Low PD-L1 Expression Research Articles

339P Analysis of first- and second-line immunotherapy efficacy in metastatic non-small cell lung cancer with low PD-L1 expression

Immune checkpoint inhibitors (ICI) improve overall survival (OS) in non-small cell lung cancer (NSCLC) but the efficacy of first- vs second-line ICI is unknown. We compared the outcomes of advanced NSCLC patients with <50% PD-L1 expression who received first- and second-line ICI.

Radioimmunotherapy study of 131I-labeled Atezolizumab in preclinical models of colorectal cancer

BackgroundProgrammed cell death 1 ligand 1(PD-L1) is overexpressed in many tumors. The radionuclide-labeled anti-PD-L1 monoclonal antibody can be used for imaging and therapy of PD-L1 overexpressing cancer. Here, we described 131I-labeled Atezolizumab (131I-Atezolizumab, targeting PD-L1) as a therapeutic agent for colorectal cancer with PD-L1 overexpression.Methods131I-Atezolizumab was prepared by the Iodogen method. The expression levels of PD-L1 in different human colorectal cells were determined by flow cytometry, western blot and cell binding assay. The immunoreactivity of 131I-Atezolizumab to PD-L1 high-expressing cells was determined by immunoreactive fraction. The killing abilities of different concentrations of 131I-Atezolizumab on cells with high and low expression of PD-L1 were detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Cerenkov luminescence imaging (CLI) and radioimmunotherapy (RIT) of 131I-Atezolizumab were performed on two human colorectal cancer models. The distribution and tumor targeting of 131I-Atezolizumab were evaluated by imaging. Tumor volume and survival time were used as indicators to evaluate the anti-tumor effect of 131I-Atezolizumab.ResultsThe expression level of PD-L1 in vitro determined by the cell binding assay was related to the data of flow cytometry and western blot. 131I-Atezolizumab can specifically bind to PD-L1 high-expressing cells in vitro to reflect the expression level of PD-L1. Immunoreactive fraction of PD-L1 high-expressing RKO cells with 131I-Atezolizumab was 52.2%. The killing ability of 131I-Atezolizumab on PD-L1 high-expressing cells was higher than that of low-expressing cells. CLI proved that the specific uptake level of tumors depends on the expression level of PD-L1. Effect of 131I-Atezolizumab RIT showed an activity-dependent tumor suppressor effect on RKO tumor-bearing mice with high PD-L1 expression. 131I-Atezolizumab (37 MBq) can improve the median survival time of mice (34 days), compared to untreated mice (27 days) (P = 0.027). Although a single activity(37 MBq) of 131I-Atezolizumab also inhibited the tumors of HCT8 tumor-bearing mice with low PD-L1 expression (P < 0.05), it could not prolong the survival of mice(P = 0.29).Conclusion131I-Atezolizumab can be used as a CLI agent for screening PD-L1 expression levels. It may be used as a radioimmunotherapy drug target for PD- L1 overexpressing tumors.

Efficacy and safety of anti-PD-1/PD-L1 therapy in the treatment of advanced colorectal cancer: a meta-analysis

BackgroundImmune checkpoint inhibitors have shown promise in microsatellite instability-high/mismatch repair deficient (MSI-H/dMMR) advanced colorectal cancer (CRC) immunotherapy, and many clinical trials have been conducted.ObjectiveTo evaluate the efficacy and safety of PD-1/PD-L1 inhibitors in advanced CRC.MethodPubMed, Web of Science, Embase, and The Cochrane Library were searched for relevant studies up to September 2021. A retrospective cross-sectional data analysis was performed and Stata 16 software was used for analyses.ResultsSixteen studies including 1503 patients were analyzed. The objective response rate (ORR) of anti-PD-1/PD-L1 was 23% (95% CI 0.14, 0.31); the overall 1-year survival rate (OSR) was 57% (95% CI 0.42, 0.73). The ORR of MSI-H/dMMR advanced CRC was 37% (95% CI 0.25, 0.48) and that of microsatellite stable/mismatch repair proficient (MSS/pMMR) disease was 11% (95% CI 0.06, 0.16). The ORR was 42% in the BRAF mutant subgroup and 19% in the RAS mutant group. The ORR was 14% in the PD-L1 ( +) subgroup and 32% in the PD-L1(-) subgroup. The rate of adverse effects was 85% (95% CI 0.80, 0.91).ConclusionAnti-PD-1/PD-L1 therapy in MSI-H/dMMR advanced CRC was associated with improved survival. Anti PD-1/PD-L1 combined with antiangiogenic drugs, targeted agents, or chemotherapy might be effective in MSS mCRC. Immunotherapy was effective for the BRAF mutant and KRAS/NRAS(RAS) mutant CRC. Low expression of PD-L1 was a potential predictive marker for positive response and outcome. The high incidence of adverse events at 85% was worthy of further investigation. Further analysis with a higher number of high-quality studies is needed to verify the conclusions.

PD-L1-Mediated Immunosuppression in Hepatocellular Carcinoma: Relationship with Macrophages Infiltration and Inflammatory Response Activity.

Immune dysfunction and pro-oncogenic inflammation play critical roles in malignant progression and non-response to immunotherapy for hepatocellular carcinoma (HCC). In particular, PD-1/PD-L1 blockade therapy could induce durable tumor remissions and improve the prognosis of patients to a certain extent. However, PD-L1, as a promising biomarker, has limited knowledge about its relevance to tumor microenvironment (TME) characterization and endogenous inflammatory immune responses. In this study, we systematically investigated and characterized the important intercommunication of PD-L1 with immunosuppressive TME and inflammatory response activity in HCC and predicted promising therapeutic drugs to improve the current therapeutic strategy for specific patients. We identified aberrant expression patterns of PD-L1 in HCC and completely different clinical and molecular characteristics among the PD-L1 subgroups. PD-L1 positively associated with immunosuppressive macrophages and macrophage-derived cytokines, which may contribute to the polarization of macrophages. Moreover, inflammatory response activity exhibited significant differences between high and low PD-L1 expression groups and had robust positive correlativity of the infiltration level of tumor-associated macrophages. Notably, given the immunosuppressive and inflammatory microenvironment in HCC, we screened four candidate drugs, including dasatinib, vemurafenib, topotecan and AZD6482, and corroborated in two pharmacogenomics databases, which might have potential therapeutic implications in specific HCC patients. Our results enhanced the understanding of linkage in PD-L1 expression patterns with macrophages and inflammation, which may provide new insight into the pathogenic mechanisms and potential therapeutic strategy for HCC.

P2.12-04 The Role of Serum Proteomic Signature in Predicting Survival in PD-L1 Low Non-small Cell Lung Cancer Receiving Immune Checkpoint Inhibitor (ICI)

The VeriStrat test is a blood-based proteomic test derived from machine learning which uses MALDI-ToF mass spectrometry based signature. Results from this test have shown prognostic utility in different stages, histologies, and treatment types for patients with non-small-cell lung cancer (NSCLC). Recently, the VeriStrat test has also shown prognostic utility in patients receiving ICI treatment. Meanwhile, the role of biomarker is not established among lung cancer patients with low PD-L1 expression, for whom the effect of ICI treatment is limited.

Association of PD-L1 Expression and Other Variables With Benefit From Immune Checkpoint Inhibition in Advanced Gastroesophageal Cancer

Approval by the US Food and Drug Administration of immune checkpoint inhibition (ICI) for advanced gastroesophageal cancer (aGEC) irrespective of PD-L1 status has generated controversy. Exploratory analyses from individual trials indicate a lack of meaningful benefit from ICI in patients with absent or low PD-L1 expression; however, analysis of a single variable while ignoring others may not consider the instability inherent in exploratory analyses. To systematically examine the predictive value of tissue-based PD-L1 status compared with that of other variables for ICI benefit in aGEC to assess its stability. MEDLINE, Embase, Scopus, Web of Science, Cochrane Central Register (2000-2022). Randomized clinical trials (RCTs) were included of adults with aGEC (adenocarcinoma [AC] or squamous cell carcinoma [SCC]) randomized to anti-PD-1 or PD-L1-containing treatment vs standard of care (SOC). Study screening, data abstraction, and bias assessment were completed independently by 2 reviewers. Of 5752 records screened, 26 were assessed for eligibility; 17 trials were included in the analysis. The prespecified primary end point was overall survival. The mean hazard ratio (HR) for ICI vs SOC was calculated (random-effects model). Predictive values were quantified by calculating the ratio of mean HRs between 2 levels of each variable. In all, 17 RCTs (9 first line, 8 after first line) at low risk of bias and 14 predictive variables were included, totaling 11 166 participants (5067 with SCC, 6099 with ACC; 77.6% were male and 22.4% were female; 59.5% of patients were younger than 65 years, 40.5% were 65 years or older). Among patients with SCCs, PD-L1 tumor proportion score (TPS) was the strongest predictor of ICI benefit (HR, 0.60 [95% CI, 0.53-0.68] for high TPS; and HR, 0.84 [95% CI, 0.75-0.95] for low TPS), yielding a predictive value of 41.0% favoring high TPS (vs ≤16.0% for other variables). Among patients with AC, PD-L1 combined positive score (CPS) was the strongest predictor (after microsatellite instability high status) of ICI benefit (HR, 0.73 [95% CI, 0.66-0.81] for high CPS; and HR, 0.95 [95% CI, 0.84-1.07] for low CPS), yielding a predictive value of 29.4% favoring CPS-high (vs ≤12.9% for other variables). Head-to-head analyses of trials containing both levels of a variable and/or having similar design generally yielded consistent results. Tissue-based PD-L1 expression, more than any variable other than microsatellite instability-high, identified varying degrees of benefit from ICI-containing therapy vs SOC among patients with aGEC in 17 RCTs.

PD-L1 Over-Expression Varies in Different Subtypes of Lung Cancer: Will This Affect Future Therapies?

Programmed death-ligand (PD-L) 1 and 2 are ligands of programmed cell death 1 (PD-1) receptor. They are members of the B7/CD28 ligand-receptor family and the most investigated inhibitory immune checkpoints at present. PD-L1 is the main effector in PD-1-reliant immunosuppression, as the PD-1/PD-L pathway is a key regulator for T-cell activation. Activation of T-cells warrants the upregulation of PD-1 and production of cytokines which also upregulate PD-L1 expression, creating a positive feedback mechanism that has an important role in the prevention of tissue destruction and development of autoimmunity. In the context of inadequate immune response, the prolonged antigen stimulation leads to chronic PD-1 upregulation and T-cell exhaustion. In lung cancer patients, PD-L1 expression levels have been of special interest since patients with non-small cell lung cancer (NSCLC) demonstrate higher levels of expression and tend to respond more favorably to the evolving PD-1 and PD-L1 inhibitors. The Food and Drug Administration (FDA) has approved the PD-1 inhibitor, pembrolizumab, alone as front-line single-agent therapy instead of chemotherapy in patients with NSCLC and PD-L1 ≥1% expression and chemoimmunotherapy regimens are available for lower stage disease. The National Comprehensive Cancer Network (NCCN) guidelines also delineate treatment by low and high expression of PD-L1 in NSCLC. Thus, studying PD-L1 overexpression levels in the different histological subtypes of lung cancer can affect our approach to treating these patients. There is an evolving role of immunotherapy in the other sub-types of lung cancer, especially small cell lung cancer (SCLC). In addition, within the NSCLC category, squamous cell carcinomas and non-G12C KRAS mutant NSCLC have no specific targetable therapies to date. Therefore, assessment of the PD-L1 expression level among these subtypes of lung cancer is required, since lung cancer is one of the few malignances wherein PD-L1 expression levels is so crucial in determining the role of immunotherapy. In this study, we compared PD-L1 expression in lung cancer according to the histological subtype of the tumor.

The Prognostic Value of Eight Immunohistochemical Markers Expressed in the Tumor Microenvironment and on Hodgkin Reed-Sternberg Cells in Pediatric Patients With Classical Hodgkin Lymphoma.

Immunohistochemical markers are associated with treatment outcome in adults with classical Hodgkin Lymphoma (cHL). Studies in children are scarce and inconsistent. We investigated in 67 children with cHL, whether the expression of CD15, CD30, PAX5, PD-1, PD-L1, CD68, CD163 and TARC at diagnosis is associated with disease free survival (DFS) and with interim remission status. Low CD15 and low TARC expression were associated with relapsed disease. Low expression of PD-L1 was associated with complete remission at interim PET-scan. Our data suggest a difference between pediatric and adult cHL. This underlines the importance of future research into specific prognostic factors in pediatric cHL, indispensable for improvement of treatment in this population.

Abstract CT008: Long-term outcomes with sotorasib in pretreated KRASp.G12C-mutated NSCLC: 2-year analysis of CodeBreaK100

Abstract Introduction: Sotorasib, a first-in-class KRASG12C inhibitor, has been FDA-approved for adults with KRAS p.G12C-mutated locally advanced or metastatic NSCLC who received prior systemic therapies based on the Phase 1/2 global, single-arm CodeBreaK100 trial. We will report the longest follow-up for a KRASG12C inhibitor, including 2-year survival, safety, and genomic profiles associated with durable clinical benefit. Methods: Sotorasib was administered orally at 960 mg once daily to patients who progressed on prior therapies and had KRAS p.G12C-mutated locally advanced, metastatic NSCLC. Primary endpoint was objective response rate (ORR) assessed by central review. Key secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. In an exploratory analysis, baseline tumor tissue and/or plasma samples were collected and analyzed for genomic alterations. Tumor samples were analyzed for PD-L1 levels to evaluate correlates with prolonged tumor response, defined as patients with PFS ≥ 12 months, in comparison with those with PFS ≤ 3 months. Results: In the first analysis of the combined Phase 1 and Phase 2 study (N=174), the median number of prior lines of therapy was 2.0 (range 1-4+). 90.2% received prior anti-PD1 or anti PD-L1 treatment; 82.8% received both prior platinum-based chemotherapy and anti-PD1/PD-L1. Updated ORR by central review was 40.7% (95% CI: 33.2, 48.4) and median DOR was 12.3 months (7.1, 14.6). Median PFS and OS was 6.3 months (95% CI: 5.3, 8.2) and 12.5 months (10.0, 17.8). One and two-year OS was 50.8% and 30.3%. Sotorasib was well-tolerated in the long-term with mild and manageable toxicities, and no new safety signals emerged. Prolonged tumor response was observed across PD-L1 expression, including in tumors with low PDL-1 expression and STK11 co-mutation that may derive less benefit from immunotherapy. Conclusions: In the longest follow-up of any KRASG12C inhibitor, sotorasib continued to demonstrate a favorable safety profile and durable efficacy, including a 2-year OS observed in 30% of patients. Current analyses continue to support long-term clinical benefit across subgroups in patients with KRAS p.G12C-mutated NSCLC, and additional biomarker data will be presented. Citation Format: Grace K. Dy, Ramaswamy Govindan, Vamsidhar Velcheti, Gerald S. Falchook, Antoine Italiano, Juergen Wolf, Adrian G. Sacher, Toshiaki Takahashi, Suresh S. Ramalingam, Christophe Dooms, Dong-Wan Kim, Alfredo Addeo, Jayesh Desai, Martin Schuler, Pascale Tomasini, Qui Tran, Simon Jones, Agnes Ang, Abraham Anderson, Antreas A. Hindoyan, David S. Hong, Bob T. Li. Long-term outcomes with sotorasib in pretreated KRASp.G12C-mutated NSCLC: 2-year analysis of CodeBreaK100 [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 CT008.

Abstract 4206: In situ vaccination with CCL21-DC sensitizes non-responsive murine NSCLCs to anti-PD-1 therapy and generates systemic tumor-specific immune memory

Abstract Decresased responses to immune checkpoint inhibitors (ICIs) in NSCLC are associated with low tumor mutational burden (TMB), impaired tumor antigen presentation, decreased baseline CD8+ T lymphocytes tumor infiltration and low PD-L1 expression in the tumor microenvironment (TME). One approach to augment the efficacy of ICIs in NSCLC is to enhance anti-tumor T cell responses by utilizing in situ vaccination (ISV) with professional antigen-presenting dendritic cells (DCs). In preclinical studies as well as a phase I clinical trial, we have shown that ISV with gene-modified DCs expressing CCL21 (CCL21-DC) promotes effector T lymphocyte infiltration into the tumor, PD-L1 upregulation, and systemic tumor-specific immune responses. We hypothesized that in situ vaccination with CCL21-DC could promote T cell priming and activation, thereby sensitizing non-responsive NSCLC tumors to ICI. Utilizing multiple syngeneic murine models of NSCLC with distinct driver mutations [KrasG12D/P53+/-/Lkb1-/- (KPL); KrasG12D/P53+/- (KP); and KrasG12D (K)] and varying TMB, we observed that CCL21-DC ISV synergizes with anti-PD-1 therapy resulting in the eradication of a subset of tumors. Evaluation of KPL tumors by flow cytometry and scRNA-seq revealed that combination therapy reprogrammed the myeloid compartment to be less immunosuppressive, and induced T cell infiltration, Th1 polarization, as well as the enrichment of stem-like TCF1+ T cells within the TME. Intratumoral (IT) CCL21-DC enhanced CD103+DC migration to the local lymph node as determined by flow cytometry. Addition of ICI to IT CCL21-DC resulted in enhanced T cell proliferation and INF-γ secretion within the TME and the spleen. Whole exome sequencing (WES) of tumors revealed immunoediting of tumor subclones after IT CCL21-DC and ICI combination therapy. Tumor rechallenge studies demonstrated the establishment of systemic tumor-specific immunity in a subset of mice that were cured of disease.In conclusion, tumor vaccination with CCL21-DC sensitizes immune-resistant murine NSCLC to anti-PD-1 therapy. Combination therapy enhances anti-tumor T cell responses, resulting in immunoediting of tumor subclones and the generation of systemic tumor-specific immunity. In an ongoing clinical trial, we are evaluating the safety and efficacy of ISV with CCL21-DC in combination with anti-PD-1, pembrolizumab, in patients with advanced NSCLC who have progressed on front-line therapy. Citation Format: Ramin Salehi-Rad, Raymond J. Lim, Linh M. Tran, Yushen Du, Camelia Dumitras, Tianhao Zhang, William Crosson, Jensen Abascal, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situ vaccination with CCL21-DC sensitizes non-responsive murine NSCLCs to anti-PD-1 therapy and generates systemic tumor-specific immune memory [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 4206.

Abstract 1709: Improved understanding of the biology and pathophysiology of the tumor microenvironment in PDAC samples revealed by InSituPlex, Imaging Mass Cytometry, and advanced image processing

Abstract Pancreatic cancer remains a deadly disease due to difficulties hindering its early diagnosis, giving way to metastasis of the tumor and resulting in poor prognosis. While there are many neoplasms of the pancreas, pancreatic invasive ductal adenocarcinoma (PDAC) is the most common, and treatment options are few, with poor overall survival. Recently, several publications have demonstrated improved outcomes with the inclusion of immunotherapy to cytotoxic drug combinations in some patients. However, optimally selecting patients as candidates for immunotherapy-chemotherapy combinations remains a critical challenge. The complexities of the tumor microenvironment (TME) have been implicated in the failure of chemotherapy, radiation therapy, and immunotherapy. The tumor microenvironment of PDAC is especially rich with multiple interactions between pancreatic epithelial/cancer cells, stromal cells, immune cells, and the extracellular matrix (ECM). PDACs are characterized by a complex ECM of desmoplastic reaction consisting of an extensive and dense fibrotic stroma that surrounds and infiltrates clusters of malignant epithelial cells, together with the loss of basement membrane integrity and an abnormal vasculature. PD-L1 is also expressed in PDACs, and its overexpression has been associated with a poor prognosis. In the present study we demonstrate a unique tissue phenotyping workflow combining complementary methods that can unravel the complexity of the tumor microenvironment. We highlight how a workflow combining multiple elements provides utility, robustness, and an ability to derive biological insights in PDAC samples. An InSituPlex® PD-L1 multiplex immunofluorescence assay (4 markers, CD8, CD68, PD-L1, Pan CK/Sox10) was used on whole slides to identify areas of high, medium, and low PD-L1 expression. Imaging Mass Cytometry™ (IMC™, 40 markers) was performed on selected regions of interest from each slide. Advanced tissue and cell segmentation followed by multiplex cellular phenotyping and spatial analyses were performed on both whole-slide InSituPlex and IMC data. These methods combine to give a detailed readout of the location and bio-distribution of specific cell phenotypes in situ in the TME of PDAC. Four-plex imaging and analysis of whole slides gives an overview of the immune status of the section, while 40-plex imaging and analysis gives a comprehensive and multiparametric exploration of cells present. These methods combine to reveal an exceptional view of the PDAC TME at the single-cell level. Citation Format: Andrew Quong, Jordan Nieto, Derek Quong, Amanda Esch, Kirsteen Maclean, Mark Rees, Devan Fleury, Gourab Chatterjee, Keith Wharton, Jeppe Thagaard, Fabian Schneider, Dan Winkowski, James Mansfield. Improved understanding of the biology and pathophysiology of the tumor microenvironment in PDAC samples revealed by InSituPlex, Imaging Mass Cytometry, and advanced image processing [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 1709.

Abstract 2530: Macrophage biomarkers CD68 and CD163 correlate with CRC patient survival

Abstract Background: Immune cells within the tumor microenvironment (TME) play a vital role in regulating tumor progression. Therefore, immunotherapies that elicit anti-tumor responses are of great interest for the treatment of various cancers. Macrophages are a current immune cell type of interest due to the ability to polarize into anti-tumor (M1) and pro-tumor (M2) phenotypes. The density and phenotype of macrophages within the tumor and TME have been linked to prognosis in multiple types of solid tumors. Our hypothesis is that colorectal cancer (CRC) patients with high immune infiltration and greater amounts of anti-tumor immune cells within the tumor compartment will have an increased time of survival compared to cancers with immune excluded or desert environments. Methods: One CRC tumor microarray (TMA) containing primary tumors, metastases, and normal tissue were stained via multiplex immunofluorescence (mIF) for 6 different immune markers: CD3, CD8, CD56, CD68, CD163, and PD-L1. The stained TMAs were analyzed utilizing Flagship Biosciences’ proprietary image analysis platform. Machine learning algorithms used cellular features to stratify cells as belonging to either the tumoral or stromal compartment. Core level expression data was pulled and represented on a whole-cohort basis. All staining and image analysis outputs were reviewed by a board-certified, MD pathologist. Kaplan-Meier curves were generated using survival data in relation to the low, medium, and high expression of CD68, CD163, and/or PDL1 in the whole tissue, as well as tumor and stromal compartments. Patients were also stratified into low (1 and 2) and high (3 and 4) cancer stages. Results: There is a correlation between patient survival and the presence or absence of macrophage markers CD68 and CD163 and lack of immune-suppressive marker PDL1. Specifically, lower levels of CD68+CD163- cells within the tumor compartment correlate with an increase in patient survival. Similarly, lower PDL1 expression in the stromal compartment positively correlate with prolonged patient survival in CRC patients. Conclusion: Data generated through Flagship Biosciences’ image analysis platform showed a strong relationship between CD68 and CD163 presence and localization with CRC patient survival. Altering the immune cells within the tumor to an anti-tumor immune environment could increase patient survival times in high stage CRC patient populations. Combining immune checkpoint inhibitors with current FDA approved therapies for CRC are of interest to further extend patient survival, as PDL1 expression negatively correlate with CRC patient survival. Further, utilizing Flagship Biosciences’ image analysis software to understand cancer immune microenvironments should be further utilized to aid in diagnosis and treatment decisions. Citation Format: Dannah Miller, Kate Hieber, Will Paces, Huong Nguyen, Adam Beharry, Roberto Gianani. Macrophage biomarkers CD68 and CD163 correlate with CRC patient survival [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 2530.

Abstract 6121: Correlation of tumor immune microenvironment status and driver genes alterations in Chinese non-small-cell lung cancer patients

Abstract Background: Patients with different driver genes show a variety of immunotherapy efficacy for non-small-cell lung cancer (NSCLC). In addition to PD-L1, the evaluation of the immune microenvironment contributes to a deeper understanding of the mechanisms of immunotherapy resistance. Methods: Tumor tissues from 2674 NSCLC patients were collected. Genetic alterations were profiled using targeted next-generation sequencing with 733 cancer-related genes panel. Immunohistochemistry was used to evaluate the PD-L1 tumor proportion score, (TPS) expression on the surface of tumor cells. Tumor-infiltrating immune cells of 120 tumor samples were labeled with multiple immunofluorescence staining. Results: Overall, CD8+ T cell infiltration level was negatively associated with both EGFR copy number gain (CNG, p &amp;lt; 0.001) and ERBB2 CNG (p = 0.021). Also, the presence of EGFR or ERBB2 CNG was related with fewer natural killer cell infiltration (EGFR: p &amp;lt; 0.001; ERBB2: p = 0.036) and a lower M1/M2 tumor-associated macrophages ratio compared to wild-type tumors (EGFR: p = 0.051; ERBB2: p = 0.036), suggesting a relatively weaker anti-tumor ability. In addition, we noted that KRAS CNV was negatively related to M2 macrophages (p = 0.027). Patients carrying ALK translocation (p &amp;lt; 0.001), ROS1 translocation (p = 0.006), KRAS mutations (p &amp;lt; 0.001) and MET (p &amp;lt; 0.001) mutations had the elevated PD-L1 TPS expressions, while those harboring EGFR (p &amp;lt; 0.001) and ERBB2 mutations (p = 0.001) had lower PD-L1 expression. Compared to the other EGFR mutations, PD-L1 expression in EGFR CNG tumors was higher, and no significant differences were found between CNG and other types of mutation in patients harboring ERBB2, KRAS, MET alterations. Conclusion: NSCLC patients with EGFR CNG and ERBB2 CNG had fewer invasion of cytotoxic T cells and NK cells and a lower M1/M2 macrophages ratio. EGFR CNG may intensively modify the tumor immune microenvironment, which is a possible mechanism contributing to EGFR-TKI resistance. Citation Format: Jindong Guo, Liwen Xiong, Haibin Yuan, Mengna Hu, Bei Zhang, Ding Zhang. Correlation of tumor immune microenvironment status and driver genes alterations in Chinese non-small-cell lung cancer patients [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 6121.

Abstract 1320: Immunogenomic analysis identifies the complement pathway as a therapeutic target in STK11 mutant non-small cell lung cancer

Abstract Background: The use of immune PD-1/-L1 checkpoint inhibitors (ICI) has dramatically altered the treatment of advanced non-small cell lung cancer (NSCLC). However, a large proportion of patients with NSCLC do not derive clinical benefit from ICI treatment. Recent studies have identified certain genomic subsets of NSCLC as drivers of primary resistance to ICI treatment. Methods: We evaluated NSCLC patients treated at Roswell Park Comprehensive Cancer Center from 2017-2020 with successful genomic and immune profiling using a CLIA-certified laboratory developed test that included targeted next-generation genomic sequencing, PD-L1 assessment by immunohistochemistry (IHC), and targeted RNA-seq of 394 immune transcripts. Results: A total of 379 treatment-naive non-squamous NSCLC subjects were identified with 113 subjects treated with an FDA approved ICI regimen and evaluable for response. Using a gene expression signature based on differential gene expression analysis in the 113 subjects with and without disease control with ICI treatment, we performed unsupervised clustering of the larger 379 subject cohort and identified a responder (R) cluster and non-responder (NR) cluster. The R cluster was associated with high PD-L1 expression, T-cell infiltration, and KRAS mutations. The NR cluster was associated with negative PD-L1 expression, lack of T-cell infiltration, and mutations in STK11, APC/CTNNB1, and NFE2L2. Of the mutation subsets, STK11 was most associated with NR with a trend towards lower PD-L1 expression by clinical IHC, but much more significantly associated by gene expression signatures with low PD-L1 and T-cell inflammation. In our gene panel, we identified elevated TRIM29 expression in STK11 mutant NSCLC tumors and confirmed TRIM29 expression by IHC on a separate 64 subject cohort with known STK11 mutation status and archival formalin fixed paraffin embedded (FFPE) tissue. A subset of STK11 mutant NSCLC cell lines also demonstrated elevated TRIM29 expression and we generated knockouts of TRIM29 with CRISPR-Cas9 in these cell lines which resulted in decreased tumor growth in xenograft models. Using Nanostring nCounter multiplex inflammation gene expression panel on both the 64 patient FFPE cohort and TRIM29 knockout cell lines, we identified the innate immune complement pathway as a target of therapeutic interest and specifically the high expression of complement component C3 by TRIM29 high tumor cells. We then generated a syngeneic mouse model where loss of Stk11 resulted in elevated expression of Trim29 and C3 for preclinical studies to evaluate the therapeutic modulation of the complement pathway on response to ICI. Conclusion: Using a large clinical cohort of non-squamous NSCLC patients we characterized the immune environment of mutational subsets of NSCLC and identified the complement pathway as a potential therapeutic target in STK11 mutant NSCLC. Citation Format: Edwin H. Yau, Bojidar Kandar, Lei Deng, Te-An Chen, Wiam Bshara, Sean Glenn, Sarabjot Pabla, Antonios Papanicolau-Sengos, Mary Nesline, Hongbin Chen, Amy Early, Carl Morrison, Grace Dy. Immunogenomic analysis identifies the complement pathway as a therapeutic target in STK11 mutant non-small cell lung cancer [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 1320.

Spatial interplay patterns of cancer nuclei and tumor-infiltrating lymphocytes (TILs) predict clinical benefit for immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) show prominent clinical activity across multiple advanced tumors. However, less than half of patients respond even after molecule-based selection. Thus, improved biomarkers are required. In this study, we use an image analysis to capture morphologic attributes relating to the spatial interaction and architecture of tumor cells and tumor-infiltrating lymphocytes (TILs) from digitized H&E images. We evaluate the association of image features with progression-free (PFS) and overall survival in non–small cell lung cancer (NSCLC) (N = 187) and gynecological cancer (N = 39) patients treated with ICIs. We demonstrated that the classifier trained with NSCLC alone was associated with PFS in independent NSCLC cohorts and also in gynecological cancer. The classifier was also associated with clinical outcome independent of clinical factors. Moreover, the classifier was associated with PFS even with low PD-L1 expression. These findings suggest that image analysis can be used to predict clinical end points in patients receiving ICI.

Application of value framework to phase III trials of immune checkpoint inhibitors in esophageal and gastric cancer.

e18817 Background: Recent clinical trials testing use of immune-checkpoint inhibitors in esophageal and gastric carcinomas have shown both positive and negative results. Given these mixed signals, we aimed to assess outcomes and tolerability of key subgroups using the ASCO Net Health Benefit Score (NHBS) and ESMO Magnitude of Clinical Benefit Scale (MCBS). Methods: A detailed search for phase III trials investigating use of FDA-approved anti PD-1 or anti PD-L1 drugs in esophageal and gastric cancer trials was completed using www.clinicaltrials.gov followed by primary literature identification on PubMed. These studies were then independently selected, reviewed, and scored using the ASCO NHBS and ESMO MCBS by two investigators, and any discrepancy was mutually resolved. The ASCO NGBS scores were compared by primary site of cancer (esophageal vs gastric) and PD-L1 expression using the Mann-Whitney test. The ESMO-MCBS grading were compared using Fisher's Exact test. Results: Of the 45 records identified, 15 clinical trials were included. Of them, six were primarily esophageal cancer trials, and nine were primarily gastric cancer trials. Of all 15, 10 stratified their analysis based on PD-L1 expression. The ASCO NHBS score was significantly higher for esophageal cancer than gastric cancer (mean 40, range 20 – 56.6 vs. mean 12, range -1.1 – 18.4, p &lt; .01). No difference, however, was observed in survival and response rate outcome endpoints between the two groups. Similarly, the ESMO MCBS was also higher for esophageal cancer group than gastric cancer (p &lt; .05). Additionally, the median scores were higher in the patients with high PD-L1 expression vs low PD-L1 expression (mean 37, range 11.2 – 66.6 vs. mean 14, range -19.5 – 43.6, p &lt; .05). Conclusions: In this cohort of drug registration trials of immune checkpoint inhibitors for upper GI cancers, we showed the ASCO NHB scores and ESMO scores were consistently higher among esophageal cancer trials than gastric cancer trials and in those with high PD-L1 expression than those with low PD-L1 expression. Oncologists should carefully discuss overall clinical value and expectations of immunotherapy benefit with their patients per cancer histology and PDL1 expression, exercising caution in cases of gastric adenocarcinoma with low PD-L1 expression.[Table: see text]

EO2401, a novel microbiome-derived therapeutic vaccine for patients with adrenocortical carcinoma (ACC): Preliminary results of the SPENCER study.

4596 Background: In advanced ACC, no significant progress has been made since introduction of mitotane and cisplatin-based therapy. EO2401 (EO) was designed to activate existing commensal memory T cells cross-reacting with tumor associated antigens (TAAs). EO includes microbial-derived, synthetically produced peptides corresponding to HLA-A2 restricted epitopes with molecular mimicry to three TAAs upregulated in ACC, IL13Rα2, BIRC5 and FOXM1, with the CD4 helper peptide UCP2 and the adjuvant Montanide. Pre-clinically EO generates strong immune responses and cross-reactive CD8 cells recognizing the TAAs. Methods: This Phase 1/2 trial (NCT04187404) investigated EO + nivolumab (N) (EO + N = EN) in pts with ACC. Cohort 1 lead-in established safety of EN. Cohort 2 includes pts with metastatic ACC, with (C2a), or without (C2b) prior systemic therapy. EN was given 4 times q2 w, followed by boosters q4 w until PD (iRECIST). Results: 33 pts with ACC started study treatment: C2a 26 pts (58% 1; 42% 2 prior lines), C2b 7 pts. Median age 47; 24% men; ECOG 0/52%, 1/42%, 2/6%; 61% ≥2 organs involved by metastases (61% liver, 76% lung). EN was well tolerated. The combination safety profile was consistent with the profile of N monotherapy except for higher local administration site reactions (any erythema/pain/induration in 35% of pts). Overall (n = 33), best RECIST response was PR 12%, SD 24%, PD 45%, NE 18%; median PFS was 1.9 mo (range 0.4-7.6+); median survival not reached, and survival rate at 6-mo 63% (median FU 4.9 mo, range 0.9-12.0). Strong CD8 T cell ELISPOT responses against the vaccine peptides (9/9 pts) and cross-reactivity against targeted TAAs (8/8 evaluable pts) was observed. Tetramer staining of specific CD8 cells for all 3 peptides was detected in 7/8 tested. When investigated, positive staining against BIRC5 was detected as early as 4 w after the first vaccination. In C2a, a group of pts (n = 10) with SD at the first CT (incl. 4 pts with PR) seemed to fare well; all investigated tumor samples in this group showed a low level of TMB, low MSI, and low PDL1 expression. In contrast, 10 pts had PD &lt; 2mo and died &lt; 6mo. There was no correlation between clinical benefit and a large panel of cytokines/chemokines. However, post-hoc analysis identified several clinical factors (prior mitotane, ECOG ≤ 1, ACC 1st diagnosis &gt; 9 mo, max lesion ≤125 mm, ≤3 organs involved, lymphocytes ≤ grade 1) that excluded 90% of pts without benefit to EN. In the post-hoc selected group (n = 14) with median FU 6.9 mo (12 pts censored) the DCR was 64% (4 PR, 5 SD), 6-mo PFS was 42% and 6-mo survival rate 93%. Conclusions: EO2401 in combination with nivolumab was well tolerated and induced a specific immune response in all tested pts. In addition, efficacy was seen in a subpopulation of pts with ACC defined by clinical parameters in a post-hoc analysis. A randomized phase 2 study based on the findings of Cohort 2a is being planned. Clinical trial information: NCT04187404.

The influence of EGFR mutation status and PD-L1 expression in stage III unresectable non-small cell lung cancer treated with chemoradiation and consolidation durvalumab.

e18810 Background: The PACIFIC trial established concurrent chemoradiation followed by one year of consolidation durvalumab as the standard of care for unresectable stage III non-small cell lung cancer (NSCLC). However, the data to support the use of durvalumab in patients with EGFR mutations and low PD-L1 expression is less clear. This study adds to the available evidence by describing the survival outcomes for patients with stage III unresectable NSCLC treated with chemoradiation followed by durvalumab with a focus on their EGFR mutation and PD-L1 status. Methods: This retrospective observational study was conducted at six sites across Sydney, Australia. It included patients diagnosed with unresectable stage III NSCLC treated with chemoradiation and at least one cycle of durvalumab between January 2018 to September 2021. Patients were stratified into EGFR wild-type and mutated tumours and PD-L1 tumour proportional score (TPS) greater than or less than 1%. Survival analyses were supplemented with cox proportional hazards regression models which estimated HRs for PFS in treatment group comparisons. The proportional hazards assumption was confirmed for all cox models. All statistical analysis was done using R version 4.1.1. Results: 146 patients were included in the analysis. The median follow-up from the start of Durvalumab was 15.1 months. The median age was 66 years in the EGFR mutated and 65 years in the EGFR wild-type cohorts. 61% (n = 89) of patients were male. 10% (n = 15) of patients had EGFR mutation. 42% (n = 61) of patients had a PD-L1 TPS of &lt; 1%. PD-L1 expression was similar in both cohorts with 47% (n = 7) of EGFR mutated patients having PD-L1 TPS of &lt; 1% compared with 45% (n = 59) in the wild-type group. The median progression-free survival (PFS) was 7.5 months for EGFR mutated patients versus 33.9 months for EGFR wild- type patients (HR: 2.7; 95% CI: 1.2 – 5.7; p = 0.01). There was no statistically significant difference in either PFS (HR 0.7, 95% CI 0.4 – 1.7, p = 0.43) or OS (HR 0.5, 95% CI 0.4 – 4.7, p = 0.16) for patients with PD-L1 TPS of &lt; 1% compared with PD-L1 TPS of &gt; 1%. Conclusions: Our data adds to the growing evidence that suggests consolidation durvalumab after definitive chemoradiation may not be as efficacious in patients with EGFR-mutated tumours. PD-L1 TPS did not affect survival outcomes.

Impact of HIV infection on clinical outcomes among people diagnosed with head and neck cancer.

e18080 Background: Incidence of non-AIDS-defining cancers, including head and neck cancer (HNC), is rising among people with HIV (PWH) in the combination antiretroviral therapy (cART) era. The following study compares demographics, clinical outcomes, and the tumor microenvironment of HIV+ and Uninfected HNC patients at a single institution. Methods: Yale Tumor Registry query identified 3,356 HNC patients from 2002 to 2018 for analysis, including 50 PWH. In addition, quantitative immunofluorescence (QIF) was performed on tumor tissue from 22 PWH and 75 matched Uninfected patients. HPV status was based on p16 staining done universally among oropharyngeal squamous cell carcinomas (OPSCC) after 2010. Results: PWH were younger at cancer presentation compared to Uninfected patients (55.5 vs. 62.0, p &lt; 0.001), with differences in race/ethnicity and insurance status (p &lt; 0.001 for both). Biologic sex, tobacco use, alcohol consumption, anatomic site, stage at presentation, stage-specific treatment, and time to initiation of treatment were comparable between HIV+ and Uninfected cohorts. 89% of the PWH were on cART, with 79% achieving viral loads ≤200 copies/mL and a median CD4 count of 341 cells/mm³. Median survival among PWH was 39.1 months, compared to 100.8 months among Uninfected patients (p &lt; 0.001). In a multivariate (MV) analysis that included age, sex, race/ethnicity, tobacco use, anatomic site, stage, time to treatment initiation and insurance status, HIV was an independent predictor of poor outcome (HR 1.88 with 95% CI: 1.25-2.81). Difference in survival was noted particularly in early stage (stages 0, I, II) cancer, with a median survival of 73.8 months in PWH compared to 141.9 months in Uninfected patients (p = 0.001). Survival was comparable among late stage (stages III, IV) HNC patients. Among PWH, increased HIV viral load was associated with poor outcome in a MV analysis (p = 0.03). Among HPV-associated OPSCC, PWH had decreased survival compared to Uninfected patients (p &lt; 0.001). In our study of the tumor microenvironment by QIF, tumors of PWH had lower tumoral CD8 T cell infiltration (p = 0.04) and lower PD-L1 expression in tumor, stroma, and combined compartments (CPS) (p = 0.01, p = 0.03, and p = 0.01, respectively) compared to Uninfected patients. Conclusions: In our single institution study, HNC patients living with HIV experienced decreased overall survival, with HIV serving as an independent predictor of poor outcome in a multivariate analysis which included insurance status and treatment approaches. Difference in outcome was significant among early stage tumors and among HPV+ OPSCC. CD8 T cell infiltration and PD-L1 expression, both associated with improved outcomes in the general population, are decreased within the TME of PWH. Our data suggest that HIV-associated HNC is associated with poorer outcomes and highlight differences in tumor biology that require further detailed characterization in large cohorts.

Landscape of fibroblast growth factor receptor (FGFR) genomic alterations (GA) in urothelial bladder cancer (UBC).

4568 Background: Urothelial bladder carcinomas (UBC) with genomic alterations (GA) in the Fibroblast Growth Factor Receptor ( FGFR) genes have been postulated to be less responsive to immune checkpoint inhibitors (ICI). Immune microenvironment of these tumors could be altered due to suppression of interferon signaling pathways. Here, we present comprehensive genomic profiling (CGP) of FGFR altered UBC to study the underlying immunogenomic mechanisms of response and resistance. Methods: 4,035 UBC underwent hybrid capture based CGP. Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined using 114 loci. Programmed death ligand (PD-L1) expression in tumor cells was assessed by IHC (Dako 22C3). Results: 894 (22%) of UBC featured FGFR GA ( FGFR1 3.7%; FGFR2 1.1%; FGFR3 17.4%). Gender and age distribution was similar in all groups. FGFR3 cases had lower GA/tumor and 14.7% GA were fusions. ERBB2 amplification was significantly higher in FGFR1/2 altered UBC compared with FGFR3 altered UBC. MTOR pathway GA were highest in FGFR3 altered UBC. FGFR3 altered UBC featured significantly higher frequencies of biomarkers predicting resistance to ICI including lower TMB, lower PD-L1 expression and higher frequencies of GA in MDM2. FGFR3 driven UBC also features significantly higher frequencies of CDKN2A/B loss and MTAP loss which have also recently been linked to IO drug resistance. Conclusions: UBC harboring FGFR GA have increased frequency of alterations that have been linked to ICI resistance. Further evaluation of FGFR-based biomarkers in UBC clinical trials focused on the assessment of the patient response to ICI appears warranted. [Table: see text]