PD-L1 IHC Research Articles

Molecular profiling METex14+ non-small cell lung cancer (NSCLC): Impact of histology

ObjectivesMET exon 14 skipping alterations (METex14+) represent a heterogeneous subgroup of non-small cell lung cancer (NSCLC) with distinct biological and genomic features. We characterized this heterogeneity in a large cohort, integrating genomic and transcriptomic profiling with clinical outcomes, to elucidate the histologic and molecular traits and survival patterns of METex14+ NSCLC. Materials and methodsNSCLC tissue samples (n = 28,739) underwent DNA-based next-generation sequencing (592 genes, NextSeq) or whole-exome sequencing (NovaSeq), RNA-sequencing including whole transcriptome sequencing (WTS, NovaSeq), and PD-L1 IHC (Dako 22C3) at Caris Life Sciences. Immune cell fractions were estimated from bulk RNA sequencing (quanTIseq). Real-world survival data (mOS) was calculated from insurance claims. Statistical analyses employed Chi-square, Fisher’s exact, or Mann-Whitney U and log-rank tests and were corrected for hypothesis testing where applicable. ResultsA total of 711 METex14+ cases were detected. Of 575 cases of defined histology, 77 (13.6 %) were squamous (Sq), 474 (82.3 %) were nSq (non-squamous), and 24 (4.1 %) were adenosquamous. Mutations in POT1 and BRCA2 were enriched, and amplifications in MDM2, HMGA2, CDK4, and MET were common in METex14+ tumors. TMB-high and TP53 mutated tumors were reduced in METex14+ independent of histology. KEAP1 (2.1 vs 14.7 %) and STK11 mutations (0.8 vs 17.1 %) were reduced only in METex14+ nSq (vs METex14+ Sq, q < 0.05). While the prevalence of PD-L1 high tumors was enriched in METex14+ independent of histology, T-cell inflamed tumors were enriched only in nSq METex14+. B-cells and CD8+ T-cells (1.07–1.43-fold) were enriched in nSq METex14+, and dendritic cells (0.32 fold) were reduced only in METex14+ Sq. METex14+ tumors had a modest improvement in mOS compared to METex14- tumors (mOS = 22.9 m vs 18.6 m, HR = 0.914, p = 0.04). Moreover, METex14+ tumors who received immunotherapy (IO) had a modest improvement in survival (mOS = 27.5 m vs 21.8 m; HR = 0.803, p = 0.03) compared to those who did not receive IO. METex14+ nSq tumors were associated with improved mOS compared to METex14+ Sq tumors (mOS = 27.7 vs 8.9 m, HR = 0.493, p < 0.0001). ConclusionMETex14+ alterations are a heterogeneous subgroup of NSCLC. Our analysis reveals that METex14+ nSq exhibit improved survival compared to METex14+ Sq. The distinct genomic and transcriptomic variations across histologies warrant clinical consideration.

PD-L1 and alternative immune checkpoints (AICs) in non-small cell lung cancer (NSCLC): Insights into the tumor immune microenvironment (TIME) and immunotherapy (IO) outcomes.

8531 Background: PD-L1 and recently identified AICs (B7x, B7-H3, and HHLA2) may function as key immune evasion mechanisms in NSCLC and influence IO efficacy. This study explores the expression patterns of PD-L1 and AICs within the TIME and their impact on IO outcomes in NSCLC, including key molecular subtypes. Methods: NSCLC samples (n=27,629) were analyzed (Caris Life Sciences) with NextGen Sequencing on DNA (592 genes or whole exome)/RNA (whole transcriptome). Tumor cell PD-L1 expression (22c3) was assessed by IHC. Gene expression profiles were analyzed for IFN-γ score and QuantiSEQ was used to assess immune cell infiltration in the TIME. Real-world overall survival (rwOS: from time of biopsy to last contact), time on treatment [TOT: from the first pembrolizumab (pembro) treatment to the last], and OS on pembro (OSpem: from pembro start to last contact) were calculated. Mann-Whitney U, X2/Fisher-Exact, and log-rank tests were applied where appropriate. Results: PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA expression had higher IFN-γ score and immune cell fractions (p&lt;0.0001 for both) vs PD-L1 IHC- and low expression, respectively. Additionally, PD-L1 IHC+ NSCLC was associated with (a/w) higher median transcript levels (TPM) of PD-L1 and B7-H3, while PD-L1 IHC- NSCLC was a/w higher TPM of HHLA2 and B7x (p&lt;0.0001 for all). AICs expression varied by molecular subtype, e.g., higher HHLA2/B7x RNA in EGFR mutant vs wild-type (WT) NSCLC. Moreover, PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA and low B7H3 RNA were a/w longer rwOS, OSpem, and TOT; high HHLA2 RNA was a/w longer rwOS and OSpem with similar TOT; and low B7x RNA expression was a/w longer OSpem and TOT with similar rwOS (Table). Conclusions: Both PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA expression are a/w a more inflammatory TIME, and superior survival and IO outcomes. Moreover, consistent with our prior HHLA2 IHC work (PMIDs: 27553831, 29374053), HHLA2 RNA expression was significantly higher in PD-L1 IHC- and EGFR mutant cases. These findings support the potential utility of transcriptomics along with protein expression for assessing novel predictive biomarkers for IO. Furthermore, the distinct expression profiles of AICs identified in our study may represent key subtypes mediating IO response in WT and EGFR mutant NSCLC. [Table: see text]

Interaction between VEGF-A and immune checkpoint targets in triple-negative breast cancer and mechanisms of immune evasion and tumor progression.

1096 Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment triple negative breast cancer (TNBC). However, not all patients with TNBC benefit from the addition of ICIs due to factors such as intrinsic and acquired resistance to ICIs, and complex interplay between tumor and immune cells in the tumor microenvironment (TME). Vascular endothelial growth factor (VEGF) promotes angiogenesis and potentially modulates tumor immune evasion. A phase I clinical trial has shown that a bispecific antibody to VEGF-A and PD-L1 produces encouraging tumor responses in advanced TNBC. In this study, we investigated the interaction between VEGF and various immune gene expression signatures including PD-L1 in a real-world breast cancer patient population. Methods: Comprehensive immune profiling, including the RNA-seq based gene expression assessment of 395 immune-associated genes, was performed on 120 formalin-fixed paraffin-embedded breast cancer samples. PD-L1 expression (Combined Positive Score, CPS ≥ 10%) was measured by immunohistochemistry (IHC; 22C3). Clinicopathologic variables were collected. Statistical associations between quantitative biomarkers, including 28 immune checkpoint genes, were calculated using Pearson correlations (r), and comparisons of quantitative biomarkers between groups were performed using the proportions test. For statistical significance, p&lt;0.05 was required. Results: The study cohort included 29 patients (24.2%) with TNBC and 91 (75.2%) with non-TNBC (including ER+/HER2- and HER2+). Most patients had metastatic tumors (n=92, 76.7%) and invasive ductal carcinoma (n=83, 69.2%). Among the TNBC group, 21 patients had PD-L1 positive tumors (72.4%), defined by CPS ≥ 10.In non-TNBC, VEGF has significant, though weak (r&lt;0.35) correlations with the expression of three checkpoint genes: AKT1 (r=0.35, p=0.00063), ICOS (r=0.34, p=0.00099), and TIM3 (r=0.29, p=0.0061). In TNBC, VEGF has significant, strong (r&gt;0.47) correlations with the expression of three other checkpoint genes: PD-1 (r=0.47, p=0.0094), PD-L1 (r=0.57, p=0.0012), and PIK3CA (r=0.6, p=0.00051) However, in TNBC, VEGF gene expression does not have a statistical association with PD-L1 IHC (CPS ≥/&lt; 10 (p=0.9)). Conclusions: Our findings suggest that there is an interplay between angiogenesis and an immunosuppressive tumor immune microenvironment in TNBC to a greater degree than in non-TNBCs. This supports the notion that angiogenesis mediators such as VEGF-A may enhance the expression of immunosuppressive checkpoint molecules, leading to immune evasion and tumor progression. Combination treatments of ICIs, especially anti-PD-1 therapy, with VEGF antibodies may be promising approaches to enhance the immune responses in TNBC, especially in tumors with CPS&lt;10, where the current frontline treatment is cytotoxic chemotherapy.

Clinical outcomes after adjuvant anti-PD1 therapy for high-risk resectable melanoma and predictors of response.

e21502 Background: Adjuvant anti-PD1 is the standard of care for high-risk resectable melanoma but many still suffer recurrence. We report a single institution observational study of patients who received adjuvant anti-PD1 with a particular focus on those who recurred. Methods: Through an approved protocol by Huntsman Cancer Institute IRB, patients with resected high-risk melanoma who first received anti-PD1 in the adjuvant setting were consented and followed. Clinical outcomes were assessed per RECIST 1.1, progression free survival (PFS), and overall survival (OS). Results: 186 eligible patients were included, 105 male and 81 females, median age at C1D1 58 (22-93), 12/161/7 at stage II/III/IV and 5 unstageable. Subtypes included 79 (43%) superficial spreading, 50 (27%) nodular, 16 (9%) lentigo/nevoid, 13 (7%) non-acral skin (NOS), 10 (5%) acral, 4 (2%) mucosal, and 14 (7%) others. With median follow up of 708 days (23-9599), 66 (35.5%) had progressive disease (PD), including 33 (50%) early PD and 33 (50%) late PD. Statistically significant predictors of worse outcome included age &gt; 50 (OS p = 0.043), acral subtype (PFS p = 0.023), PD-L1 IHC &lt; = 1% (OS p = 0.052, PFS p = 0.002), duration of treatment &lt; 3 months (OS p = 0.005, PFS p = 1.8E-07), early PD (OS p = 0.003, PFS p = 1.5E-6) and distant recurrence (OS p = 0.002). Trends that were not significant included: male gender (worse OS), BRAF mutation (better PFS), NF1 mutation (better PFS). No correlation between outcomes and TMB, NRAS, TERT mutation, and primary location. In the early/late PD groups, median age was 57/55 (range 28-79 / 25-84), 64/61% male, 1/0, 29/31, 3/2 stage II, III, IV. 17/13 were superficial spreading, 6/10 nodular, 2/5 acral, 2/2 lentigo, 1/0 mucosal and 4/3 other non-acral skin. Primary site was 12/12 head/neck, 9/5 trunk, 4/7 upper limb, 8/9 lower limb. BRAF was 9/11 wildtype, 15/9 mutant and 9/13 unknown. PD-L1 &gt; = 1% by IHC was 4/2. TMB mut/mb &gt; 10 was 6/5. Sites of recurrence included 6/12 local, 7/7 regional LN, 20/14 distal (3/1 in brain) relapses. For unresectable PD (13/13 early/late) response to next line systemic therapy is shown in Table 1. Conclusions: One third of patients developed PD after adjuvant anti-PD1 for high-risk melanoma. PD-L1 negativity and short duration of treatment was associated with worse outcome. Early PD corresponded to more distant metastasis, poorer OS and response to subsequent systemic therapy. Importantly most late PD can still have a favorable response by resuming anti-PD1 therapy. Further molecular study is ongoing to investigate mechanisms of resistance. [Table: see text]

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

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

Exploration of different immunogenomic TMEs in gastric cancer based on HER2 and PD-L1 expression.

e16073 Background: Recent results from the KEYNOTE-811 study showed that first-line pembrolizumab was superior to placebo with respect to progression-free survival (PFS) in combination with trastuzumab and chemotherapy in patients with HER2 positive metastatic gastric cancer (GC). Interestingly, In the subgroup analysis, the PFS benefit with pembrolizumab was significant in patients with tumors with PD-L1 CPS ≥1, whereas there was no benefit in the small subgroup of patients with tumors with CPS &lt; 1. Therefore, this study is to investigate immune-genomic differences in the tumor microenvironment according to the composition of surrounding cells and the expression status of HER2 and PD-L1. Methods: Utilizing single cell RNA sequencing (scRNA seq) and TCR sequencing, we examined 14 HER2 positive and 18 negative primary GC samples with CPS score based on PD-L1 IHC 22C3 pharmDx. Among the HER2-positive group, there were 4 samples in the CPS &lt; 1 (negative) and 10 samples in the CPS ≥1 (positive) group. In the HER2 negative group, there were 6 samples in the CPS &lt; 1 and 12 samples in the CPS ≥1. Results: First, when analyzing the distribution of total cells according to HER2 status, in the HER2 positive group, more endothelial cells ( p= 0.034) and fibroblasts ( p= 0.054) appeared in the PD-L1 negative group compared to the PD-L1 positive group, while there was no difference in the HER2 negative group. Furthermore, Through TCR analysis, the exhausted and cytotoxic score exhibit contrasting trends between HER2 negative and positive groups. In HER2-positive patients, the exhausted and cytotoxic score appear significantly higher in the CPS negative group. Next, we evaluated the proportion of immune cells according to HER2 status. The observed of CD4 Treg ( p= 0.084) and CD8 exhausted T (Tex), ( p= 0.036) cells was increased in the HER2 positive compared to the negative. When regrouped according to PD-L1 expression, only the HER2 positive group showed a tendency to increase the observation of CD4 Treg and CD8 Tex cells compared to positive in PD-L1 negative. In addition, we investigated whether the expression pattern of other immune checkpoint molecules differed depending on the PD-L1 expression pattern in both HER2 positive and negative patients. In the HER2 negative group, the expression of immune checkpoint proteins was similar regardless of the CPS expression group, or the expression of some proteins was higher in the CPS positive group. In other hands, in HER2 positive, CPS &lt; 1 group had higher expression of immune checkpoint proteins, such as PDCD1, LAG2, BTLA, VISTA and TIGHT. Conclusions: The high proportion of CD4 Tregs, CD8 Tex and the elevated exhausted score in the subgroup with CPS &lt; 1 in HER2 positive GCs suggesting immune exclusive environment compared to her-2 and PD-L1 coexpressing group. Considering increased expression of immune checkpoint proteins other than PD-1/PD-L1, for patients with HER2-positive and CPS &lt; 1, combination therapy with other immune checkpoint inhibitors rather than anti-PD-1/PD-L1 may be suggested.

Immune checkpoint inhibitor treatment in PD-L1-negative gastroesophageal cancer tumors.

e16062 Background: We aimed to investigate whether there is a subgroup of patients with gastroesophageal cancers who, despite having IHC-PD-L1 negative tumors, might still benefit from ICI treatment. Furthermore, the impact of ICI treatment on patients with tumors that are PD-L1 negative by IHC but exhibit high CD274 (PD-L1) expression remains undefined. Methods: 1,416 esophageal adenocarcinomas (EA), 486 esophageal squamous carcinomas (ES), 859 esophagogastric junction adenocarcinomas (EJA), and 1,613 gastric adenocarcinoma (GA) samples were analyzed using NGS of DNA (NextSeq, 592 genes and NovaSeq, WES) and RNA (NovaSeq, WTS) (Caris Life Sciences, Phx, AZ). PD-L1 IHC positivity was defined as &gt; 1% CPS (Agilent's 22c3). Correlation between PD-L1 CPS and CD274 expression (transcripts per million – TPM) were assessed by Spearman’s coefficient. Tumors were divided into CD274-H (high expression) and CD274-L (low expression) cohorts, representing the top 25% and bottom 25% of expression levels, respectively. Real-world overall survival (OS) data were obtained from insurance claims records and analyzed using Cox proportional hazards with hazard ratios (HR) and log-rank tests. Results: Weak correlations were observed between PD-L1 CPS and CD274 expression in adenocarcinoma histology tumors (EA: 0.374, EJA: 0.358, GA: 0.387), while a stronger correlation was found in squamous tumors (ES: 0.537). Median CD274 expression was significantly higher in PD-L1+ compared to PD-L1- ES tumors (2.65-fold) and in EA (1.68-fold), EJA (1.92-fold), and GA (1.96-fold) tumors (p &lt; 0.001). In PD-L1 IHC-negative ES, there was no association between CD274-H expression and OS (H 12.4 vs L 10.6 mo; HR 0.90, p = 0.48). However, in adenocarcinomas, CD274-H showed a slightly improved OS (12.8 vs 11.3 mo; HR 0.79, p &lt; 0.05). CD274-H trended towards worse post-Carboplatin/paclitaxel (CP) OS (ES: 13.8 vs 18.2 mo; HR 1.60; p = 0.22; Adenocarcinoma: 18.2 vs 27.4 mo; HR 1.50, p = 0.19). Interestingly, CD274-H was associated with improved post-CP/nivolumab survival in ES (96.5 vs 28 mo; HR 0.32; p = 0.047), with a similar but not statistically significant pattern in Adenocarcinoma EGC (Not Reached vs 30.2 mo; HR 0.39, p = 0.23). Furthermore, in CD274-L GEC, there was no difference in post-treatment survival with the addition of Nivolumab to CP (Squamous: 30 vs 18.2 mo; HR 0.91, p = 0.83; Adenocarcinoma: 30.2 vs 27.4 mo; HR 1.14; p = 0.76). In contrast, CD274-H showed improved survival with the addition of Nivolumab (Squamous: 96.5 mo vs 13.8 mo; HR 0.15; p = 0.01; Adenocarcinoma: NR vs 18.2 mo; HR 0.30; p = 0.08). Conclusions: High CD274 expression was associated with improved survival when Nivolumab was added to chemotherapy in patients with PD-L1-negative tumors. Further investigation is needed to confirm the potential benefits of Nivolumab treatment in gastroesophageal cancer patients with tumors that are PD-L1-negative by IHC but exhibit high mRNA expression.

Abstract PO4-25-03: Comprehensive immune profiling reveals factors associated with neoadjuvant chemotherapy response in triple negative breast cancer

Abstract Background: KEYNOTE-522 has resulted in FDA approval of the immune checkpoint blocker pembrolizumab with neoadjuvant chemotherapy for patients with high-risk triple negative breast cancer (TNBC), given the remarkable improvement in pCR rate to 65% along with improvement in event free survival, while with chemotherapy alone, the pCR rate is 40-50%. Unfortunately, use of pembrolizumab is associated with several immune related adverse events (irAE), some of which can be life-threatening and debilitating, including cardiomyositis, encephalitis, and adrenal insufficiency, among others. Hence, there is an unmet need to identify biomarkers in the tumor microenvironment which could predict patients who may attain pCR with chemotherapy alone and be spared the side effects from the added PD-1 inhibition. Methods: Comprehensive immune profiling, including PD-L1 IHC and the expression of 395 immune genes by RNA-seq, was performed on 1:2 matched FFPE tumor samples from 23 stage 1-3 TNBC patients (2 stage 1, 6 stage 2, 15 stage 3). All patients were female with an average age of 48 years (range: 25-79 years). 14 patients were treated with neoadjuvant chemotherapy alone (NAC) and 9 were treated with neoadjuvant chemotherapy combined with the checkpoint inhibitor pembrolizumab (NAC+I). mRNA expression signatures of tumor inflammation (TIGS, weak/moderate/strong), cell proliferation (CP, poor/moderate/high), and cancer testis antigen expression burden (CTAB) were determined by RNA-sequencing. Statistical comparisons of quantitative biomarkers between groups were calculated using the Wilcoxon Rank-Sum test, overrepresentation analysis of categorical variables between groups was calculated by proportions test, and survival differences were quantified by Cox proportional hazards analysis. Pathological responses were documented as pathological complete response (pCR) vs. non-pCR. Results: Across the entire cohort, patients with a BMI &amp;lt; 30 had significantly higher PD-L1 expression (assessed by RNA) than those with a BMI ³ 30 [p=0.047]. While not a statistically significant association, the NAC+I group tended to have a higher pCR rate compared to the NAC group [44.4% vs. 28.6%, p=0.66]. Across the entire cohort, tumors with high CTA expression, designated as CTAB high, had a higher pCR rate than CTAB low tumors [54.5% vs 16.7%, p=0.089]. No significant difference in pCR rate between TIGS or CP groups was detected for the NAC group, but in the NAC+I group, highly proliferative tumors had a lower pCR rate than moderately proliferative tumors [0% vs. 80%, p=0.048]. The pCR and non-pCR groups for each treatment type also exhibited distinct gene expression profiles. Among the NAC group, underexpression of FOXP3 and overexpression of MAPK14 and CD44 were associated with pCR [p≤0.031], while underexpression of IFNB1, MAPK14, and CD44 was associated with non-pCR [p≤0.029]. Among the NAC+I group, overexpression of CXCR4, TNFR and CCL20 was associated with non-pCR [p≤0.046]. Interestingly, SDHA was significantly overexpressed in the non-pCR subset of patients in the NAC group and significantly underexpressed in the non-pCR subset of patients in the NAC+I group [p≤0.031]. Conclusions: The development of biomarkers of treatment response is essential to the integration of immunotherapy with chemotherapy as a combined cancer treatment. Our study profiled the immune context of both NAC and NAC+I and identified several key microenvironmental differences underlying divergent treatment response in both groups. A comprehensive understanding of these factors could potentially predict pCR to chemotherapy alone, enabling the avoidance of the unnecessary treatment of these tumors with immunotherapy. Citation Format: Robert Seager, Heidi Ko, Sarabjot Pabla, Maria-Fernanda Senosain, Pawel Kalinski, Erik Van Roey, Shuang Gao, Kyle Strickland, Rebecca Previs, Mary Nesline, Stephanie Hastings, Shengle Zhang, Jeffrey Conroy, Taylor Jensen, Marcia Eisenberg, Brian Caveney, Eric Severson, Shakti Ramkissoon, Shipra Gandhi. Comprehensive immune profiling reveals factors associated with neoadjuvant chemotherapy response in triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-03.

Abstract PO5-06-09: Metastatic triple negative breast cancer has distinct tumor immune landscape

Abstract Background: Triple negative breast cancer (TNBC) is considered the most immunogenic breast cancer subtype due to higher levels of tumor infiltrating immune cells (TILS), elevated tumor mutational burden and PD-L1 expression, providing a rationale for immunotherapy. Here we aimed to investigate the differences in the immune microenvironment of primary vs. metastatic sites in TNBC vs. non-TNBC and their impact on survival. Methods: Comprehensive immune profiling, including PD-L1 IHC and the expression of 395 immune genes, was performed on 147 real-world FFPE breast cancer samples (32 primary, 115 metastatic). 37 samples were, by definition, triple negative for ER, PR, and HER2 overexpression. PD-L1 (CPS positive ≥1% and CPS positive ≥10%) was determined using immunohistochemistry (22C3). mRNA expression signatures of tumor inflammation (TIGS, weak/moderate/strong) and cell proliferation (CP, poor/moderate/high) were determined by RNA-sequencing. Demographic and clinicopathologic variables were collected. Statistical comparisons of biomarkers between groups were calculated using the Wilcoxon Rank-Sum test for continuous variables and the proportions test for categorical variables (p≤0.05 for significance). Survival differences were quantified by Cox proportional hazards analysis (p≤0.05 for significance). Results: Triple negative status was associated with current use of alcohol [p=0.03]. Black patients were also more likely to have TNBC, constituting 24% of TNBC patients while only making up 14% of the total cohort [p=0.04]. Conversely, white patients were less likely to have TNBC, constituting 73% of TNBC patients while making up 84% of the total cohort [p=0.04]. Comparing TNBC and non-TNBC cases, TNBC cases were observed to have significantly higher TIGS [p=0.014] and PD-L1 expression [p=4.4 × 10−7]. Additionally, TNBC cases trended towards exhibiting greater cell proliferation [p=0.069]. These differences were found to be driven primarily by metastatic tumors, among which TNBC tumors showed significantly higher TIGS [p=0.015], cell proliferation [p=0.021], and PD-L1 expression [p=2.9 × 10−7] than non-TNBC tumors, while none of these significant associations were observed among primary tumors. Triple negative status was significantly associated with PD-L1 expression (assessed by IHC), with a majority of PD-L1 positive cases determined to be triple negative [70% of CPS≥1% cases, p=5 × 10-5; 59% of CPS≥10% cases, p=6 × 10−4]. Several immune-related genes and immune checkpoint molecules were over-expressed in triple negative breast cancer, including CD8, GZMB, PRF1, CCL5, IFNG, TIGIT and CXCL10. A number of genes were also significantly overexpressed in metastatic tumors: KRT5 [p=0.02], TFRC [p=0.04], ABCF1 [p=0.04], FCRLA [p=0.05], LAMP3 [p=0.05], and underexpressed in metastatic tumors: ENTPD1 [p=0.05], IGSF6 [p=0.03], and ITGA1 [p=0.03]. Conclusions: Our comprehensive biomarker analyses showed that metastatic TNBC has a more inflamed tumor microenvironment and higher checkpoint target expression compared to non-TNBC. Further analysis of immune expression by site-specific metastases and correlation with immunotherapy outcomes is warranted to guide clinicians in selection of the ideal metastatic site to biopsy for therapeutic decision making. However, in a collective TNBC context, these data support the use of immunotherapy in metastatic TNBC. Citation Format: Robert Seager, Heidi Ko, Sarabjot Pabla, Maria-Fernanda Senosain, Erik Van Roey, Shuang Gao, Kyle Strickland, Rebecca Previs, Mary Nesline, Stephanie Hastings, Shengle Zhang, Jeffrey Conroy, Taylor Jensen, Marcia Eisenberg, Brian Caveney, Eric Severson, Shakti Ramkissoon, Shipra Gandhi. Metastatic triple negative breast cancer has distinct tumor immune landscape [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-09.

Abstract PO5-14-08: External Reproducibility of PD-L1 IHC 22C3 pharmDx on Breast Cancer Specimens at CPS ≥ 1 and CPS ≥ 10 Cutoffs

Abstract Background: PD-L1 IHC 22C3 pharmDx is an approved companion diagnostic for pembrolizumab treatment in triple-negative breast cancer (TNBC). Clinical evidence for pembrolizumab approval in TNBC was demonstrated by KEYNOTE-355 (NCT02819518), which investigated the clinical validity of PD-L1 IHC 22C3 pharmDx in identifying patients with PD-L1 expressing Combined Positive Score (CPS) ≥ 10 in previously untreated locally recurrent unresectable or metastatic TNBC.1 The role of immune checkpoint inhibitors for breast cancer as a whole is not as well defined as it is for TNBC. In this study, we show evidence of the reproducibility of PD-L1 IHC 22C3 pharmDx in PD-L1 expression determination in breast cancer specimens at the CPS ≥ 1 and CPS ≥ 10 cutoffs documenting robust assay performance in breast cancer. Methods: A blinded, randomized study was conducted at three external sites using formalin-fixed, paraffin-embedded breast cancer specimens. Reproducibility of PD-L1 IHC 22C3 pharmDx was assessed at the inter-site, intra-site/inter-day, inter-observer, and intra-observer endpoints at the CPS ≥ 1 and CPS ≥ 10 cutoffs. To assess inter-site and intra-site reproducibility, five replicate sets of pre-qualified unstained specimens were stained and evaluated at each of the three external sites. To assess inter-observer and intra-observer reproducibility, one set of pre-stained specimens was evaluated three times by each of three external pathologists. Negative percent agreement (NPA), positive percent agreement (PPA), and overall percent agreement (OA), based on comparisons to consensus diagnostic outcome, were computed with corresponding two-sided 95% percentile bootstrap confidence intervals. Acceptance criteria required at least 85.0% for the lower-bound (LB) of the two-sided 95% confidence interval (CI) on NPA, PPA, and OA for all endpoints. Results: All but one endpoint in the external reproducibility study met pre-determined acceptance criteria. Both the inter- and intra-site reproducibility endpoints had 95% CI LB values of 98.8% for NPA, 95.9% for PPA, and 97.9% OA at the CPS ≥ 1 cutoff. At the CPS ≥ 10 cutoff, the inter-site reproducibility endpoint had 95% CI LB values of 88.0% for NPA, 87.1% for PPA, and 89.5% OA, and the intra-site reproducibility endpoint had 95% CI LB values of 96.7% for NPA, 92.2% for PPA, and 95.6% OA. The inter-observer reproducibility endpoint had 95% CI LB values of 95.3% for NPA, 92.3% for PPA, and 94.7% OA at the CPS ≥ 1 cutoff. At the CPS ≥ 10 cutoff, the inter-observer reproducibility endpoint had 95% CI LB values of 84.9% for NPA, 86.8% for PPA, and 87.3% OA. The intra-observer reproducibility endpoint had 95% CI LB values of 97.0% for NPA, 95.0% for PPA, and 96.6% OA at the CPS ≥ 1 cutoff. At the CPS ≥ 10 cutoff, the intra-observer reproducibility endpoint had 95% CI LB values of 92.2% for NPA, 89.2% for PPA, and 91.7% OA. Conclusions: Acceptance criteria were met for all endpoints in the breast cancer external reproducibility study of PD-L1 IHC 22C3 pharmDx at the CPS ≥ 1 and CPS ≥ 10 cutoffs, except for inter-observer at the CPS ≥ 10 cutoff, which had a 95% CI LB value of 84.9% for NPA.

Clinical and Biomarker Analysis of a Phase I/II Study of PDR001 Plus Imatinib for Advanced Treatment-Refractory Gastrointestinal Stromal Tumors.

In this phase Ib/II study, we aimed to evaluate the safety and efficacy of PDR001, an anti-PD1 antibody, in combination with imatinib in patients with treatment-refractory gastrointestinal stromal tumor (GIST). Patients with advanced GIST whose disease had progressed on imatinib, sunitinib, and regorafenib were enrolled. In phase Ib, the standard 3 + 3 dose escalation scheme was applied. Intravenous administration of PDR001 at 400 mg for every 4 weeks plus imatinib (300 and 400 mg daily for dose levels I and II, respectively) was given. The primary outcome for phase II was the disease control rate at 12 weeks. Exploratory biomarker analysis was performed based on PDL1 IHC, next-generation sequencing, and multiplexed IHC. No dose-limiting toxicity was observed in the phase Ib part (n = 10), and dose level II was selected as the recommended phase II dose. In the phase II part (n = 29), there was no objective response, and the disease control rate at 12 weeks was 37.9%, not meeting the primary efficacy endpoint. For patients in phase Ib-dose level II and phase II (n = 36), the median progression-free survival (PFS) and overall survival were 2.3 and 9.5 months, respectively. The most common grade 3 to 4 adverse event was anemia. Exploratory biomarker analysis indicated that a higher CD8+ T-cell density was associated with a favorable PFS but to a limited degree. Tumor mutational burden and PDL1 were not associated with better PFS. In patients with treatment-refractory GIST, PDR001 in combination with imatinib was generally tolerable, but it was not effective.

Abstract CT061: Pharmacodynamic analysis from a phase 1/2 study of subasumstat (TAK-981) as single agent in patients (pts) with advanced/refractory metastatic solid tumors

Abstract Background Subasumstat (TAK-981, suba) is a first-in-class innate immunity enhancer that unlocks innate/adaptive immune responses to target tumors through SUMOylation inhibition, which may enhance antitumor activity by restoring endogenous type 1 IFN signaling in immune cells. In the phase 1/2 study (NCT03648372), suba showed dose-dependent upregulation of an IFN-1 gene signature in peripheral blood (PB) and plasma levels of several IFN-1-induced cytokines, and an increase in activated NK, CD8, and CD4 T cells in PB. Here, we report pharmacodynamic data from skin and tumor biopsy samples from this study. Methods In phase 1, 84 pts received suba IV at escalating doses (3-120 mg) twice-weekly (BIW) or once-weekly in 21-day cycles. In phase 2, 25 pts with refractory NSCLC, cervical cancer, or CRC received suba 90 mg BIW. Skin and tumor biopsies were collected at screening and on cycle 1, day 8 (C1D8, skin) or C2D8 (+7 days; tumor). Target engagement (TE) and levels of SUMO2/3-conjugates were assessed using IHC. Immune activation in the tumor microenvironment (TME) was measured by IHC and multiplex IF. Results In skin samples collected 1-3 hours after D8 dosing, suba ≥10 mg demonstrated dose-dependent TE as seen by suba-SUMO adduct formation and dose-dependent SUMO2/3 inhibition at ≥60 mg. Assays of paired tumor samples from pts with CPI-naïve MSS-CRC (90 mg BIW, n=3), cervical cancer (90 mg BIW, n=1), CPI-exposed non-squamous NSCLC (90 mg BIW, n=2), and melanoma (25 mg BIW, n=1) showed robust TE in the post-dose sample, along with SUMO2/3 inhibition or stabilization. Suba’s effects on the TME were assessed through PD-L1 IHC. There was an increase in PD-L1 combined positive score in most on-treatment tumors, consistent with suba’s ability to enhance IFN production, leading to induction of an inflammatory landscape in the TME. Multiplex IF analysis revealed significant CD8+ and CD4+ T cell infiltration in samples from CPI-naïve (cervical cancer) and CPI-exposed (melanoma and NSCLC) pts. In contrast, biopsies obtained from MSS-CRC pts did not exhibit an increase in T cell density, suggesting that “cold” tumors may be less susceptible to suba-mediated immune remodeling. Accordingly, biopsies from pts with cervical cancer and melanoma displayed a significant increase in T cell activation and density of activated CD11c+HLA-DR+ myeloid dendritic cells within the TME, as well as a marked increase in HLA-class I expression. Conclusion Suba-mediated inhibition of SUMOylation led to marked changes in the TME across different indications, supporting the hypothesized immune-activating mechanism of action of the drug. Our findings support continued development of suba in solid tumors. Suba is being evaluated in combination with pembrolizumab in NSCLC, cervical cancer, and melanoma (NCT04381650). Citation Format: Dina Stroopinsky, Slawomir Mandzuik, Daniel Anderson, José-Ángel Hernández-Rivas, Hadeel Assad, Allison Berger, Aleksander Chudnovsky, Qi Dong, Afshin Dowlati, Dejan Juric, Razelle Kurzrock, Iwona Lugowska, Kelly Richter, Jordi Rodón, David Schröder, Siddha Kasar, Anthony Olzanski. Pharmacodynamic analysis from a phase 1/2 study of subasumstat (TAK-981) as single agent in patients (pts) with advanced/refractory metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT061.

Abstract CT283: Phase 3 MK-2870-007 study: MK-2870 (SKB264) plus pembrolizumab vs pembrolizumab alone as first-line treatment for metastatic non-small-cell lung cancer with PD-L1 tumor proportion score (TPS) ≥50%

Abstract Background: Pembrolizumab (anti-PD-1) monotherapy is a standard of care first-line treatment for advanced or metastatic non-small-cell lung cancer (NSCLC) with PD-L1 TPS ≥50% without actionable genetic alterations; however, some patients do not respond to therapy or experience disease progression. Trophoblast cell-surface antigen 2 (TROP2) is another candidate for anticancer treatment and overexpression of TROP2 is associated with poor prognosis in patients with NSCLC. MK-2870 (SKB264) is an antibody-drug conjugate (ADC) composed of the humanized TROP2 monoclonal antibody, a hydrolytically cleavable linker, and the cytotoxic drug KL610023. In patients with relapsed or refractory locally advanced or metastatic NSCLC, MK-2870 monotherapy showed encouraging antitumor activity with a manageable safety profile. The complementary mechanisms of action of MK-2870 and pembrolizumab may provide more potent antitumor activity when given in combination compared with each therapy alone. To investigate this further, the MK-2870-007 study will evaluate the efficacy and safety of the addition of MK-2870 to pembrolizumab vs pembrolizumab alone in patients with previously untreated metastatic NSCLC with PD-L1 TPS ≥50%. Trial Design: Approximately 614 eligible patients aged ≥18 y with previously untreated histologically or cytologically confirmed stage IV (per American Joint Committee on Cancer v8.0) NSCLC, PD-L1 TPS ≥50% and no EGFR, ALK, or ROS1 alterations (nonsquamous only); ECOG PS 0 or 1; and measurable disease per RECIST v1.1 will be enrolled. Patients with well-controlled HIV are permitted. Patients will be randomized 1:1 to receive either pembrolizumab 400 mg Q6W for up to 18 cycles plus MK-2870 4 mg/kg Q2W or pembrolizumab 400 mg IV Q6W alone until progressive disease, intercurrent illness, patient withdrawal, unacceptable toxicity, prolonged interruption of study drug, or until maximum number of cycles (18 cycles for pembrolizumab). Randomization stratification factors include ECOG PS (0 vs 1), histology (squamous vs nonsquamous), TROP2 expression (low vs medium vs high), and geographic region (East Asia vs North America/Western Europe/Australia vs Rest of World). The primary endpoint is OS. Secondary endpoints include PFS, ORR, and duration of response per RECIST v1.1 by blinded independent central review, patient-reported outcomes, and safety. Tumor imaging will occur at baseline, Q6W until wk 48, and Q12W thereafter until PD, start of new anticancer treatment, or withdrawal of consent. PD-L1 TPS will be assessed centrally using PD-L1 IHC 22C3 pharmDx (Agilent Technologies, Carpinteria, CA). AEs will be graded according to National Cancer Institute Common Terminology Criteria for Adverse Events v5.0. Enrollment began in December 2023 and is currently ongoing. Citation Format: Nikolaj Frost, Razi Ghori, Ananya Roy, Ana Tablante Nunes, James Chih-Hsin Yang. Phase 3 MK-2870-007 study: MK-2870 (SKB264) plus pembrolizumab vs pembrolizumab alone as first-line treatment for metastatic non-small-cell lung cancer with PD-L1 tumor proportion score (TPS) ≥50% [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT283.

Abstract CT235: RNA-seq molecular subtypes and gene expression signatures in the phase 3 IMpower151 study of 1L bevacizumab + pemetrexed/paclitaxel ± atezolizumab in metastatic NSCLC

Abstract Introduction IMpower151 (NCT04194203) was a phase 3 study to evaluate the efficacy and safety of atezolizumab, bevacizumab, carboplatin, plus paclitaxel or pemetrexed (ABCP) vs BCP, as 1L treatment for metastatic nsqNSCLC. IMpower151 did not meet its primary endpoint of INV-PFS. The objectives of this biomarker analysis were to characterize the molecular subtypes of metastatic nsqNSCLC and to explore the relationship between molecular subtype, predefined gene expression signatures, PD-L1 status and clinical outcomes with ABCP and BCP. Methods RNA sequencing of 250 nsqNSCLC tumor samples from IMpower151 was analyzed in the ACBP (n=123) and BCP (n=127) treatment arms. Non-negative matrix factorization (NMF) was applied to variably expressed genes to define molecular subtypes. Gene expression signatures were defined based on previously published associations with their respective biology. Tumor PD-L1 levels were determined by SP263 immunohistochemistry (Ventana). Results No PD-L1 dependent difference of survival benefit of ABCP compared to BCP was observed at the cutoff of TC 1% or TC 50%. Four molecular subtypes (NMF1-NMF4) were identified based on RNA sequencing. NMF1 (n=78) were immune-depleted and had low tumor proliferation. NMF2 (n=73) were characterized by low level of anti-tumor immune cells, increased expression of stromal signatures and high tumor proliferation. NMF3 (n=65) had the most inflamed transcriptomic profile with high enrichment of immune cells, and increased expression of stromal signatures as well. NMF4 (n=34) had the enrichment of granulocytes, a less inflamed immunophenotype and decreased expression of stromal signatures. The positive rate of PD-L1 expression with TC&amp;gt;=1% was significantly lower in NMF1 compared to other subtypes. None of patients with the four NMF subtypes showed significantly longer PFS or OS with ABCP than BCP. Overall, patients with NMF2 showed numerically shorter PFS and patients with NMF3 showed numerically longer PFS than other subtypes. Gene expression signatures of antitumor cytokines are correlated with survival benefit of ABCP compared to BCP. Patients with high expression level of antitumor cytokines showed substantially improved PFS and OS benefit from ABCP than BCP in both ITT population and EGFR mutation population. Conclusion Four molecular subtypes of nsqNSCLC with different immune-cell and tumor microenvironmental features were characterized. NMF2 may identify a fibrotic and immune-depleted subgroup correlated with worse prognosis. NMF3 may identify an immune-inflamed subgroup correlated with better prognosis. Neither NMF subtypes nor PD-L1 IHC level showed a correlation with survival benefit of ABCP controlled by BCP. The gene expression signature of antitumor cytokines could serve as a potential predictive biomarker for survival benefit of ABCP in metastatic nsqNSCLC including EGFR mutant patients. This finding is based on the exploratory analysis of a single clinical trial with limited sample size. It needs further investigation in other cohorts of tumor tissues and with larger clinical potential in blood samples as well. Citation Format: Caicun Zhou, Charlie Sun, Stacey Huang, Xiaorong Dong, Gongyan Chen, Zhehai Wang, Xianghua Wu, Yu Yao, Yiping Zhang, Ying Cheng, Hongming Pan, Xiaodong Zhang, Jiuwei Cui, Lifeng Wang, Xi Chen, Xiaoling Li, Ziping Wang, Qiming Wang, Jianxing He, Mengzhao Wang, Li Qian, Habib Hamidi, Shanshan Li, Marcus Ballinger, David S. Shames, Minu Srivastava. RNA-seq molecular subtypes and gene expression signatures in the phase 3 IMpower151 study of 1L bevacizumab + pemetrexed/paclitaxel ± atezolizumab in metastatic NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT235.

Abstract CT095: Phase 1 evaluation of triple therapy with boserolimab (anti-CD27 agonist), pembrolizumab, and chemotherapy in patients with triple-negative breast cancer (TNBC) with PD-L1 combined positive score (CPS) &amp;lt;10: Safety, antitumor activity, and association between biomarkers and response

Abstract Background: Pembrolizumab (pembro) plus chemotherapy (chemo) is an approved therapy for patients (pts) with locally recurrent unresectable or metastatic TNBC whose tumors express PD-L1 (CPS ≥10) based on results from KEYNOTE-355. However, there is unmet need for effective treatment options in pts with PD-L1 CPS &amp;lt;10. In a first-in-human, open-label, phase 1 study (NCT03396445), treatment with escalating doses of the humanized anti-CD27 agonist boserolimab (MK-5890) alone and with pembro had acceptable safety and preliminary evidence of antitumor activity in pts with advanced solid tumors. We report results for a dose-expansion cohort of this study in pts with metastatic TNBC and PD-L1 CPS &amp;lt;10 tumors who received first-line triple therapy with boserolimab, pembro, and chemo. Exploratory analyses in this cohort assessed the association of baseline T-cell-inflamed gene expression profile (TcellinfGEP) and tumor mutational burden (TMB) with ORR. Methods: This dose-expansion cohort enrolled pts aged ≥18 y with previously untreated locally recurrent inoperable or metastatic TNBC, measurable disease per RECIST v1.1, ECOG PS of 0/1, and tumors expressing PD-L1 at the cutoff of CPS &amp;lt;10 per PD-L1 IHC 22C3 pharmDx (local or central testing). Pts received boserolimab 30 mg IV Q6W plus pembro 400 mg IV Q6W for 18 cycles plus nab-paclitaxel 100 mg/m2 (3 weeks on [days 1, 8, and 15]/1 week off). Primary objectives were safety/tolerability. A secondary objective was ORR per RECIST v1.1 by investigator assessment. Baseline TcellinfGEP was assessed using the NanoString PCI assay (N=31); TMB was assessed using the Illumina TSO500 mutational panel assay (N=21). Results: 41 pts were enrolled in the dose-expansion cohort. Median study follow-up at data cutoff (Apr 25, 2023) was 11.1 (range, 2.2-20.5) mo. The most common treatment-related AEs were fatigue (51.2%), pruritus (51.2%), and alopecia (46.3%). Grade 3/4 treatment-related AEs occurred in 56.1% of pts (23/41); no grade 5 treatment-related AEs occurred. 9.8% of pts (4/41) discontinued ≥1 study treatment due to treatment-related AEs. Confirmed ORR was 46.3% (95% CI, 30.7%-62.6%), with 2 CRs and 17 PRs; 14 pts had SD, 5 had PD, and 3 had no postbaseline assessment at data cutoff. Median PFS was 10.3 (95% CI, 4.2-not reached) mo. No clear association between TcellinfGEP or TMB and confirmed ORR was observed; responses were observed in pts with non-TcellinfGEP-high (less than −0.318) and non-TMB-high (&amp;lt;10 mut/Mb) tumors. Conclusions: In pts with TNBC and tumors expressing PD-L1 at the cutoff of CPS &amp;lt;10, triple therapy with boserolimab, pembro, and chemo had acceptable safety and showed preliminary evidence of antitumor activity regardless of baseline TcellinfGEP expression or TMB status. Citation Format: Carlos Rojas, Bernard Gaston Doger de Spéville, Seock-Ah Im, Ronnie Shapira-Frommer, María De Miguel, Lucía González Cortijo, Margarita Romeo Marin, Maja de Jonge, Mark Ayers, Yiwei Zhang, Song Zhai, Elliot Chartash, Konstantin Dobrenkov, Joohyuk Sohn. Phase 1 evaluation of triple therapy with boserolimab (anti-CD27 agonist), pembrolizumab, and chemotherapy in patients with triple-negative breast cancer (TNBC) with PD-L1 combined positive score (CPS) &amp;lt;10: Safety, antitumor activity, and association between biomarkers and response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT095.

Soluble immune-checkpoint factors: a potential immunotherapy biomarker.

There is unmet need for additional biomarkers to better select patients with non-small cell lung cancer (NSCLC) that are likely to benefit from immunotherapy in order to improve patient outcomes, reduce patient toxicity, and relieve the growing burden of healthcare costs. In this issue of the JCI, Hayashi and colleagues evaluated soluble forms of the immune checkpoint molecules PD-L1, PD-1, and CTLA-4 in the plasma of patients with advanced NSCLC who had been treated with anti-PD-1/L1 therapy. The findings suggest that these soluble immune-checkpoint factors may provide a complementary biomarker to PD-L1 IHC, although application into the clinic may not be straightforward.

Abstract 7526: Landscape of TIGIT and PD-L1 co-expression in solid tumors

Abstract Background: Combination treatment of solid tumors with anti-PD-1/PD-L1 and anti-TIGIT agents have shown promise in recent clinical trials. However, TIGIT co-expression with other checkpoints has not been comprehensively characterized to support identification and prioritization of patients most likely to benefit from anti-TIGIT drugs and guide clinical trial design. In this study, we investigate TIGIT and other PD-1/PD-L1 axis checkpoint targets across various solid tumors. Methods: A discovery cohort (DC) of 24,186 solid tumors across 35 tumor types was evaluated by comprehensive immune profiling. TIGIT gene expression was normalized and ranked yielding a percentile rank values [0-100]. TIGIT rank was classified as high (rank &amp;gt;e 75) and low (rank &amp;lt; 25). We studied the distribution of TIGIT expression, cellular proliferation signature, and co-expression with PD-1/PD-L1 axis and other checkpoint targets using Spearman correlation(rs). Kaplan-Meier analysis and Chi-squared assessed associations between TIGIT expression/checkpoint co-expression and overall survival (OS), progression free survival (PFS) and objective response rate (ORR) differences in a separate cohort of 72 pembrolizumab treated non-small cell lung cancer (LC) patients treated with pembrolizumab. Results: The DC confirmed a large range of TIGIT expression with highest median expression observed in NSCLC (median = 61), head &amp; neck (HNSCC) (median = 59), and cervical cancer (median = 55). Significant association between PD-L1 IHC positivity (TPS≥1%, CPS≥1) and TIGIT expression (p&amp;lt;0.001) was discovered, where 69% (n=3574) of TIGIT high cases were PD-L1 positive, whereas 66% (n=3606) of TIGIT low cases were PD-L1 negative. By RNA-seq measurements, significant TIGIT co-expression (p&amp;lt;0.001) with various checkpoint blockade targets including PD-1 (rs=0.5), PD-L1 (rs=0.5), and TIM3 (rs=0.6) was confirmed. In the LC, patients with TIGIT high tumors had significantly improved OS (median OS = 44 months; p=0.01) and PFS (median PFS = 35 months; p=0.006) compared TIGIT low group (median OS = 23 months; median PFS = 18 months). Similar results were observed for response to pembrolizumab, where TIGIT high cases had significantly higher response (ORR=60%, p= 0.001) vs. TIGIT low cases (ORR=39%). This was confirmed in TIGIT high and PD-L1-IHC positive group with improved OS (p=0.013), PFS (p=0.017) and ORR (p=0.016). Conclusion: In an ultra-large cohort of clinically tested solid tumors, TIGIT was differentially expressed across tumor histologies of solid tumors. TIGIT expression was strongly co-expressed with PD-1/PD-L1 axis, supporting the improved outcomes for patients with NSCLC treated with a combination of TIGIT and PD-1/PD-L1 inhibitors from recent clinical trials. This finding of co-expression pattern of TIGIT and PD-1/PD-L1 axis highlights the potential use of immune profiling for combination therapy strategies and clinical trial design in solid tumors. Citation Format: Sarabjot Pabla, Maria-Fernanda Senosain, R.J. Seager, Hardik Parikh, Erik Van Roey, Shuang Gao, Yamuna Pulivendula, Paul DePietro, Mary Nesline, Durgapras Dash, Jeffrey M. Conroy, Stephanie Hastings, Heidi Ko, Kyle C. Strickland, Rebecca A. Previs, Eric Severson, Marcia Eisenberg, Brian Caveney, Taylor J. Jensen, Shakti Ramkissoon. Landscape of TIGIT and PD-L1 co-expression in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7526.

Abstract 5151: High serum kidney injury marker-1 and high baseline tumor PD-L1 protein expression levels are independently associated with treatment effect in adjuvant nivolumab plus ipilimumab vs placebo in localized clear cell renal cell carcinoma

Abstract Introduction: CheckMate 914 (CM-914) Part A is a double-blind, phase III randomized trial of the Nivolumab (NIVO) plus Ipilimumab (IPI) vs placebo (PBO) in localized ccRCC. Our prior report from this study suggested a disease-free survival (DFS) benefit for NIVO+IPI among patients with Fuhrman grade 4, TNM stages PT2a and PT4, or sarcomatoid features, although the sample size was limited. It is known that high circulating KIM-1 is associated with worse DFS after nephrectomy. In this exploratory post hoc analysis, we investigated whether high KIM-1 may help identify a subset of patients who benefit from adjuvant NIVO+IPI. Methods: Patients (n=816) with RCC after nephrectomy were randomized in CM-914 Part A to receive NIVO+IPI or PBO as previously described. Assessment of KIM-1 levels was performed using enzyme linked immunoassay (ELISA) on pre-treatment (n=584) and matched on-treatment blood samples (n=584). We used pre-treatment tumor samples to assess PD-L1% tumor cell expression (%TC) in an PD-L1 IHC 28-8 pharm Dx assay. The association between biomarkers and DFS outcomes was investigated by Kaplan-Meier (KM) and Cox proportional hazards analysis. Results: Median baseline serum KIM-1 level was 102 (9.9 - 1055.7) pg/mL. Serum KIM-1 levels were higher in males vs females, ≥65 yrs vs &amp;lt;65 yrs, Asian vs white patients, and patients with partial vs radical nephrectomy. In the PBO arm, subjects with highest quartile of pre-treatment KIM-1 had significantly worse DFS than those from the three lower quartiles. In contrast, this DFS risk among the subjects within the highest KIM-1 quartile was mitigated with NIVO+IPI treatment. Among patients within the highest quartile of pre-treatment KIM-1, there was trend for better DFS for NIVO+IPI versus PBO, HR=0.6 (0.34-1.04). Increase in KIM-1 during study therapy was positively associated with higher DFS rate in both arms. Multivariable analysis showed that PD-L1 %TC was predictive at predefined PD-L1 cutoffs (&amp;gt;=1%, &amp;gt;=5%, and &amp;gt;=10%), associating with improved DFS compared to placebo. Subjects with high PD-L1 expression had a DFS benefit from NIVO+IPI independent of KIM-1. Conclusion: Circulating KIM-1 and tumor PD-L1 expression may enrich for benefit from IO therapy in adjuvant ccRCC and hence holds promise for informing risk stratification and patient inclusion in neoadjuvant or adjuvant clinical trials. Citation Format: Sai Vikram Vemula, Wenxin Xu, Yu Wang, Xiaowen Liu, Jorge Ruiz de Somocurcio, David McDermott, Jun Li, Rupal Bhatt, Chung-Wei Lee, Burcin Simsek, Saurabh Gupta, Robert Motzer. High serum kidney injury marker-1 and high baseline tumor PD-L1 protein expression levels are independently associated with treatment effect in adjuvant nivolumab plus ipilimumab vs placebo in localized clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5151.

Abstract 3629: Prevalence of claudin18.2 expression in gastric/gastroesophageal junction adenocarcinoma among patients in TranStar101 and TranStar102 clinical trials

Abstract Background: Claudin18.2 (CLDN18.2) is a tight junction protein highly specific to gastric mucosa, and a validated target for gastric cancer (GC) treatment. Immune checkpoint therapy targeting PD-1 combined with chemotherapy has been approved as the first line therapy of GC. Understanding the expression profiles of CLDN18.2 and PD-L1 could guide the development of combination therapies to maximize the benefits of these two agents. This study investigated the prevalence of CLDN18.2 expression in gastric/gastroesophageal junction adenocarcinoma (G/GEJC) screening samples from studies Transtar101 (NCT04396821 in US) and TranStar102 (NCT04495296 in China), and its correlation with various clinical characteristics and PD-L1 expression. Methods: CLDN18.2 expression in formalin-fixed, paraffin-embedded (FFPE) G/GEJC tissue samples was prospectively detected by an immunohistochemistry-based LDT using an in-house anti-CLDN18.2 antibody (Clone14G11) on the Leica Bond III stainer. CLDN18.2 expression was assessed by scoring the staining intensity (0, 1+, 2+, 3+) and the percentage of positive tumor cells. Positive CLDN18.2 expression is defined as the cutoff at ≥10% of tumor cell with ≥1+ staining intensity for this evaluation. PD-L1 expression was assessed by combined positive score (CPS) using Agilent’s PD-L1 IHC 28-8 pharmDx. Both assays were conducted in CAP/CLIA certified LabCorp central lab. Results: 547 G/GEJC patient samples had CLDN18.2 results as part of the screening procedures for the clinical trials. Of these patients (454 GC/93 GEJC), 439 (80%) were Asian, 29 (5%) were Caucasian, 2 (0.4%) were other ethnic group, and 77 (14%) were not recorded; 434 (79%) were primary tumors and 113 (21%) were metastasis; 201 (37%) were core needle biopsies (CNB), 108 (20%) were surgical resections (SR) and 238 (44%) had no information. 311 (57%) samples had positive CLDN18.2 expressions (≥10%/≥1+). No significant difference in CLDN18.2 positive rates were found between TranStar101 and TranStar102 studies (p=0.801), Asian and Caucasian (p=0.612), GC and GEJC (p=0.373), primary and metastatic specimens (p=0.055) or CNBs and SRs (p=0.715). Out of 83 TranStar102 specimens that had both CLDN18.2 and PD-L1 results, 15 (18%) had PD-L1 CPS≥5, 59 (71%) had positive CLDN18.2, and 10 (15%) had both positive CLDN18.2 and PD-L1 CPS≥5. No correlation (p=0.393) was observed between PD-L1 scores (CPS&amp;lt;5 or CPS≥5) and CLDN18.2 expression (≥10%/≥1+ or &amp;lt;10%/≥1+). Conclusions: Data suggested CLDN18.2 expression levels were independent of PD-L1 status, and support the use of Transcenta 14G11 antibody for CLDN18.2 detection regardless of sample collection methods, location, and patient demographics. An anti-CLDN18.2 companion diagnostic device based on 14G11 is being developed (CLDN18.2 IHC 14G11 pharmDx, Agilent Technologies, Inc.). Citation Format: Li Shen, Carol Mao, Alan Lin, Ying Gu, Jenny Milata, Lijuan Zhang, Chuan Qi, Jenny Yao, Steven Yu, Changjun Yue, Lin Shen, Yelena Janjigian, Xueming Qian, Wen-I Chang, Caroline Germa. Prevalence of claudin18.2 expression in gastric/gastroesophageal junction adenocarcinoma among patients in TranStar101 and TranStar102 clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3629.

Abstract A007: Picturing progression: A new role of whole slide imaging in comprehensive clinical genomic datasets

Abstract Introduction: Real world data is needed to understand cancer evolution based on changing standards of care. While Next Generation Sequencing (NGS) is the preferred standard for confirmatory testing of mutational status, its use varies tremendously across settings and countries, with the most influential variable being cost. In this research, we examined the utility of Whole Slide Images (WSI) as a discrete data type to predict mutational and other statuses to inform assessments of clinical progression. This could include initial mutational status or the evolution of resistance to a specific treatment and the emergence of a new driver mutation. Experimental Procedures: We have curated datasets from 4000 NSCLC and 2000 CRC patients with clinically tested WES, WTS and WSI tumors with deeply curated longitudinal clinical electronic medical records were compiled through a partnership between ConcertAI and Caris Life Science. This de-identified clinical dataset is drawn from a diverse range of U.S. community and academic oncology practices. The practice patterns reflect the real-world variability of treatment and utilization of molecular testing. Additional stratification was performed based on lines of therapy and evidence of clinical progression for patients with information available. Results: Features from whole slide H&amp;E images were extracted and compared between subcohorts of interest. We demonstrated the correlation of the WSI features with TMB, MSI-H, molecular variants, differential gene expression and PD-L1 IHC of these cohorts within the context human interpretable features (HIFs) of whole slide images. Conclusion: The availability of whole slide images (WSI) is a dimensionality requirement for real world longitudinal comprehensive clinical datasets. Digital images should be considered as a key deliverable of clinical genomic datasets and have the potential to inform treatment selection and outcomes. In settings where NGS is not the standard of care, WSI could be used to predict the value of a test for treatment decisions or determination of prospective clinical trial eligibility. Where the cost of NGS is fundamentally prohibitive, such as less developed economies, WSI is a much lower cost modality that could have very high utility in guiding treatment decisions with better predicted outcomes. In all cases WSI imaging could be used to assess potential status or acquired resistance and the evolution of a new driver mutation. All critical in the decision to continue or discontinue a specific treatment. Citation Format: Elizabeth G. Sweeney, Yawei J. Wang, Jacob Picard, Jim Abraham, Eghbal Amidi, Michael R. Rossi, Sergey Klimov, Claudio D'Ambrosio, Ming Chen, Jeff Elton, George Sledge. Picturing progression: A new role of whole slide imaging in comprehensive clinical genomic datasets [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A007.