Tumor Cell PD-L1 Research Articles

Photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes to promote PD-L1 multivalent binding for effective immune checkpoint blockade therapy

Immune checkpoint blockade (ICB) therapy targeting PD-L1 via monoclonal antibody (mAb) has shown extensive clinical benefits in the diverse types of advanced malignancies. However, most patients are completely refractory to ICB therapy owing to the PD-L1 recycling mechanism. Herein, we propose photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes (immune checkpoint blockade liposomes; ICB-LPs) to promote PD-L1 multivalent binding for inducing lysosomal degradation of PD-L1 in tumor cells. The ICB-LPs are prepared by formulation of DC8,9PC with photo-polymerized diacetylenic moiety, 1,2-dipalmitoylphosphatidylcholine (DPPC) and anti-PD-L1 peptide (D-form NYSKPTDRQYHF)-conjugated DSPE-PEG2k (anti-PD-L1-DSPE-PEG2k) in a molar ratio of 45:45:10, followed by cross-linking of liposomal bilayer upon UV irradiation. The 10 mol% anti-PD-L1-DSPE-PEG2k incorporated ICB-LPs have a nano-sized lipid bilayer structure with an average diameter of 137.7 ± 1.04 nm, showing a high stability in serum condition. Importantly, the ICB-LPs efficiently promote the multivalent binding with PD-L1 on the tumor cell membrane, which are endocytosed with aim to deliver PD-L1 to the lysosomes, wherein the durable PD-L1 degradation is observed for 72 h, in contrast to anti PD-L1 mAbs showing the rapid PD-L1 recycling within 9 h. The in vitro co-culture experiments with CD8+ T cells show that ICB-LPs effectively enhance the T cell-mediated antitumor immune responses against tumor cells by blocking the PD-L1/PD-1 axis. When ICB-LPs are intravenously injected into colon tumor-bearing mice, they efficiently accumulate within the targeted tumor tissues via both passive and active tumor targeting, inducing a potent T cell-mediated antitumor immune response by effective and durable PD-L1 degradation. Collectively, this study demonstrates the superior antitumor efficacy of crosslinked and anti-PD-L1 peptide incorporated liposome formulation that promotes PD-L1 multivalent binding for trafficking of PD-L1 toward the lysosomes instead of the recycling endosomes.

Research progress in the regulation of tumor cell PD-L1 expression by N6-methyladenosine modification

Research progress in the regulation of tumor cell PD-L1 expression by N6-methyladenosine modification

Cbl induced ubiquitination of HER2 mediate immune escape from HER2-targeted CAR-T.

Breast cancer (BC) with high HER2 expression has higher recurrence rate and worse prognosis, and its immunotherapy is promising.Based on the high expression of HER2, develop Chimeric Antigen Receptor T-cell (CAR-T) and PDL-1 immunotherapy, and study the molecular pathways of related immune cells and recurrence.HER2-CAR-T cells were constructed using retroviruses, and their specific recognition and immune effects on HER2+ BC cells were verified by in vivo and in vitro experiments. PDL-1 was used as adjuvant immunotherapy, knocking down PDL-1 in tumor cells or dendritic cells, or depleted macrophages to study immune pathways. The negative regulation of HER2 by cbl was determined by IP, ubiquitination experiments, and segmented plasmids, elucidating the molecular mechanism of HER2+ BC recurrence after immunotherapy.HER2-CAR-T specifically recognizes HER2-positive tumor cells and inhibits tumor growth in vivo and in vitro, and anti-PDL1 treatment enhances the therapeutic effect of HER2-CAR-T on tumors. HER2-CART therapy eradicated solid tumors after PDL1 knockdown in dendritic cells. Immunotherapy of relapsed tumors lost HER2 expression by upregulating cbl.HER2-CAR-T shows specific recognition of HER2+ cells and can mediate immune response therapy with the cooperation of PDL-1.

Biomarkers of response to first-line nivolumab therapy in patients with advanced renal cell carcinoma (RCC) enrolled in the HCRN GU16-260 trial.

4549 Background: The HCRN GU16-260 study showed that nivolumab (nivo) monotherapy is active in treatment-naïve patients (pts) with RCC. Although efficacy correlated with tumor cell (TC) PD-L1 expression, more clinically relevant biomarkers are needed. We previously showed that levels of CD8+ tumor-infiltrating lymphocytes (TIL) expressing PD-1 but not TIM-3 and LAG-3 (CD8+ PD1+ TIM3- LAG3-) were associated with response to nivo in previously treated clear cell RCC (ccRCC) pts (Pignon, 2019; Ficial, 2021). Here, we sought to validate these findings in the first-line setting. Methods: Primary tumor tissues from pts with ccRCC (n= 67) and non-ccRCC (n= 13) were analyzed by multiparametric immunofluorescence (IF), and the density of CD8+PD-1+TIM-3-LAG-3- TIL was quantified by image analysis. The correlation of the log-transformed biomarkers with objective response rate (ORR) and progression-free survival (PFS) was assessed in the ccRCC cohort and the combined (ccRCC + non-ccRCC) cohort (n=80) using Cox or logistic regression analysis, or Chi-square/Fisher’s exact test (as appropriate) for statistical inference – alpha level was set at 5% (1-sided). Results: In both the ccRCC and the combined cohort, density of CD8+PD-1+TIM-3-LAG-3- TIL (IF biomarker) measured as continuous variable was associated with ORR (ccRCC: OR = 1.54, p = 0.017; combined: OR = 1.35, p = 0.040) and PFS (ccRCC: HR = 0.79, p = 0.015; combined: HR = 0.85, p = 0.016). At a cutoff optimized for maximum sensitivity for ORR, ccRCC patients with a high (61/67) vs low (6/67) IF biomarker had higher ORR (44.3% vs. 0% p = 0.039) and longer median PFS (10.9 months vs. 4.2 months, p = 0.016). Of note, when combined with the IF biomarker, TC PD-L1 expression (≥1%) further separated the clinical outcomes of ccRCC pts. Conclusions: High levels of CD8+ PD1+ TIM-3-LAG-3- TILs were associated with response to first-line nivo in pts with advanced RCC and the combination with TC PD-L1 status further separated the outcomes of pts with ccRCC, validating our previous findings. Further investigations into the role of myeloid inflammation as determinant of clinical outcome to nivo therapy are ongoing. Clinical trial information: NCT03117309 . [Table: see text]

Clinical characterization of MYC family proto-oncogene amplification in solid tumors from Chinese patients.

e15140 Background: The dysregulation of the MYC family oncogenes ( c-MYC, MYCN and MYCL) play critical roles in tumorigenesis, prognosis and immune escape. MYC inactivation can result in sustained tumour regression and many therapeutic agents that directly target MYC are under development. MYC signaling is associated with tumor cell PD-L1, overall immune cell infiltration. Herein, we explore MYC family proto-oncogene amplification profiles and clinical characterization in chinese solid tumors. Methods: This research comprehensively characterized gene mutations by next-generation sequencing (NGS) in 23990 chinese solid tumors tissues to reveal the prevalence of MYC family proto-oncogene amplification(MYC AMP) and the association with Tumor mutational burden(TMB) and microsatellite instability(MSI). Results: The prevalence of MYC AMP (copy number, CN≥5) in the cohort was 2.1% (504/23,990), in which ovarian cancer (7.6%, 22/289) showed the highest prevalence, followed by breast cancer (6.1%, 26/429), esophagus cancer (5.9%, 17/287). Only one glioma patient(pt) carried co-amplification of MYCN and c-MYC. In 504 MYC AMP pts, c-MYC AMP accounted for 93.7%(472/504), MYCN and MYCL AMP accounted for 6.5%(33/504) in total. The CN was significantly higher in MYCN and MYCL AMP pts than c-MYC AMP pts (22.9 vs 7.6, p < 0.0001). MSI-H showed a lower detection rate in MYC AMP pts other than Non-MYC AMP pts (0% vs 1.2%, p < 0.05). The proportion of TMB-L in MYC AMP pts was similar to Non-MYC AMP pts (90.9% vs. 90.1%, p > 0.05). Conclusions: In totally, 2.1% of chinese solid tumor pts had MYC high level AMP, mainly c-MYC Amp. The CN was higer in MYCN/ MYCL AMP pts than c-MYC AMP pts. In addition, MYC AMP pts tended to have MSS and TMB-L, suggesting that MYC may be a novel target for tumor immunotherapy. MYC inhibitor combines with immunotherapy may be an important direction for the treatment of MYC AMP pts.

The metabolic activity assessed by 18F-FDG PET and its correlation with tumor immune status and prognosis in advanced biliary tract cancer patients treated with 1L GemCis plus durvalumab +/-tremelimumab.

4092 Background: Gemcitabine/Cisplatin (GemCis) in combination with durvalumab have been established as a global new standard of care in first-line (1L) advanced biliary tract cancer (BTC) in the TOPAZ-1 trial (Oh et al. NEJM Evid, 2022). In our phase 2 study (NCT03046862, Oh et al. Lancet Gastroenterol Hepatol, 2022) exploring GemCis/durvalumab +/- tremelimumab, dynamics of PD-L1 expression after one cycle of treatment has been suggested as a predictor for clinical outcomes. Tumor metabolic activity assessed with 18F-FDG PET is usually associated with prognosis in solid tumors. However, the role of metabolic activity in patients treated with the combination of chemotherapy and immunotherapy has not yet been much studied, in terms of correlation with tumor immune landscape and prognosis. Methods: This study recruited 1L, advanced BTC patients in 3 different cohorts. 18F-FDG PET was conducted at baseline (pre-treatment) and at the time of first tumor response evaluation (post-treatment). Metabolic indices of the main tumor lesion were measured, including maximum standardized uptake values (SUVmax), metabolic tumor volume (MTV), and total lesion glycolysis (TLG). Metabolic response evaluation was assessed according to EORTC criteria. Tumor biopsies were done at baseline and after 1 cycle of treatment. Results: Among total 124 enrolled patients, 118 patients were included in this PET analysis. The early metabolic response rate was 54.3% (mCR 10.2%, mPR 44.1%, mSD 36.4%, mPD 9.3%). Pre-treatment metabolic parameters (SUVmax, MTV, TLG) were associated with disease status, previous operation history, and CRP level. High pre-treatment SUVmax was associated with poor progression-free survival (PFS) and overall survival (OS). Low post-treatment metabolism and large reduction in metabolism between pre- and post-treatment were associated with responder (vs non-responder), long PFS and long OS. In terms of PD-L1 expression and tumor metabolism, Tumor cell (TC) PD-L1 at pre-treatment was not associated with metabolic parameters; however, high immune cell (IC) PD-L1 expression showed significant association with lower MTV and TLG. In terms of dynamics of PD-L1 and tumor metabolism, post-treatment SUVmax was higher in patients with TC PD-L1 decrease. In patients with decrease of PD-L1 on both TC and IC (worst prognostic group), smaller reduction in metabolism between pre- and post-treatment was observed than the other patients. Conclusions: In BTC patients treated with 1L GemCis plus durvalumab +/- tremelimumab, tumor metabolic activity and its dynamics conferred prognostic impact. Furthermore, tumor metabolic activity has a potential to be associated with tumor immune landscape. Clinical trial information: NCT03046862 .

B7H3 expression in sarcoma.

e23517 Background: Targeted therapy development in sarcoma has been burdened by the heterogeneity of this rare group of tumors. B7H3 is an immune checkpoint molecule in the same family as PD-L1 (also known as B7H1). Its limited expression in non-cancerous tissues and over-expression in numerous tumor types makes it an attractive target for cancer immunotherapy. Available data suggests high expression of B7H3 in certain sarcoma subtypes, including rhabdomyosarcoma, osteosarcoma, liposarcoma, Ewing sarcoma, synovial sarcoma and chondrosarcoma. This study was conducted to further explore B7H3 expression patterns across sarcoma subtypes and the relationship, if any, with PD-1 and PD-L1 expression. Methods: This single-institution, retrospective analysis evaluated soft tissue sarcoma Formalin-Fixed Paraffin-Embedded tumor specimens in patients with a variety of different subtypes. Specimens were evaluated for expression of B7H3, vessel positivity for B7H3, B7H3 staining pattern, and expression of PD-L1 and PD-1. The association between B7H3 expression and baseline and tumor factors was tested using the Mantel Haenszel test or two-sample T-test. Overall survival was estimated using the Kaplan-Meier method where survival time was calculated from diagnosis to death (or last follow-up for those last known to be alive). Results: 156 sarcoma specimens encompassing 16 subtypes were included. B7H3 was broadly expressed across sarcoma subtypes, with some degree of expression found in 91% of samples and 61% demonstrating high levels of B7H3 expression. All tumor samples expressing B7H3 exhibited the biomarker in at least 80% of tumor cells per sample (with a mean of 92.34% of cells testing positive for B7H3). All samples positive for B7H3 showed both cytoplasmic and membranous expression, and B7H3 vessel positivity was found in 90% (137/152) of samples. There were no significant differences in expression or level of expression when stratified by sarcoma subtype, prior treatment(s), tumor size, tumor grade or patient age. No significant difference in overall survival was seen regardless of B7H3 expression or expression levels across sarcoma subtypes. There was no correlation between B7H3 expression and expression of PD-L1 or PD-1 in tumor cells. Conclusions: This is the most comprehensive analysis of B7H3 expression in sarcoma to date. This data shows high levels of B7H3 expression across sarcoma subtypes, suggesting its feasibility as a therapeutic target for future sarcoma treatments. Future clinical trials are needed to evaluate its numerous actions and if manipulation of B7H3 can be employed to improve outcomes in sarcoma. [Table: see text]

CDH1-mutated clinically advanced urothelial bladder cancer (UBC): A genomic landscape and real-world clinical outcome study (RWCOS).

4587 Background: CDH1 mutated bladder cancers are characterized by plasmacytoid histology and are associated with an aggressive clinical course. Methods: Cohort 1: 6,676 clinically advanced UBC patients underwent hybrid capture based comprehensive genomic profiling (CGP) to evaluate all classes of genomic alterations (GA) as well as microsatellite instability (MSI), tumor mutational burden (TMB), and genomic loss of heterozygosity (gLOH, high ≥16%). Tumor cell PD-L1 expression was determined by immunohistochemistry (Dako 22C3). GAs were compared between CDH1 mutated and wild-type (WT) patients using Chi-square. Cohort 2: 586 UBC patients underwent a RWCOS using the de-identified Flatiron Health-Foundation Medicine urothelial clinicogenomic database. The de-identified data originated from approximately 280 US cancer clinics (~800 sites of care) between January 2011 and April 2022. Differences in real-world progression-free survival (rwPFS) and overall survival (rwOS) were evaluated by Cox proportional hazard models. Results: Cohort 1: 217 UBC patients featured a CDH1 short variant mutation with 65.2% featuring plasmacytoid histology (PLC). The PLC cohort was slightly younger, with a higher proportion of male patients, and MSI-High status. Cell-Cycle regulatory GAs were significantly higher in the non-PLC cohort, specifically CDKN2A (25.5% vs 9.7%, p=0.01) and CDKN2B (23.6% vs 5.8%, p<0.01). CDH1-mutated UBCs featured a higher MSI high frequency (2.7% vs 0.8%; p=.002), mean TMB (14.8 vs 9.9mut/Mb p<.0001), RB1 GA (52.5% vs 20.3%; p<.0001), PTEN GA (9.2% vs 4.3%; p=.006) and PIK3CA GA (29.5% vs 21.8%; p=.02), but lower gLOH (6.8% vs 15.9%; p=.009), CDKN2A loss (12.4% vs 38.3%; p<.0001), MTAP loss (10.1% vs 25.1%; p<.0001), and FGFR3 GA (9.7% vs 18.1%; p=.002). TP53 GAs and PD-L1 expression levels were similar between groups. From cohort 2: 22 (3.7%) patients featured CDH1 mutations. When compared with the CDH1 WT patients, the age, gender, ethnicity and ECOG status were similar. CDH1 mutation was associated with less favorable outcomes for 270 UBC patients treated with immune checkpoint inhibitors (ICPI) including rwPFS (2.8 vs 3.5 months; p=.096) and rwOS (3.3 vs 9.5 months; p=.03). Similar comparisons for 316 UBC patients treated with chemotherapy showed no significant adverse impact of CDH1 mutation status on either rwPFS (7.9 vs 6.2 months; p=.11) and rwOS (13.4 vs 13.4 months; p=0.83). Conclusions: In addition to its classic association with PLC histology, CDH1-mutated UBC features an unique genomic landscape including higher MSI and TMB, activating GAs in the MTOR pathway, but lower frequency of FGFR3 GAs. RWCOS further supports that CDH1 mutation predicts resistance to ICPI-based treatment, but does not systemic chemotherapy. These findings support CGP to guide therapeutic approaches based on the personalized genomic signature of UBC.

Association of biomarkers (BMs) with efficacy of adjuvant nivolumab (NIVO) vs placebo (PBO) in patients with resected stage IIB/C melanoma (CA209-76K).

9504 Background: Adjuvant NIVO significantly prolonged recurrence-free survival (RFS) over PBO in stage IIB/C melanoma in the randomized, phase III Checkmate 76K trial (HR; 95% CI: 0.42 [0.30–0.59]). For the first time, select BMs and their association with RFS were analyzed in early-stage melanoma treated with an anti-PD-1 agent. Methods: Baseline primary tumor and serum BMs were analyzed, including interferon gamma-related gene expression signature (IFNγ-sig), TMB, BRAF mutation, % intratumoral CD8+ T cells, tumor cell PD-L1 expression, and serum CRP levels. BMs were evaluated as continuous variables (high vs low levels) and dichotomized by median or prespecified cutoffs. HRs were estimated using a Cox proportional hazards model. Results: Analysis of continuous BM levels within treatment arms showed that higher IFNγ-sig, TMB, and % CD8+ T cells, and lower CRP levels were associated with prolonged RFS with NIVO but not PBO. The same trend was observed in the dichotomized analyses. No clear association between PD-L1 expression and RFS was identified in either arm. Benefit of NIVO over PBO was seen across all BM subgroups, including by BRAFV600 mutation status. Patients with higher IFNγ-sig, TMB, and % CD8+ T cells, and lower CRP levels showed numerically greater relative benefit with NIVO, although CIs overlapped (Table). Conclusions: Higher IFNγ-sig, TMB, and % CD8+ T cells, and lower CRP levels were associated with longer RFS with NIVO treatment. No BMs assessed were prognostic for RFS in the PBO arm, in contrast to previous reports in melanoma. Prolonged RFS was seen in patients treated with NIVO over PBO across levels of BMs, and irrespective of BRAF mutational status. Composite analyses of BMs with clinical and histopathologic factors will be evaluated for their potential to optimize risk:benefit stratification for NIVO in this patient population. Clinical trial information: NCT04099251 . [Table: see text]

Afatinib combined with anti-PD1 enhances immunotherapy of hepatocellular carcinoma via ERBB2/STAT3/PD-L1 signaling.

Afatinib is mainly used to treat advanced non-small cell lung cancer, but its therapeutic effect on hepatocellular carcinoma is still unclear. Over 800 drugs were screened by CCK8 technology and afatinib was found to have a significant inhibitory effect on liver cancer cells. The expression of PDL1 in tumor cells treated with drugs were detected by qRT-PCR and Weston Blot experiments. The effects of afatinib on the growth, migration and invasion of HCC cells were evaluated using wound healing, Transwell, and cell cloning assays. The in vivo effects of afatinib in combination with anti-PD1 were evaluated in C57/BL6J mice with subcutaneous tumorigenesis. Bioinformatics analysis was performed to explore the specific mechanism of afatinib's inhibition of ERBB2 in improving the expression level of PD-L1, which was subsequently verified through experiments. Afatinib was found to have a significant inhibitory effect on liver cancer cells, as confirmed by in vitro experiments, which demonstrated that it could significantly suppress the growth, invasion and migration of HCC cells. qRT PCR and Weston Blot experiments also showed that Afatinib can enhance the expression of PD-L1 in tumor cells. In addition, in vitro experiments confirmed that afatinib can significantly enhance the immunotherapeutic effect of hepatocellular carcinoma. Afatinib's ability to increase PD-L1 expression is mediated by STAT3 activation following its action on HCC cells. Afatinib enhances PD-L1 expression in tumor cells through the STAT3/PD-L1 pathway. The combination of afatinib and anti-PD1 treatment significantly increases the immunotherapeutic effect of HCC.

Abstract CT005: AEGEAN: A phase 3 trial of neoadjuvant durvalumab + chemotherapy followed by adjuvant durvalumab in patients with resectable NSCLC

Abstract Background: Recent trials have demonstrated the clinical benefit of immunotherapy in either the neoadjuvant or adjuvant resectable (R) NSCLC setting. AEGEAN (NCT03800134) is a randomized, double-blind, placebo (PBO)-controlled trial assessing neoadjuvant durvalumab (D) + chemotherapy (CT) followed by surgery (Sx) and adjuvant D in patients (pts) with R-NSCLC. Methods: Adults with treatment (Tx)-naïve R-NSCLC (stage II-IIIB[N2]; AJCC 8th ed) and ECOG PS 0/1 were randomized (1:1) to receive D 1500 mg or PBO IV + platinum-based CT (every 3 weeks [Q3W] for 4 cycles) before Sx, then further D 1500 mg or PBO IV (Q4W, up to 12 cycles). Pts were stratified by disease stage (II vs III) and PD-L1 tumor cell expression (<1% vs ≥1%, Ventana SP263). Pts with documented EGFR/ALK aberrations were excluded from the modified intent-to-treat (mITT) population for efficacy analyses. The primary endpoints were pathological complete response (pCR), evaluated centrally, and event-free survival (EFS; using RECIST v1.1), evaluated by BICR. Safety was assessed in all pts who received ≥1 study Tx dose. Results: Between Jan 2, 2019, and Apr 19, 2022, 802 pts were randomized to the ITT population (n=740 in the mITT population) of whom 799 received Tx (D arm, n=400; PBO arm, n=399). Baseline characteristics were largely balanced (mITT). Overall, 84.7% in the D arm and 87.2% in PBO arm completed 4 cycles of platinum-doublet CT and 77.6% and 76.7%, respectively, completed Sx (mITT). As of Nov 10, 2022 (data cutoff), median EFS follow-up in censored pts was 11.7 months (mITT). The pCR rate was significantly higher and EFS significantly prolonged in the D vs PBO arms (mITT) (Table). In the safety analysis set, maximum grade 3/4 any-cause AEs occurred in 42.3% vs 43.4% in the D and PBO arms, respectively, during the overall Tx period. Conclusions: The AEGEAN trial met both of its primary endpoints of improved pCR and EFS. Perioperative D plus neoadjuvant CT was associated with a manageable safety profile. Clinical trial identification: NCT03800134 (release date: January 11, 2019) Endpoint D arm PBO arm Tx effect P value pCR n/N: 63/366 (17.2%) n/N: 16/374 (4.3%) Difference in pCR (95% CI), %: 13.0 (8.7-17.6)a 0.000036 (assessed at IA)b EFS n events/N: 98/366 (26.8%) n events/N: 138/374 (36.9%) HR (95% CI): 0.68 (0.53-0.88)d 0.003902e Median (95% CI), months: NR (31.9-NR)c Median (95% CI), months: 25.9 (18.9-NR)c aCIs by stratified Miettinen and Nurminen’s method. bStatistical significance was achieved at the IA (402 pts; data cutoff, Jan 14, 2022); no testing was performed at FA. The statistically significant p-value of 0.000036 was based on a Cochran-Mantel-Haenszel test. cKaplan-Meier method. dStratified Cox proportional hazards model. eStratified log-rank test. CI, confidence interval; FA, final analysis; HR, hazard ratio; IA, interim analysis; NR, not reached. Citation Format: John V. Heymach, David Harpole, Tetsuya Mitsudomi, Janis M. Taube, Gabriella Galffy, Maximilian Hochmair, Thomas Winder, Ruslan Zukov, Gabriel Garbaos, Shugeng Gao, Hiroaki Kuroda, Jian You, Kang-Yun Lee, Lorenzo Antonuzzo, Mike Aperghis, Gary J. Doherty, Helen Mann, Tamer M. Fouad, Martin Reck. AEGEAN: A phase 3 trial of neoadjuvant durvalumab + chemotherapy followed by adjuvant durvalumab in patients with resectable NSCLC [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 CT005.

Reproducibility in PD-L1 Immunohistochemistry Quantification through the Tumor Proportion Score and the Combined Positive Score: Could Dual Immunostaining Help Pathologists?

We studied the pathologists’ agreements in quantifying PD-L1 expression through the tumor proportion score (TPS) and the combined positive score (CPS) using single PD-L1 immunohistochemistry (S-IHC) and double immunohistochemistry (D-IHC) combining PD-L1 staining and tumor cell markers. S-IHC and D-IHC were applied to 15 cancer samples to generate 60 digital IHC slides (30 whole slides images and 30 regions of interest of 1 mm2) for PD-L1 expression quantification using both TPS and CPS, twice by four pathologists. Agreements were estimated calculating intraclass correlation coefficients (ICC). Both S-IHC and D-IHC slides analyses resulted in excellent (for TPS, ICC > 0.9) to good (for CPS, ICC > 0.75) inter- and intra-pathologist agreements with slightly higher ICC with D-IHC than with S-IHC. S-IHC resulted in higher TPS and CPS than D-IHC (+5.6 and +6.1 mean differences, respectively). High reproducibility in the quantification of PD-L1 expression is attainable using S-IHC and D-IHC.

Novel Strategy to Regulate PD-L1 Expression and Enhance Anti-Tumor Effect

Abstract The programmed death 1/PD ligand 1(PD-1/PD-L1 axis is a major player in the tumor immune evasion strategy. We show that the immunomodulatory small tellurium molecules, AS101 [ammonium trichloro(dioxoethylene-o, o′)tellurate] and SAS [octa-O-bis-(R,R)-tartarate ditellurane], suppress PD-L1 expression on a variety of human and mouse malignant cells by inhibition of the α4β1 VLA-4 (very late antigen-4) integrin activity. As a consequence, pAkt and its downstream effector pNFκB are inhibited. VLA-4/pAkt/pNFκB signaling blockade was found crucial for decreasing PD-L1 expression. Moreover, the tellurium compounds had physiological effects. SAS augmented killing of human and mouse malignant cells by stimulated syngeneic splenocytes or CD8+ T cells. Furthermore, SAS prevented the development of chemoresistance in malignant cells. IL-10 production by tumor cells, the inhibition of which by AS101 we showed suppressed chemoresistance, is shown here to be closely associated with inhibition of VLA-4 and PD-L1 expression suggesting that AS101 and SAS compounds may, at least partially, induce chemosensitivity by virtue of inhibition of the VLA-4/IL-10/PD-L1 pathway. AS101 or SAS treatment of B16 melanoma-bearing mice decreased tumor cell PD-L1 expression, led to increased CD8+ T-cell infiltration into the tumors and tumor shrinkage. Combined treatment with αPD-1 antibody and either of the tellurium compounds significantly increased antitumor effects. These results collectively suggest that VLA-4 integrin signaling serves as a critical component of cancer evasion and is a potential target for cancer treatment. Furthermore, AS101 or SAS may be suitable for effective combinatorial cancer immunotherapy with αPD-1.

Abstract CT080: Tremelimumab (T) + durvalumab (D) + chemotherapy (CT) in 1L metastatic NSCLC: Outcomes by blood tumor mutational burden (bTMB) in POSEIDON

Abstract Background: In the Phase 3 POSEIDON study (NCT03164616), 1L T plus D and platinum-based CT demonstrated statistically significant improvements in both PFS (HR, 0.72; 95% CI, 0.60-0.86; p=0.0003; data cutoff [DCO] July 24, 2019) and OS (HR, 0.77; 95% CI, 0.65-0.92; p=0.0030; DCO March 12, 2021) vs CT in patients (pts) with EGFR/ALK wild-type metastatic (m) NSCLC. On the basis of these results, T+D+CT was approved by the FDA in November 2022 for use in this setting. Here we report outcomes in POSEIDON pt subgroups defined by a range of bTMB values, including a pre-specified cutoff of 20 mutations/megabase (mut/Mb). Methods: Pts were randomized 1:1:1 to 1L T+D+CT (platinum-based), D+CT or CT, with stratification by tumor cell (TC) PD-L1 expression (TC ≥50% vs <50%; VENTANA PD-L1 [SP263] assay), disease stage (IVA vs IVB) and histology (squamous vs non-squamous). bTMB was assessed using the GuardantOMNI platform. OS, PFS and ORR with T+D+CT vs CT were determined for pts with bTMB ≥ vs <20 mut/Mb. Outcomes across additional bTMB cutoffs (10, 12 and 16 mut/Mb) were also explored. Results: Plasma samples were available for 958/1013 pts from the intention-to-treat (ITT) population. Of those with available plasma samples, 81.8% (784/958) were evaluable for bTMB, including 277 in the T+D+CT arm and 241 in the CT arm. Similar demographic characteristics and OS were observed in the bTMB evaluable vs ITT populations. Consistent with previous reports, the proportion of never smokers was higher in the bTMB <20 mut/Mb subgroup than in the bTMB ≥20 mut/Mb subgroup. Across all bTMB cutoffs analyzed, OS and PFS benefit for T+D+CT vs CT were generally consistent with the ITT population in both bTMB high and bTMB low subgroups. However, at each bTMB cutoff, OS and PFS benefit appeared more prominent among pts in the bTMB high subgroups. Median OS (mOS) was longer with T+D+CT vs CT in both the bTMB ≥20 mut/Mb and <20 mut/Mb subgroups and HRs suggested more pronounced benefit in the bTMB high group. mOS was 13.5 months with T+D+CT vs 10.3 months with CT (unstratified HR, 0.61; 95% CI, 0.42-0.88) for pts with bTMB ≥20 mut/Mb and 12.6 months vs 10.9 months (unstratified HR, 0.79; 95% CI, 0.63-0.99) for pts with bTMB <20 mut/Mb. PFS and ORR showed similar trends to OS (data will be presented). In both subgroups with PD-L1 TC ≥1% or <1%, HRs suggested more pronounced OS/PFS benefit in pts with bTMB ≥20 (vs <20) mut/Mb (data will be presented). Conclusions: In pts with mNSCLC, treatment with a limited course of T plus D (until progression) and four cycles of CT consistently improved clinical outcomes vs CT alone in both bTMB high and low subgroups, supporting the use of this regimen as a 1L treatment option for pts with mNSCLC. The clinical benefit vs CT appeared to be greater in pts with higher bTMB over a range of cutoffs, consistent with expectations based on mechanistic biology and previous clinical data. Citation Format: Solange Peters, Hisani Madison, Kelly Oliner, Anne L’Hernault, Marianne Ratcliffe, Karen Barrett, Xiaojin Shi, Edward B. Garon, Tony Mok, Melissa L. Johnson. Tremelimumab (T) + durvalumab (D) + chemotherapy (CT) in 1L metastatic NSCLC: Outcomes by blood tumor mutational burden (bTMB) in POSEIDON [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 CT080.

Abstract 5358: Digital SP263 PD-L1 tumor cell scoring in non-small cell lung cancer achieves comparable outcome prediction to manual pathology scoring

Abstract Background: Tumor cell (TC) PD-L1 expression is predictive of response to PD-L1-targeted immunotherapy, and accurate scoring is crucial for treatment selection. Scoring relies on manual assessment of immunohistochemically labeled tissue and is subject to subjective variation due to pathologist assessment. As a digital alternative, a clone-agnostic AI-based model for PD-L1 quantification in non-small cell lung cancer (AIM-PD-L1 NSCLC) was developed1. AIM-PD-L1 was deployed on samples from a Phase 3 study of anti-PD-L1 atezolizumab combination therapy with carboplatin and paclitaxel, and/or bevacizumab in Stage IV NSCLC (IMpower150; NCT02366143). Digital and manual PD-L1 TC scores were compared and interrogated for their respective potential to predict efficacy to atezolizumab combination treatments. Methods: AIM-PD-L1 was deployed on images (n=768) digitized from SP263-labeled slides with available manual pathologist TC scores to quantify tissue regions and individual TCs. PD-L1 expression status was determined for each tumor cell and a slide-level TC score was computed. Overall survival (OS) and progression free survival (PFS) analyses of patients at selected cutoffs of 1%, 50%, and across a continuum of cutoffs from 0% to 100% PD-L1 TC by digital and manual methods were conducted, comparing groups treated with or without atezolizumab in combinations with carboplatin and paclitaxel, and/or bevacizumab. Digital and manual scores were compared using agreement rates, Lin's concordance and Spearman’s correlation coefficients, and hazard ratios (HRs) were calculated for OS and PFS analysis. Results: At the slide level, correlation between continuous digital and manual scores was high (r 0.84 [95% CI 0.81-0.86]). Digital assessment of PD-L1 positivity at the 1% cutoff identified more positive patients than manual scoring (70% vs. 55% prevalence, respectively), with comparable treatment benefit (digital OS HR 0.69 [0.53-0.9], and manual OS HR 0.72 [0.54-0.98], digital PFS HR 0.67 [0.54-0.83], and manual PFS HR 0.66 [0.52-0.84]). Continuous digital PD-L1 TC scores showed that treatment benefit improved for patients with scores ≥50% compared to manual scoring. At the ≥50% PD-L1 TC cutoff, numerical improvement was observed in OS and PFS by digital scoring compared to manual (digital OS HR 0.5 [0.29-0.86] compared to manual OS HR 0.64 [0.4-1.02], and digital PFS HR 0.37 [0.24-0.57], compared to manual PFS HR 0.53 [0.37-0.76]). Conclusions: AIM-PD-L1 scoring was as effective at predicting outcomes as manual using the ≥1% PD-L1 TC expression and at the ≥50% TC cutoff, digital scoring identified a subgroup with enriched efficacy compared to manual. Further evaluation of the accuracy and reproducibility of PD-L1 scoring by digital pathology as well as its potential use for patient enrollment or stratifications in clinical trials is needed. 1Griffin et. al. AACR (2022) Citation Format: Hen Prizant, John Shamshoian, John Abel, Andrew Beck, Laura Chambre, Stephanie Hennek, Hartmut Koeppen, Daniel Ruderman, Meghna Das Thakur, Michael Montalto, Benjamin Trotter, Ilan Wapinski, Wei Zou, Minu K. Srivastava, Jennifer Giltnane. Digital SP263 PD-L1 tumor cell scoring in non-small cell lung cancer achieves comparable outcome prediction to manual pathology scoring. [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 5358.

Abstract 5636: Multiplex digital spatial profiling (DSP) of proteins in the tumor microenvironment in response to GSK-3 inhibition by 9-ING-41 (elraglusib) correlates with novel immunostimulatory effects observed in vivo

Abstract Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase with key roles in myriad biological processes such as tumor progression, and inhibition of GSK-3 using the small molecule elraglusib has shown promising preclinical antitumor activity in multiple tumor types. Our preclinical experiments showed that elraglusib treatment increased tumor cell PD-L1 expression, downregulated angiogenic and immunosuppressive signaling pathways, and increased anti-tumor immune responses in vitro and in vivo. Subsequent studies showed that several circulating factors were predictive of response to PD-1/PD-L1 blockade and GSK-3 inhibition in a murine model of colorectal cancer. To determine the translational relevance of our prior results we evaluated tumor biopsies and plasma samples from patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating elraglusib (NCT03678883). Plasma samples were collected from patients at baseline and 24 hours post-IV administration of elraglusib and were analyzed using Luminex technology. Paired FFPE tumor biopsies from patients with colorectal or pancreatic cancer before and after treatment were selected to analyze the tumor microenvironment using NanoString GeoMx DSP technology. The region of interest (ROI) selection strategy focused on mixed tumor and immune cell segments and ROIs were segmented using panCK+ and CD45+ morphology stains. Cytokine analysis revealed that elevated baseline plasma levels of IL-1 beta and reduced levels of VEGF correlated with improved progression-free survival (PFS) and overall survival (OS). PFS was also found to be positively correlated with elevated plasma levels of immunostimulatory analytes such as Granzyme B, IFN-gamma, and IL-2 at 24 hours post-treatment with elraglusib. CD45+ tumor-infiltrating immune cells had lower expression of VISTA, PD-1, and IDO-1 inhibitory checkpoint proteins and higher expression of OX40L and B7-H3 stimulatory checkpoint proteins in post-treatment biopsies as compared to pre-treatment biopsies. Moreover, time-on-study length negatively correlated with CD39 expression in PanCK+ segments and positively correlated with CD163 expression in CD45+ segments. This ongoing study, to our knowledge, represents the first digital spatial analysis of tumor biopsies from patients treated with elraglusib. These novel circulating biomarkers of response to GSK-3 inhibition could provide significant clinical utility and the spatial proteomics data may give us insights into the immunomodulatory mechanisms of GSK-3 inhibition. Citation Format: Kelsey E. Huntington, Christoph Schorl, Shaolei Lu, Daniel Newhouse, Benedito A. Carneiro, Wafik S. El-Deiry. Multiplex digital spatial profiling (DSP) of proteins in the tumor microenvironment in response to GSK-3 inhibition by 9-ING-41 (elraglusib) correlates with novel immunostimulatory effects observed in vivo. [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 5636.

Abstract 5466: Functional status of tumor-associated macrophages impacts clinical outcome of durvalumab in patients with advanced NSCLC as revealed by proteomics mass spectrometry

Abstract Introduction: Predictive biomarkers of anti‒PD-(L)1 therapies have largely focused on the tumor-T cell axis where tumor cell PD-L1 expression has demonstrated its clinical utility in predicting overall survival (OS) in patients with advanced NSCLC. In prior work using computational image analysis of multiplex immunofluorescence (mIF), we have shown the positive impact of CD68+ PD-L1+ macrophages in combination with CD8+ T cells in predicting long-term OS benefit in NSCLC patients treated with durvalumab (anti-PD-L1), highlighting the impact of the myeloid compartment on IO responses. In part, using proteomics mass spectrometry we sought to investigate further the functional impact of myeloid cells on the IO response and in particular, suppressive (M2) tumor associated macrophages (TAMs). Methods: Pre-treatment tumor samples from 66 patients with advanced NSCLC patients enrolled in durvalumab nonrandomized phase 1/2 trial (10 mg/kg Q2W, CP1108/NCT01693562) were processed for global and targeted proteomics. A pathologist determined the tumor area and a single FFPE tumor tissue section was used for laser-capture macrodissection and protein extraction followed by mass spectrometry analysis using label-free, data-independent and parallel reaction monitoring. Results: Among the immune associated proteins detected by proteomics mass spectrometry, we evaluated CD163 protein expression, a well-described marker of suppressive (M2) macrophages, and its impact on durvalumab clinical outcomes, alone and in relationship with PD-L1 and immune infiltration. While proteomics-based biomarker evaluable population (BEP) shows shorter median (m) OS compared to intended to treat population, we validated that high expression of PD-L1 as measured by IHC or proteomics associates with greater OS benefit as expected when treating with durvalumab. Building on this, the evaluation of protein associated with suppressive M2 macrophages, revealed that approximately 30% of NSCLC patients with high CD163 protein expression show poor OS benefit (5 months mOS) following durvalumab treatment. In contrast, patients with PD-L1 TC ≥50% and low CD163 expression derive greater OS benefit (13.4 months mOS) than those with high CD163 expression (5 months mOS) or with PD-L1 TC <50% regardless of level of CD163 expression (4.1-7.4 months mOS). Moreover, patients with poorer outcome have significantly higher baseline CD163 protein expression as a proportion of total CD45 protein expression. Conclusion: This analysis further confirms the importance of myeloid cell function within the tumor microenvironment (TME) in determining the outcome to T cell-directed IO therapy and highlights the utility of proteomics mass spectrometry in assessing more broadly and quantitatively the TME complementing findings based on RNAseq and IHC/mIF assays. Citation Format: Anna Margaret Hansen, Vivian Ying Wang, Andrew Chambers, David Chain, Jaime Rodriguez Canales, Jorge Blando, Steve Sweet, Zachary A. Cooper, Ashok Gupta, Carl Barrett, Yeoun Jin Kim, Ikbel Achour. Functional status of tumor-associated macrophages impacts clinical outcome of durvalumab in patients with advanced NSCLC as revealed by proteomics mass spectrometry. [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 5466.

MAX transcriptionally enhances PD-L1 to inhibit CD8+ T cell-mediated killing of lung adenocarcinoma cells.

Immune checkpoint blockade (ICB) therapies, such as monoclonal antibodies against the PD-1/PD-L1 immune checkpoint pathway, have been a major breakthrough in the treatment of lung cancer especially lung adenocarcinoma (LUAD), but their effectiveness is limited. High expression of PD-L1 in tumor cells is one of the key reasons evading immune surveillance, yet the mechanisms that regulate PD-L1 expression are not fully understood. By analyzing the chromatin immunoprecipitation sequencing data of MYC-associated X-factor (MAX) based on lung cancer cell lines, we found that the transcriptional regulator MAX is able to bind to the promoter region of the PD-L1 gene. Further, we performed several molecular biology experiments to determine that MAX promotes PD-L1 transcription in LUAD cells, which in turn assists LUAD cells to evade killing by CD8+T cells, an effect that can be reversed by anti-PD-L1 antibody. In LUAD, the expression of MAX is positively correlated with PD-L1 and the infiltration of CD8+T cells. Importantly, we further identified that high expression of the MAX/PD-L1 axis is associated with poor overall survival and fist progression of patients with LUAD. Thus, this study sheds light on the mechanism by which MAX inhibits CD8+T cell-mediated killing of LUAD cells by activating PD-L1 transcription, and MAX may serve as a potential combinatorial target for ICB therapies that block the PD-1/PD-L1 pathway in LUAD.

LRPPRC facilitates tumor progression and immune evasion through upregulation of m6A modification of PD-L1 mRNA in hepatocellular carcinoma.

LRPPRC is a newly discovered N6-methyladenosine (m6A) modification reader, which potentially affects hepatocellular carcinoma (HCC) progression. PD-L1 in tumor cells is essential for tumor immune evasion. This work investigated the LRPPRC-mediated m6A-modification effect on PD-L1 mRNA and immune escape in HCC. Expression and clinical implication of LRPPRC and PD-L1 were measured in human HCC cohorts. The influence of LRPPRC on malignant behaviors of HCC cells was investigated through in vitro assays and xenograft tumor murine models. The posttranscriptional mechanism of LRPPRC on PD-L1 and anti-tumor immunity was elucidated in HCC cells via RIP, MeRIP-qPCR, RNA stability, immunohistochemical staining, and so forth. LRPPRC exhibited the notable upregulated in human HCC tissues, which was in relation to advanced stage and worse overall survival and disease-free survival. Impaired proliferative capacity and G2/M phage arrest were found in LRPPRC-knockout cells, with increased apoptotic level, and attenuated migratory and invasive abilities. In HCC patients and murine models, LRPPRC presented a positive interaction with PD-L1, with negative associations with CD8+, and CD4+ T-cell infiltrations and chemokines CXCL9, and CXCL10. LRPPRC loss downregulated the expression of PD-L1 and its m6A level in HCC cells. Moreover, LRPPRC suppression mitigated tumor growth in murine models and improved anti-tumor immunity and immune infiltration in tumors. This work unveiled that LRPPRC may posttranscriptionally upregulate PD-L1 partially with an m6A-dependent manner for heightening mRNA stabilization of PD-L1 and provided a new mechanism for m6A regulator-mediated immunosuppression in HCC.

Genomic landscape of metastatic breast cancer (MBC) patients with methylthioadenosine phosphorylase (MTAP) loss

Homozygous deletion of MTAP upregulates de novo synthesis of purine (DNSP) and increases the proliferation of neoplastic cells. This increases the sensitivity of breast cancer cells to DNSP inhibitors such as methotrexate, L-alanosine and pemetrexed. 7,301 cases of MBC underwent hybrid-capture based comprehensive genomic profiling (CGP). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. Tumor cell PD-L1 expression was determined by IHC (Dako 22C3). 208 (2.84%) of MBC featured MTAP loss. MTAP loss patients were younger (p = 0.002) and were more frequently ER- (30% vs. 50%; p < 0.0001), triple negative (TNBC) (47% vs. 27%; p < 0.0001) and less frequently HER2+ (2% vs. 8%; p = 0.0001) than MTAP intact MBC. Lobular histology and CDH1 mutations were more frequent in MTAP intact (14%) than MTAP loss MBC (p < 0.0001). CDKN2A (100%) and CDKN2B (97%) loss (9p21 co-deletion) were significantly associated with MTAP loss (p < 0.0001). Likely associated with the increased TNBC cases, BRCA1 mutation was also more frequent in MTAP loss MBC (10% vs. 4%; p < 0.0001). As for immune checkpoint inhibitors biomarkers, higher TMB >20 mut/Mb levels in the MTAP intact MBC (p < 0.0001) and higher PD-L1 low expression (1-49% TPS) in the MTAP loss MTAP (p = 0.002) were observed. MTAP loss in MBC has distinct clinical features with genomic alterations (GA) affecting both targeted and immunotherapies. Further efforts are necessary to identify alternative means of targeting PRMT5 and MTA2 in MTAP-ve cancers to benefit from the high-MTA environment of MTAP-deficient cancers.