Search

Introduction Anti-PD-(L)1 immune checkpoint inhibitors (ICI) improve survival in patients with advanced non-small cell lung cancer (aNSCLC). The clinical features, survival, and burden of toxicities of patients with aNSCLC alive >1 year from ICI initiation are poorly understood. Materials and Methods We defined ICI survivors as patients alive >1 year after ICI start and retrospectively reviewed demographics, treatment, and immune-related adverse events (irAEs). Long-term irAEs were defined as ongoing irAEs lasting >1 year; burden of toxicity measures were based on percentage of days a patient experienced toxicity. Using linear and logistic regression, we evaluated association between demographics and disease characteristics with burden of toxicity. Results We identified 114 ICI survivors from 317 patients with aNSCLC. Half (52%) experienced an irAE of any grade, and 23.7% developed long-term irAEs. More ICI survivors with irAES in the first year had never smoked (P = .018) or received ICIs as frontline therapy (P = .015). The burden of toxicity in the first year significantly correlated with the burden of toxicity afterward (ρ = 0.72; P < .001). No patients with progressive disease had a high burden of toxicity, and they experienced 30.6% fewer days with toxicity than those with stable disease. Increased duration of therapy was associated with higher odds of experiencing toxicity. Half of ICI survivors with irAEs were still receiving treatment for unresolved irAEs at time of death or last follow-up. Conclusion Significant proportions of ICI survivors have unresolved long-term toxicities. These data support a growing need to understand long-term toxicity to optimize management of those treated with ICIs.

Supplementary Tables and Figure Legends_Unmarked

Supplementary Figure 2

Supplementary Figure 1

Purpose:Nintedanib enhances the activity of chemotherapy in metastatic non–small cell lung cancer (NSCLC). In this phase I/II study, we assessed safety and efficacy of nintedanib plus neoadjuvant chemotherapy, using major pathologic response (MPR) as primary endpoint.Patients and Methods:Eligible patients had stage IB (≥4 cm)–IIIA resectable NSCLC. A safety run-in phase was followed by an expansion phase with nintedanib 200 mg orally twice a day (28 days), followed by three cycles of cisplatin (75 mg/m2), docetaxel (75 mg/m2) every 21 days plus nintedanib, followed by surgery. With 33 planned patients, the study had 90% power to detect an MPR increase from 15% to 35%.Results:Twenty-one patients (stages I/II/III, N = 1/8/12) were treated. One of 15 patients treated with nintedanib 200 mg achieved MPR [7%, 95% confidence interval (CI), 0.2%–32%]. Best ORR in 20 evaluable patients was 30% (6/20, 95% CI, 12%–54%). Twelve-month recurrence-free survival and overall survival were 66% (95% CI, 47%–93%) and 91% (95% CI, 79%–100%), respectively. Most frequent treatment-related grade 3–4 toxicities were transaminitis and electrolyte abnormalities. On the basis of an interim analysis the study was discontinued for futility. Higher levels of CD3+ and cytotoxic CD3+CD8+ T cells were found in treated tumors of patients who were alive than in those who died (652.8 vs. 213.4 cells/mm2, P = 0.048; 142.3 vs. 35.6 cells/mm2, P = 0.018).Conclusions:Although tolerated, neoadjuvant nintedanib plus chemotherapy did not increase MPR rate compared with chemotherapy historical controls. Additional studies of the combination in this setting are not recommended. Posttreatment levels of tumor-infiltrating T cells were associated with patient survival. Use of MPR facilitates the rapid evaluation of neoadjuvant therapies.See related commentary by Blakely and McCoach, p. 3499

PURPOSE To compare clinical outcomes in a cohort of patients with advanced non–small-cell lung cancer (NSCLC) with targetable genomic alterations detected using plasma-based circulating tumor DNA (ctDNA) or tumor-based next-generation sequencing (NGS) assays treated with US Food and Drug Administration–approved therapies at a large academic research cancer center. METHODS A retrospective review from our MD Anderson GEMINI database identified 2,224 blood samples sent for ctDNA NGS testing from 1971 consecutive patients with a diagnosis of advanced NSCLC. Clinical, treatment, and outcome information were collected, reviewed, and analyzed. RESULTS Overall, 27% of the ctDNA tests identified at least one targetable mutation and 73% of targetable mutations were EGFR-sensitizing mutations. Among patients treated with first-line epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapies, there were no significant differences in progression-free survival of 379 days and 352 days ( P value = .41) with treatment based on tissue (n = 40) or ctDNA (n = 40), respectively. Additionally, there were no differences in progression-free survival or objective response rate among those with low (n = 8, 0.01%-0.99%) versus high (n = 16, ≥ 1%) levels of ctDNA of the targetable mutation as measured by variant allele frequency (VAF). Overall, there was excellent testing concordance (n = 217 tests) of > 97%, sensitivity of 91.7%, and specificity of 99.7% between blood-based ctDNA NGS and tissue-based NGS assays. CONCLUSION There were no significant differences in clinical outcomes among patients treated with approved EGFR-TKIs whose mutations were identified using either tumor- or plasma-based comprehensive profiling and those with very low VAF as compared with high VAF, supporting the use of plasma-based profiling to guide initial TKI use in patients with metastatic EGFR-mutant NSCLC.

Background: T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an inhibitory immune checkpoint receptor expressed on subsets of T-cells and natural killer (NK) cells. TIGIT inhibits T and NK cell function by binding to CD155 and CD112. SEA-TGT is an investigational human, nonfucosylated monoclonal antibody directed against TIGIT, blocking TIGIT's interaction with CD155 and CD112. SGNTGT-001 (NCT04254107) is a phase 1, open-label, multicenter study of SEA-TGT in patients (pts) with advanced malignancies (solid tumors and lymphomas). In this abstract, we present data from the dose-escalation phase (Part A). Methods: Pts received SEA-TGT ranging from 0.01 to 6.0 mg/kg of body weight intravenously every 3 weeks (Q3W). The primary objectives were to evaluate the safety and tolerability of SEA-TGT and identify the maximum tolerated dose (MTD) or the recommended dose and schedule of SEA-TGT. The optimal biological dose was defined using the Clinical Utility Index. Secondary objectives included the evaluation of antitumor activity, pharmacokinetics (PK) and immunogenicity. Antitumor activity was based on Response Evaluation Criteria in Solid Tumors version 1.1 or the Lugano classification criteria with Lymphoma Response to Immunomodulatory Therapy Criteria. Results: From June 12, 2020, to October 5, 2022, 41 pts were enrolled (2, 4, 5, 11, 12, and 7 pts in the 0.01, 0.1, 0.3, 1, 3, and 6 mg/kg cohorts, respectively). Among the 39 pts who were treated 23 pts had solid tumors and 16 had lymphomas. The median number of prior therapies across all cohorts was 5. Treatment emergent adverse events of any grade were reported in 100% of pts, and treatment-related adverse events (TRAEs) of any grade were reported in 69.2%. TRAEs seen in ≥10% of the pts were infusion-related reaction (38.5%), chills (25.6%), pyrexia (17.9%), fatigue (12.8%), maculopapular rash (12.8%), and rash (10.3%). Seven pts (17.9%) reported TRAEs grade ≥3, and rash (5.1%) was the most frequently reported. No grade 4 or 5 TRAEs were reported. One dose-limiting toxicity, pruritic rash, was observed in 1 pt in the 6 mg/kg cohort, and MTD was not exceeded. Based on the PK and pharmacodynamic data, 1 mg/kg Q3W was selected as the optimal biological dose for the expansion cohorts. One partial response was observed in a pt with gastric cancer, and 2 partial metabolic responses were seen in a pt with Hodgkin’s lymphoma and a pt with diffuse large B-cell lymphoma. Conclusions: Overall, clinical data for SEA-TGT from the SGNTGT-001 trial suggest a manageable and tolerable safety profile. Preliminary antitumor activity warrants further clinical evaluation of SEA-TGT. SGNTGT-001 is ongoing and evaluating monotherapy and combination therapies. Citation Format: Elena Garralda Cabanas, Elisa Fontana, Honey Kumar Oberoi, Nataliya V. Uboha, Ecaterina E. Dumbrava, Emiliano Calvo, Giuseppe Curigliano, Diwakar Davar, Bridget K. Keenan, Vincent K. Lam, Sudhir Manda, Amitkuma Mehta, Anna Minchom, Alison Moskowitz, Ravi Paluri, Vincent Ribrag, Donald Richards, Lillian Siu, Paul Swiecicki, Trisha Wise-Draper, Jasmine Zain, Andres Forero-Torres, Ping Xu, Stephen Ansell. Phase 1 dose-escalation study of SGN-TGT monotherapy in patients with advanced malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT265.

Introduction: There is a critical need to incorporate molecular assessments of minimal residual disease (MRD) during neoadjuvant immunotherapy, in order to identify individuals at high risk for disease recurrence based on analyses of circulating cell-free tumor DNA (ctDNA) landscapes. Here we employed longitudinal liquid biopsies to dynamically assess clinical outcomes with neoadjuvant immuno-chemoradiotherapy in patients with esophageal/gastroesophageal junction (E/GEJ) cancer. Methods: We utilized targeted error-correction sequencing to perform high-depth ctDNA next-generation sequencing for 141 serial plasma and 32 matched white blood cell (WBC) DNA samples from 32 patients with operable stage II/III E/GEJ cancer that received neoadjuvant immune checkpoint blockade (ICB) with chemoradiotherapy prior to surgery (NCT03044613). ctDNA analyses were performed at baseline, post-ICB induction, after completion of chemoradiotherapy (pre-op), and post-operatively (post-op). Using a tumor-agnostic WBC DNA-informed panel NGS approach we determined the cellular origin of plasma variants, filtering out germline and clonal hematopoiesis (CH) variants and evaluated ctDNA clonal dynamics over time. Molecular MRD was evaluated post-ICB, pre-op and post-op and correlated with recurrence-free (RFS) and overall survival (OS). Results: Twenty out of 32 patients had detectable ctDNA at any timepoint. Of the 12 patients with undetectable ctDNA, 9 had only CH- and/or germline-derived variants, while 3 patients had no detectable variants of any origin. ctDNA clearance post-ICB was correlated with tumor regression >80% at the time of resection (Fischer’s exact p=0.04). The subset of patients that did not attain complete pathologic response was heterogeneous with respect to ctDNA dynamics; such that ctDNA clearance pre-op identified patients with longer OS despite residual tumor of >0% at the time of resection (log rank p=0.06). Patients with undetectable ctDNA or ctDNA clearance pre-op had a longer RFS (log rank p=0.007) and OS (log rank p=0.03). Molecular MRD was associated with RFS and OS such that patients with ctDNA clearance post-op had longer RFS (log-rank p=0.007) and OS (log-rank p=0.017). Conclusion: ctDNA clearance post-ICB, pre-op and post-op reflects differential clinical outcomes for patients with E/GEJ cancer receiving neoadjuvant immuno-chemoradiotherapy. Understanding ctDNA dynamics and their relationship with pathological response and long-term outcomes can help identify patients at higher risk for recurrence and open a therapeutic window for future intervention. Citation Format: Blair V. Landon, Ronan J. Kelly, Ali H. Zaidi, Archana Balan, Jenna V. Canzoniero, Gavin Pereira, Zineb Belcaid, Russell K. Hales, K Ranh Voong, Richard J. Battafarano, Blair A. Jobe, Stephen C. Yang, Stephen Broderick, Jinny Ha, Kellie N. Smith, Elizabeth Thompson, Fyza Y. Shaikh, James R. White, Cynthia L. Sears, Eun J. Shin, Ali I. Amjad, Benny Weksler, Josephine L. Feliciano, Chen Hu, Vincent K. Lam, Valsamo Anagnostou. Circulating cell-free tumor DNA dynamics capture minimal residual disease with neoadjuvant immune checkpoint blockade plus chemoradiotherapy for patients with operable esophageal/gastroesophageal junction cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3374.

Introduction: Targeted next-generation sequencing (NGS) of cell-free DNA in plasma, referred to as liquid biopsy, has become a valuable diagnostic tool in clinical oncology. However, detection of variants related to clonal hematopoiesis (CH) is a major confounder that significantly impairs the clinical utility of liquid biopsies. Here we developed a machine-learning model to determine tumor versus CH origin of variants identified in plasma-only NGS. Methods: We assembled a training cohort of 352 variants identified by targeted deep plasma sequencing from 199 patients with stage I-IV breast, colorectal, esophageal, lung, and ovarian cancer, coupled with matched white blood cell (WBC) and tumor tissue NGS to allow determination of the reference origin for each plasma variant. We employed Extreme Gradient Boosting (XGBoost) to integrate fragment, variant, gene, and patient level features to predict tumor versus CH plasma variant origin, evaluating the performance of this approach within the training cohort using 10-fold cross-validation. We applied the fixed model to two independent validation cohorts: a small cell lung cancer (SCLC) cohort comprising of 74 variants from targeted plasma NGS from 26 patients and a multi-cancer cohort of 409 variants detected using the MSK-Impact panel from 74 patients with breast, colorectal, and prostate cancer. Results: Variant allele frequencies (VAF) did not differentiate tumor from CH variants, as the VAFs between tumor (median VAF 0.53%) and CH (median VAF 0.409%) variants in the training cohort were largely overlapping (area under the ROC curve-AUC 0.54, 95% confidence interval-CI 0.48-0.61). Similarly, individual fragmentomic features (mutant fragment length, cut points, and endpoint motifs) had limited ability to distinguish tumor from CH variants (AUC range 0.51-0.76). Using serial plasma samples, we identified stable statistical measures of differences in fragment feature distributions between mutant and wild type fragments; these were subsequently incorporated into an XGBoost machine-learning model along with variant, gene and patient features to predict tumor versus CH variant origin. Our model predicted variant origin with an AUC of 0.95 (95% CI 0.87-1) from 10-fold cross validation in the training cohort. The performance of the model was tested in independent SCLC and multi-cancer validation cohorts; the fixed model predicted plasma variant origin with an AUC of 0.87 (95% CI 0.73-1) and 0.89 (95% CI 0.86-0.92) respectively. Conclusion: We developed a machine-learning model that integrates patient, gene, variant and fragment features to predict tumor versus CH origin of plasma variants across solid tumors and NGS sequencing platforms. The ability to identify bona fide tumor variants in plasma-only sequencing fills a critical need in the clinical implementation of liquid biopsy-guided cancer therapy by reducing misinterpretation due to CH contamination. Citation Format: Jenna V. Canzoniero, Archana Balan, Jillian Phallen, Blair V. Landon, Lavanya Sivapalan, Benjamin Green, Zineb Belcaid, Susan C. Scott, Gavin Pereira, Vincent K. Lam, Ali H. Zaidi, Ronan J. Kelly, Christine L. Hann, Wade T. Iams, Christine M. Lovly, Patrick M. Forde, Gerrit A. Meijer, Geraldine R. Vink, Remond J. Fijneman, The MEDOCC Group, Victor E. Velculescu, Robert B. Scharpf, Valsamo Anagnostou. A machine learning approach to determine the cellular origin of variants in liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3366.

Consolidation durvalumab immunotherapy following definitive chemoradiation (CRT) for unresectable stage III non-small cell lung cancer (NSCLC) improves overall survival. As therapeutic options for patients with KRAS-driven disease evolve, more understanding regarding genomic determinants of response and patterns of progression for durvalumab consolidation is needed to optimize outcomes.We conducted a single-institutional retrospective analysis of real-world patients with locally advanced, unresectable NSCLC who completed CRT and received durvalumab consolidation. Kaplan-Meier analyses compared progression-free survival (PFS) and overall survival (OS) from start of durvalumab consolidation between patients with KRAS-mutated and non-mutated tumors. Fisher's exact test was used to compare rates of intrathoracic or extrathoracic progression.Of 74 response-evaluable patients, 39 had clinical genomic profiling performed. 18 patients had tumors with KRAS mutations, 7 patients had tumors with non-KRAS actionable alterations (EGFR, ALK, ERBB2, BRAF, MET, RET, or ROS1), and 14 patients had tumors without actionable alterations. Median PFS for the overall cohort was 16.1 months. PFS for patients with KRAS-mutated NSCLC was 12.6 months versus 12.7 months for patients with non-actionable tumors (P= 0.77, log-rank). Fisher's exact test revealed a statistically significantly higher rate of extrathoracic progression versus intrathoracic-only progression for patients with KRAS-driven disease compared to patients with non-actionable tumors (P= 0.015).Patients with KRAS-mutated NSCLC derived similar benefit from durvalumab as patients with non-actionable tumors. A higher rate of extrathoracic progression was also observed among the patients with KRAS-mutated NSCLC compared to patients with non-actionable tumors. This highlights the potential unmet needs for novel systemic therapies and surveillance methods for KRAS-mutated stage III NSCLC.

BackgroundThe phase I first-in-human study ENGAGE-1 evaluated the humanized IgG1 OX40 agonistic monoclonal antibody GSK3174998 alone (Part 1 (P1)) or in combination with pembrolizumab (Part 2 (P2)) in patients with advanced solid tumors.MethodsGSK3174998 (0.003–10 mg/kg) ± pembrolizumab (200 mg) was administered intravenously every 3 weeks using a continuous reassessment method for dose escalation. Primary objectives were safety and tolerability; secondary objectives included pharmacokinetics, immunogenicity, pharmacodynamics, and clinical activity.Results138 patients were enrolled (45 (P1) and 96 (P2, including 3 crossovers)). Treatment-related adverse events occurred in 51% (P1) and 64% (P2) of patients, fatigue being the most common (11% and 24%, respectively). No dose-toxicity relationship was observed, and maximum-tolerated dose was not reached. Dose-limiting toxicities (P2) included Grade 3 (G3) pleural effusion and G1 myocarditis with G3 increased troponin. GSK3174998 ≥0.3 mg/kg demonstrated pharmacokinetic linearity and >80% receptor occupancy on circulating T cells; 0.3 mg/kg was selected for further evaluation. Limited clinical activity was observed for GSK3174998 (P1: disease control rate (DCR) ≥24 weeks 9%) and was not greater than that expected for pembrolizumab alone (P2: overall response rate 8%, DCR ≥24 weeks 28%). Multiplexed immunofluorescence data from paired biopsies suggested that increased infiltration of natural killer (NK)/natural killer T (NKT) cells and decreased regulatory T cells (Tregs) in the tumor microenvironment may contribute to clinical responses: CD16+CD56–CD134+ NK /NKT cells and CD3+CD4+FOXP3+CD134+ Tregs exhibited the largest magnitude of change on treatment, whereas CD3+CD8+granzyme B+PD-1+CD134+ cytotoxic T cells were the least variable. Tumor gene expression profiling revealed an upregulation of inflammatory responses, T-cell proliferation, and NK cell function on treatment with some inflammatory cytokines upregulated in peripheral blood. However, target engagement, evidenced by pharmacologic activity in peripheral blood and tumor tissue, did not correlate with clinical efficacy. The low number of responses precluded identifying a robust biomarker signature predictive of response.ConclusionsGSK3174998±pembrolizumab was well tolerated over the dose range tested and demonstrated target engagement. Limited clinical activity does not support further development of GSK3174998±pembrolizumab in advanced cancers.Trial registration numberNCT02528357.

BackgroundADP-A2M10 specific peptide enhanced affinity receptor (SPEAR) T cells (ADP-A2M10) are genetically engineered autologous T cells that express a high-affinity melanoma-associated antigen A10 (MAGE-A10)-specific T-cell receptor (TCR) targeting MAGE-A10+ tumors in the context of human leukocyte antigen (HLA)-A*02. ADP-0022-003 was a phase I dose-escalation trial that aimed to evaluate the safety and antitumor activity of ADP-A2M10 in non-small cell lung cancer (NSCLC) (NCT02592577).MethodsEligible patients were HLA-A*02 positive with advanced NSCLC expressing MAGE-A10. Patients underwent apheresis; T cells were isolated, transduced with a lentiviral vector containing the TCR targeting MAGE-A10, and expanded. Patients underwent lymphodepletion with varying doses/schedules of fludarabine and cyclophosphamide prior to receiving ADP-A2M10. ADP-A2M10 were administered at 0.08–0.12×109 (dose group 1), 0.5–1.2×109 (dose group 2), and 1.2–15×109 (dose group 3/expansion) transduced cells.ResultsEleven patients (male, n=6; female, n=5) with NSCLC (adenocarcinoma, n=8; squamous cell carcinoma, n=3) were treated. Five, three, and three patients received cells in dose group 1, dose group 2, and dose group 3/expansion, respectively. The most frequently reported grade ≥3 adverse events were lymphopenia (n=11), leukopenia (n=10), neutropenia (n=8), anemia (n=6), thrombocytopenia (n=5), and hyponatremia (n=5). Three patients presented with cytokine release syndrome (grades 1, 2, and 4, respectively). One patient received the highest dose of lymphodepletion (fludarabine 30 mg/m2 on days –5 to –2 and cyclophosphamide 1800 mg/m2 on days −5 to −4) prior to a second infusion of ADP-A2M10 and had a partial response, subsequently complicated by aplastic anemia and death. Responses included: partial response (after second infusion; one patient), stable disease (four patients), clinical or radiographic progressive disease (five patients), and not evaluable (one patient). ADP-A2M10 were detectable in peripheral blood and in tumor tissue. Peak persistence was higher in patients who received higher doses of ADP-A2M10.ConclusionsADP-A2M10 demonstrated an acceptable safety profile and no evidence of toxicity related to off-target binding or alloreactivity. There was persistence of ADP-A2M10 in peripheral blood as well as ADP-A2M10 trafficking into the tumor. Given the discovery that MAGE-A10 and MAGE-A4 expression frequently overlap, this clinical program closed as trials with SPEAR T cells targeting MAGE-A4 are ongoing.

Purpose: Nintedanib enhances the activity of chemotherapy in metastatic non–small cell lung cancer (NSCLC). In this phase I/II study, we assessed safety and efficacy of nintedanib plus neoadjuvant chemotherapy, using major pathologic response (MPR) as primary endpoint. Patients and Methods: Eligible patients had stage IB (≥4 cm)–IIIA resectable NSCLC. A safety run-in phase was followed by an expansion phase with nintedanib 200 mg orally twice a day (28 days), followed by three cycles of cisplatin (75 mg/m2), docetaxel (75 mg/m2) every 21 days plus nintedanib, followed by surgery. With 33 planned patients, the study had 90% power to detect an MPR increase from 15% to 35%. Results: Twenty-one patients (stages I/II/III, N = 1/8/12) were treated. One of 15 patients treated with nintedanib 200 mg achieved MPR [7%, 95% confidence interval (CI), 0.2%–32%]. Best ORR in 20 evaluable patients was 30% (6/20, 95% CI, 12%–54%). Twelve-month recurrence-free survival and overall survival were 66% (95% CI, 47%–93%) and 91% (95% CI, 79%–100%), respectively. Most frequent treatment-related grade 3–4 toxicities were transaminitis and electrolyte abnormalities. On the basis of an interim analysis the study was discontinued for futility. Higher levels of CD3+ and cytotoxic CD3+CD8+ T cells were found in treated tumors of patients who were alive than in those who died (652.8 vs. 213.4 cells/mm2, P = 0.048; 142.3 vs. 35.6 cells/mm2, P = 0.018). Conclusions: Although tolerated, neoadjuvant nintedanib plus chemotherapy did not increase MPR rate compared with chemotherapy historical controls. Additional studies of the combination in this setting are not recommended. Posttreatment levels of tumor-infiltrating T cells were associated with patient survival. Use of MPR facilitates the rapid evaluation of neoadjuvant therapies. See related commentary by Blakely and McCoach, p. 3499

Introduction The efficacy of targeted therapies in oncogene-driven lung adenocarcinomas (LUADs) has been well established; however, the benefit for EGFR-mutant or ALK-rearranged lung squamous cell carcinomas (LUSCs) is less known, partially owing to the rarity of the incidence. Methods We reviewed the database of the MD Anderson Cancer Center and identified metastatic LUSC with classic EGFR or ALK alterations. Results There were eight patients with EGFR-mutant LUSC (median age = 58 y) and six patients with EML4-ALK LUSC (median age = 50 y) who received tyrosine kinase inhibitors (TKIs) that were identified. Of the 14 patients, 11 (79%) were females and 12 (86%) were never smokers, similar to the demographics of EGFR or ALK LUAD. With TKI treatment, seven of eight cases of EGFR LUSC and four of six cases of ALK LUSC achieved partial response or stable disease, but the progression-free survival was 4.9 months and 2.9 months for EGFR-mutant and ALK-rearranged LUSC, respectively. In addition, we compared comutation profile of EGFR-mutant LUAD (The Cancer Genome Atlas, n = 46) versus LUSC (n = 19) and found that the comutation patterns are more consistent with squamous disease with a higher incidence of PIK3CA (p = 0.02) and KRAS or BRAF (p = 0.04) alterations. Conclusions EGFR or ALK alterations occur in patients with LUSC, especially never-smoker females. TKI treatments render clinical benefit in disease control, but the duration was considerably truncated compared with those historically observed in LUAD.

PURPOSE Targeted therapies against non–small-cell lung cancer (NSCLC) harboring HER2 mutations remain an unmet need. In this study, we assessed the efficacy and safety of poziotinib in patients with HER2 exon 20 mutant advanced NSCLC in a single-arm, open-label, phase II study. PATIENTS AND METHODS Patients with advanced HER2 exon 20 mutant NSCLC were enrolled to receive poziotinib at a dose of 16 mg/d for 28-day cycles. The primary end point was objective response rate per RECIST version 1.1. Confirmatory scans were performed at least 28 days from initial radiologic response. RESULTS Thirty patients received poziotinib treatment. At baseline, 90% of patients received prior platinum-based chemotherapy and 53% had two lines or more prior systemic therapies. As of data cutoff on March 1, 2021, the confirmed objective response rate was 27% (95% CI, 12 to 46). Responses were observed across HER2 exon 20 mutation subtypes. The median duration of response was 5.0 months (95% CI, 4.0 to not estimable). The median progression-free survival was 5.5 months (95% CI, 4.0 to 7.0). The median overall survival was 15 months (95% CI, 9.0 to not estimable). The most common grade 3 treatment-related adverse events were skin rash (47%) and diarrhea (20%). There was one possible treatment-related death because of pneumonitis. CONCLUSION Poziotinib showed promising antitumor activity in patients with HER2 exon 20 mutant NSCLC including patients who had previously received platinum-based chemotherapy.

Introduction Tumor mutation profiling is standard-of-care in lung carcinoma patients. However, comprehensive molecular profiling of small specimens, including core needle biopsy (CNB) and fine-needle aspiration (FNA) specimens, may often be inadequate due to limited tissue. Centrifuged FNA supernatants, which are typically discarded, have emerged recently as a novel liquid-based biopsy for molecular testing. In this study, we evaluate the use of lung carcinoma FNA supernatants for detecting clinically relevant mutations. Methods Supernatants from lung carcinoma FNA samples (n = 150) were evaluated. Samples were further analyzed using next-generation sequencing (NGS) and ultrasensitive droplet digital PCR (ddPCR). Mutation profiles in a subset of samples were compared with results derived from paired tissue samples from the same patient (n = 67) and available plasma liquid biopsy assay (n = 45). Results All 150 samples yielded adequate DNA and NGS were carried out successfully on 104 (90%) of 116 selected samples. Somatic mutations were detected in 82% of the samples and in 50% of these patients a clinically relevant mutation was identified that would qualify them for targeted therapy or a clinical trial. There was high overall concordance between the mutation profiles of supernatants and the corresponding tissue samples, with 100% concordance with concurrent FNA and 96% with concurrent CNB samples. Comparison of actionable driver mutations detected in supernatant versus plasma samples showed 84% concordance. Conclusions FNA supernatants can provide a valuable specimen source for genotyping lung carcinoma especially in patients with insufficient tumor tissue, thereby reducing multigene mutation profiling failure rates, improving turnaround times, and avoiding repeat biopsies.

Introduction Immune checkpoint blockade (ICB) has revolutionized the treatment of NSCLC, but only approximately 15% of patients achieve durable benefit. Understanding mechanisms of resistance to ICB is pivotal in developing more effective treatment strategies. Recent studies showed that human leukocyte antigen (HLA) class I heterozygosity might be important in mediating benefit from ICB. We aimed to investigate the impact of HLA class I genotype on outcomes of patients with NSCLC treated with ICB. Methods We collected HLA typing, genomic, and clinical data from patients with advanced NSCLC treated with ICB at M. D. Anderson Cancer Center. We compared HLA class I–heterozygous and HLA class I–homozygous patients for progression-free survival (PFS) and overall survival (OS). HLA I supertype/alleles were also analyzed. To validate our findings, we also analyzed two previously published independent cohorts of patients with NSCLC (the CheckMate-012 and Chowell cohorts). Results No significant correlations were observed for HLA class I zygosity and PFS or OS in the M. D. Anderson Cancer Center (n = 200), CheckMate-012 (n = 75), or Chowell (n = 371) cohorts. No HLA class I supertype/allele was consistently shown to be correlated with PFS or OS. Predictors of worse outcome across the three cohorts included presence of targetable driver mutation, serine/threonine kinase 11 gene (STK11) mutation, negative programmed death ligand 1 expression, and low tumor mutational burden. Conclusions HLA class I genotype is not correlated with survival in advanced NSCLC treated with ICB. This suggests that the impact of HLA class I diversity may be disease specific and that tumor genomic and immune markers are more impactful in predicting benefit from ICB in NSCLC.

Background: Preclinical and clinical data indicate that neoadjuvant chemoradiotherapy (CRT) may prime an anti-tumor immunological response in esophageal cancer driven by intratumoral CD8+ T cells and PD-L1 expression. LAG-3 is also highly expressed in esophagogastric cancers. The microbiome, a novel and potentially modifiable, biomarker of IO response, has not yet been examined in the neoadjuvant setting in esophageal cancer and is the goal of our study. Methods: Fecal samples were collected from patients with stage II/III esophageal or gastroesophageal junction carcinoma eligible for curative resection treated with the standard of care regimen of carboplatin paclitaxel (50mg/m2), radiation 50.4 Gy in 28 fractions and an Ivor-Lewis esophagectomy 6-10 weeks after last CRT and immunotherapy (IO) dose. Patients on arm A (n=11) received 2 cycles of induction with nivolumab plus 3 additional cycles on week 1, 3 and 5 of CRT. Patients on arm B (n=8) received nivolumab plus relatlimab on the same schedule (Clinical trial: NCT03044613). We examined longitudinal fecal samples from n=19 patients across both arms (n=90 samples) using 16S rRNA amplicon sequencing. Patients were classified based on pathological response: complete response (CR) and grades 1, 2, and 3 (G1, G2, G3) with increasing residual tumor visible in the resected specimen. Sequencing data was trimmed and filtered for contaminants, followed by high-resolution taxonomic assignment and normalization of reads across all samples. Analysis was performed using multiple metrics for alpha diversity and beta-diversity, with principal coordinates analysis/PERMANOVA, and pathway analysis using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Results: Patients with improved response in the neoadjuvant setting (CR/G1 vs G2/G3) grouped in distinct clusters using Bray-Curtis (p < 0.001). Patients with CR had higher alpha diversity, using both measures of richness and evenness, compared to patients with a G3 responses (p < 0.03). Specifically, family Bacteroidaceae and genus Bacteroides were enriched in patients with CR vs G3 (p < 0.02). At the species level, B. finegoldii, B. ovatus, and B. uniformis were enriched in patients with CR vs G3 (p < 0.02). In contrast, genus Klebsiella and Clostridium termitidis were enriched in patients with a poor response, G3 (p Conclusions: Patients with operable esophageal cancer and improved responses to combined CRT and IO had distinct microbiome profiles enriched in multiple Bacteroides species. Further analyses and validation efforts are underway to confirm metabolomic pathways. Citation Format: Fyza Y. Shaikh, James R. White, Ronan J. Kelly, Ali H. Zaidi, Jenna V. Canzoniero, Josephine L. Feliciano, Russell K. Hales, K Ranh Voong, Richard J. Battafarano, Blair A. Jobe, Stephen C. Yang, Stephen Broderick, Jinny Ha, Kellie N. Smith, Elizabeth Thompson, Eun J. Shin, Ali I. Amjad, Patrizia Guerrieri, Benny Weksler, Chen Hu, Valsamo Anagnostou, Vincent K. Lam, Cynthia L. Sears. Patients with operable esophageal cancer and improved responses to combined chemoradiotherapy and immunotherapy display distinct microbiome profiles enriched in multiple Bacteroides species [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1973.

9077 Background: New tyrosine kinase inhibitors (TKIs) targeting ALK and ROS1 alterations in non-small cell lung cancer (NSCLC) have emerged over the last decade. Given the rarity of these genetic changes in NSCLC, data on long term outcomes with sequential therapies are limited. Methods: We conducted a multicenter retrospective cohort study of patients with metastatic NSCLC and ALK or ROS1 alterations across 12 Academic Thoracic Oncology Medical Investigators Consortium (ATOMIC) sites between 1/29/2007 and 3/31/2021. Data were abstracted from the electronic medical record. Median time to treatment discontinuation (TTD) of 1st TKI, overall survival (OS), and time to brain metastases were estimated using Kaplan-Meier methodology from start date of 1st TKI. Results: 566 patients with ALK (n = 464) or ROS1 (n = 102) were included. The majority (ALK: 426/464, 92%; ROS1: 88/102, 86%) received a TKI at some point during therapy (1st line TKI n = 262 ALK, 48 ROS1). Crizotinib was the most common 1st TKI (ALK: 57%; ROS1: 88%). Following crizotinib, alectinib (64%) and lorlatinib (41%) were the most common subsequent TKIs for ALK and ROS1, respectively. Alectinib (38%) and entrectinib (10.2%) were the 2nd most common initial TKIs used in ALK and ROS1, respectively. Additional treatment patterns presented in table. With a median follow up time of 31.1 (ALK, 95% CI, 27.6-35.0) and 32.6 (ROS1, 95% CI, 25.7-39.6) months, median OS from start of 1st TKI was 53.3 (ALK, 95% CI, 40.0-68.9) and 42.0 (ROS1, 95% CI, 31.8-NA) months. Out of the 321 patients with brain imaging prior to 1st line therapy, 40% (105/262, ALK) and 39% (23/59, ROS1) had CNS disease. Median time to development of brain metastases from start of 1st TKI in those without previous CNS disease (ALK: 278; ROS1: 58) was 30.0 (ALK, 95% CI, 25.3-39.1) and 27.0 (ROS1, 95% CI, 18.2-NA) months. Median TTD of 1st TKI was 11.2 (ALK) and 10.8 (ROS1) months. Conclusions: This is the largest retrospective cohort of NSCLC patients with ALK or ROS1 rearrangements treated in the real world setting. CNS metastases are common and subset analyses by agent and by year of diagnosis will be presented. Median time to CNS metastasis of > 2 years supports revision of the NCCN guidelines to include regular surveillance brain MRIs in this population. [Table: see text]

e16092 Background: Brain metastasis (BRM) in gastroesophageal (GE) cancer is uncommon, but contributes to poor prognosis and significant symptom burden. While HER2 overexpression has been suggested as a potential driver of BRM, clinicopathologic and molecular determinants of BRM in GE cancer and their impact on clinical outcome remain incompletely understood. Methods: We retrospectively analyzed clinicopathologic data from 323 advanced esophageal/GEJ patients (pts) at Johns Hopkins from 2003 to 2021. Univariate and multivariate logistic regression were performed to investigate the association between several clinical and molecular features and the occurrence of BRM, with particular focus on HER2 overexpression (by IHC and FISH) and PD-L1 expression (by IHC) as measured by the combined positive score (CPS). Overall survival was estimated using the Kaplan-Meier method. Results: Median age was 64 and most pts were white (90.4%). Tumors were 73.7% esophageal primary, 82.7% adenocarcinoma, 43.8% HER2+, and 11.1% CPS ≥ 1. Cumulative incidence of BRM in overall cohort and within HER2+ subgroup was 14.6% (47 pts) and 25.4% (16 pts), respectively. Pts with BRM (not already present at time of stage IV diagnosis) had median onset of 10.8 months. Seventeen (36.2%) pts had a solitary brain lesion and 10 (21.7%) were asymptomatic. On univariate analysis, adenocarcinoma and HER2 overexpression were associated with increased risk of BRM (OR 3.45; 95% CI 0.09-0.96; p = 0.043 and OR 2.72; 95% CI 1.11-6.67; p = 0.028, respectively). Notably, the HER2 association was strengthened when HER2 equivocal tumors were re-classified as positive in an exploratory analysis (OR 3.55; 95% CI 1.38-9.14; p = 0.009). Adenocarcinoma and HER2 overexpression were collinear in the multivariate model. In pts with BRM, OS was improved in HER2+ pts (16.72 months; 95% CI 12.3-21.1 months; log-rank p = 0.039 and OR 0.33; 95% CI 0.11-0.99; Cox regression p = 0.049). CPS and primary tumor location were not associated with increased BRM. Conclusions: HER2 overexpression identifies an esophageal/GEJ molecular subtype that is significantly associated with increased risk of BRM; though with improved OS (compared to non-HER2+ BRM) and later onset CNS progression, likely reflecting durable extra-CNS control on targeted systemic therapy and a potential HER2+ subgroup with relatively indolent biology. Prevalence of asymptomatic and solitary brain lesions supports increased brain surveillance for this population.

TPS4169 Background: Cyclin dependent kinases (CDKs) are serine/threonine kinases that are responsible for phosphorylating the intracellular proteins that coordinate cell-cycle progression. The Cancer Genome Atlas (TCGA) molecular analysis of esophageal/gastroesophageal junction (E/GEJ) carcinomas has highlighted significant dysregulation of CDKN2A, the gene coding for the tumor suppressor p16, through deletion, mutation or epigenetic silencing in up to 76% of cases and the associated significant upregulation of the CDK4/6-Cyclin D-axis (The Cancer Genome Atlas Research Network. Integrated genomic characterization of oesophageal carcinoma. Nature 2017). Preclinical experiments demonstrated that single agent abemaciclib has potent antitumor efficacy both in vitro and in vivo in esophageal adenocarcinoma with direct pathway inhibition (Kosovec J et al. CDK4/6 dual inhibitor abemaciclib demonstrates compelling preclinical activity against esophageal adenocarcinoma: a novel therapeutic options for a deadly disease. Oncotarget 2017 Nov 1;8[59]). Significant activity was seen in the OE33 (p=0.048) and FLO1 (p=0.043) esophageal cancer cell lines while western blot revealed downregulation of Cyclin D, phospho-pRb, E2F1, and Cyclin A2 (Kosovec J et al, 2017). In vivo experiments , utilizing the Levrat model of end-to-side esophagojejunostomy performed on Sprague-Dawley rats demonstrated that 78.9% of animals given abemaciclib demonstrated >20% tumor volume decrease (placebo 0%). Post treatment comparison between placebo and abemaciclib of qRT-PCR gene expression demonstrated downregulation of pathway correlates, including CDK4 (p=0.023), CDK6 (p=0.068), E2F1 (p=0.005), Rb1 (p=0.067), Cyclin D (p=0.031), and Cyclin A (p=0.039) (Kosovec J et al, 2017). The JPBJ phase 1b study has previously demonstrated the safety of combining abemaciclib with ramucirumab in NSCLC. Methods: Subjects with previously treated metastatic esophageal or gastroesophageal junction adenocarcinomas are eligible for this study which is currently open and enrolling patients. Squamous cell and mixed histology with neuroendocrine features are excluded. A total of 30 patients will be enrolled across 3 sites at Baylor University Medical Center in Dallas, The Allegheny Health Network in Pittsburgh and at Johns Hopkins Hospital in Baltimore. The primary objective is to describe the safety profile of abemaciclib (150mg po bid) and ramucirumab (8mg/kg iv every 2 weeks) in this patient population as assessed according to Common Terminology Criteria for Adverse Events version 4.0. Secondary objectives are to assess response rate, progression free and overall survival. In addition, we will determine the rate of stable disease at 3 months post targeted therapy. Correlative studies investigating changes in the expression of selected serum/tissue genomic markers of interest will be performed. Clinical trial information: NCT04921904.

321 Background: The phase III CheckMate 577 study resulted in adjuvant nivolumab becoming a new standard of care for patients with completely resected E/GEJ cancer with residual pathologic disease post neoadjuvant chemoradiotherapy. We evaluated if neoadjuvant nivolumab (N) or nivolumab/relatlimab (N/R) combined with chemoradiotherapy (CRT) can further improve patient outcomes. Methods: Patients with stage II/III E/GEJ carcinoma eligible for curative resection were treated with standard of care regimen of carboplatin (AUC2), paclitaxel (50mg/m2), RT 41.1Gy in 23 fractions and an Ivor-Lewis esophagectomy (E/MIE) 6-10 weeks after last CRT/IO dose. Patients on arm A (n=16) received 2 cycles of induction N (240mg q2 wks) plus three additional cycles of N on week 1, 3 and 5 of CRT. After safety and feasibility evaluation of arm A, patients on arm B (n=16) received N (240mg q 2 wks) plus R (80mg q 2 wks) following the same schedule. The primary endpoints of the study were safety and feasibility. Secondary endpoints include pCR, MPR (

Introduction Plasma-based circulating tumor DNA (ctDNA) is an established biomarker for molecular profiling with emerging applications in disease monitoring in multiple tumor types, including, NSCLC. However, determinants of ctDNA shedding and correlation with tumor burden are incompletely understood, particularly in advanced-stage disease. Methods We retrospectively analyzed ctDNA-based and tissue-based genomic data and imaging from 144 patients with NSCLC. Tumor burden was quantified with computed tomography (CT) and brain magnetic resonance imaging for the overall cohort and 18F-fludeoxyglucose positron emission tomography-CT in a subset of patients. Results There was a moderate but statistically significant correlation between ctDNA variant allele frequency and multiple imaging measures of tumor burden such as CT volume (rho = 0.34, p ≤ 0.0001) and metabolic tumor volume (rho = 0.36, p = 0.003). This correlation was strongest in KRAS-mutant tumors (rho = 0.56, p ≤ 0.001), followed by TP53 mutants (rho = 0.43, p ≤ 0.0001), and weakest in EGFR-mutated (EGFR+) tumors (rho = 0.24, p = 0.077). EGFR+ tumors with EGFR copy number gain had significantly higher variant allele frequency than EGFR+ without copy number gain (p ≤ 0.00001). In multivariable analysis, TP53 and EGFR mutations, visceral metastasis, and tumor burden were independent predictors of increased ctDNA shedding. Conclusions Levels of detectable ctDNA were affected not only by tumor burden but also by tumor genotype. The genotype-specific differences observed may be due to variations in DNA shedding and cellular turnover. These findings have implications for the emerging use of ctDNA in NSCLC disease monitoring and early detection.

Identifying locoregional gastric cancer patients who are at high risk for relapse after resection could facilitate early intervention. By detecting molecular residual disease (MRD), circulating tumor DNA (ctDNA) has been shown to predict post-operative relapse in several cancers. Here, we aim to evaluate MRD detection by ctDNA and its association with clinical outcome in resected gastric cancer. This prospective cohort study enrolled 46 patients with stage I–III gastric cancer that underwent resection with curative intent. Sixty resected tumor samples and 296 plasma samples were obtained for targeted deep sequencing and longitudinal ctDNA profiling. ctDNA detection was correlated with clinicopathologic features and post-operative disease-free (DFS) and overall survival (OS). ctDNA was detected in 45% of treatment-naïve plasma samples. Primary tumor extent (T stage) was independently associated with pre-operative ctDNA positivity (p = 0.006). All patients with detectable ctDNA in the immediate post-operative period eventually experienced recurrence. ctDNA positivity at any time during longitudinal post-operative follow-up was associated with worse DFS and OS (HR = 14.78, 95%CI, 7.991–61.29, p p = 0.002, respectively), and preceded radiographic recurrence by a median of 6 months. In locoregional gastric cancer patients treated with curative intent, these results indicate that ctDNA-detected MRD identifies patients at high risk for recurrence and can facilitate novel treatment intensification studies in the adjuvant setting to improve survival.

Subclonal architecture and genomic evolution of small-cell lung cancer (SCLC) under treatment has not been well studied primarily due to lack of tumor specimens, particularly longitudinal samples acquired during treatment. SCLC is characterized by early hematogenous spread, which makes circulating cell-free tumor DNA (ctDNA) sequencing a promising modality for genomic profiling. Here, we perform targeted deep sequencing of 430 cancer genes on pre-treatment tumor biopsies, as well as on plasma samples collected prior to and during treatment from 22 SCLC patients. Similar subclonal architecture is observed between pre-treatment ctDNA and paired tumor DNA. Mean variant allele frequency of clonal mutations from pre-treatment ctDNA is associated with progression-free survival and overall survival. Pre- and post-treatment ctDNA mutational analysis demonstrate that mutations of DNA repair and NOTCH signaling pathways are enriched in post-treatment samples. These data suggest that ctDNA sequencing is promising to delineate genomic landscape, subclonal architecture, and genomic evolution of SCLC., Small cell lung cancer (SCLC) may evolve under treatment. But tumor tissues are often not available to study evolution of SCLC. Here, the authors utilize circulating tumor DNA to investigate the genomic evolution and subclonal architecture of SCLC during therapy.

Introduction: The magnitude of benefit from immune checkpoint inhibitors (ICI) may be sex-dependent, however the mechanisms underlying this sex dimorphism in ICI response are unknown. Methods: To explore sex-specific genomic features linked with ICI response, we analyzed whole-exome sequence data of 89 patients with non-small cell lung cancer (NSCLC) treated with ICI. Sequence alteration, mutation signatures and clonality analyses were combined with HLA class I and II genotyping, and computationally derived mutation-associated neoantigens were evaluated based on the peptide-HLA binding affinity (2]. Missense sequence alterations were characterized as putatively immunogenic mutations (IMM), and HLA class I and II matched IMM loads were calculated. Durable clinical benefit ≥6 months was used to define response. Our findings were validated in an independent cohort of ICI treated NSCLC (n=34). Results: There were no differences in the genomic features assessed or in clinical outcomes between male (n=46) and female (n=43) patients. Female responders had higher tumor mutational burden (TMB; p=0.004), in contrast to male patients (p=0.15). Similarly, a mutation spectra-derived smoking signature was associated with response in females (p=0.001) but not in males (p=0.2). Responding female patients had a more clonal TMB compared to non-responders (p=0.008). Importantly, female responders harbored a significantly higher class I and II IMM load (p=0.008 and p=0.004 respectively), which was not appreciated in males. These findings were supported by analyses of a second independent cohort, where HLA class I and II IMM load differentiated responders from non-responders only in the female patient group (p=0.008 and p=0.004 for class I and II IMM, respectively). In the primary cohort, germline HLA class I zygosity was not associated with outcome for either sex, however a trend towards HLA DQ-B1 homozygosity and worse outcome was evident only in females. Importantly, when HLA class II diversity was combined with class II IMM load, a survival benefit was noted for tumors harboring a high IMM load and maximal HLA II germline diversity in males (log rank p=0.015) but not in females. Similarly, HLA class I diversity was important in males when combined with class I IMM load, such that low IMM load combined with reduced antigen presentation capacity was predictive of non-durable clinical benefit (p=0.003 vs p=0.68 for males and females respectively) and was linked with shorter overall survival (log rank p= 0.062 vs p=0.58 for males and females respectively). Conclusions: Our findings support a sex dimorphism in the genomic determinants of response to immune checkpoint blockade, which may point to differences in immune surveillance between males and females in the context of therapy and may be used to tailor therapeutic delivery. Citation Format: Susan C. Scott, Xiaoshan M. Shao, Noushin Niknafs, Archana Balan, Mara Lanis, James White, Patrick Forde, Kristen Marrone, Vincent Lam, Josephine Feliciano, Benjamin Levy, Julie Brahmer, David Ettinger, Victor Velculescu, Rachel Karchin, Christine Hann, Jarushka Naidoo, Valsalmo Anagnostou. Sex-specific genomic determinants of response to immunotherapy [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 1617.

We examined the utility of serial liquid biopsies to monitor clonal dynamics and predict pathologic response in patients with esophageal/gastroesophageal junction (E/GEJ) cancer undergoing treatment with neoadjuvant immunotherapy and concurrent chemoradiation (CA209-906; NCT03044613). Methods: Using Targeted Error Correction sequencing (TEC-Seq), we performed high-depth next generation sequencing on 79 serial plasma samples from 16 patients with operable stage II/III E/GEJ cancer undergoing treatment with neoadjuvant nivolumab, followed by nivolumab plus chemo-radiation and surgery as part of the CA209-906 trial. Liquid biopsies were evaluated pre-treatment, after each of two cycles of neoadjuvant nivolumab, and after concurrent nivolumab and chemoradiation immediately prior to surgery, for an average of 4 time points per patient. Deep sequencing of matched leukocyte DNA and whole exome sequencing (WES) of pre-treatment tumors was performed. The origin of plasma variants was determined by comparison to tumor WES (tumor-derived) and leukocyte TEC-Seq (clonal hematopoiesis (CH)-derived or germline) and their clonal dynamics over time were evaluated. Resected tumors were reviewed by central pathology and percent residual tumor was estimated. Patients were followed for an average of 2 years post-surgery. Results: Eight of 16 patients had detectable circulating tumor-derived DNA (ctDNA) at any time point. Additionally, 13 CH-derived mutations were detected in plasma of 8 patients. The number of CH-derived mutations was correlated with increasing patient age. Identification and removal of CH-derived mutations via comparison to matched leukocyte sequencing allowed for accurate assessment of kinetics of bona fide tumor-derived mutations in plasma. Tumor mutation burden as determined by tumor WES was not associated with pathological response (mean baseline mutations per exome 90 vs 81 for patients with residual disease >20% and 20% (50% vs 23% with or without detectable ctDNA respectively). ctDNA clearance, defined as detectable ctDNA at one or more earlier time points that subsequently becomes undetectable before surgery, occurred in 5 patients and was associated with improved pathologic response (80% of patients with ctDNA clearance had residual tumor Conclusion: Comprehensive analyses of ctDNA from E/GEJ patients undergoing neoadjuvant immunotherapy with concurrent chemoradiation revealed ctDNA dynamics that were associated with pathologic response and disease recurrence. These approaches may be used to identify patients at high risk for disease progression. Citation Format: Jenna VanLiere Canzoniero, Vincent Lam, Zineb Belcaid, Mara Lanis, Lamia Rhymee, Blair Landon, Archana Balan, Kristen Marrone, Patrick Forde, Benjamin Levy, David Ettinger, Heather Schneider, Hanika Rodavia, Richard Battafarano, Stephen Yang, Stephen Broderick, Jinny Ha, Blair Jobe, Ali Zaidi, Elizabeth Thompson, Julie Brahmer, Victor Velculescu, Ronan Kelly, Josephine Feliciano, Valsamo Anagnostou. Liquid biopsy approaches for determining pathologic response to neoadjuvant immunotherapy in esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 538.

Given the success of immunotherapy (IO), multiple IO-based options exist for advanced non-small cell lung cancer (NSCLC). Currently used biomarkers do not fully predict clinical outcomes and response assessment remains limited to radiological evaluation. Dynamic biomarkers that evaluate both tumor and host immune responses to IO are needed. We studied patients with NSCLC that received IO and chemo-IO to identify predictive and longitudinal biomarkers of clinical response using circulating tumor DNA (ctDNA) and plasma proteomic dynamics. We conducted deep targeted error-correction sequencing (TEC-Seq) of plasma cell-free DNA (cfDNA) and matched white blood cell (WBC) genomic DNA (gDNA) to identify ctDNA variants. We performed multiplexed antibody-based proximity extension assays of serial plasma samples to detect immune-related proteins. From separate training (n=31) and validation (n=29) cohorts, a total of 288 plasma cfDNA and 52 WBC gDNA specimens underwent TEC-Seq. A total of 260 variants were detected in plasma cfDNA and 78 variants in WBC gDNA. Almost a third of the plasma cfDNA variants (32%, n=61 of 188) were also found in WBC gDNA and filtered out. ctDNA variants were identified in 82% of patients (n=50). In the training cohort, longitudinal decreases in ctDNA variant levels were observed in patients with durable clinical benefit (DCB). Molecular response, defined as the loss of detectable ctDNA, was associated with longer progression-free (PFS; p=0.0004, log-rank) and overall survival (OS; p=0.017, log-rank). We incorporated on-treatment ctDNA dynamics including molecular response, recrudescence, and emergence of new variants into a logistic regression model to predict clinical benefit. This integrative model predicted DCB at a sensitivity of 84%, specificity of 76%, and with an area under the curve (AUC) of 0.90, better than baseline tumor PD-L1 expression alone (AUC 0.70, p=0.044, bootstrap method). In the validation cohort, molecular response was associated with longer PFS (p=0.00098, log-rank) and OS (p=0.0037, log-rank) and the integrative model predicted DCB at a sensitivity of 100%, specificity of 78%, and AUC of 0.89. In a subset (n=28 of 31) of the training cohort, plasma proteomics analysis revealed that increased baseline levels of IL15 and DCN were independently associated with DCB (p Citation Format: Joseph C. Murray, Karlijn Hummelink, Lamia Rhymee, Alessandro Leal, Leonardo Ferreira, Mara Lanis, James R. White, Noushin Niknafs, Kristen Marrone, Jarushka Naidoo, Benjamin Levy, Samuel Rosner, Christine Hann, Josephine Feliciano, Vincent Lam, David Ettinger, Qing K. Li, Peter Illei, Kim Monkhorst, Hatim Husain, Julie R. Brahmer, Victor Velculescu, Patrick Forde, Robert B. Scharpf, Valsamo Anagnostou. Longitudinal dynamics of circulating tumor DNA and plasma proteomics predict clinical outcomes to immunotherapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1668.

Background: Treatment-induced anti-tumor immune response may be reflected in peripheral blood leukocyte composition and shifts. The relative abundance of immune cell subsets captured by neutrophil-to-lymphocyte ratio (NLR) is a promising biomarker of response to immune checkpoint inhibition (ICI). However early dynamics and integration with circulating tumor DNA (ctDNA) kinetics during ICI have not been comprehensively studied. Methods: We evaluated the predictive role of early dynamic changes of peripheral immune cell subsets in 239 ICI treated metastatic non-small cell lung cancers (mNSCLC) and combined those with ctDNA dynamics in 18 early stage NSCLCs (esNSCLC) treated with neoadjuvant ICI. Blood cell counts were analyzed at baseline, 4 weeks on treatment, and at first radiographic follow up (mNSCLC) or preoperatively (esNSCLC). mNSCLC patients without progression at 6 months were considered to have durable clinical benefit (DCB). We employed eXtreme Gradient Boosting, a decision-tree based machine learning algorithm, to integrate baseline values and early changes in immune cell subsets. To reduce modeling overfitting, we trained an ensemble of models, incorporating 10-fold cross-validation that included feature selection (training n=171) and tested the model in an unseen independent set (n=68). For esNSCLCs, major pathologic response (MPR; ≤10% residual tumor; RT) was determined. We performed Targeted Error Correction next generation sequencing on 60 serial plasma and matched leukocyte DNA samples to assess ctDNA clonal dynamics. Changes in immune cell subsets were correlated with RT, MPR, progression-free (PFS) and overall survival (OS). Results: Our machine learning integrative model predicted DCB with an area under the ROC curve (AUC) of 0.96 for training, 0.72 for cross-validation testing and 0.74 for unseen testing datasets. Feature importance analysis revealed that NLR at 4 weeks, first radiographic follow up, and relative change in NLR from baseline were the strongest predictors of outcome together with relative eosinophil and lymphocyte count. Our model's performance was superior to TMB (AUC=0.52) or PD-L1 (AUC=0.54). For esNSCLCs, change in NLR at 4 weeks after ICI initiation was predictive of tumor regression (p=0.02), such that those with MPR showed significant decrease in NLR. This was also predictive of PFS (log rank p=0.004) and OS (log rank p=0.02). Furthermore, higher eosinophil count at 4 weeks was correlated with MPR (p=0.006) and decreased RT (p=0.006). Immune cell subset kinetics were concordant with ctDNA clearance in all but two patients and importantly NLR dynamics at 4 weeks were predictive of MPR even when ctDNA was undetectable. Conclusions: Early changes in peripheral immune cell subsets together with ctDNA are reflective of anti-tumor immune response during ICI and may more accurately predict ICI response than currently used biomarkers. Citation Format: Michael Hwang, Jenna Canzoniero, Samuel Rosner, Guangfan Zhang, Mara Lanis, Lamia Rhymee, Alexandria Curry, Gavin Pereira, Kristen Marrone, Joshua Reuss, Jarushka Naidoo, Christine Hann, Vincent Lam, Benjamin Levy, David Ettinger, Patrick Forde, Julie Brahmer, Victor Velculescu, Tanguy Seiwert, Valsamo Anagnostou. Early dynamics in peripheral blood immune cell subsets and ctDNA are predictive of outcome to immunotherapy [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 27.

4042 Background: Improving immunotherapy efficacy remains an unmet need in esophagogastric cancer and a deeper understanding of tumor and immune system dynamics during therapy may tailor immuno-oncology approaches. Methods: We performed whole exome sequencing (WES) and bulk RNA sequencing (RNAseq) of 70 serial tumor samples from 23 patients with stage II/III esophageal/gastroesophageal junction (E/GEJ) cancer treated on a phase 1B clinical trial with neoadjuvant nivolumab with or without relatlimab (anti-LAG-3) and chemoradiation followed by surgery (NCT03044613; CA209-906). Pathologic response was measured by tumor regression at the time of resection. Median follow up was 23 months post-surgery. Serial tumor samples were collected prior to therapy, after 2 cycles of induction immune checkpoint blockade (ICB), and at the time of resection. Twenty-two baseline tumor/normal DNA pairs were analyzed by WES and 48 serial tumor samples were analyzed by RNAseq. WES data was analyzed to identify somatic mutations, generate tumor mutation burden (TMB) estimates and assess the fraction of expressed mutations in conjunction with RNAseq data. Immune cell subset composition was determined by RNAseq data deconvolution by CIBERSORT and gene set enrichment analyses were performed utilizing GSEA. B-cell density was inferred by immunoglobulin rearrangements detected by RNAseq. Results: Gene set enrichment expression analyses revealed an upregulation of effector pro-inflammatory cytokines after induction ICB. Interferon-gamma, interferon-alpha and TNF-alpha related genes were significantly upregulated after induction ICB compared to baseline (p < 0.0001). In contrast, significant downregulation of E2F targets (p = 0.002), G2M checkpoint genes (p = 0.005) and DNA damage repair genes (p = 0.004) was observed post ICB; enrichment analyses were independent of response to therapy and treatment arm. While TMB was not predictive of pathologic response (p = 0.22), patients with tumors harboring a higher number of expressed mutations were more likely to achieve a pathologic complete response (pCR; p = 0.026). RNAseq deconvolution analyses revealed a higher B-cell density post ICB induction in tumors with pCR (p = 0.018). Furthermore, an increased baseline content of intra-tumoral activated M1 macrophages differentiated tumors from patients achieving a pCR (p = 0.0034), which was further exemplified post induction ICB. Conclusions: Neoadjuvant immunotherapy induces an inflammatory immune response in the tumor microenvironment that is linked with tumor elimination and pathologic response. Our findings highlight the importance of nuanced multi-omics analyses to understand the wiring of response to immunotherapy and guide therapy for E/GEJ cancer.

BACKGROUND Neutrophil-lymphocyte ratio (NLR) is a measure of systemic inflammation that appears prognostic in localized and advanced non-small cell lung cancer (NSCLC). Increased systemic inflammation portends a poorer prognosis in cancer patients. We hypothesized that low NLR at diagnosis is associated with improved overall survival (OS) in locally advanced NSCLC (LANSCLC) patients. PATIENTS AND METHODS Records from 276 patients with stage IIIA and IIIB NSCLC treated with definitive chemoradiation with or without surgery between 2000 and 2010 with adequate data were retrospectively reviewed. Baseline demographic data and pretreatment peripheral blood absolute neutrophil and lymphocyte counts were collected. Patients were grouped into quartiles based on NLR. OS was estimated using the Kaplan-Meier method. The log-rank test was used to compare mortality between groups. A linear test-for-trend was used for the NLR quartile groups. The Cox proportional hazards model was used for multivariable analysis. RESULTS The NLR was prognostic for OS (p

To determine the prognostic effect of Body Mass Index (BMI) in definitively treated locally advanced NSCLC patients.In this single institution retrospective cohort study, we evaluated 291 patients who were treated for locally advanced NSCLC from 2000 to 2010. They were stratified into four BMI groups based on World Health Organization criteria: underweight (18.5kg/m2), normal weight (18.5 to25kg/m2), overweight (25 to30kg/m2), and obese (≧30kg/m2). Overall survival was analyzed by BMI group.Baseline patient characteristics and treatment parameters were similar between obese and normal weight patients. Increasing BMI was associated with improved overall survival (P=0.011), even when underweight cases were excluded. There was a sustained 31%-58% reduction in mortality of obese relative to normal weight patients (HR 0.68±0.21, 0.61±0.19, and 0.42±0.19, for each year post-treatment respectively). Statin use after diagnosis was highly associated with increasing BMI (P0.001) and predicted improved survival in a multivariate analysis (HR 0.60, 95% CI 0.41-0.89, P=0.011).Obese patients in this retrospective study had significantly improved survival relative to normal weight patients. Our data suggest that the protective effect of obesity in locally advanced NSCLC is not solely due to short-term treatment effects, decreased smoking exposure, or poor prognostic factors from underweight patients. Notably, statin use was also associated with improved survival. Additional studies are needed to clarify the mechanisms and possible concomitant factors underlying the obesity paradox in NSCLC.