Tissue Tumor Mutational Burden Research Articles

Plasma versus tissue tumor mutational burden as biomarkers of durvalumab plus tremelimumab response in patients with metastatic colorectal cancer in the CO.26 trial.

Tissue derived tumor mutation burden (TMB) of ≥10 mutations/Mb is a histology agnostic biomarker for the immune checkpoint inhibitor (ICI) pembrolizumab. However, the dataset on which this was validated lacked colorectal cancers (CRCs), and there is limited evidence for immunotherapy benefit in CRC using this threshold. CO.26 was a randomized phase II study of 180 patients comparing durvalumab and tremelimumab (D+T, n=119 patients) versus best supportive care (BSC, n=61 patients). ctDNA sequencing was available for 168 patients (n=118 D+T, n=50), of which 165 had evaluable plasma TMB (pTMB). Tissue sequencing was available for 108 patients. Optimal thresholds for stratifying patients based on overall survival were determined using a minimal p-value approach. This report includes the final overall survival analysis. Tissue TMB ≥10 mutations/Mb was not predictive of benefit from D+T compared to BSC in microsatellite stable (MSS) metastatic CRC (HR 0.71 [95% CI:0.28-1.80], p=0.47). No tissue TMB threshold could identify a high TMB group that benefited from ICI. In contrast, plasma TMB (pTMB) ≥28 mutations/Mb was predictive of benefit from D+T (HR=0.34 [95%CI:0.13-0.85], p=0.022), as was clonal pTMB ≥10.6 mutations/Mb (HR=0.10 [95%CI:0.014-0.79], p=0.029) and subclonal pTMB ≥25.9/Mb (HR=0.20 [95% CI:0.061-0.69], p=0.010). Higher pTMB was associated with length of time on cytotoxic agents (p=0.021) and prior anti-EGFR exposure (p=2.44x10-06). pTMB derived from either clonal or subclonal mutations may identify a group more likely to benefit from immunotherapy, though validation is required. Tissue TMB provided no predictive utility for immunotherapy in this trial.

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.

POLE variant phenotypic classification to enable identification of patients with colorectal cancer (CRC) who may benefit from immune checkpoint inhibition (ICI).

194 Background: Pathogenic POLE (p POLE) variants interfere with the exonuclease activity of POLε during DNA replication, resulting in somatic ultramutation and conferring susceptibility to ICI. Historical classification of p POLE relies on localization to the exonuclease domain (ED). We present the landscape of p POLE variants in CRC according to a proposed phenotypic classification model. Methods: Comprehensive genomic profiling was performed on tissue (TBx) and liquid (LBx) biopsies during the course of clinical care. A POLE-specific variant classification model including tumor mutational burden (TMB), mutational signatures, and germline frequency was used to differentiate p POLE from benign POLE (b POLE) variants. Results: POLE variant status was evaluated in 455,965 TBx and 41,804 LBx pan-tumor. POLE alterations, including p POLE and variants of unknown significance, were detected in 3.9% of samples with 7,404 discrete alleles observed. After applying the classification model, 30 variants were classified as p POLE, representing 3.6% (751/20,601) of observed POLE variants. All p POLE were localized to the ED and a majority were missense substitutions (29/30). There were an additional 620 ED variants categorized as b POLE. CRC had the second highest prevalence of p POLE variants (0.5%, 302/60,547). p POLE variants were associated with ultramutation in CRC where the median tissue TMB was 183.0 mut/Mb, 1-2 orders of magnitude higher than for CRC harboring b POLE variants (7.8 mut/Mb) or without POLE variants (3.5 mut/Mb). 32.2% of p POLE CRC also had evidence of high microsatellite instability (MSI-H). Combined p POLE + MSI-H was associated with markedly elevated TMB compared to either signature alone (344.4 versus 53.1 and 151.3 mut/Mb for b POLE + MSI-H and p POLE + MS-Stable, respectively). A small minority of CRC tumors with p POLE variants (7/298, 2.3%) had low TMB (<10 mut/Mb). However, this was associated with a low p POLE variant allele frequency (VAF; median 2.8% versus 24.0% for tumors w/ TMB ≥10, p < .0001) pointing to low tumor purity and likely an underestimation of TMB. While targetable alterations (e.g., homologous recombination repair [HR] gene variants) were enriched in p POLE tumors, these tumors had a low rate of HR deficiency (HRD) as determined by a computational algorithm (HRDsig), suggesting these are likely incidental monoallelic variants. Conclusions: A POLE phenotype-specific classification model identified p POLE variants associated with ultramutation in CRC. Co-occurrence of p POLE and MSI-H in CRC is common and has a synergistic effect leading to even higher TMB. We hypothesize that p POLE detection predicts ultramutation and response to ICI even when TMB is under-detected due to low tumor purity. Caution is advised for therapy selection based on co-alterations in the setting of p POLE due to an abundance of incidental variants.

In Search of Lost Biomarker for Immunotherapy in Small Cell Lung Cancer.

Chemo-immunotherapy is the current standard of care for extensive-stage small cell lung cancer, but predictive biomarkers are lacking. In a recent article, the authors report the predictive role of programmed death ligand-1 expression and tissue tumor mutational burden on durvalumab ± tremelimumab + platinum-etoposide efficacy. See related article by Paz-Ares et al., p. 824.

Genomic profiling of tissue and blood predicts survival outcomes in patients with resected pleural mesothelioma

PurposePleural mesothelioma (PM) is an aggressive tumor still considered incurable, in part due to the lack of predictive biomarkers. Little is known about the clinical implications of molecular alterations in resectable PM tissues and blood. Here, we characterized genetic alterations to identify prognostic and predictive biomarkers in patients with resected PM. Experimental DesignTargeted next-generation sequencing was performed in retrospective pleural tumor tissue and paired plasma samples from stage IB-IIIB resected PM. Association between prognosis and presence of specific mutations was validated in silico. ResultsThirty PM tissues and paired blood samples from 12 patients were analyzed. High tissue tumor mutational burden (TMB) (>10 mutations/Mb), tissue median minor allele frequency (MAF) (>9 mutations/Mb), and blood TMB (>6 mutations/Mb), tissue KMT2C, PBRM1, PKHD1,EPHB1 and blood LIFR mutations correlated with longer disease-free survival and/or overall survival. High concordance (>80%) between tissue and blood was found for some mutations. ConclusionsTissue TMB and MAF, blood TMB, and specific mutations correlated with outcomes in patients with resected PM and should be further studied to validate their role as prognostic biomarkers and potentially predictive factors for combinations with immune-checkpoint inhibitors. This suggest that molecular profiling could identify longer survivors in patients with resected PM.

Validity and utility of blood tumor mutational burden (bTMB) is dependent on circulating tumor DNA (ctDNA) shed: SCRUM-Japan MONSTAR-SCREEN

BackgroundThe tumor mutational burden (TMB) is a genomic biomarker associated with the benefits from immune checkpoint inhibitors (ICIs) cancer therapy. An elevated blood TMB (bTMB) in circulating tumor DNA (ctDNA) represents a compelling non-invasive diagnostic approach; however, the validity and utility of this emerging biomarker across cancer types have not been examined. Patient and methodsThe blood and tissue TMB was measured in a large pan-tumor clinical cohort and the MONSTAR-SCREEN observational study (UMIN000036749) using the FoundationOne Liquid CDx and FoundationOne CDx assays. A subset of the MONSTAR-SCREEN cohort was used to evaluate the association between the bTMB and the efficacy of ICIs therapy. ResultsThe majority of cancer types showed similar prevalence of TMB≥10 mutations/megabase and bTMB≥10. There was high concordance between bTMB and TMB in blood and tissue biopsy from the same patient when the plasma tumor fraction (TF) was at least 1% (Spearman's coefficient 0.74 and > 80% sensitivity to detect TMB-high). High microsatellite instability (MSI-H) was detected by ctDNA with 79% sensitivity when TF was at least 1%, but only 6% sensitivity when TF was <1%. Among patients with bTMB≥14 and elevated TF≥10% treated with ICIs, there was a trend toward a longer progression-free survival and overall survival compared with patients with bTMB<14 and elevated TF≥10% (HR, 0.62 [95%CI, 0.39–0.98]; p = 0.04 and HR, 0.60 [95%CI, 0.34-1.1]; p = 0.05). ConclusionsOur findings suggest that an elevated bTMB is correlated with elevated TMB and represents a pragmatic biomarker for assessing ICIs benefits. The utility of this biomarker is likely to be associated with high TF levels, informing future prospective investigations.

Exploration of biomarkers in potentially resectable stage IIIB NSCLC patients receiving neoadjuvant chemoimmunotherapy.

e20591 Background: Non–small cell lung cancer (NSCLC) is one of the main leading causes of cancer mortality in the world. NSCLC is incurable in most patients with potentially resectable locally advanced stage IIIB disease. Numerous studies suggest that advantageous chemoimmunotherapy is a promising treatment for resectable NSCLC. The determination of PD-L1, T-cell receptor (TCR) repertoire and tumor mutational burden (TMB) have been reported to predict the response to immunotherapy. However, the value of pathologic response determination in the context of neoadjuvant chemoimmunotherapy in resectable stage IIIB NSCLC is limited. Therefore, it is necessary to identify biomarkers for the prediction of the benefit from neoadjuvant chemoimmunotherapy in patients with potentially resectable stage IIIB NSCLC. Methods: A total of 15 patients with potentially resectable clinical stage IIIB NSCLC were enrolled into the study receiving neoadjuvant sintilimab (200mg IV Q3W) plus chemotherapy therapies for 4-6 weeks followed by surgery. All peripheral blood and tissue samples were collected and detected by using TCR repertoire sequencing before and after chemoimmunotherapy. The patients were divided into MPR group (n=5) and non-MPR group (n=10) according to MPR (&lt;10% viable tumour at resection) as an index. Furthermore, TMB spectra of 1021-gene panels and PD-L1 immunohistochemical staining were performed in paraffin-embedded human NSCLC tissue before neoadjuvant chemoimmunotherapy. The Pearson correlation coefficient method was used to analyze correlation. The ROC analysis were performed using “pROC” R package. Results: In baseline tissue samples, the levels of TMB and NAD were higher in MPR compared with non-MPR groups (P=0.0328, P=0.015, respectively). The negative correlation was existed between pretreatment tissue TCR richness and TMB in non-MPR group (P=0.028, R=-0.66). In MPR group, TCR clones and evenness showed significant difference (P=0.00237, P=0.04658, respectively). In non-MPR group, the TCR repertoire of tissue in posttreatment showed significant difference compared with those of pretreatment, including clones (P=1.04x10-6), shannon’s diversity (P=0.00142), evenness (P=0.00433), richness (P=0.00591). The clinical data showed that MPR patients with DDR mutation had longer DFS after sintilimab plus chemotherapy therapies (18.4±5.41 months, X-squared=9.8, P=0.02034). DDR mutation, TMB and TCR richness revealed better sensitivity and specificity to classify patients achieving MPR after neoadjuvant chemoimmunotherapy (AUC=1.000, AUC=0.796, AUC=0.773, respectively). Conclusions: This study confirmed the superiority of neoadjuvant chemoimmunotherapy in patients with potentially resectable stage IIIB NSCLC in terms of MPR. TCR repertoire, TMB and DDR mutation were associated with pathologic response to neoadjuvant chemoimmunotherapy as biomarkers for effective anti-tumor immunity. Clinical trial information: ChiCTR2000040673 .

Abstract CT216: Response to first-line (1L) pembrolizumab (pembro) + chemotherapy (chemo) in non-small cell lung cancer (NSCLC) by blood tumor mutational burden (bTMB): the phase 2 KEYNOTE-782 trial

Abstract Background: 1L pembro + platinum-based chemo has shown clinical activity in metastatic NSCLC regardless of tissue tumor mutational burden (tTMB) status. bTMB assessed using circulating tumor DNA in plasma may be a surrogate for tTMB. The single-arm, phase 2 KEYNOTE-782 study (NCT03664024) evaluated the correlation of bTMB with efficacy of 1L pembro + chemo in nonsquamous NSCLC. Methods: Eligible patients (pts) had histologically or cytologically confirmed stage IV nonsquamous NSCLC with measurable disease per RECIST v1.1 and were not eligible for EGFR-, BRAF-, ROS1-, or ALK-directed therapy. Pts were not previously treated for advanced or metastatic disease, had an ECOG PS of 0 or 1, and had an evaluable biopsy sample for biomarker analysis. All pts received intravenous pembro 200 mg Q3W + platinum chemo doublet Q3W (pemetrexed 500 mg/m2 + 4 cycles of carboplatin AUC 5 mg/mL/min or cisplatin 75 mg/m2). The primary objective was to evaluate the association of baseline bTMB with ORR per RECIST v1.1 by investigator assessment. Secondary objectives were to determine safety and the association of baseline bTMB with PFS per RECIST v1.1 by investigator assessment and OS. A study-specific next-generation sequencing-based assay using a 1.9 Mb/654-gene cancer panel, which includes specific lung cancer-associated gene targets, was used to measure bTMB (continuous scale) in cell-free DNA extracted from baseline plasma samples. Paired white blood cell DNA sequencing was also performed to eliminate potential clonal hematopoiesis-derived somatic mutations. Database cutoff: November 5, 2021. Results: 117 pts were enrolled; median age was 64.0 years (range, 37-85), and the majority were male (60.7%), had an ECOG PS of 1 (69.2%), and no brain metastases at baseline (92.3%). 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; 6 CRs, 41 PRs), 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 adverse events (TRAEs) occurred in 113 pts (96.6%) and grade 3-5 TRAEs occurred in 56 (47.9%). Eight pts (6.8%) died due to a TRAE (febrile neutropenia and pneumonitis [n = 2 each], and septic shock, pulmonary sepsis, general physical health deterioration, and neutropenia [n = 1 each]). bTMB data were available for 101 pts. The area under the receiver operating curve for bTMB as a continuous variable to discriminate response was 0.47 (95% CI, 0.36-0.59). The posterior probabilities for a positive association of bTMB with PFS and OS were 16.8% and 7.8%, respectively. Conclusions: Baseline bTMB showed no evidence of an association with ORR, PFS, or OS in pts with nonsquamous NSCLC treated with 1L pembro + chemo. These findings indicate no clinical utility of bTMB in this patient population and treatment setting. No new safety signals were observed. 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 B. Rao, D. Ross Camidge, Giovanna Speranza, Steven M. Townson, Julie Kobie, Mark Ayers, Elisha J. Dettman, Robert McDaniel, Byoungsok Jung, David Burkhardt, Ruth Mauntz, Tibor Csőszi. Response to first-line (1L) pembrolizumab (pembro) + chemotherapy (chemo) in non-small cell lung cancer (NSCLC) by blood tumor mutational burden (bTMB): the phase 2 KEYNOTE-782 trial [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 CT216.

Abstract 1027: Assessment of tumor mutation burden (TMB) based on whole exome sequencing using extracellular vesicles-derived DNA obtained from bronchoalveolar lavage fluid (BALF) in advanced NSCLC patients treated with pembrolizumab

Abstract Background: Although multiple immune checkpoint inhibitors showed improved outcomes in patients with high tumor mutational burden (TMB) as well as high PD-L1 expression, obtaining adequate tumor tissue for various molecular testing including next-generation sequencing (NGS) is not a simple issue in patients with advanced non-small cell lung cancer (NSCLC). We aimed to assess TMB based on whole exome sequencing (WES) using extracellular vesicles (EV)-derived DNA isolated from bronchoalveolar lavage fluid (BALF) and determine the correlation of TMB between BALF and tissue. Methods: Patients diagnosed with advanced NSCLC and who were treated with pembrolizumab between December 2018 and December 2021 were included in this multicenter translational cohort study. The correlation of TMB between BALF and tissue measured by WES was evaluated by Spearman’s rank correlation coefficient in matched BALF and tissue samples from 62 patients. We analyzed the association of BALF TMB level with progression-free survival (PFS) and response to pembrolizumab. Results: Of 62 patients, 47 (75.8%) had evaluable tissue TMB and 57 (91.9%) had evaluable BALF TMB. There were no significant differences between BALF and tissue with regards to DNA amounts, estimated library size, mean depth, and uniformity. Median tissue TMB and BALF TMB were 6.02 and 2.16 mut/Mb, respectively. There was a weak positive correlation between tissue TMB and BALF TMB (r=0.23, p &amp;lt; 0.001), whereas BALF TMB found higher correlations with tissue TMB in patients with squamous cell carcinoma (r=0.77, p &amp;lt; 0.001). Neither tissue TMB nor BALF TMB showed significant differences PFS or ORR benefit for treatment with pembrolizumab, however the BALF TMB of patients who had been treated with pembrolizumab for more than 6 months was higher than that of those who did not (p=0.039). Conclusions: The findings suggest that NGS using BALF EV DNA is feasible for TMB calculation to overcome the matter of small biopsy issue in advanced NSCLC patients, which may play a supplementary serve as a potential biomarker of clinical benefit in patients with NSCLC treated with pembrolizumab. Citation Format: Heejoung Kim, Jaeyoung Hur, Wan Seop Kim, In Ae Kim, Dong Il Park, In-Jae Oh, Kye Young Lee. Assessment of tumor mutation burden (TMB) based on whole exome sequencing using extracellular vesicles-derived DNA obtained from bronchoalveolar lavage fluid (BALF) in advanced NSCLC patients treated with pembrolizumab [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 1027.

Abstract 2313: Real-world concordance between tumor mutation burden from blood and tissue

Abstract Background: Tissue tumor mutational burden (tTMB) is known to have predictive value for response to immune checkpoint inhibitor treatment of cancer. Utilization of next-generation sequencing of circulating tumor DNA added another data point of blood tumor mutational burden (bTMB). However, the concordance between tTMB and bTMB is not well understood. Method: A total of 119 patients with solid tumors who had both tTMB and bTMB (collected from 10/2020 to 10/2022) results were included in the analysis. Patients' tTMB and bTMB concordance were analyzed according to the interval between tTMB and bTMB collection, tumor type, and site of tTMB collection using the Pearson correlation coefficient. Result: Median tTMB was 4.2 mut/Mb and median bTMB was 9.61mut/Mb. bTMB was moderately correlated with tTMB (Pearson r 0.54) in total patient population. The median interval between tTMB and bTMB collection was 27 days (Q1:14, Q3:1834). Concordance did not significantly change when the interval was under 30 days (Pearson r 0.51). Patients with metastatic disease had higher concordance compared to patients without metastasis of cancer (Pearson r 0.58 vs 0.39). bTMB and tTMB were most strongly correlated when tTMB was collected from metastatic lesions (Pearson r 0.92). No significant correlation was observed when tTMB was collected from lymph nodes (Pearson r 0.20). Regarding cancer types, adenocarcinoma of the lung had a moderate correlation between bTMB and tTMB (Pearson r 0.63) while small cell lung cancer did not demonstrate a significant correlation between bTMB and tTMB (Pearson t -0.30). Correlation between tTMB and bTMB Total study population 119 Interval between bTMB and tTMB collection &amp;lt;30days 64 Pearson R (p value, CI) 0.55 (0.00, 0.41-0.66) Pearson R (p value, CI) 0.52 (0.00, 0.27-0.70) R squared 0.30 R squared 0.27 Patients with metastatic disease 89 Patients without metastatic disease 30 Pearson R (p value, CI) 0.58 (0.00, 0.42-0.70) Pearson R (p value, CI) 0.39 (0.03, 0.04-0.66) R squared 0.33 R squared 0.16 tTMB collected from primary lesion 51 Lung adenocarcinoma 53 Pearson R (p value, CI) 0.52 (0.00, 0.29-0.70) Pearson R (p value, CI) 0.63 (0.00, 0.45-0.77) R squared 0.27 R squared 0.41 tTMB collected from metastatic lesion 29 Lung squamous cell carcinoma 16 Pearson R (p value, CI) 0.92 (0.00, 0.84-0.96) Pearson R (p value, CI) 0.56 (0.02, 0.09-0.82) R squared 0.85 R squared 0.31 tTMB collected from lymph nodes 34 Lung small cell carcinoma 10 Pearson R (p value, CI) 0.20 (0.25, -0.14-0.50) Pearson R (p value, CI) -0.30 (0.38, -0.79-0.40) R squared 0.04 R squared 0.09 Citation Format: Leeseul Kim, Youjin Oh, Jewel Park, Ilene Hong, Lena Chae, Pedro Viveiros. Real-world concordance between tumor mutation burden from blood and tissue [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 2313.

Associations of tissue tumor mutational burden and mutational status with clinical outcomes in KEYNOTE-042: pembrolizumab versus chemotherapy for advanced PD-L1-positive NSCLC

We evaluated whether tissue tumor mutational burden (tTMB) and STK11, KEAP1, and KRAS mutations have clinical utility as biomarkers for pembrolizumab monotherapy versus platinum-based chemotherapy in patients with programmed death ligand 1 (PD-L1)-positive (tumor proportion score ≥1%) advanced/metastatic non-small-cell lung cancer (NSCLC) without EGFR/ALK alterations in the phase III KEYNOTE-042 trial. This retrospective exploratory analysis assessed prevalence of tTMB and STK11, KEAP1, and KRAS mutations determined by whole-exome sequencing of tumor tissue and matched normal DNA and their associations with outcomes in KEYNOTE-042. Clinical utility of tTMB was assessed using a prespecified cut point of 175 mutations/exome. Of 793 patients, 345 (43.5%) had tTMB ≥175 mutations/exome and 448 (56.5%) had tTMB <175 mutations/exome. No association was observed between PD-L1 expression and tTMB. Continuous tTMB score was associated with improved overall survival (OS) and progression-free survival among patients receiving pembrolizumab (Wald test, one-sided P < 0.001) but not those receiving chemotherapy (Wald test, two-sided P > 0.05). tTMB ≥175 mutations/exome was associated with improved outcomes for pembrolizumab versus chemotherapy, whereas tTMB <175 mutations/exome was not {OS: hazard ratio, 0.62 [95% confidence interval (CI) 0.48-0.80] and 1.09 (95% CI 0.88-1.36); progression-free survival: 0.75 (0.59-0.95) and 1.27 (1.04-1.55), respectively}. Improved OS [hazard ratio (95% CI)] for pembrolizumab versus chemotherapy was observed regardless of STK11 [STK11 mutant (n= 33): 0.37 (0.16-0.86), STK11 wild-type (n= 396): 0.83 (0.65-1.05)]; KEAP1 [KEAP1 mutant (n=64): 0.75 (0.42-1.35), KEAP1 wild-type (n= 365): 0.78 (0.61-0.99)], or KRAS [KRAS mutant (n= 69): 0.42 (0.22-0.81); KRAS wild-type (n= 232): 0.86 (0.63-1.18)] mutation status. tTMB with a cut point of ≥175 mutations/exome is a potential predictive biomarker for pembrolizumab monotherapy for advanced/metastatic PD-L1 tumor proportion score ≥1% NSCLC. Pembrolizumab is a standard first-line treatment in this setting regardless of STK11, KEAP1, or KRAS mutation status.

Characteristics of the immunogenicity and tumor immune microenvironment in HER2-amplified lung adenocarcinoma.

Besides breast and gastric cancer, HER2 amplification/mutation are also found in lung adenocarcinoma (LUAD). However, the correlation between HER2 variations and the phenotype of immunogenicity and tumor immune microenvironment (TIME) in LUAD compared with breast and gastric cancer has yet to be fully elucidated. We integrated public databases (discovery set) and internal data (validated set) of 288 patients representing three distinct HER2-altered tumors. Genomic data were used to identify somatic mutations, copy number variations, and calculate tumor mutational burden (TMB) and microsatellite instability score. RNA sequencing was conducted to estimate immune gene signatures and contents of tumor-infiltrating immune cell populations. Finally, IHC was used to determine PD-L1 expression and the tumoral-infiltration of immune cells in 50 HER2-variant tumor specimens with no prior therapeutic regimens. Compared with HER2-amplified breast and gastric cancers, patients with HER2-amplified LUAD showed higher immunogenicity, mainly manifested in immune checkpoints expression and tissue/blood TMB. Additionally, HER2-amplified LUAD exhibited an inflamed TIME with remarkably increased genes encoding HLAs, T-cell activity and immune cell-type, and accompanied with tumor-infiltrating lymphocytes. In LUAD, patients with HER2 amplification possessed higher tissue TMB than HER2 mutation, whereas no difference was observed in PD-L1 expression. HER2 amplification (primary) was associated with significantly higher PD-L1 expression and TMB than acquired HER2 amplification after resistance to EGFR-TKIs. Patients with HER2-amplified LUAD have better immunogenicity and/or an inflamed TIME among HER2-aberrant tumors. Our study may provide clues for establishing the benefits and uses of ICIs for patients with this disease.

Associations of Tissue Tumor Mutational Burden and Mutational Status With Clinical Outcomes With Pembrolizumab Plus Chemotherapy Versus Chemotherapy For Metastatic NSCLC

IntroductionWe evaluated tissue tumor mutational burden (tTMB) and mutations in STK11, KEAP1, and KRAS as biomarkers for outcomes with pembrolizumab plus platinum-based chemotherapy (pembrolizumab-combination) for NSCLC among patients in the phase 3 KEYNOTE-189 (ClinicalTrials.gov, NCT02578680; nonsquamous) and KEYNOTE-407 (ClinicalTrials.gov, NCT02775435; squamous) trials. MethodsThis retrospective exploratory analysis evaluated prevalence of high tTMB and STK11, KEAP1, and KRAS mutations in patients enrolled in KEYNOTE-189 and KEYNOTE-407 and the relationship between these potential biomarkers and clinical outcomes. tTMB and STK11, KEAP1, and KRAS mutation status was assessed using whole-exome sequencing in patients with available tumor and matched normal DNA. The clinical utility of tTMB was assessed using a prespecified cutpoint of 175 mutations/exome. ResultsAmong patients with evaluable data from whole-exome sequencing for evaluation of tTMB (KEYNOTE-189, n = 293; KEYNOTE-407, n = 312) and matched normal DNA, no association was found between continuous tTMB score and overall survival (OS) or progression-free survival for pembrolizumab-combination (Wald test, one-sided p > 0.05) or placebo-combination (Wald test, two-sided p > 0.05) in patients with squamous or nonsquamous histology. Pembrolizumab-combination improved outcomes for patients with tTMB greater than or equal to 175 compared with tTMB less than 175 mutations/exome in KEYNOTE-189 (OS, hazard ratio = 0.64 [95% confidence interval (CI): 0.38‒1.07] and 0.64 [95% CI: 0.42‒0.97], respectively) and KEYNOTE-407 (OS, hazard ratio = 0.74 [95% CI: 0.50‒1.08 and 0.86 [95% CI: 0.57‒1.28], respectively) versus placebo-combination. Treatment outcomes were similar regardless of KEAP1, STK11, or KRAS mutation status. ConclusionsThese findings support pembrolizumab-combination as first-line treatment in patients with metastatic NSCLC and do not suggest the utility of tTMB, STK11, KEAP1, or KRAS mutation status as a biomarker for this regimen.

Analytical and Clinical Validation of Cell-Free Circulating Tumor DNA Assay for the Estimation of Tumor Mutational Burden.

Ultra-deep sequencing to detect low-frequency mutations in circulating tumor-derived DNA (ctDNA) increases the diagnostic value of liquid biopsy. The demand for large ctDNA panels for comprehensive genomic profiling and tumor mutational burden (TMB) estimation is increasing; however, few ctDNA panels for TMB have been validated. Here, we designed a ctDNA panel with 531 genes, named TMB500, along with a technical and clinical validation. Synthetic reference cell-free DNA materials with predefined allele frequencies were sequenced in a total of 92 tests in 6 batches to evaluate the precision, linearity, and limit of detection of the assay. We used clinical samples from 50 patients with various cancers, 11 healthy individuals, and paired tissue samples. Molecular barcoding and data analysis were performed using customized pipelines. The assay showed high precision and linearity (coefficient of determination, r2 =0.87) for all single nucleotide variants, with a limit of detection of 0.24%. In clinical samples, the TMB500 ctDNA assay detected most variants present and absent in tissues, showing that ctDNA could assess tumor heterogeneity in different tissues and metastasis sites. The estimated TMBs correlated well between tissue and blood, except in 4 cases with extreme heterogeneity that showed very high blood TMBs compared to tissue TMBs. A pilot evaluation showed that the TMB500 assay could be used for disease monitoring. The TMB500 assay is an accurate and reliable ctDNA assay for many clinical purposes. It may be useful for guiding the treatment of cancers with diverse genomic profiles, estimating TMB in immune therapy, and disease monitoring.

Tislelizumab Versus Docetaxel in Patients With Previously Treated Advanced NSCLC (RATIONALE-303): A Phase 3, Open-Label, Randomized Controlled Trial

IntroductionThe phase 3 RATIONALE-303 trial (NCT03358875) investigated the efficacy and safety of tislelizumab versus docetaxel in pretreated patients with advanced NSCLC. Here, we report the efficacy and safety results and describe the exploratory biomarker analyses. MethodsA total of 805 patients aged more than or equal to 18 years with locally advanced or metastatic squamous or nonsquamous NSCLC were randomized 2:1 to intravenous tislelizumab 200 mg or docetaxel 75 mg/m2 every 3 weeks. Co-primary end points were overall survival (OS) in the intent-to-treat (ITT) and programmed death-ligand 1 (PD-L1) tumor cell expression greater than or equal to 25% populations. The exploratory biomarker analyses included PD-L1 expression, tumor mutation burden, and gene expression profile. ResultsAt the prespecified interim analysis (August 10, 2020), the co-primary end point of OS in the ITT population was met, with a statistically significant and clinically meaningful improvement in OS with tislelizumab versus docetaxel (median 17.2 versus 11.9 mo, respectively; hazard ratio [HR] = 0.64, p < 0.0001). At the final analysis (July 15, 2021), the other co-primary end point of OS in the PD-L1 tumor cell greater than or equal to 25% population was further met (median 19.3 versus 11.5 mo, respectively; HR = 0.53, p < 0.0001), and OS continued to improve in the ITT population (median 16.9 versus 11.9 mo, respectively, HR = 0.66). Exploratory biomarker analyses revealed the potential association of NOTCH1–4 mutations with improved tislelizumab efficacy for both OS and progression-free survival, whereas tissue tumor mutation burden correlated with progression-free survival benefit, but not OS benefit. No new safety signals were identified. ConclusionsTislelizumab was found to have a significantly improved and long-term clinical benefit in OS versus docetaxel in pretreated patients with advanced NSCLC, regardless of PD-L1 expression.

O19-4 Outcomes in Japanese patients with TMB-high and non-TMB-high solid tumors treated with pembrolizumab: KEYNOTE-158

Tissue tumor mutational burden (tTMB) was correlated with response to pembrolizumab (pembro) monotherapy in the global phase 2 KEYNOTE-158 study (NCT02628067). We report an analysis of the relationship between the response to pembro and tTMB in patients with advanced solid tumors in the KEYNOTE-158 study enrolled in Japan.

Abstract CT022: CheckMate 848: A randomized, open-label, phase 2 study of nivolumab in combination with ipilimumab or nivolumab monotherapy in patients with advanced or metastatic solid tumors of high tumor mutational burden

Abstract High tumor mutational burden assessed in tissue biopsies (tTMB-H) or blood (bTMB-H) is associated with clinical efficacy in patients treated with immunotherapies. CheckMate 848 (NCT03668119) is a prospective phase 2 study of nivolumab (NIVO) with or without ipilimumab (IPI) in patients with advanced or metastatic solid tumors that are tTMB-H or bTMB-H (≥ 10 mutations/megabase) who were immunotherapy-naive and refractory to standard local therapies. The primary endpoint was objective response rate (ORR) in patients with tTMB-H or bTMB-H, assessed by FoundationOne® CDx-based and Clinical Trial Assays (Foundation Medicine), respectively. The study was not powered to compare NIVO + IPI vs NIVO. We present the interim and final analyses for the tTMB-H and bTMB-H cohorts, respectively (≥ 12 months follow-up, database lock June 2021). Of 1954 screened patients, 212 were randomized 2:1 to NIVO 240 mg Q2W + IPI 1 mg/kg Q6W or NIVO 480 mg Q4W for ≤ 24 months, and 201 (135 tTMB-H; 147 bTMB-H) were refractory to standard therapies. Of &amp;gt; 40 tumor types, colorectal (10.8%), small-cell lung (7.5%), breast (7.1%), and uterine (7.1%) were the most common. ORR and survival outcomes with NIVO + IPI were improved in patients with tTMB-H. The responses were independent of bTMB-H status in the tTMB-H cohort but improved with tTMB-H status in the bTMB-H cohort (Table). The safety profile of NIVO + IPI was manageable, and clinical outcomes with NIVO were comparable with previous studies. The impact of TMB cutoff, PD-L1 expression, and microsatellite instability were explored. In conclusion, NIVO + IPI demonstrated clinical efficacy with a manageable safety profile in patients with advanced or metastatic solid tumors that are tTMB-H or bTMB-H and refractory to standard therapies, with increased efficacy observed in patients with tTMB-H. NIVO + IPI tTMB-H cohort bTMB-H cohorta Patients, n (%)b,c 68 (32.1) 80 (37.7) Number of prior treatments, median (range) 2 (0–7) 2 (1–9) ORR, n (%)c, 95% CI 24 (35.3), 24.1–47.8 18 (22.5), 13.9–33.2 ORR in patients with bTMB-H by tTMBc: &amp;lt; 10 mut/Mb (n = 31), n (%), 95% CI NA 3 (9.7), 2.0–25.8 ≥ 10 mut/Mb (n = 39), n (%), 95% CI NA 13 (33.3), 19.1–50.2 ≥ 10 to &amp;lt; 16 mut/Mb (n = 18), n (%), 95% CI NA 3 (16.7), 3.6–41.4 ≥ 16 mut/Mb (n = 21), n (%), 95% CI NA 10 (47.6), 25.7–70.2 ORR in patients with tTMB-H by bTMBc: &amp;lt; 10 mut/Mb (n = 20), n (%), 95% CI 7 (35.0), 15.4–59.2 NA ≥ 10 mut/Mb (n = 43), n (%), 95% CI 16 (37.2), 23.0–53.3 NA ≥ 10 to &amp;lt; 16 mut/Mb (n = 12), n (%), 95% CI 3 (25.0), 5.5–57.2 NA ≥ 16 mut/Mb (n = 31), n (%), 95% CI 13 (41.9), 24.5–60.9 NA Percentage of responders (≥ 9 months) (95% CI) 91 (68–98) 88 (61–97) Median PFS, months (95% CI)c 4.1 (2.8–11.3) 2.8 (2.3–3.0) Median OS, months (95% CI)c 14.5 (7.7–NE) 8.5 (5.8–10.5) aThe bTMB cohort was randomized prior to December 20, 2019. bOut of 212 randomized patients; data presented in this table are from patients who were refractory to standard therapies. cMinimum follow-up 12 months. bTMB, blood tumor mutational burden; NA, not applicable; NE, not evaluable; PFS, progression-free survival; OS, overall survival; tTMB, tissue tumor mutational burden. Citation Format: Michael Schenker, Mauricio Burotto, Martin Richardet, Tudor Ciuleanu, Anthony Goncalves, Neeltje Steeghs, Patrick Schöffski, Paolo A. Ascierto, Michele Maio, Iwona Lugowska, Lorena Lupinacci, Alexandra Leary, Jean-Pierre Delord, Julieta Grasselli, David S. Tan, Jennifer E. Friedmann, Jacqueline Vuky, Marina Tschaika, Ruta Slepetis, Georgia D. Kollia, Misena Pacius, Ning Huang, Parul Doshi, Jonathan Baden, Massimo Di Nicola. CheckMate 848: A randomized, open-label, phase 2 study of nivolumab in combination with ipilimumab or nivolumab monotherapy in patients with advanced or metastatic solid tumors of high tumor mutational burden [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 CT022.

Abstract LB512: RATIONALE-304: The association of tumor mutational burden (TMB) with clinical outcomes of tislelizumab (TIS) + chemotherapy (chemo) versus chemo alone as first-line treatment for advanced non-squamous non-small cell lung cancer (nsq-NSCLC)

Abstract Background: In the primary analysis of RATIONALE-304 (NCT03663205), TIS + platinum-based chemo significantly improved clinical outcomes over chemo alone in treatment-naïve advanced nsq-NSCLC (median progression-free survival [PFS] by IRC [9.7 vs 7.6 months, HR=0.645, p=0.0044]). Here we report biomarker analysis of baseline tissue and blood TMB (tTMB and bTMB, respectively). Methods: Patients with nsq-NSCLC were randomized 2:1 to TIS + platinum + pemetrexed or platinum + pemetrexed. TMB scores were evaluated on baseline tumor and blood samples by OncoScreen Plus®. The Spearman’s rank correlation of tTMB with bTMB was assessed. PFS by independent review committee (primary endpoint) was assessed within subgroups defined by TMB status, using a Cox proportional hazard model with disease stage and programmed death-ligand 1 (PD-L1) expression as stratification factors. Interaction p-values &amp;lt; 0.05 were considered statistically significant without multiplicity adjustment. Results: Of 325 patients treated in RATIONALE-304, without an EGFR sensitizing mutation, 177 (54.5%) had evaluable tTMB and 107 (32.9%) had evaluable bTMB. Median tTMB and bTMB were 7.2 and 3.1 mut/Mb, respectively. There was a modest correlation between tTMB and bTMB (r=0.71, p &amp;lt; 0.001). Prolonged PFS benefit of adding TIS to chemo was oberved in patients with TMB-high status compared with TMB-low status (Table). Interaction analysis showed that neither tTMB nor bTMB significantly differentiated treatment-specific PFS benefit (interaction p-values &amp;gt; 0.05; Table). Conclusions: In this retrospective analysis, neither tTMB nor bTMB was significantly associated with PFS benefit, suggesting limited clinical utility of tTMB and bTMB in the setting of TIS + chemo as first-line therapy for advanced nsq-NSCLC. Association of TMB with PFS benefit of TIS + chemo vs chemo tTMB bTMB Cutoffs mut/Mb N HR (95% CI) Interaction Cutoffs mut/Mb N HR (95% CI) Interaction p-value p-value BEP 177 0.76 (0.46, 1.25) NA BEP 107 0.48 (0.26, 0.87) NA ≥ 8 (TMB-high) 80 0.52 (0.25, 1.10) 0.208 ≥ 4 (TMB-high) 47 0.30 (0.12, 0.75) 0.212 &amp;lt; 8 (TMB-low) 97 0.98 (0.51, 1.88) &amp;lt; 4 (TMB-low) 60 0.64 (0.29, 1.39) BEP, biomarker evaluable population; bTMB, blood tumor mutational burden; CI, confidence interval; HR, hazard ratio; Mb, megabase; mut, mutation; NA, not applicable; PFS, progression-free survival; TIS, tislelizumab; TMB, tumor mutational burden; tTMB, tissue tumor mutational burden Citation Format: Shun Lu, Meili Sun, Yunpeng Liu, Yanping Hu, Yanyan Xie, Zhehai Wang, Dong Wang, Zhenzhou Yang, Liang Liang, Yi Huo, Yun Zhang, Ruiqi Huang, Yang Shi, Zhirong Shen, Yan Yu. RATIONALE-304: The association of tumor mutational burden (TMB) with clinical outcomes of tislelizumab (TIS) + chemotherapy (chemo) versus chemo alone as first-line treatment for advanced non-squamous non-small cell lung cancer (nsq-NSCLC) [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 LB512.

Genetic alteration profiling of Chinese lung adenocarcinoma: A targeted and immunotherapy biomarker analysis.

e20572 Background: Lung adenocarcinoma is the most common primary lung cancer, comprises about 30% of lung cancers. So far, little is known about an in-depth molecular characterization for lung adenocarcinoma in Chinese patients. The genomic characteristics, microsatellite instability (MSI) status, programmed cell death-1 (PD-L1) and tissue tumor mutational burden (tTMB) in Chinese lung adenocarcinoma patients will benefit to understanding drug resistance-related mechanisms. Methods: 591 Formalin-Fixed Paraffin-Embedded (FFPE) lung adenocarcinoma samples were collected and detected by Next Generation Sequencing 603-gene panel. The biomarkers of immunotherapy (TMB-High [≥10/Mb], MSI-High, and PD-L1 22C3 [TPS] were also calculated. Results: Of 591 patients, 312 patients (52.8%) were male and 279 (47.2%) patients were female. The median age of the patients was 60.7 (range 28-99). The most commonly mutated genes were EGFR (45.5%), TP53 (22.1%), KRAS (7.8%), PIK3CA (5.3%), ERBB2 (2.5%), BRAF (2.2%), CTNNB1 (1.5%), SMAD4 (1.5%),NRAS (1.0%), SMAD4 (1.5%), NRAS (1%), ATM (1%), RB1 (1%), NF1(1%), IDH2 (0.67%). Three genes mutations (EGFR, TP53, KRAS) were identified significantly different between male and female. EGFRmutations were significantly more frequent in females (54.12%) than in males (37.5%;p = 0.000051). In contrast, TP53 mutations were significantly more frequent in males (11.53%) than in females. KRASmutations were significantly more frequent in males (25.6%) than in females (17.9%;p = 0.023). (3.6%;p = 0.0003). The aging of patients was associated with a higher PTEN mutations (&lt; 50 years-old, p = 0.039), RAF1 mutations (&gt; 76 years-old, p = 0.038), BRCA1 mutations (&lt; 42 years-old, p = 0.032). The average of TMB was 5.3 mut/Mb (range 0-54.8). Immunotherapy markers were presented in 36.4% of cases (TMB-High in 15.91%, PD-L1+(TPS &gt; 1%) in 47.12%,PD-L1+(TPS &gt; 50%) in 5.17%, MSI-High in 0.17%). PD-L1 expression and TMB score were not significantly correlated in lung adenocarcinoma. The TMB score was associated with a higher TP53 mutations(TMB &gt; 6.6, p &lt; 0.0001), KRAS(TMB &gt; 6.1, p = 0.034), NF1(TMB &gt; 9.8, p = 0.0018), IDH2(TMB &gt; 11.3, p = 0.0028). Conclusions: These findings may be helpful for identifying therapeutic targets strategies in lung adenocarcinoma patients that were previously unavailable to clinicians in China.

173P Genomic characteristics and its correlations with tumor mutational burden and PD-L1 expression in Chinese lung squamous cell carcinoma

The emergence of immunotherapy brings new opportunities for the treatment of LSCC. The present study aims to analyze the genomic characteristics of Chinese lung squamous cell carcinoma (LSCC) and to investigate its correlation with tissue tumor mutational burden (tTMB) and PD-L1 expression.