Blood Tumor Mutational Burden Research Articles

Abstract B049: Validation of the PGDx elio™ plasma complete for comprehensive genomic profiling of solid tumors through liquid biopsy with Epic Sciences

Abstract Introduction: To help guide treatment decisions and enable clinical trial matching for cancer patients, tissue-based comprehensive genomic profiling (CGP) is an essential precision oncology tool. Liquid biopsy may be used as a complementary approach to assess tumor-specific DNA alterations circulating in the blood when tissue is limited or unavailable. PGDx’s elio™ plasma complete is a next-generation-sequencing (NGS), cell-free DNA (cfDNA) CGP assay that identifies clinically significant variants from the plasma of patients with advanced and metastatic solid cancers. Methods: The PGDx elio plasma complete is a hybrid-capture, DNA-based kitted solution, optimized for 25 ng of cfDNA input with a targeted panel covering coding exons for 521 genes, and is designed to detect single nucleotide variants (SNVs), small insertions and deletions (indels), copy number amplifications (CNAs), and translocations. It also evaluates blood MSI and tumor mutational burden (bTMB) to guide immunotherapy selection. This study presents the validation of the PGDx elio plasma complete assay to enable the processing of patient blood samples in Epic Sciences' CAP/CLIA-certified laboratory. In the validated configuration, patient plasma will be received and isolated at Epic Sciences and subsequently shipped to PGDx/Labcorp Oncology for cfDNA extraction, library preparation, sequencing, and bioinformatics analysis. Finally, clinical report generation will be carried out by Velsera’s CGW software. Analytical validation was assessed using 105 replicates of SeraCare® (n=21) and Horizon (n=3) reference materials to establish analytical sensitivity, accuracy, and reproducibility and repeatability. Specificity was assessed using 20 non-cancerous plasma samples that were also tested orthogonally. For clinical validation a cohort of >150 cancer patient plasma samples were evaluated, including lung, breast, colorectal, head and neck, pancreatic, and prostate cancers, and compared to Illumina’s TSO500 ctDNA or Labcorp Plasma Focus results. Results: Analytical sensitivity and specificity were evaluated for SNVs, indels, CNAs, fusions, MSI and bTMB. Across all variant types, sensitivity was ≥90%, and specificity was ≥95% at LoDs. The reproducibility and repeatability study resulted in ≥95% agreement for all variant types. Analytical sensitivity was calculated using vendor-verified variants in each control. SeraCare® controls and Horizon reference materials were used to assess variant concordance with an aggregate analytical performance across all control samples with ≥95% PPA and NPA for all variant types. Conclusions: The validation of the PGDx elio plasma complete liquid biopsy assay as part of the Epic Sciences' CAP/CLIA diagnostic workflow for cfDNA-based NGS demonstrates that the liquid biopsy approach is highly sensitive, specific, accurate, reproducible, and robust for comprehensive genomic profiling to complement tissue- based testing and inform clinical decision making. Citation Format: Cynthia Maddox, Kenneth C Valkenburg, Cesar Nalvarte, Amanda Harvey, Amy Greer, Ellen L Verner, Renee L Sears, Martin Blankford, Eric A Severson, Taylor J Jensen, Marcia Eisenberg, Shakti Ramkissoon. Validation of the PGDx elio™ plasma complete for comprehensive genomic profiling of solid tumors through liquid biopsy with Epic Sciences [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B049.

Sintilimab combined with anlotinib and chemotherapy as second-line or later therapy in extensive-stage small cell lung cancer: a phase II clinical trial

Treatment options for patients with relapsed extensive-stage small cell lung cancer (ES-SCLC) remain scarce. This study aims to evaluate the efficacy and safety of combining anlotinib and sintilimab plus chemotherapy as a second line or later therapy for ES-SCLC patients. This is a phase II clinical trial (ChiCTR2100049390) conducting at Shandong Cancer Hospital. Patients with ES-SCLC and received at least one prior systemic treatment were enrolled. The trial design involved a combination therapy (sintilimab, anlotinib, and nab-paclitaxel) administered over six 21-day cycles, followed by maintenance sintilimab therapy. The primary endpoint was objective response rate (ORR). Circulating tumor DNA sequencing was employed for exploratory analysis. From July 2021 to April 2023, 25 eligible patients were enrolled. The confirmed ORR was 60% (95% CI: 38.7–78.9%) and the DCR was 76% (95% CI: 54.9–90.6%). The mPFS was 6.0 months (95% CI: 5.4–9.7), and the 6-month PFS rate was 49.2%. The mOS was 13.4 months (95% CI: 11.8-NR), with a 12-month survival rate of 62.2%. Treatment-related adverse events (TRAEs) of any grade occurred in 80% of patients, with the most common being fatigue (40%) and nausea (32%). TRAEs of Grade 3 or higher were reported in 12% of patients. ctDNA analysis indicated that low on-treatment blood tumor mutation burden was associated with longer PFS and OS and a potential role of KMT2D mutation in treatment resistance. This combination therapy shows promising efficacy and a manageable safety profile as a second-line or later treatment for ES-SCLC, with genomic insights providing potential biomarkers for treatment response.

Blood Tumor Mutational Burden Alone Is Not a Good Predictive Biomarker for Response to Immune Checkpoint Inhibitors in Patients With Gastrointestinal Tumors.

There has been a controversy about the predictive value of tissue-TMB-H for immune checkpoint inhibitors (ICIs) with limited data regarding blood-TMB (bTMB) in GI tumors. We aim to evaluate the predictive value of bTMB compared with MSI-H in GI tumors. Patients with unresectable/metastatic GI cancer, harboring either MSS with bTMB-H (≥10mut/Mb) or dMMR/MSI-H who received ICI were included. We compared ICIs' efficacy between MSS-bTMB-H (N=45) versus MSI-H (N=50) in GI tumors. Ninety-five patients were identified with the majority having colorectal (49.5%) or esophagogastric (34.7%) cancers. MSS-bTMB-H group had more esophagogastric cancer and later-line ICI recipients, with no significant differences in other known prognostic variables. At a median follow-up of 9.4 months, MSI-H group showed superior ORR (58.0% vs. 26.7%), DCR (84.0% vs. 42.2%), DoR (not-reached vs. 7.6mo), PFS (22.5 vs. 3.8mo), and OS (Not-reached vs. 10.1mo) compared with MSS-bTMB-H. Multivariable analysis showed that MSI-H was an independent favorable factor over MSS-bTMB-H for PFS (HR=0.31, CI 0.15-0.63, P =0.001) and OS (HR=0.33, CI 0.14-0.80, P =0.014). MSI-H group showed favorable outcomes compared with MSS-bTMB-16+ (ORR: 58.0% vs. 26.9%; DCR: 84.0% vs. 42.3%; PFS:22.5 vs. 4.0mo) and MSS-bTMB-20+ (ORR: 58.0% vs. 31.6%; DCR: 84.0% vs. 42.1%; PFS:22.5 vs. 3.2mo). There was no difference between MSS-bTMB10-15 compared with MSS-bTMB-16+ in ORR, DCR, and PFS, or between MSS-bTMB10-19 compared with MSS-bTMB20+. Regardless of bTMB cutoff at 10, 16, or 20, bTMB-H did not appear to be a predictive biomarker in MSS GI tumors in this retrospective analysis.

Identification and Application of Emerging Biomarkers in Treatment of Non-Small-Cell Lung Cancer: Systematic Review.

Non-small-cell lung cancer (NSCLC) comprises approximately 85% of all lung cancer cases, often diagnosed at advanced stages, which diminishes the effective treatment options and survival rates. This systematic review assesses the utility of emerging biomarkers-circulating tumor DNA (ctDNA), microRNAs (miRNAs), and the blood tumor mutational burden (bTMB)-enhanced by next-generation sequencing (NGS) to improve the diagnostic accuracy, prognostic evaluation, and treatment strategies in NSCLC. Analyzing data from 37 studies involving 10,332 patients from 2020 to 2024, the review highlights how biomarkers like ctDNA and PD-L1 expression critically inform the selection of personalized therapies, particularly beneficial in the advanced stages of NSCLC. These biomarkers are critical for prognostic assessments and in dynamically adapting treatment plans, where high PD-L1 expression and specific genetic mutations (e.g., ALK fusions, EGFR mutations) significantly guide the use of targeted therapies and immunotherapies. The findings recommend integrating these biomarkers into standardized clinical pathways to maximize their potential in enhancing the treatment precision, ultimately fostering significant advancements in oncology and improving patient outcomes and quality of life. This review substantiates the prognostic and predictive value of these biomarkers and emphasizes the need for ongoing innovation in biomarker research.

Prediction accuracy of biomarkers for response to immune checkpoint inhibitors in advanced non-small cell lung cancer: A systematic review and meta-analysis.

e14652 Background: To compare the predictive accuracy of PD-L1 IHC, tissue or blood tumor mutation burden (tTMB, bTMB), gene expression profiling (GEP), driver gene mutation, and combined marker for immunotherapy response of advanced non-small cell lung cancer (NSCLC). Methods: Systematic searches of PubMed, Cochrane CENTRAL, and Embase were conducted from inception to July 6, 2023. Clinical trials involved with predictive biomarkers exploration for immune checkpoint inhibitors (ICIs) in advanced NSCLC were eligible. The response according to the biomarkers were extracted. The primary outcome was the area under the curve (AUC) of the summary receiver operating characteristic (SROC). Sensitivity, specificity, likelihood ratios and predictive values were evaluated. The subgroup analysis of different PD-L1 cut-off, TMB test and driver gene mutation were performed. Results: 36 articles of 59 biomarker tests were included. The AUC of combined marker (0.75; 95%CI, 0.71-0.78) was higher than PD-L1 (0.64; 95%CI, 0.60-0.68), tTMB (0.64; 95%CI, 0.60-0.68), bTMB (0.68; 95%CI, 0.64-0.72), GEP (0.67; 95%CI, 0.63-0.71), and driver gene mutation (0.51; 95%CI, 0.47-0.55). Combined marker also had higher specificity, positive likelihood ratio and positive predictive value than single biomarkers (Table). In subgroup analysis, PD-L1 with cut-off 50% had higher AUC (0.67; 95%CI, 0.63-0.71) than cut-off 1% (0.62; 95%CI, 0.58-0.66); EGFR mutation had higher AUC (0.69; 95%CI, 0.65-0.73) than KRAS (0.53; 95%CI, 0.49-0.58); tTMB measured by Whole Exosome Sequencing (WES) had approximate AUC to that measure by targeted gene panel. The predictive accuracy of the biomarkers was further compared for patients receiving ICI-based combination therapy. The AUC of combined marker (0.70; 95%CI, 0.66-0.74) remained the highest for combination therapy. Conclusions: Combined marker (PD-L1+tTMB/bTMB/GEP) has superior predictive accuracy than single biomarkers for immunotherapy response of NSCLC. bTMB is a promising liquid biopsy biomarker. The prescription of ICI for patients with EGFR mutation should be cautious. Further investigation is warranted to precisely identify biomarkers in various clinical settings. [Table: see text]

Prognostic significance of ctDNA mutation analysis in patients with HER2-positive breast cancer undergoing neoadjuvant chemotherapy.

e12654 Background: Neoadjuvant chemotherapy (NACT) combined with dual anti-HER2 agents followed by surgical resection is the standard treatment for stage II-III HER2- positive breast cancer (BC). Despite the availability of effective therapies such as ado-trastuzumab emtansine (T-DM1) and neratinib, identifying patients at high risk of disease recurrence remains challenging. To evaluate whether circulating tumor DNA (ctDNA) can be used as a biomarker to assess treatment response in patients with HER2-positive EBC treated with neoadjuvant anti-HER2 therapy. ctDNA burden can serve as a useful determinant for escalating or de-escalating (neo)adjuvant strategy in EBC patients. This study aimed to assess the role of ctDNA mutation analysis using low-pass whole-genome sequencing (lpWGS) and whole exome sequencing (WES) in evaluating blood copy-number burden (bCNB), genomic alterations, mutational signatures, blood tumor mutational burden (bTMB), and minimal residual disease (MRD) monitoring. Methods: A total of 46 HER2-positive BC patients who underwent NACT combined with dual anti-HER2 agents and surgical resection were enrolled, including 28 clinical stage II and 17 III patients. Plasma samples were collected at four time points: before NACT, after 3 cycles, at the completion of NACT, and after surgery. lpWGS was utilized to assess bCNB, while WES was performed to evaluate genomic alterations, mutational signatures, and bTMB. Mutations selected from the baseline sample were monitored for MRD. In cases where lpWGS yielded negative results, targeted NGS sequencing was conducted. Somatic mutations are identified by whole exome sequencing across 20,000 genes (with boosted sequencing coverage for 600 cancer-related genes). Between 4-50 personalized somatic mutations or fusions are selected for each patient. This personalized panel together with a fixed MRD core panel is utilized for MRD detection and monitoring. Results: Among the enrolled patients, nodal metastasis was observed in 29 patients and 29 were hormone receptor-positive, and 36 achieved pCR (78.2%). Positive ctDNA was detected 58.8% of patients. Higher nodal stage was associated with ctDNA positivity at baseline. MRD positivity after chemotherapy was observed in 4 patients and 2 patients achieved pCR TP53 was the most frequently altered gene, followed by PIK3CA, MAP3K1, REV3L, MYC, MAP2K4, ERBB3, ATRX, ATM and ALK. This pattern is comparable to that observed in previous studies on BC. Non-PCR patients commonly exhibit MAP3K1 and MAP2K4 alteration is their ctDNA. Conclusions: The identification of MRD, regardless of pCR status, can be valuable for risk stratification and personalized treatment decision-making. Further studies are warranted to validate these findings and explore the clinical implications of ctDNA mutation analysis in HER2-positive BC management.

Abstract PO4-01-04: Clinical significance of Circulating tumor DNA (ctDNA) analysis in predicting pathologic complete remission in HER2-positive Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy

Abstract Background: Neoadjuvant chemotherapy (NACT) combined with dual anti-HER2 agents followed by surgical resection is the standard treatment for stage II-III HER2-positive breast cancer. Despite the availability of effective therapies as an adjuvant treatment such as ado-trastuzumab emtansine (T-DM1) and neratinib, pathologic complete remission (pCR) strongly predicts survival. This study aimed to investigate the role of ctDNA analysis in predicting pCR and clinical outcome using comprehensive ctDNA analysis. Methods: A total of 34 HER2-positive breast cancer patients who underwent NACT combined with dual anti-HER2 agents and surgical resection were enrolled, including 22 clinical stage II and 12 III patients. Plasma samples were collected at four-time points: before neoadjuvant chemotherapy, after 3 cycles, at the completion of neoadjuvant chemotherapy, and after surgery. low-pass whole-genome sequencing (lpWGS) and whole exome sequencing (WES) in evaluating blood copy-number burden (bCNB), genomic alterations, mutational signatures, blood tumor mutational burden (bTMB). Mutations selected from the baseline sample were monitored for Molecular residual disease (MRD) monitoring. lpWGS yielded negative results, targeted NGS sequencing was conducted. Results: Among the enrolled patients (N=34), 21 patients (61.8%) had nodal metastasis and 22 patients (64.7%) were hormone receptor-positive. A total 26 patients (76.5%) achieved pCR. Positive ctDNA was detected in 20 patients (58.8%) at baseline. Higher nodal stage was significantly associated with ctDNA positivity at baseline. After surgery, MRD positivity was observed in 3 patients (8.8%). Among the 26 patents who acheived pCR, only 1 patient (3.8%) showed MRD positivity, while 2 non-pCR patients (25%) demonstrated MRD positivity. Conclusion: The presence of MRD was significantly associated with non-pCR status. The presence of ctDNA at baseline was associated with a higher nodal stage, suggesting its potential as a prognostic marker. Further studies with larger cohorts are warranted to validate these findings and explore the utility of ctDNA analysis for personalized treatment decisions in HER2-positive breast cancer. Citation Format: Byeongju Kang, Soo Jung Lee, Jeeyeon Lee, In Hee Lee, Jee-Young Park, Ho Yong Park, Tiantian Zheng, Lu Tan, Liam Il-Young Chung, Shidong Jia, Pan Du, Young Kwang Chae, Yee Soo Chae. Clinical significance of Circulating tumor DNA (ctDNA) analysis in predicting pathologic complete remission in HER2-positive Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-01-04.

Abstract LB107: Association of circulating free DNA (cfDNA) maximum variant allele frequency (mVAF) levels with clinical outcomes in patients (pts) with metastatic nonsquamous non-small cell lung cancer (NSCLC) treated with pembrolizumab (pembro) + chemotherapy (chemo) in the phase 2 KEYNOTE-782 trial

Abstract Background: The phase 2 KEYNOTE-782 trial (NCT03664024) was designed to prospectively evaluate plasma-derived biomarkers of response to first-line pembro + chemo in pts with metastatic nonsquamous NSCLC. This exploratory analysis of KEYNOTE-782 evaluated the association of cfDNA mVAF levels with clinical outcomes. Methods: Adults with untreated stage IV nonsquamous NSCLC received pembro 200 mg Q3W + chemo (pemetrexed + carboplatin or cisplatin). cfDNA was isolated from plasma samples collected at baseline (BL) and cycle 2 (C2) and sequenced using a targeted NGS-based assay previously used to assess blood tumor mutational burden from cfDNA for the primary analysis. cfDNA mVAF was quantified for each patient. The associations of BL cfDNA mVAF (all pts) and change in cfDNA mVAF from BL at C2 (pts with ≥1 detectable variant at BL; cfDNA+) as continuous variables with ORR, PFS, and OS were evaluated; 1-sided α was prespecified at 0.05 after multiplicity adjustment. ORR, PFS, and OS were also descriptively evaluated by cfDNA bins (BL mVAF, >0 vs 0; mVAF change from BL, ≥median vs <median). Results: Of 101 pts with evaluable cfDNA samples at BL, 83 (82.2%) were cfDNA+; 74 of these 83 pts had evaluable cfDNA samples at C2. Low BL mVAF was associated with improved ORR, PFS, and OS (P <0.01, each). The area under the receiver operating characteristic curve (AUROC) for discriminating response was 0.61 (95% CI, 0.50-0.72). Among pts with BL mVAF >0 (n = 83) vs 0 (n = 18), ORR (95% CI) was 38.6% (28.1-49.9) vs 55.6% (30.8-78.5), median PFS (95% CI) was 6.6 mo (4.9-9.0) vs 19.7 mo (9.8-not reached [NR]), and median OS (95% CI) was 13.6 mo (9.4-22.5) vs 31.6 mo (20.1-NR). Larger reductions in mVAF from BL at C2 were associated with improved ORR, PFS, and OS (P <0.05, each). The AUROC for discriminating response was 0.69 (95% CI, 0.57-0.82). After adjustment for best overall response, change in mVAF from BL at C2 was not associated with PFS or OS (P >0.05). In a descriptive evaluation for pts with a mVAF change from BL at C2 ≥median of 0.15 (n = 37) vs <median (n="37)," orr="" (95%="" ci)="" was="" 18.9%="" (8.0-35.2)="" vs="" 64.9%="" (47.5-79.8),="" median="" pfs="" 5.1="" mo="" (4.2-11.1)="" 7.2="" (6.2-21.7),="" and="" os="" 12.8="" (7.9-18.1)="" 25.9="" (15.3-nr).="" conclusions:="" in="" pts="" with metastatic nonsquamous NSCLC treated with pembro + chemo, low cfDNA mVAF levels at BL and larger reductions in cfDNA mVAF levels were associated with improved clinical outcomes. However, after correction for clinical response, the prognostic value of cfDNA mVAF reduction was not significant. Further studies are warranted to elucidate the utility of cfDNA for disease monitoring in this setting. Citation Format: Jair Bar, Emilio Esteban, Delvys Rodríguez-Abreu, Santiago Ponce-Aix, Zsuzsanna Szalai, Enriqueta Felip, Maya Gottfried, Mariano Provencio Pulla, Andrew Robinson, Andrea Fülöp, Suman Bannur Rao, D. Ross Camidge, Giovanna Speranza, Nathan Hunkapiller, Robert McDaniel, Byoungsok Jung, David Burkhardt, Ruth Mauntz, Cai Chen, Carol Pena, Razvan Cristescu, Elisha J. Dettman, Steven M. Townson, Julie Kobie, Tibor Csőszi. Association of circulating free DNA (cfDNA) maximum variant allele frequency (mVAF) levels with clinical outcomes in patients (pts) with metastatic nonsquamous non-small cell lung cancer (NSCLC) treated with pembrolizumab (pembro) + chemotherapy (chemo) in the phase 2 KEYNOTE-782 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB107.

Dynamic bTMB combined with residual ctDNA improves survival prediction in locally advanced NSCLC patients with chemoradiotherapy and consolidation immunotherapy

BackgroundLiquid biopsy-based biomarkers, including circulating tumor DNA (ctDNA) and blood tumor mutational burden (bTMB), are recognized as promising predictors of prognoses and responses to immune checkpoint inhibitors (ICIs), despite insufficient sensitivity of single biomarker detection. This research aims to determine whether the combinatorial utility of longitudinal ctDNA with bTMB analysis could improve the prognostic and predictive effects. MethodsThis prospective two-center cohort trial, consisting of discovery and validation datasets, enrolled unresectable locally advanced non-small-cell lung cancer (LA-NSCLC) patients and assigned them to chemoradiotherapy (CRT) or CRT + consolidation ICI cohorts from 2018 to 2022. Blood specimens were collected pretreatment, 4 weeks post-CRT, and at progression to assess bTMB and ctDNA using 486-gene next-generation sequencing. Dynamic ∆bTMB was calculated as post-CRT bTMB minus baseline bTMB levels. Decision curve analyses were performed to calculate Concordance index (C-index). ResultsOne hundred twenty-eight patients were enrolled. In the discovery dataset (n = 73), patients treated with CRT and consolidation ICI had significantly longer overall survival (OS; median not reached [NR] vs 20.2 months; P < 0.001) and progression-free survival (PFS; median 25.2 vs 11.4 months; P = 0.011) than those without ICI. Longitudinal analysis demonstrated a significant decrease in ctDNA abundance post-CRT (P < 0.001) but a relative increase with disease progression. Post-CRT detectable residual ctDNA correlated with significantly shorter OS (median 18.3 months vs NR; P = 0.001) and PFS (median 7.3 vs 25.2 months; P < 0.001). For patients with residual ctDNA, consolidation ICI brought significantly greater OS (median NR vs 14.8 months; P = 0.005) and PFS (median 13.8 vs 6.2 months; P = 0.028) benefit, but no significant difference for patients with ctDNA clearance. Dynamic ∆bTMB was predictive of prognosis. Patients with residual ctDNA and increased ∆bTMB (∆bTMB > 0) had significantly worse OS (median 9.0 vs 23.0 months vs NR; P < 0.001) and PFS (median 3.4 vs 7.3 vs 25.2 months; P < 0.001). The combinatorial model integrating post-CRT ctDNA with ∆bTMB had optimal predictive effects on OS (C-index = 0.723) and PFS (C-index = 0.693), outperforming individual features. In the independent validation set, we confirmed residual ctDNA predicted poorer PFS (median 50.8 vs 14.3 months; P = 0.026) but identified more consolidation ICI benefit (median NR vs 8.3 months; P = 0.039). The combined model exhibited a stable predictive advantage (C-index = 0.742 for PFS). ConclusionsThe multiparameter assay integrating qualitative residual ctDNA testing with quantitative ∆bTMB dynamics improves patient prognostic risk stratification and efficacy predictions, allowing for personalized consolidation therapy for LA-NSCLC.

Prediction performance comparison of biomarkers for response to immune checkpoint inhibitors in advanced non-small cell lung cancer.

The aim of the present study was to compare the predictive accuracy of PD-L1 immunohistochemistry (IHC), tissue or blood tumor mutation burden (tTMB, bTMB), gene expression profile (GEP), driver gene mutation, and combined biomarkers for immunotherapy response of advanced non-small cell lung cancer (NSCLC). In part 1, clinical trials involved with predictive biomarker exploration for immunotherapy in advanced NSCLC were included. The area under the curve (AUC) of the summary receiver operating characteristic (SROC), sensitivity, specificity, likelihood ratio and predictive value of the biomarkers were evaluated. In part 2, public datasets of immune checkpoint inhibitor (ICI)-treated NSCLC involved with biomarkers were curated (N = 871). Odds ratio (OR) of the positive versus negative biomarker group for objective response rate (ORR) was measured. In part 1, the AUC of combined biomarkers (0.75) was higher than PD-L1 (0.64), tTMB (0.64), bTMB (0.68), GEP (0.67), and driver gene mutation (0.51). Combined biomarkers also had higher specificity, positive likelihood ratio and positive predictive value than single biomarkers. In part 2, the OR of combined biomarkers of PD-L1 plus TMB (PD-L1 cutoff 1%, 0.14; cutoff 50% 0.13) was lower than that of PD-L1 (cutoff 1%, 0.33; cutoff 50% 0.24), tTMB (0.28), bTMB (0.48), EGFR mutation (0.17) and KRAS mutation (0.47), for distinguishing ORR of patients after immunotherapy. Furthermore, positive PD-L1, tTMB-high, wild-type EGFR, and positive PD-L1 plus TMB were associated with prolonged progression-free survival (PFS). Combined biomarkers have superior predictive accuracy than single biomarkers for immunotherapy response of NSCLC. Further investigation is warranted to select optimal biomarkers for various clinical settings.

Abstract 7491: ctDNA liquid biopsy result among gynecologic cancer patients: Does it give additional information

Abstract Objectives: There are many target molecules for precisional cancer therapy Precision diagnosis-based target treatment is believed to be better survival approach compared than conventional treatment. Predictive markers are variant and vast. This study evaluated the initial experience of ctDNA liquid biopsy as a precision tumor marker for gynecologic cancer patients? Methods: we retrospectively identified ovary/endometrial and cervical cancer patients who approved ctDNA test for various purpose. Two methods were used. Guardant 360 and Foundation One Liquid CDx. Results: There were 31 cases. Median age was 60(46-84). Half of cases were ovarian cancer(17/31 55%).Cervix and endometrial cancer are 12(39%) and 2(6%).We found about ten percentages of actional genetic mutation patients(3/31).Two BRCA1 mutation(each patients have variant allele frequency(VAF) percentages are 50.4,49.6).One high bTMB(blood tumor mutational burden)was 21.There was no mutation in 6/31(19 %). Conclusions: It seems additional supportive information may be acquired through well-established ctDNA liquid biopsy test among this population. Role of ctDNA liquid biopsy test for precision tumor marker should be investigated for future study. Keywords: Liquid biopsy; Precision medicine; Tumor marker Citation Format: Min Kyu Kim. ctDNA liquid biopsy result among gynecologic cancer patients: Does it give additional information [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7491.

Abstract 6557: Longitudinal cell-free tumor load dynamics represent an early endpoint for immunotherapy response in non-small cell lung cancer

Abstract Background: Liquid biopsies can inform treatment strategies by rapidly and accurately assessing tumor burden; with emerging data supporting the clinical utility of circulating cell-free tumor DNA (ctDNA) as an early endpoint of immunotherapy response. Methods: We performed targeted error-correction sequencing of ctDNA (n=330) and matched white blood cell (WBC) DNA (n=109) from 109 patients with advanced non-small cell lung cancer (NSCLC) who received immune checkpoint inhibitor monotherapy (n=79) or immunotherapy-containing combination regimens (n=30). Variant cellular origin was determined using a tumor-independent WBC DNA-informed approach. The maximum mutant allele fraction (maxMAF) of tumor-derived mutations was tracked longitudinally to assess changes in total cell-free tumor load (cfTL). Results: Among 3,418 variants with resolved cellular origin, 2,452 (72%) were tumor-derived, 125 (4%) were germline, while 841 (25%) were related to clonal haematopoiesis (CH). Cell-free DNA fragments harbouring tumor-derived mutations were significantly shorter than fragments containing wild-type alleles; however, fragment length alone was insufficient to distinguish CH variants from wild-type. Overall, 97 (89%) patients with &amp;gt;1 detectable tumor mutation were considered for cfTL tracking and molecular response assessment. Clearance of cfTL indicated molecular response (mR) whereas persistence indicated molecular progression (mPD). Time-course analyses, aiming to establish the optimal timing for molecular response assessment, revealed that early cfTL clearance within 3-9 weeks was significantly linked to improved survival outcomes. Notably, blood tumor mutation burden above or below 16 mutations/megabase showed no association with overall survival (OS; log rank p=0.98) or progression-free survival (PFS; log rank p=0.95). Of 81 patients evaluated for early on-therapy molecular responses, 28 (35%) were classified as having an mR, while 53 (65%) patients were classified in the mPD category. The median time to mR was 5 weeks in patients receiving immune checkpoint monotherapy and 6 weeks in patients receiving chemo-immunotherapy combination regimens. Patients in the mR group achieved longer OS (log rank p=0.001) and PFS (log rank p=0.0003) compared to those with mPD. Assessment of landmark PFS at 6 months demonstrated a significant association between ctDNA molecular response and durable clinical benefit (DCB) in patients receiving immune checkpoint monotherapy (p &amp;lt; 0.001, Fisher’s exact test), and a numerical association with DCB in patients on chemo-immunotherapy (p=0.07, Fisher’s exact test). Conclusions: Longitudinal tracking of ctDNA provides a real-time and accurate approach for monitoring immunotherapy response. These findings underscore the clinical utility of employing ctDNA molecular response to inform clinical decision-making. Citation Format: Lavanya Sivapalan, Bahar Alipanahi, Archana Balan, Jamie E. Medina, Noushin Niknafs, Erica Peters, Jaime Wehr, Alissa Konicki, Mimi Najjar, Bryan Chesnick, Gavin Pereira, Sarah Rongione, Nisha Rao, Michael Rongione, Brittany Shavatt, Lorenzo Rinaldi, Benjamin Levy, Victor E. Velculescu, Patrick M. Forde, Valsamo Anagnostou. Longitudinal cell-free tumor load dynamics represent an early endpoint for immunotherapy response in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6557.

Abstract 6469: The association of mismatch repair gene alterations with elevated tumor mutational burden in microsatellite stable gastrointestinal cancers

Abstract Background: Benefit from single-agent immune checkpoint inhibitor has been observed in 5-10% of the patients with microsatellite stable (MSS) gastrointestinal (GI) cancers. However, a reliable predictive marker to immune checkpoint inhibitor has not been discovered in this patient population. We sought to assess the incidence of mismatch repair (MMR) gene alterations and their association with tumor mutational burden (TMB) in MSS GI cancers. Methods: This is a retrospective cohort study of patients with advanced GI cancer who had molecular profiling of the tumor using either Guardant360 blood-based or Tempus tissue next generation sequencing (NGS) platforms between 2020 and 2023. We assessed four MMR genes including MLH1, MSH2, MSH6 and PMS2 for alterations. Continuous variables were summarized as median and interquartile range (IQR) and compared using Mann-Whitney test. Categorical variables were summarized as counts and proportions and compared using Fisher’s exact test. Results: A total of 2920 patients with Guardant360 were included with a median age of 65 (IQR: 56-73) years. 870 (30%) patients had pancreas cancer; 846 (29%) patients had colorectal cancer; 688 (24%) patients had biliary tract cancers; and the rest had other GI cancers. Among patients evaluable for microsatellite instability (MSI) status, 58 patients (2%) had MSI-high GI cancers, 31 (53%) of whom had MMR gene alterations. 2445 (98%) patients had MSS GI cancers, 130 (5.3%) of whom had MMR gene alterations. Patients with MSS GI cancers bearing MMR gene alterations had a median age of 70 (IQR: 62-75) years, significantly older than those without MMR gene alterations (p&amp;lt;0.001). In addition, patients with MMR gene alterations had significantly higher median blood TMB of 11 (IQR: 7.4-16) mut/Mb compared to 7.7 (IQR: 4.8-11) mut/Mb in patients without MMR gene alterations. In MSS GI cancers, MMR gene alterations are more commonly associated with the presence of alterations in APC (p=0.03), TP53 (p=0.01), CTNNB1 (p&amp;lt;0.001) and SMAD4 (p=0.01). 1453 patients with GI cancer and Tempus tissue NGS data were included as a validation cohort. They had comparable demographics with the Guardant360 cohort. 14 (1.6%) of 896 patients with MSS GI cancers had MMR gene alterations, significantly fewer (p&amp;lt;0.001) than those found in the Guardant360 cohort. Patients with MMR gene alterations and wild type POLE had significantly higher (p=0.001) median TMB of 9.8 (IQR: 4.8-14) mut/Mb compared to 4.7 (IQR: 3.2-6.3) mut/Mb in patients without MMR gene alterations. Conclusions: MMR gene alterations can be identified by clinical NGS platforms in a small proportion of patients with MSS GI cancers. They are associated with elevated TMB, which may suggest a hypermutated profile serving as a basis for potential role of immune checkpoint inhibitor in MSS GI cancers. Citation Format: Conor D. O'Donnell, Mojun Zhu, Hao Xie. The association of mismatch repair gene alterations with elevated tumor mutational burden in microsatellite stable gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6469.

Outcomes in biomarker-selected subgroups from the KESTREL study of durvalumab and tremelimumab in recurrent or metastatic head and neck squamous cell carcinoma

BackgroundSelective biomarkers may improve outcomes in patients with recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) treated with immune checkpoint inhibitor therapy. We investigated three independent biomarkers for association with efficacy in the randomized, phase III KESTREL study (NCT02551159) of first-line durvalumab monotherapy or durvalumab plus tremelimumab versus the EXTREME regimen: programmed cell death ligand-1 (PD-L1) immunohistochemistry, blood tumor mutational burden (bTMB) via circulating tumor DNA, and neutrophil-to-lymphocyte ratio (NLR).MethodsTumor or blood samples from patients enrolled in the KESTREL study were analyzed for PD-L1, bTMB, and NLR. Associations with overall survival (OS) or objective response rates (ORRs) were evaluated based on prespecified cut-offs for PD-L1 (tumor cell [TC] ≥ 50%/immune cell ≥ 25% or TC ≥ 25%), bTMB (≥ 16 mutations [mut] per megabase [Mb]), and NLR (≤ 7). Ad hoc analyses of exploratory cut-offs were performed.ResultsPrespecified or exploratory cut-offs for PD-L1 did not enrich for ORR or OS for durvalumab monotherapy or durvalumab plus tremelimumab versus EXTREME. In the bTMB ≥ 16 mut/Mb subgroup, OS hazard ratios (95% confidence interval) for durvalumab monotherapy and durvalumab plus tremelimumab versus EXTREME were 0.90 (0.48–1.72) and 0.69 (0.39–1.25), respectively. Complete response rates were 8.6% with durvalumab plus tremelimumab and 4.3% with EXTREME (≥ 16 mut/Mb subgroup). No improvement in OS was observed for durvalumab monotherapy or durvalumab plus tremelimumab versus EXTREME at prespecified or exploratory NLR cut-offs.ConclusionsbTMB demonstrated potential utility for selecting patients with R/M HNSCC who benefited from durvalumab with or without tremelimumab versus EXTREME.Trial registration ClinicalTrials.gov identifier NCT02551159.

Blood tumor mutational burden and response to pembrolizumab plus chemotherapy in non–small cell lung cancer: KEYNOTE-782

BackgroundFirst-line pembrolizumab plus chemotherapy has shown clinical benefit in patients with metastatic non–small cell lung cancer (NSCLC) regardless of tissue tumor mutational burden (tTMB) status. Blood tumor mutational burden (bTMB), assessed using plasma-derived circulating tumor DNA (ctDNA), may be a surrogate for tTMB. The KEYNOTE-782 study evaluated the correlation of bTMB with the efficacy of first-line pembrolizumab plus chemotherapy in NSCLC. MethodsPreviously untreated patients with stage IV nonsquamous NSCLC received pembrolizumab 200 mg plus pemetrexed 500 mg/m2 and investigator’s choice of carboplatin area under the curve 5 mg/mL/min or cisplatin 75 mg/m2 for 4 cycles, then pembrolizumab plus pemetrexed for ≤31 additional cycles every 3 weeks. Study objectives were to evaluate the association of baseline bTMB with objective response rate (ORR) (RECIST v1.1 by investigator assessment; primary), progression-free survival (PFS; RECIST v1.1 by investigator assessment), overall survival (OS), and adverse events (AEs; all secondary). A next-generation sequencing assay (GRAIL LLC) with a ctDNA panel that included lung cancer-associated and immune gene targets was used to measure bTMB. Results117 patients were enrolled; median time from first dose to data cutoff was 19.3 months (range, 1.0–35.5). ORR was 40.2 % (95 % CI 31.2–49.6 %), median PFS was 7.2 months (95 % CI 5.6–9.8) and median OS was 18.1 months (95 % CI 13.5–25.6). Treatment-related AEs occurred in 113 patients (96.6 %; grade 3–5, n = 56 [47.9 %]). Of patients with evaluable bTMB (n = 101), the area under the receiver operating characteristics curve for continuous bTMB to discriminate response was 0.47 (95 % CI 0.36–0.59). Baseline bTMB was not associated with PFS or OS (posterior probabilities of positive association: 16.8 % and 7.8 %, respectively). ConclusionsAEs were consistent with the established safety profile of first-line pembrolizumab plus chemotherapy in NSCLC. Baseline bTMB did not show evidence of an association with efficacy.

Association of candidate alterations with primary resistance to KRAS G12D targeting in colorectal cancer.

170 Background: KRAS-mutant colorectal cancer (CRC) has a worse prognosis and greater resistance to therapy, attracting tremendous efforts to develop both allele-specific and pan-KRAS targeting agents including KRAS G12C and G12D inhibitors. CRC has demonstrated both primary and acquired resistance to KRAS G12C inhibitors, which can be better evaluated by circulating tumor DNA (ctDNA)-based next generation sequencing (NGS) methods without tissue sampling bias. Here, we utilized clinical ctDNA data to delineate the landscape of candidate alterations associated with primary resistance to KRAS G12D targeting in CRC. Methods: Samples from advanced CRC patients with Guardant360 data in 2021 were categorized into a KRAS G12D group and comparator groups of KRAS G12C and KRAS not detected (ND) according to KRAS mutation status. Demographics, MSI-H status, and blood tumor mutational burden (bTMB) were summarized and compared between groups using t-test for continuous variables and Fisher’s exact test for categorical variables. Co-occurrence patterns of alterations between KRAS and other genes were assessed. Primary resistance mutations were inferred by acquired resistance findings to KRAS G12C inhibitors reported by Awad et al. N Engl J Med. 2021 and Tanaka et al. Cancer Discov. 2021. Results: Among approximately 2500 samples, 16.8% had KRAS G12D, the most common KRAS mutation in CRC. The mean allele frequency of KRAS G12D (11.4%) is comparable to that of KRAS G12C (10.9%). Mean age (61.6 years), MSI-H frequency (3.8%), and mean bTMB (14.2 Mut/Mb) of KRAS G12D group is comparable to those of KRAS ND group. This contrasts with KRAS G12C with significantly lower mean age (60.2 years, p=0.01), lower MSI-H frequency (0.56%, p=0.03), and higher mean bTMB (16.3 Mut/Mb, p=0.02) compared to KRAS ND. Both KRAS G12D and G12C significantly co-occur with APC, PIK3CA, and SMAD4 alterations. FBXW7, AR, and KEAP1 alterations were unique co-occurrences to KRAS G12D, while KIT was unique to KRAS G12C. Candidate alterations associated with primary resistance in KRAS G12D CRC included simultaneously present mutations of KRAS Q61H (14.1%), G12V (6.4%), G12C (5.9%), G13D (3.6%), BRAF V600E (2.7%) and others, amplification of MET (2%), MYC (1.6%), and KRAS (1.4%), and FGFR3-TACC3 translocation (Table). Conclusions: KRAS G12D is associated with unique co-occurring molecular alterations compared to KRAS G12C in CRC. ctDNA-based NGS platforms can survey candidate alterations associated with primary resistance to KRAS G12D targeting in CRC. Further validation in preclinical models is needed. [Table: see text]

Clinical/molecular features and prognostic significance of RAS mutant-low versus RAS mutant-high in patients with metastatic colorectal cancer.

187 Background: Addressing tumor heterogeneity is one of the main advantage of liquid biopsy over tissue biopsy, as liquid biopsy is likely to better reflect the global molecular profile (primary tumor site and metastatic disease). Variant Allele Frequency (VAF) represents the fraction of sequencing reads in which a variant is observed. The aim was to evaluate the prognostic value of circulating tumor (ct) VAF for KRAS and NRAS genes in patients with ctRAS mutant metastatic colorectal cancer (MCRC). Methods: Circulating comprehensive genomic profiling using FoundationOne Liquid CDx (FO-Liq) was proposed to patients with histologically proven or highly suspected solid tumor whatever treatment line. FO-Liq is a next generation sequencing panel of 310 cancer related genes and 3 genomic signatures (blood tumor mutational burden (bTMB), microsatellite instability (MSI) and tumor fraction (TF)). The optimal threshold for ctVAF was determined by calculating Harrell’s C discrimination index extended for survival data. The primary endpoint was overall survival (OS). Results: From October 2020 to June 2023, 740 patients were screened for circulating molecular profiling. In the colorectal cohort (N=201), 198 (98.5%) patients had metastatic disease (ctRAS/BRAF wild-type, N=76 (38.4%) ; ctRAS mutant, N=106 (53.5%) and ctBRAF V600E mutant, N=16 (8.1%)). The optimal threshold for ctVAFRAS was 5% (C-index 0.67, 95% CI 0.62-0.73 ; P&lt;0.0001). Among patients with ctRAS mutant MCRC, 43 (40.6%) were ctRAS mutant-Low (VAFRAS&lt;5%) and 63 (59.4%) were ctRAS mutant-High (VAFRAS≥5%). Median follow-up was 18.5 months (95% CI 15.3-34.5). There was a significant increase in the the risk of death for RAS mutant-High compared to RAS mutant-Low (HR 2.81, 95% CI 1.70-4.63; P&lt;0.001). Patients with RAS mutant-low variants had the same prognosis that RAS/BRAF wild-type profile (HR 1.22 ; P=0.573), and patients with RAS mutant-high variants had the same prognosis that BRAF mutant profile (HR 0.82, P=0.563). The factors associated with RAS mutant-High profile were : bTMB-High, TF-High, RAS G12A and G13D variants. MAP kinase, TGFb and Wnt signaling pathways and BRCA1-2 genes were more frequently altered in patients with ctRAS mutant-High tumors. We observed a transient decrease of VAFRAS in second-line setting. Conclusions: The circulating VAF of RAS genes enables to split RAS mutant-Low (favorable prognosis similar to RAS/BRAF wild-type) from RAS mutant-High (poorer prognosis similar to BRAF mutant) metastatic colorectal cancer. These results provide further insights into prognostication and therapeutic strategies in patients with RAS mutant metastatic colorectal cancer.

Characterization of the Thyroid Cancer Genomic Landscape by Plasma-Based Circulating Tumor DNA Next-Generation Sequencing.

Background: The limited availability of targeted therapies in thyroid cancer (TC) has challenged conventional treatment algorithms and has established urgency for the identification of targetable genomic abnormalities. In addition to widely adopted tissue-based next-generation sequencing (NGS), plasma-based circulating tumor DNA (ctDNA) NGS is rapidly emerging as a genomic biomarker detection method and is steadily gaining utility across solid tumors. To date, plasma-based genomic alterations in TC have not been determined. Herein, we profile potential actionable mutations detected through ctDNA in patients with TC subtypes. Methods: A retrospective data analysis of the Guardant Health, Inc. database was performed using the commercially available Guardant360® plasma-NGS test on TC samples from adult patients collected between 2016 and 2021. The landscape of genomic alterations and blood tumor mutation burden (bTMB) were analyzed in patients with different types of TC: anaplastic TC (ATC), papillary TC (PTC), follicular TC (FTC), oncolytic carcinoma of the thyroid (OCA), poorly differentiated TC (PDTC), medullary TC (MTC), and TC not otherwise specified (TC NOS). Results: Of the 1094 patients included most of the patients n = 876 had TC NOS, and 20% had a specific diagnosis (92 ATC, 62 PTC, 14 FTC, 16 OCA, 2 PDTC, and 32 MTC patients). The median age was 65 (range 10-98) and 47.3% were male. 78.3% of patients had one or more genomic alteration detected by ctDNA NGS. TP53 (46.9%) was the most common mutation detected among all TC. BRAFV600E was detected in 27.2% of ATC, 35.7% of PTC, and in none of FTC. RAS was detected in 18.5% of ATC, 11.9% of PTC, and 62.5% of FTC. RET, ALK, and NTRK fusions were seen in 1.1%, 0.5%, and 0.2% of all TC, respectively. RET mutations were detected in 66.7% of MTC. bTMB analysis was performed on 159 patients. The mean bTMB was higher in ATC compared with other types of TC (p = 0.0011, 0.0557, and <0.0001, respectively). Conclusions: Plasma-based comprehensive NGS is a promising NGS method in TC; however, future validation of the clinical utility by analysis of paired tumor and plasma samples is needed.

Efficacy of immune checkpoint inhibitors in non-small cell lung cancer with NTRK family mutations

BackgroundThe efficacy of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) patients harboring neurotrophin receptor kinase (NTRK) family mutations remains obscure.MethodsThe Zehir cohort from cBioPortal was used to analyze the mutations (MT) frequency of NTRK family in patients with NSCLC, and their correlation with clinical characteristics and patient survival. The influence of NTRK MT on ICIs efficacy was evaluated in ICIs-treated patients from Samstein cohort and further validated by use of data from OAK/POPLAR cohort.ResultsIn the Zehir cohort, a significant difference was observed in median overall survival (mOS) between patients with NTRK MT and wild-type (WT) (mOS: 18.97 vs. 21.27 months, HR = 1.34, 95%CI 1.00-1.78; log-rank P = 0.047). In Samstein cohort, the mOS of NTRK mutant patients receiving ICIs has improved compared to WT patients (mOS: 21.00 vs. 11.00 months, log-rank P = 0.103). Notably, in subgroup analysis, ICIs significantly prolonged mOS in patients with NTRK3 MT than in WT patients (mOS: not available vs. 11.00 months, HR = 0.36, 95%CI 0.16–0.81; log-rank P = 0.009). Identical mOS between NTRK MT and WT patients receiving ICIs treatment (mOS: 13.24 vs. 13.50 months, log-rank P = 0.775) was observed in OAK/POPLAR cohort. Moreover, a similar programmed death ligand 1 (PD-L1) expression, but higher tumor mutational burden (TMB), blood TMB (bTMB) and enriched anti-tumor immunity were observed in NTRK MT compared to WT (P < 0.05).ConclusionTaking high TMB or bTMB into consideration, patients with NTRK mutant NSCLC could benefit from ICIs treatment.

Analyzing the Predictive Effects of PD-L1 Expression, Early Changes of bTMB and Circulated CD8+T Cells during Treatment for Responses of RT Combined with ICI in NSCLC

The beneficial role of immunotherapy and the clinical relevance of current biomarkers remain inconclusive; thus, appropriate strategies and reliable predictors need further definition. A rational combination of biomarkers is needed. Here, we estimated potential predictive factors for responses of radiotherapy (RT) combined with immune checkpoint inhibitor (ICI) in a phase II trial to determine the efficacy and safety of combination of moderate hypofractionated RT with ICI in patients with oligometastatic NSCLC (NCT03557411). Pretreatment tumor tissue samples and longitudinal blood were collected for immune and tumor biomarker analysis. We examined pre-treatment (pre-ICI) PD-L1 expression in tumor cells. Circulating tumor cell (CTC), PD-L1+CTC, blood tumor mutation burden (bTMB), CD8+T cells, CD4+T cells, NK cells, B cells in circulation were acquired pre-ICI and 1 month after ICI starting (1-mth). In addition, early changes of CTC (CTC), PD-L1+CTC (PD-L1+CTC), bTMB (bTMB), CD8+T cells (CD8+T cells), CD4+T cells (CD4+T cells), NK cells (NK cells), B cells (B cells) were also analyzed to estimate the predictive effects for treatment. High pre-ICI bTMB and increased CD8+T cells at 1 month was associated with better PFS (p = 0.016; p = 0.006). Interaction analyses revealed that each combination of two markers in the 5 markers including PD-L1, pre-ICI bTMB, 1-mth bTMB, 1-mth CD8+T cells and CD8+T cells was significantly associated with PFS, except for CTC, PD-L1+CTC, CD4+T cells, NK cells and B cells in circulation due to low power. Unsupervised cluster analysis based on these markers revealed three sub-cohorts. Cohort-1 was overrepresented by patients with progressive disease (81%) of whom were negative for 3-4 of the 5 biomarkers. Cohort-3 was overrepresented by patients with partial response (70%) of whom were positive for 3-4 of the 5 biomarkers. Survival analyses of the 3 cohorts indicated a significant association with PFS (p = 0.017). This study suggests that a combination of PD-L1 expression, early changes of bTMB and circulated CD8+T cells as a better predictive biomarker for response to RT combined with ICI. Consequently, refinement of this set of biomarkers and validation in a larger set of patients is warranted.