controlled Phase II Study of First-line Intercalated Gefitinib and Pemetrexed-Cisplatin Chemotherapy for Never-Smoker Lung Adenocarcinoma Patients

BackgroundWe aimed to evaluate whether intercalated combination of EGFR tyrosine kinase inhibitor gefitinib and chemotherapy improves survival outcomes in never-smokers with advanced lung adenocarcinoma. Patients and MethodsNever-smokers with chemo-naive stage IIIB/IV lung adenocarcinoma were randomly assigned to receive either gefitinib or placebo on days 5 to 18 of a 3-weekly cycle of pemetrexed and cisplatin. Chemotherapy was given up to 9 cycles, after which gefitinib or placebo was given daily. Patients in the placebo arm who had progression were crossed over to receive gefitinib. ResultsBetween June 2012 and December 2014, 76 patients with median age of 58.0 years were randomized, 39 on gefitinib and 37 on the placebo arm. EGFR mutation was positive in 34 (44.7%) patients. Baseline characteristics were well balanced between the 2 arms. The gefitinib arm had a better response rate (79.5% vs. 51.4%, P = .010) and median progression-free survival (PFS) (12.4 vs. 6.7 months, hazard ratio [HR] 0.49, P = .005) than the placebo arm; however, there was no statistically significant difference in median overall survival between the 2 arms (31.8 vs. 22.9 months, HR 0.78, P = .412). The PFS benefit of intercalated use of gefitinib over placebo was more apparent for patients with EGFR-mutant tumors (13.3 vs. 7.8 months, P = .025) than those with EGFR–wild-type tumors (8.2 vs. 6.6 months, P = .063). Overall, there was no difference in the frequency of severe adverse effect between the 2 arms. ConclusionsIntercalated combination of gefitinib with pemetrexed and cisplatin was well tolerated and improved PFS in never-smoker patients with lung adenocarcinoma.

Association of metastatic pattern and molecular status in stage IV non-small cell lung cancer adenocarcinoma.

The aim of our study was to investigate the association between driver oncogene alterations and metastatic patterns on imaging assessment, in a large cohort of metastatic lung adenocarcinoma patients. From January 2010 to May 2017, 550 patients with stage IV lung adenocarcinoma with molecular analysis were studied retrospectively including 135 EGFR-mutated, 81 ALK-rearrangement, 47 BRAF-mutated, 141 KRAS-mutated, and 146 negative tumors for these 4 mutations (4N). After review of the complete imaging report by two radiologists (junior and senior) to identify metastatic sites, univariate correlation analyzes were performed. We found differences in metastatic tropism depending on the molecular alteration type when compared with the non-mutated 4N group: in the EGFR group, pleural metastases were more frequent (32% versus 20%; p = 0.021), and adrenal and node metastases less common (6% versus 23%; p < 0.001 and 11% versus 23%; p = 0.011). In the ALK group, there were more brain and lung metastases (respectively 42% versus 29%; p = 0.043 and 37% versus 24%; p = 0.037). In the BRAF group, pleural and pericardial metastases were more common (respectively 47% versus 20%; p < 0.001 and 11% versus 3%; p = 0.04) and bone metastases were rarer (21% versus 42%; p = 0.011). Lymphangitis was more frequent in EGFR, ALK, and BRAF groups (respectively 6%, 7%, and 15% versus 1%); p = 0.016; p = 0.009; and p < 0.001. The application of these correlations between molecular status and metastatic tropism in clinical practice may lead to earlier and more accurate identification of patients for targeted therapy. • Bone and brain metastasis are the most common organs involved in lung adenocarcinoma but the relative incidence of each metastatic site depends on the molecular alteration. • EGFR-mutated tumors preferentially spread to the pleura and less commonly to adrenals, ALK-rearrangement tumors usually spread to the brain and the lungs, whereas BRAF-mutated tumors are unlikely to spread to bones and have a serous (pericardial ad pleural) tropism. • These correlations could help in the clinical management of patients with metastatic lung adenocarcinoma.

PD-L1 expression and T cells infiltration in patients with uncommon EGFR-mutant non-small cell lung cancer and the response to immunotherapy

ObjectivesThe efficacy of immunotherapy in epidermal growth factor receptor (EGFR)-activating non-small cell lung cancer (NSCLC) is limited. However, a series of patients with uncommon EGFR alterations was reported to derive clinical benefit from PD-1 blockade. To characterize the tumor immune microenvironment, we retrospectively evaluated tumor PD-L1 expression and T cells infiltration among NSCLC patients with uncommon EGFR mutations. Materials and methodsImmunohistochemistry was used to analyze the expression of PD-L1 and the abundance of CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs). Chi-square test and Cox proportional hazards regression were conducted to investigate the correlations between the immune microenvironment features and clinicopathological variables and survival, as well as to explore the potential of immunotherapy in this patient population. ResultsAmong 600 NSCLC patients with EGFR alterations, we identified 49 (8.2 %) bearing uncommon mutations, including G719X, L861Q, S768I, and Ex20 ins. In total, 49.0 % (24/49) of these patients showed positive PD-L1 expression in tumor cells, markedly higher than the proportion in patients with classic sensitive mutations (19del and L858R, 12.2 % [67/551], P < 0.05). Furthermore, PD-L1 expression was associated with relatively short overall survival (OS; 15.2 vs 29.3 months, P = 0.006) and was identified as an independent predictor of OS (hazard ratio=2.57, 95 % confidence interval: 1.03–7.12, P = 0.045). Additionally, PD-L1 positivity was predominantly observed among tumors with CD8+ TILs infiltration (P = 0.001). Uncommon EGFR-mutant tumors had a high frequency (36.7 %) of concurrent PD-L1 expression and abundant CD8 + TILs infiltration. Moreover, this dual-positive group exhibited the most unfavorable prognosis (P = 0.023). Notably, patients with PD-L1 and CD8 dual positivity showed a favorable response to PD-1 inhibitors. ConclusionsHigh rates of concomitant PD-L1 expression and CD8 + TILs within the tumor microenvironment were observed in NSCLC patients with uncommon EGFR mutations. Further investigations are needed to confirm the therapeutic sensitivity to PD-1 blockade in this subgroup.

A20 Estrogen Metabolism in Patients with EGFR-Mutated and ALK-Mutated Non-Small Cell Lung Cancer (NSCLC)

Prior studies have shown that the human lung can extensively metabolize estrogen to reactive catechols. Of greatest concern is 4-hydroxyestrogen (4-OHE), a putative carcinogen, produced by cytochrome P450 1B1 (CYP1B1). In contrast, CYP1A1 metabolizes parent estrogens to 2-hydroxyestrogen (2-OHE), which are less reactive and converted to derivatives that may be antiproliferative. Data from this group strongly suggest that 4-OHE contributes to lung tumorigenesis, though its role in driver-mutated NSCLC has not been investigated. This study assessed estrogen metabolite profiles in EGFR-mutated and ALK-mutated NSCLC patients as compared to cancer-free subjects. Advanced-stage NSCLC patients with tumors that possessed either an EGFR (n = 14) or ALK (n = 8) mutation and cancer-free subjects (n = 17) were recruited from Fox Chase Cancer Center. Tumor mutation status of NSCLC patients was determined by tissue biopsy. All study participants were 50 years of age or older to circumvent any confounding influence of young age or premenopausal status on estrogen levels. NSCLC patients included 14 females and 8 males. Cancer-free subjects serving as controls were never-smoking women. Urine specimens were collected from study participants and urinary estrogen species (E1, E2, E3, 4-OHEs, 2-OHEs, 2-OMEs) were quantified using UPLC-MS/MS. Medians were calculated and the Wilcoxon rank-sum test was used to compare estrogen metabolite measures (4-OHEs/total estrogen, 2-OHE/total estrogen, and the ratio of 4-OHEs/2-OHEs) between NSCLC patients and control subjects. EGFR-mutated NSCLC patients had a significantly higher proportion of 4-OHEs/total estrogen (0.18 vs. 0.05, p-value = 0.048) and a trend towards lower 2-OHEs/total estrogen (0.18 vs. 0.26, p-value = 0.084) as compared to cancer-free control subjects. The ratio of 4-OHEs/2-OHEs was higher in EGFR-mutated NSCLC patients as compared to cancer-free controls (0.90 vs. 0.16, p = 0.053). Differences were not seen between ALK-mutated NSCLC patients and cancer-free subjects for the measures of 4-OHE/total estrogen (0.09 vs. 0.05, p-value = 0.842), 2-OHE/total estrogen (0.20 vs. 0.26, p-value = 0.238), and the ratio of 4-OHEs/2-OHEs (0.34 vs. 0.16, p-value = 0.669). The greater relative level of 4-OHE to 2-OHE in EGFR-mutated NSCLC patients suggests that enhanced production of 4-OHE may contribute to the development of EGFR-mutated lung tumors. Targeting CYP1B1, the enzyme responsible for 4-OHE production, may be of therapeutic interest. Research is ongoing to validate these findings in a larger cohort of EGFR-mutated NSCLC patients.