Biomarkers For Immune Checkpoint Inhibitors Research Articles

Immunologic Significance of CD80/CD86 or Major Histocompatibility Complex-II Expression in Thymic Epithelial Tumors

IntroductionUnresectable or recurrent thymic epithelial tumors (TETs) have a poor prognosis, and treatment options are limited. This study aimed to investigate the immunologic significance of CD80/CD86 or major histocompatibility complex class II (MHC-II) expression in TETs, as potential predictive biomarkers for immune checkpoint inhibitors (ICIs). MethodsWe analyzed CD80, CD86, MHC class I (MHC-I), and MHC-II expression in TETs using immunohistochemistry and investigated their association with T-cell infiltration or ICI efficacy. In addition, we generated CD80- or MHC-II–expressing mouse tumors, evaluated the effects of ICIs, and analyzed tumor-infiltrating lymphocytes. We also performed tumor-rechallenge experiments in vivo. ResultsWe found that approximately 50% and 30% of TETs had high expression of CD80/CD86 and MHC-II in tumor cells, respectively, and that this expression was related to T-cell infiltration in clinical samples. In mouse models, both CD80 and MHC-II increase the effects of ICIs. In addition, senescent T cells and long-lived memory precursor effector T cells were significantly decreased and increased, respectively, in tumor-infiltrating lymphocytes from CD80-expressing tumors, and rechallenged tumors were completely rejected after the initial eradication of CD80-expressing tumors by programmed cell death protein 1 blockade. Indeed, patients with CD80-high thymic carcinoma had longer progression-free survival with anti–programmed cell death protein 1 monoclonal antibody. ConclusionsHalf of the TETs had high expression of CD80/CD86 or MHC-II with high T-cell infiltration. These molecules could potentially increase the effects of ICIs, particularly inducing a durable response. CD80/CD86 and MHC-II can be predictive biomarkers of ICIs in TETs, promoting the development of drugs for such TETs.

Utility of Tumor Mutational Burden as a Biomarker for Response to Immune Checkpoint Inhibition in the VA Population.

Immune checkpoint inhibitors (ICIs) are used for an increasing number of indications across various tumor types, as well as several tumor-agnostic indications in patients with advanced cancer. Although many patients benefit from ICI therapy, others do not, highlighting a need for better predictive biomarkers. Tumor mutational burden (TMB) reflects the global number of mutations within a tumor and has been widely explored as a predictive biomarker of ICI response. The current tumor type-agnostic US Food and Drug Administration approval of pembrolizumab for metastatic solid tumors defines high TMB (TMB-H) as ≥10 mut/Mb as measured by FoundationOne CDx. This fixed cutoff may not be the ideal value across all solid tumors. We performed a retrospective analysis of the association of survival outcomes with TMB in patients treated with ICI for five major cancer types, using real-world data from the VA. Survival was measured from initiation of ICI, and Kaplan-Meier survival curves were compared by log-rank test. Overall survival (OS) was significantly longer for patients with TMB-H versus TMB low tumors in non-small-cell lung cancer (NSCLC; n = 1,593), head and neck (H&N) cancer (n = 222), and urothelial cancer (n = 332). OS was not significantly different based on TMB status in melanoma (n = 207) or esophageal/gastric cancer (n = 248). Consistent with previous studies, a predictive value of TMB ≥10 mut/Mb for ICI response was found in NSCLC and H&N, but not in esophageal/gastric cancer. Although inconclusive in the literature, significant association was found in urothelial cancer. The predictive value of TMB in melanoma was inconclusive. Our analysis does not support the use of a fixed threshold for TMB as a standalone predictive biomarker for ICI across all solid tumors.

Biomarkers for Immune Checkpoint Inhibitors in Renal Cell Carcinoma.

Immune checkpoint inhibitor (ICI) therapy has revolutionized renal cell carcinoma treatment. Patients previously thought to be palliative now occasionally achieve complete cures from ICI. However, since immunotherapies stimulate the immune system to induce anti-tumor immunity, they often lead to adverse autoimmunity. Furthermore, some patients receive no benefit from ICI, thereby unnecessarily risking adverse events. In many tumor types, PD-L1 expression levels, immune infiltration, and tumor mutation burden predict the response to ICI and help inform clinical decision making to better target ICI to patients most likely to experience benefits. Unfortunately, renal cell carcinoma is an outlier, as these biomarkers fail to discriminate between positive and negative responses to ICI therapy. Emerging biomarkers such as gene expression profiles and the loss of pro-angiogenic proteins VHL and PBRM-1 show promise for identifying renal cell carcinoma cases likely to respond to ICI. This review provides an overview of the mechanistic underpinnings of different biomarkers and describes the theoretical rationale for their use. We discuss the effectiveness of each biomarker in renal cell carcinoma and other cancer types, and we introduce novel biomarkers that have demonstrated some promise in clinical trials.

Screening biomarkers for predicting the efficacy of immunotherapy in patients with PD-L1 overexpression

PurposeImmunotherapy plays an important role in non-small cell lung cancer (NSCLC); in particular, immune checkpoint inhibitors (ICIs) therapy has good therapeutic effects in PD-L1-positive patients. This study aims to screen NSCLC patients with PD-L1-positive expression and select effective biomarkers for ICI immunotherapy.MethodsCollected tumor samples from the Affiliated Cancer Hospital of Xinjiang Medical University and 117 patients with stage III–IV NSCLC were included in the study. All patients were on first- or second-line therapy and not on targeted therapy. Based on the molecular profiles and clinical features, we screened biomarkers for predicting the efficacy of immunotherapy in patients with PD-L1 overexpression.Results117 NSCLC patients receiving ICIs immunotherapy were enrolled. First, we found that immunotherapy was more effective in patients with positive PD-L1 expression. Second, we found that ROS1 gene mutations, KRAS gene mutations, tumor stage, and the endocrine system diseases history are independent prognostic factors for PD-L1 positive patients. Then we combined independent risk factors and constructed a new Nomogram to predict the therapeutic efficacy of ICIs immunotherapy in PD-L1 positive patients. The Nomogram integrates these factors into a prediction model, and the predicted C-statistic of 3 months, 6 months and 12 months are 0.85, 0.84 and 0.85, which represents the high predictive accuracy of the model.ConclusionsWe have established a model that can predict the efficacy of ICIs immunotherapy in PD-L1 positive patients. The model consists of ROS1 gene mutations, KRAS gene mutations, tumor staging, and endocrine system disease history, and has good predictive ability.

POTEE mutation as a potential predictive biomarker for immune checkpoint inhibitors in lung adenocarcinoma.

Precise selection of patients who could benefit from immune checkpoint inhibitors (ICIs) is an important challenge for immunotherapy in lung cancer. POTEE (POTE Ankyrin Domain Family Member E) is a member of one primate-specific gene family which have been identified as cancer-related antigens and potential target for immunotherapy of cancer. Here, we investigated the correlation between POTEE mutation and the clinical outcome of ICIs treatment in non-small cell lung cancer (NSCLC). We merged three NSCLC cohorts (n = 165) to assess predictive value of POTEE mutation of immunotherapy efficacy in NSCLC. The prognostic analysis and the potential molecular mechanism exploration were conducted based on the data from The Cancer Genome Atlas (TCGA) database. In the merged cohort, patients with POTEE-mutation (POTEE-Mut) had a significantly higher objective response rate (ORR) (100% vs 27.7%; P < 0.001) and longer progression-free survival (PFS) (P = 0.001; HR 0.08; 95% CI 0.01 - 0.54) compared to patients with POTEE wild-type (POTEE-WT) in NSCLC. Also, patients with POTEE-Mut showed higher ORR (100% vs 27.2%; P < 0.001) and longer PFS (P = 0.001; HR 0.07; 95% CI 0.01 - 0.52) in lung adenocarcinoma (LUAD). POTEE mutation was significantly associated with higher tumor mutational burden (TMB) and higher neoantigen load (NAL), but not with PD-L1 expression in LUAD. Gene set enrichment analyses (GSEA) analysis revealed prominent enrichment of signatures related to DNA repair in POTEE-Mut group (P < 0.001) in LUAD. Our results indicate that POTEE mutation could serve as a potential predictive biomarker for ICIs in LUAD. However, prospective cohort studies are still needed for further validation.

Evaluation of microsatellite instability status, a definitive predictive biomarker for immune checkpoint inhibitors (ICI), in underrepresented minorities (URM) with gastrointestinal (GI) cancers.

3538 Background: Mismatch repair deficiency (dMMR) results in MSI-H state and is the first tumor type-agnostic biomarker predictive of ICI response. Among GI cancers, MSI-H is most frequent in colorectal cancer (CRC 15%), gastroesophageal (GEC, 5%) and other (small bowel, hepatopancreatobiliary; 1%). For CRC, MSI-H can be attributed to germline mutation (Lynch syndrome, 3%) or somatic inactivation (sporadic, 12%) of foundational MMR genes. Studies evaluating ICI efficacy in dMMR cancers focus primarily on non-Hispanic White (NHW) patients (pts). We present prevalence, tumor genomic features, and outcomes in pts from a large cohort at Memorial Sloan Kettering (MSK). Methods: Retrospective analysis of MSI-H GI cancers from MSK-IMPACT database. Pts were grouped by self-reported race and ethnicity into 4 study arms: NHW, Asian, non-Hispanic Black (NHB), and Hispanic. Age, tumor type, tumor mutation burden (TMB), and MMR genes were analyzed. Overall survival (OS) estimated with Kaplan-Meier. Results: Of 776 pts with MSI-H GI cancers: 623 (80.3 %) NHW, 60 (7.7 %) Hispanic, 50 Asian (6.5 %), and 43 (5.5 %) NHB. CRC (76%), GEC (14%), other cancers (10%). We present initial evaluation of CRC and GEC: Median age, TMB, and most frequently altered MMR genes (MMR gene FA) are in table. Median OS (mOS) in NHW/URM by receipt of ICI in MSI-H CRC were 38.5m/25.3m (p 0.07) in no-ICI group, 34.2m/28.7m (p 0.64) in +ICI group; MSI-H GEC 43.4m/30m (p 0.44) in no-ICI group, 28.8m/26.7m in +ICI group. Conclusions: Number of URM MSI-H CRC/GEC pts is 7 to 15-fold less than NHW, with no such difference in % MSI-H/MSS between groups; reflecting significant undertesting in URM pts. In MSI-H CRC, median age (m-Age) at sequencing was younger in URM compared to NHW; pronounced in Asian and Hispanic patients, who were 10+ years younger than NHW. No such age difference seen in GEC. No difference in mOS detected between NHW and URM, however a non-significant trend towards worse mOS in URM was observed in the no-ICI group. Next steps include validation of clinico-genomics of MSI-H GI cancers in other large cohorts, including TEMPUS (N = 768) which is ongoing. [Table: see text]

Identification of RPTOR mutation as a novel predictor of efficacious immunotherapy in melanoma.

9524 Background: mTOR pathway is known to influence cancer immunity by metabolism reprogramming. RPTOR (Regulatory Associated Protein of MTOR Complex 1), one of mTOR pathway genes, which is involving in control of mammalian target of mTORC1 activity for regulating cell growth and survival. Whether mutations of RPTOR are associated with clinical efficacy of immune checkpoint inhibitors (ICIs) in melanoma is still ambiguous. Methods: In discovery cohort, we retrospective analyzed the genomic data of 418 melanoma samples which derived from seven immunotherapy studies ( http://www.cbioportal.org/ ) to evaluate the relationship between RPTOR mutation status and efficacy of immunotherapy. Then in validation cohort, the predictive value of RPTOR mutation was confirmed in 320 melanomas from MSKCC cohort (http://www.cbioportal.org/). TMB was calculated as the total count of nonsynonymous mutations in coding sequence. We used the CIBERSORT to evaluate the 22 types immune cell infiltration status in TCGA melanoma cohort. Results: The TMB level of RPTOR-mutant patients was higher than RPTOR-wildtype patients in both discovery (Median [IQR]: 43.11[23.76-140.15] vs. 6.13[2.07-13.66], P &lt; 0.001) and validation (Median [IQR]: 37.38[20.17-84.95] vs. 8.85[3.35-21.07], P &lt; 0.001) cohort. In discovery cohort, compared to RPTOR-wildtype patients, the RPTOR-mutant patients achieved prolonged OS (median OS: not reach, NR vs 22.7 months, HR (95%CI): 0.47(0.22-0.99), P = 0.043). This result was confirmed in validation cohort (median OS: NR vs. 42; HR (95%CI): 0.34 (0.11-1.0); P = 0.049). According to analysis of immune cell infiltration status, the mechanism of the predictive values of RPTOR mutations to ICI efficacy may be activated CD4 memory T cell more abundant in RPTOR-mutant tumors. Conclusions: RPTOR mutation is associated with higher TMB in ICI-treated melanoma patients. Survival analysis shows RPTOR mutation have a good link with longer OS after immunotherapy. These findings indicate that RPTOR mutation may serve as a potential predictive biomarker for ICIs in melanoma.

Biomarkers to predict the efficacy of immune checkpoint inhibitors in patients with non-small-cell lung cancer with actionable genetic alterations.

e21175 Background: Although the efficacy of immune checkpoint inhibitors (ICI) in patients with actionable genetic alterations (AGAs) in non-small-cell lung cancer (NSCLC) has been known to be modest, some patients demonstrated improved survival. Thus, this study aimed to find predictive biomarkers for ICI in patients with NSCLC with various AGAs and to identify the predictive biomarkers for ICI efficacy. Methods: We compared the progression-free survival (PFS) of 324 advanced NSCLC patients who received ICI monotherapy (as over second-line therapy) by different AGAs from January 2018 and July 2022 at the Samsung Medical Center. To identify predictive markers of ICI, we adjusted the impacts on PFS of ICI in smoking status, PD-L1 expression, TP53, KEAP, STK11 mutation status, and steroid use during ICI monotherapy. Results: The median age was 63.1 years (range, 27.8–84.8) and 53.1% of patients was male, and 46.3% of patients was an ex-/or current smoker. A total of 324 patients were included with the following AGAs: EGFR mutation (n = 149, 46.0%), ALK rearrangement, (n = 12, 3.7%), KRAS mutation (n = 72, 22.2%), HER2 mutation or amplification (n = 34, 10.5%), and MET ex14 skipping or amplification (n = 32, 9.9%), ROS1 rearrangement (n = 9, 2.8%), BRAF V600E (n = 9, 2.8%), and RET rearrangement (n = 7, 2.2%). The median PFS was 2.0 months (95% confidence interval, 1.8–2.2) in the total AGA group. The 6-month PFS rate was 25.8% (19.5–34.3) in the group with KRAS/ BRAF V600E/ MET/ HER2 and 12.8% (8.6–19.1) in the group with EGFR/ ALK/ ROS1/ RET (P &lt; 0.01). In the group with KRAS/ BRAF V600E/ MET/ HER2, KEAP, or STK11 wild-type (P = 0.03), PD-L1 (≥1%) (P &lt; 0.01) and steroid use during ICI monotherapy due to immune-related adverse events (IRAE) (P &lt; 0.01) were related to a favorable PFS of ICI; however, no statistical significance was found for TP53 mutation and smoking status for PFS of ICI. In the group with EGFR/ ALK/ ROS1/ RET, PD-L1(≥ 50%) was the only factor that was related to a favorable PFS, and not TP53 mutation status, KEAP, or STK11. Conclusions: This study showed a difference in PFS among each type of AGAs. Although there are limitations due to the small number of patients, the KRAS/ BRAF V600E/ MET/ HER2 group had a favorable PFS compared with the EGFR/ ALK/ ROS1/ RET group . KEAP/STK11/PD-L1 status and steroid use due to IRAE predicted favorable PFS of ICI in the KRAS/ BRAF V600E/ MET/ HER2 group .

Pan-cancer analysis of tumor mutation burden and other key ICI biomarkers in the Indian cohort.

e18872 Background: Tumor Mutation Burden has been correlated with therapy outcomes in patients receiving immune checkpoint inhibitors (ICI). However, tumor specific TMB cut-offs are not well established in the Indian cohort. Genomic alterations are shown to be vital determinants of response, resistance, and hyper-progression post-ICI administration and information about these alterations in Indian patients is scarce. Further, there is limited evidence of reliable predictive biomarkers in ‘immunologically cold’ tumors representing a crucial challenge in this setting. Uncovering key molecular alterations along with TMB and other genomic signatures can aid in selecting potential candidates for immunotherapy. Methods: The study cohort comprised 1383 tumor tissue specimens from solid tumors across twenty three organs including the bladder, breast, lung, ovary, colon, stomach, esophagus, kidney, liver, pancreas, etc. Targeted next-generation sequencing was carried out for more than 400 genes. dMMR/MSI status was evaluated in a subset of samples. Results: Of the 1383 tumor samples analyzed, 81.6% showed TMB-Low ( &lt; 10 mut/Mb), while 18.4% samples had TMB-High (≥10 mut/Mb) and 6.1% had TMB ≥16 mut/Mb. Bladder and cervical cancer sub-cohorts showed the highest median TMB of 8. Breast cancer was the largest subcohort comprising 255 samples with a median TMB of 5. The lowest median TMB of 4 was seen in thyroid cancer. Highest degree of variability was observed within cervical cancer subcohort with TMBs ranging from 1 – 39 mut/Mb, followed by thyroid (1 – 29 mut/Mb), prostate (2 – 31 mut/Mb), and breast (1 – 88 mut/Mb) subcohorts. The least variation in TMB was observed in tumors of the pancreaticobiliary tract (1- 12 m/Mb) and small intestine (3 – 12 m/Mb). Among TMB-H tumors, markers of potential hyper-progression were analyzed. MDM2 amplification (CNV ≥8 copies) was detected in 2.4% (6/254) and MYC amplification (CNV ≥6 copies) was detected in 7.1% (18/254) cases. In a breast cancer subcohort, PIK3CA mutation-linked immune-cold tumor microenvironment was detected in 36% of TMB-H tumors against 29% of TMB-L tumors. Only 0.8% (6/751) samples were diagnosed as MSI-H/dMMR, at a significantly lower incidence against the reported incidence in the western population. All 6 MSI-H patients were also TMB-H; while 95.9% (139/145) patients with TMB-H were found to be MSI-stable. Conclusions: This study gives insights into key ICI biomarkers with TMB profiles of Indian patients and reinforces the varied range of TMB depending on tumor type. The incidence of key genes involved in hyper-progression may support active disease monitoring on ICI therapy in such patients and co-application of multi-pathway therapies to address these resistant pathways. These findings suggest that wider prospective studies to establish cancer-specific thresholds concerning tumor type and patient outcomes should be undertaken.

AI-based radiomic biomarkers to predict PD-(L)1 immune checkpoint inhibitor response within PD-L1 high/low/negative expression categories in stage IV NSCLC.

1517 Background: Recent efforts exploring the utility of quantitative imaging (radiomic) biomarkers to predict response to immune checkpoint inhibitors (ICI) have shown promise to provide a more accurate and scalable method than biopsy-based PD-L1 IHC and tumor mutation burden. One of the challenges of evaluating ICI biomarkers on retrospective real-world data is the inherent association between a physician’s treatment choice and PD-L1 expression status. This work investigates radiomics-based multi-modal biomarkers within patient cohorts receiving first-line therapy (ICI monotherapy, ICI plus chemotherapy, and chemotherapy), and in cohorts of all patients receiving ICI (all-lines) subdivided by PD-L1 expression. Methods: Using a large multi-institutional real-world dataset, we analyzed radiomic characteristics of 6,295 primary and metastatic lesions from 1,206 stage IV NSCLC patients treated with PD-(L)1 ICIs from nine institutions across the US and Europe. Patients with unavailable imaging follow-up or with EGFR/ALK oncogenic driver mutations were excluded from analysis, resulting in a total dataset of 791 subjects randomly assigned to training (N = 541) and validation sets (N = 250). Radiomic data was extracted from baseline CT scans capturing tumor heterogeneity, spicularity, and burden in the lung, lymph nodes, and liver. A multi-modal ensemble classifier combining demographic features, PD-L1 TPS, and radiomic data was developed to predict response to ICI therapy per RECIST 1.1 criteria. The model's performance was evaluated in terms of the area under the receiver operating characteristic curve (ROC-AUC) and compared to PD-L1 IHC using the two-tailed DeLong test. Results: In first-line cohorts, the model identified responders with an ROC-AUC of 0.83 (0.72-0.94, P = 0.005; N = 80, 81% PD-L1 &gt; = 50%) in ICI monotherapy, 0.65 (0.47-0.83, P = 0.28; N = 78, 71% PD-L1 &lt; 50%) in ICI plus chemotherapy, and 0.38 (0.002-0.76, P = 0.53; N = 37) in chemotherapy. In all-lines cohorts subdivided into PD-L1 high/low/negative expression categories, the model identified responders with an ROC-AUC of 0.72 (0.57-0.87, P = 0.005, N = 102), 0.74 (0.58-0.89, P = 0.003, N = 71), and 0.60 (0.46-0.74, P = 0.16, N = 77), respectively. Conclusions: Our study demonstrates that multi-modal models can predict ICI response with high performance. The stronger performance in patients receiving ICI therapy alone indicates the model’s predictive, rather than prognostic, power. Furthermore, the model demonstrated good performance in identifying ICI responders within patient sub-cohorts defined by PD-L1 expression status. These insights may be used to inform clinical decision-making, such as escalation or de-escalation of concurrent chemotherapy in stage IV NSCLC patients. In our future work, we will investigate these questions in larger cohorts and prospective studies.

Progress and Challenges of Immunotherapy Predictive Biomarkers for Triple Negative Breast Cancer in the Era of Single-Cell Multi-Omics.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with a poor prognosis. Despite conventional treatments, including surgery, radiation, and chemotherapy, the overall response rate to PD-1/PD-L1 immune checkpoint inhibitors remains low, with limited predictive significance from current biomarkers such as PD-L1 expression, tumor-infiltrating lymphocytes (TILs), and tumor mutational burden (TMB). To address this challenge, recent advancements in single-cell sequencing techniques have enabled deeper exploration of the highly complex and heterogeneous TNBC tumor microenvironment at the single-cell level, revealing promising TNBC predictive biomarkers for immune checkpoint inhibitors. In this review, we discuss the background, motivation, methodology, results, findings, and conclusion of multi-omics analyses that have led to the identification of these emerging biomarkers. Our review suggests that single-cell multi-omics analysis holds great promise for the identification of more effective biomarkers and personalized treatment strategies for TNBC patients.

Radiomics models based on multisequence MRI for predicting PD-1/PD-L1 expression in hepatocellular carcinoma

The purpose of this study was to explore the effectiveness of radiomics based on multisequence MRI in predicting the expression of PD-1/PD-L1 in hepatocellular carcinoma (HCC). One hundred and eight patients with HCC who underwent contrast-enhanced MRI 2 weeks before surgical resection were enrolled in this retrospective study. Corresponding paraffin sections were collected for immunohistochemistry to detect the expression of PD-1 and PD-L1. All patients were randomly divided into a training cohort and a validation cohort at a ratio of 7:3. Univariate and multivariate analyses were used to select potential clinical characteristics related to PD-1 and PD-L1 expression. Radiomics features were extracted from the axial fat-suppression T2-weighted imaging (FS-T2WI) images and the arterial phase and portal venous phase images from the axial dynamic contrast-enhanced MRI, and the corresponding feature sets were generated. The least absolute shrinkage and selection operator (LASSO) was used to select the optimal radiomics features for analysis. Logistic regression analysis was performed to construct single-sequence and multisequence radiomics and radiomic-clinical models. The predictive performance was judged by the area under the receiver operating characteristic curve (AUC) in the training and validation cohorts. In the whole cohort, PD-1 expression was positive in 43 patients, and PD-L1 expression was positive in 34 patients. The presence of satellite nodules served as an independent predictor of PD-L1 expression. The AUC values of the FS-T2WI, arterial phase, portal venous phase and multisequence models in predicting the expression of PD-1 were 0.696, 0.843, 0.863, and 0.946 in the training group and 0.669, 0.792, 0.800 and 0.815 in the validation group, respectively. The AUC values of the FS-T2WI, arterial phase, portal venous phase, multisequence and radiomic-clinical models in predicting PD-L1 expression were 0.731, 0.800, 0.800, 0.831 and 0.898 in the training group and 0.621, 0.743, 0.771, 0.810 and 0.779 in the validation group, respectively. The combined models showed better predictive performance. The results of this study suggest that a radiomics model based on multisequence MRI has the potential to predict the preoperative expression of PD-1 and PD-L1 in HCC, which could become an imaging biomarker for immune checkpoint inhibitor (ICI)-based treatment.

Distinct circulating cytokine/chemokine profiles correlate with clinical benefit of immune checkpoint inhibitor monotherapy and combination therapy in advanced non-small cell lung cancer.

An ever-increasing number of efforts are focused on identifying effective biomarkers for immune checkpoint inhibitors (ICIs). Cytokines and chemokines are critical to tumor growth, metastasis, tumor angiogenesis, and the immune response against tumor cells. In the study here, we determined the correlation between circulating cytokines/chemokines and the clinical benefit of ICIs for non-small cell lung cancer (NSCLC) patients. Peripheral blood samples were collected before and during treatment (at 12th week). Plasma levels of cytokines/chemokines and specific stress response markers were measured using the Bio-Plex Pro Human Cytokines Grp I Panel (27-plex), an APEX1 detection kit, and a human LAP(TGF-β1) immunoassay kit. A Mann-Whitney U-test or Wilcoxon signed-rank test and a Cox proportional hazards model were employed for statistical analysis. In the ICI monotherapy cohort, a high level of IL-6 at pretreatment or an elevation of IL-6, IL-8, FGF2, CXCL10, CCR1, PDFGB, TNF, and APEX1 posttreatment was associated with poor progress-free survival (PFS). A posttreatment elevation (defined herein as change rate) of CXCL10 was also associated with poor overall survival (OS). In the combinational therapy group, a high level of IL-12, IL-17A, FGF2, VEGF, and APEX1 at pretreatment and an elevation of CCL2 posttreatment were associated with poor PFS. A high level of IL-9, FGF2, PDFGB, CCL4, TFGB, and APEX1 at pretreatment and an elevation of IL-13, CSF2, and CCL2 at posttreatment were associated with poor OS of patients receiving combination therapy. The study here suggests that circulating cytokines/chemokines are feasible, noninvasive biomarkers for predicting clinical benefit of ICI treatment for NSCLC. Distinct circulating factor profiles were observed in individuals receiving ICI monotherapy or combination therapy.

Role of β-Catenin Activation in the Tumor Immune Microenvironment and Immunotherapy of Hepatocellular Carcinoma

Recently, the therapeutic combination of atezolizumab and bevacizumab was widely used to treat advanced hepatocellular carcinoma (HCC). According to recent clinical trials, immune checkpoint inhibitors (ICIs) and molecular target agents are expected to be key therapeutic strategies in the future. Nonetheless, the mechanisms underlying molecular immune responses and immune evasion remain unclear. The tumor immune microenvironment plays a vital role in HCC progression. The infiltration of CD8-positive cells into tumors and the expression of immune checkpoint molecules are key factors in this immune microenvironment. Specifically, Wnt/β catenin pathway activation causes “immune exclusion”, associated with poor infiltration of CD8-positive cells. Some clinical studies suggested an association between ICI resistance and β-catenin activation in HCC. Additionally, several subclassifications of the tumor immune microenvironment were proposed. The HCC immune microenvironment can be broadly divided into inflamed class and non-inflamed class, with several subclasses. β-catenin mutations are important factors in immune subclasses; this may be useful when considering therapeutic strategies as β-catenin activation may serve as a biomarker for ICI. Various types of β-catenin modulators were developed. Several kinases may also be involved in the β-catenin pathway. Therefore, combinations of β-catenin modulators, kinase inhibitors, and ICIs may exert synergistic effects.

Clinical markers of immunotherapy outcomes in advanced sarcoma

BackgroundDespite immunotherapy’s promise in oncology, its use for sarcoma remains challenging. There are no sarcoma-specific biomarkers for immune checkpoint inhibitors (ICI). Previously, we reported our institutional experience highlighting ICI activity in 29 patients with sarcoma. In this study, we explore responses to ICI based on ICI regimen and other covariates to identify significant clinical factors in advanced sarcoma outcomes.MethodsPatients in The Ohio State University Sarcoma Clinics were enrolled in the Sarcoma Retrospective ICI database from January 1, 2015 through November 1, 2021. Data included treatment regimen (single-agent ICI or ICI + combination) along with clinical covariates. ICI + combination was further categorized into ICI + medication, ICI + radiation, ICI + surgery, or ICI + multiple (more than 2 modalities). Statistical analysis included log-rank tests and proportional hazard regression. The primary objective was to evaluate overall survival (OS) and progression-free survival (PFS).ResultsOf the patients in the database, 135 met inclusion criteria. We demonstrated improved OS in patients treated with ICI + combination (p = 0.014, median 64 weeks), but no effect on PFS (p = 0.471, median 31 weeks). Patients with a documented immune-related adverse event (irAE) of dermatitis had improved OS, but only in the ICI + combination cohort (p = 0.021). Patients who received single-agent ICI and whose change in the neutrophil-to-lymphocyte ratio (NLR) was less than 5 had an improved OS (p = 0.002); this was not seen in patients who received ICI + combination therapy (p = 0.441). There were no differences in OS based on age, gender, histology, or subcategories of ICI + combination. This was not the case for PFS; patients who received any ICI regimen and were younger than 70 had a worse PFS (p = 0.036) compared with their older counterparts in this dataset. Patients who developed an irAE, specifically colitis (p = 0.009), hepatitis (p = 0.048), or dermatitis (p = 0.003), had an improved PFS. There were no differences in PFS based on ICI regimen (or subcategories of ICI + combination), gender, histology, change in NLR, or grade of irAE.ConclusionsThis retrospective study demonstrates that ICI + combination therapy can improve OS in some patients with advanced sarcoma. This is consistent with our prior results of ICI in sarcoma.

Abstract 3321: Development of a CTC-based novel diagnostic technology for unresectable non-small cell lung cancer

Abstract BACKGROUND: PD-L1 in pathological tissue samples is known as a biomarker for immune checkpoint inhibitors (ICI) to unresectable non-small cell lung cancer (NSCLC) patients. However, collecting pathological tissue is highly invasive, and lower invasive test is needed. As an alternative diagnostic target, circulating cancer cells (CTCs) that are known to exist in the peripheral blood of cancer patients are garnering attention. PURPOSE: The goal of this study is to verify the utility of CTC analysis isolated by a novel system (Sysmex, Hyogo) against pathological testing of tumor samples. The primary objective is to determine whether CTCs can be detected in unresectable NSCLC patients, and the secondary objective is to evaluate the relationship between CTCs and clinicopathological features or clinical outcome. METHODS: Blood samples were collected from 45 patients with unresectable NSCLC and 17 healthy controls. CTCs were size-dependently enriched from whole blood, immunofluorescent stained with pan-CK (AE1/AE3), PD-L1, CD45/34/16, and Hoechst, and analyzed by Molecular Imaging Flow Cytometry (MI-1000). Fluorescence intensity and cell images of each marker were obtained by MI-1000. RESULTS: Cells positive for Hoechst but negative for blood cell markers were defined as non-blood cells (NBCs). Pan-CK positive NBCs were defined as CTCs. A similar number of NBCs were detected in both patients and healthy controls. When using a threshold set at two standard deviations above the mean fluorescence intensity of pan-CK and PD-LI of NBCs in healthy controls, 28.9% of patients had two or more CTCs. On the other hands, PD-L1 positive NBCs were only detected in patients, and the sensitivity of detecting CTCs or PD-L1 positive NBCs was 86.7%. Concordance with PD-L1 expression of tumor tissue was evaluated for three parameters: the number of PD-L1 positive NBCs, the median and the mean fluorescence intensities. The AUC in each case was low, indicating no concordance between PD-L1 expression of tumor tissue and CTCs. Additionally, no relation was found with the overall survivals and progression-free survival when comparing patients divided into two groups: those with two or more PD-L1 positive cells and those with less than two. CONCLUSION: The detected rate of pan-CK positive cells was low (&amp;lt;30%), but increased to 86.7% when pan-CK negative and PD-L1 positive NBCs, which are not found in healthy controls, were included. However, further studies are needed to evaluate whether these NBCs are CTCs. In addition, there was no association between PD-L1 positive NBCs and PD-L1 expression of tumor tissue or clinical outcome in this study. Recently, we have verified that the detected ratio of CTCs could be improved by adding a CK8/18 marker and hence, plan to verify the clinical usefulness of detecting CTCs using CK8/18. Citation Format: Mami Onishi, Eri Morita, Tetsuya Sakai, Yoshitaka Zenke, Masatoshi Yanagida, Koichi Goto. Development of a CTC-based novel diagnostic technology for unresectable non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3321.

Abstract 600: Tumour and local lymphoid tissue interaction determines prognosis in high grade serous ovarian cancer

Abstract Tertiary lymphoid structure (TLS) is an ectopic lymphoid organ found in some tumors. The presence of TLS is recently proposed as a predictive biomarker for immune checkpoint inhibitors (ICI) in mutation-driven tumors. It is also associated with prognosis in copy number-driven tumors, including high grade serous ovarian cancer (HGSOC), although the function of TLS and its interaction with copy-number alterations in HGSOC is not fully understood. In the current study, we comprehensively analyze immune profile, copy number profile and quantitative imaging profile from 242 HGSOC patients (HH cohort), and combine these with two public single cell RNA-sequencing datasets (Lambrechts cohorts; n=9, 29,999 cells). TLS-high HGSOC patients show significantly better progression free survival in both HH and TCGA cohorts. We show that presence of TLS in HGSOC tumors is associated with B-cell maturation, resulting in higher and more specific immunoglobulin production; TLS also leads to more cytotoxic tumor-specific T-cells activation and proliferation. Through integrated analysis of single cell RNA-sequencing and whole exome sequencing data, we reveal that the copy number loss of IL15 and CXCL10 may limit TLS formation in HGSOC; we also propose a list of genes that may dysregulate TLS function. A candidate gene, DCAF15, is found to confer resistance to NK-mediated cytotoxicity in HGSOC cell lines. Lastly, we develop a radiomics-based signature to predict presence of TLS, which independently predicts PFS in both HGSOC patients and ICI-treated NSCLC patients. Overall, we reveal that TLS coordinates intratumoral B-cell and T-cell response against HGSOC tumor, while cancer genome evolves to counteract TLS formation and function. Citation Format: Haonan Lu, Georg Wengert, Hantao Lou, Reema Paudel, Naina Patel, Saral Desai, Bill Crum, Kristofer Linton-Reid, Mitchell Chen, Dongyang Li, Jacey Ip, Francesco Mauri, David J. Pinato, Andrea Rockall, Susan J. Copley, Sadaf Ghaem-Maghami, Eric O. Aboagye. Tumour and local lymphoid tissue interaction determines prognosis in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 600.

Heterogeneity of tumour mutational burden in metastatic NSCLC demonstrated by endobronchial ultrasound sampling.

Tumour mutational burden (TMB) is an important emerging biomarker for immune checkpoint inhibitors (ICI). The stability of TMB values across distinct EBUS tumour regions is not well defined in advanced lung cancer patients. This study included a whole-genome sequencing cohort (n=11, LxG cohort) and a targeted Oncomine TML panel cohort (n=10, SxD cohort), where paired primary and metastatic samples were obtained by endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA). The LxG cohort displayed a strong correlation between the paired primary and metastatic sites, with a median TMB score of 7.70 ± 5.39 and 8.31 ± 5.88 respectively. Evaluation of the SxD cohort demonstrated greater inter-tumoural TMB heterogeneity, where Spearman correlation between the primary and metastatic sites fell short of significance. Whilst median TMB scores were not significantly different between the two sites, 3 out of 10 paired samples were discordant when using a TMB cut-off of 10 mutations per Mb. In addition, PD-L1 copy number and KRAS mutations were assessed, demonstrating the feasibility of performing multiple molecular tests relevant to ICI treatment using a single EBUS sample. We also observed good consistency in PD-L1 copy number and KRAS mutation, where cut-off estimates were consistent across the primary and metastatic sites. Assessment of TMB acquired by EBUS from multiple sites is highly feasible and has the potential to improve accuracy of TMB panels as a companion diagnostic test. We demonstrate similar TMB values across primary and metastatic sites, however 3 out of 10 samples displayed inter-tumoural heterogeneity that would alter clinical management.

Phase II study of the combination of durvalumab, tremelimumab, and paclitaxel as second-line chemotherapy in biomarker-selected patients with metastatic gastric cancer.

401 Background: Currently, ramucirumab plus paclitaxel is the standard second-line chemotherapy in metastatic gastric cancer (mGC), however, its efficacy is very limited. MSI, PD-L1 (CD274), and EBV have been suggested as predictive biomarkers for immune checkpoint inhibitors in mGC. This study was conducted to evaluate efficacy and safety of the combination of durvalumab, tremelimumab, and paclitaxel as a second-line chemotherapy in mGC with potential biomarkers for immune checkpoint inhibitors. Methods: The combination of durvalumab, tremelimumab, and paclitaxel consisted of 1500 mg of i.v. durvalumab on day 1, every 4 weeks for 13 cycles, 75 mg of i.v. tremelimumab on day 1 every 4 weeks for 4 cycles, and 60 mg/m2 of i.v. paclitaxel on days 1, 8, and 15, every 4 weeks until disease progression or unacceptable toxicities. Patients (pts) with MSI-high mGC, EBV-positive mGC, or mGC with CD274 amplification, mutations of mismatch repair or POL gene, or tumor mutation burden (TMB) &gt;5/Mb were included in second-line setting. Results: Forty-eight pts were enrolled. Overall response rate (ORR) was 52.1% with complete response in 4 (8.3%) pts, partial response in 21 (43.8%), stable disease in 14 (29.2%), and progressive disease in 8 (16.7%), and with response not evaluable in 1 (2.0%). With a median follow-up of 18.0 months (range, 7.1-39.4), the median progression-free survival (PFS) and overall survival (OS) were 5.3 months (95% CI, 3.7-6.9) and 13.1 months (95% CI, 5.2-21.0 ), respectively. Compared to mGC with other genetic alterations (n=25), mGC with TMB &gt;20/Mb or MSI-high mGC (n=23) tended to have a higher ORR (56.5% vs 48%), and a prolonged PFS (median 7.2 vs 4.5 months; 44.0 vs 20.8% at 1 year). The combination chemotherapy was generally well tolerable; treatment-related grade 3 or 4 adverse events with frequency &gt;5% included only neutropenia (10.4%) and anemia (8.3%), and there was no treatment-related death. Conclusions: The combination chemotherapy of durvalumab, tremelimumab, and paclitaxel showed encouraging efficacy, especially in mGC with TMB &gt;20/Mb or MSI-high mGC, as a second-line chemotherapy with manageable toxicities in biomarker-selected mGC. Clinical trial information: NCT03751761 .

The efficacy of immune checkpoint inhibitors in biliary tract cancer with KRAS mutation.

598 Background: Biliary tract cancer (BTC) is rare cancer but has a poor prognosis with only 10% of 5-year overall survival (OS). The treatment of advanced BTC remains challenging with limited standard options. With a 15% of incidence, KRAS is one of the most common mutations in BTC. The immune checkpoint inhibitor as salvage therapy has modest activity in BTC. Methods: We conducted molecular profiling including tumor mutational burden in BTC patients with immune checkpoint inhibitors (ICI) as salvage therapy. Programmed death ligand 1 (PD-L1) expression on tumor cells and on tumor-infiltrating lymphocytes (TIL) was also assessed. Results: Between 2020 to 2022, a total of 62 patients were analyzed. The median age was 68 (58.0-73.0). 47 patients (75.8%) received pembrolizumab and 15 (24.2%) received nivolumab as salvage therapy. All patients received gemcitabine plus cisplatin as the frontline therapy, and 53.2% had fluoropyrimidine plus oxaliplatin (FOLFOX) before ICIs. The median lines of prior chemotherapy were 2.5 (2.0-3.0). KRAS mutation was found in 14 patients (22.6%). 28 patients (45.2%) were positive for PD-L1. There was no statistical correlation between KRAS mutation and PD-L1 expression. The median OS and PFS of patients were 5.2 and 3.5 months respectively. There were no statistically significant differences in PFS according to the status of KRAS mutation (Mutant type vs. Wild type) and PD-L1 expression (Positive vs Negative). In subgroup analysis, patients with KRAS mutation and PD-L1 positivity had longer PFS as compared to patients with KRAS mutation and PD-L1 negative (6.5 vs 2.6 months, p=0.047). This finding was not shown in patients without KRAS mutation. Conclusions: The PD-L1 expression might be a novel biomarker for ICIs in BTC patients with KRAS mutation but not in those with the wild type of KRAS.