Immune Oncology Research Articles

OPtimal Treatment by Invoking biologic clusters in renal cell carcinoma (OPTIC RCC)

Abstract Background The first-line treatment for metastatic clear cell renal cell carcinoma (mccRCC) includes immune oncology (IO) based combination therapy. The current standard includes a PD-1 inhibitor plus either an anti-CTLA-4 inhibitor (IO/IO) or an anti-vascular endothelial growth factor receptor tyrosine kinase inhibitor (VEGF TKI) (IO/TKI). Currently, there is no evidence to guide a physician’s choice between an IO/IO versus an IO/TKI combination. The phase III IMmotion 151 trial performed RNA-seq from 823 ccRCC tumors and established seven biologically distinct gene expression clusters of ccRCC (Motzer and Rini et al., Cancer Cell 2020). The seven clusters showed differential responses to immune checkpoint inhibitor and may serve as a predictive biomarker to select clusters to assign patients with mccRCC to either an IO/IO (ipilimumab/ nivolumab) or an IO/TKI (nivolumab/cabozantinib) regimen. Methods Patients diagnosed with mccRCC without prior systemic therapy (including in the neoadjuvant or adjuvant setting) and at least one measurable lesion as defined by RECIST 1.1 are eligible for enrollment. RNA-seq will be performed on metastatic tumor specimens and used to assign tumor clusters. Patients with cluster 1/2 tumors will be assigned to the nivolumab/cabozantinib arm. Patients with cluster 4/5 tumors will be assigned to the ipilimumab/nivolumab arm. Cluster 3/6/7 will be excluded. The primary endpoint is overall response rate (ORR) per RECIST 1.1. Key secondary endpoints include progression-free survival, depth of response >80%, and rate of immune-related adverse events (irAEs). The hypothesis is that use of tumor clusters to assign front-line therapy to either nivolumab/cabozantinib or ipilimumab/nivolumab will lead to a 20% greater ORR compared to unselected historical controls in CheckMate 9ER (ORR 55%) or CheckMate 214 (ORR: 40%). This trial adopts Simon’s MiniMax two-stage design (power: 80%, one-sided alpha: 0.1). For the nivolumab/cabozantinib arm, stage I will enroll 12 eligible patients. If there are 7 or more responders in the first 12 patients, stage II will enroll an additional 14 patients (n=26). For the ipilimumab/ nivolumab arm, stage I will enroll 16 eligible patients. If there are 7 or more responders in the first 16, stage II will enroll an additional 12 patients (n=28). The primary endpoint will be met if there are 15 or more responders (ORR>60%). This trial is open and enrolling with 12 patients on study at Vanderbilt University Medical Center and additional sites to open including UT Southwestern, City of Hope, University Hospitals Seidman Cancer Center, and Cleveland Clinic. This trial is funded by the Department of Defense Kidney Cancer Research Program Clinical Trial Award (W81XWH-22-1-1033) (NCT05361720). CDMRP DOD Funding: yes

Machine learning reveals genetic modifiers of the immune microenvironment of cancer

Heritability in the immune tumor microenvironment (iTME) has been widely observed yet remains largely uncharacterized. Here, we developed a machine learning approach to map iTME modifiers within loci from genome-wide association studies (GWASs) for breast cancer (BrCa) incidence. A random forest model was trained on a positive set of immune-oncology (I-O) targets, and then used to assign I-O target probability scores to 1,362 candidate genes in linkage disequilibrium with 155 BrCa GWAS loci. Cluster analysis of the most probable candidates revealed two subfamilies of genes related to effector functions and adaptive immune responses, suggesting that iTME modifiers impact multiple aspects of anticancer immunity. Two of the top ranking BrCa candidates, LSP1 and TLR1, were orthogonally validated as iTME modifiers using BrCa patient biopsies and comparative mapping studies, respectively. Collectively, these data demonstrate a robust and flexible framework for functionally fine-mapping GWAS risk loci to identify translatable therapeutic targets.

Discovery of quinazoline HPK1 inhibitors with high cellular potency

Hematopoietic progenitor kinase 1 (HPK1) is regarded as a highly validated target in pre-clinical immune oncology. HPK1 has been described as regulating multiple critical signaling pathway in both adaptive and innate cells. In support of this role, HPK1 KO T cells show enhanced sensitivity to TCR activation and HPK1 KO mice display enhanced anti-tumor activity. Taken together, inhibition of HPK1 has the potential to induce enhanced anti-tumor immune response. Herein, we described the discovery of highly potent HPK1 inhibitors starting form a weak HTS hit. Using a structure-based drug design, HPK1 inhibitors exhibiting excellent cellular single-digit nanomolar potency in both proximal (pSLP76) and distal (IL-2) biomarkers along with sustained elevation of IL-2 cytokine secretion were discovered.

Real-world Outcome of Patients with Advanced Renal Cell Carcinoma and Intermediate- or Poor-risk International Metastatic Renal Cell Carcinoma Database Consortium Criteria Treated by Immune-oncology Combinations: Differential Effectiveness by Risk Group?

BackgroundRenal c carcinoma (RCC) is one of the most common urinary cancers worldwide, with a predicted increase in incidence in the coming years. Immunotherapy, as a single agent, in doublets, or in combination with anti–vascular endothelial growth factor receptor tyrosine kinase inhibitors (TKIs), has rapidly become a cornerstone of the RCC therapeutic scenario, but no head-to-head comparisons have been made. In this setting, real-world evidence emerges as a cornerstone to guide clinical decisions. ObjectiveThe objective of this retrospective study was to assess the outcome of patients treated with first-line immune combinations or immune oncology (IO)-TKIs for advanced RCC. Design, setting, and participantsData from 930 patients, 654 intermediate risk and 276 poor risk, were collected retrospectively from 58 centers in 20 countries. Special data such as sarcomatoid differentiation, body mass index, prior nephrectomy, and metastatic localization, in addition to biochemical data such as hemoglobin, platelets, calcium, lactate dehydrogenase, neutrophils, and radiological response by investigator's criteria, were collected. Outcome measurements and statistical analysisOverall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. The median follow-up was calculated by the inverse Kaplan-Meier method. Results and limitationsThe median follow-up time was 18.7 mo. In the 654 intermediate-risk patients, the median OS and PFS were significantly longer in patients with the intermediate than in those with the poor International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) criteria (38.9 vs 17.3 mo, 95% confidence interval [CI] p < 0.001, and 17.3 vs 11.6 mo, 95% CI p < 0.001, respectively). In the intermediate-risk subgroup, the OS was 55.7 mo (95% CI 31.4–55.7) and 40.2 mo (95% CI 29.6–51.6) in patients treated with IO + TKI and IO + IO combinations, respectively (p = 0.047). PFS was 30.7 mo (95% CI 16.5–55.7) and 13.2 mo (95% CI 29.6–51.6) in intermediate-risk patients treated with IO + TKI and IO + IO combinations, respectively (p < 0.001). In the poor-risk subgroup, the median OS and PFS did not show a statistically significant difference between IO + IO and IO + TKI. Our study presents several limitations, mainly due to its retrospective nature. ConclusionsOur results showed differences between the IO + TKI and IO + IO combinations in intermediate-risk patients. A clear association with longer PFS and OS in favor of patients who received the IO + TKI combinations compared with the IO-IO combination was observed. Instead, in the poor-risk group, we observed no significant difference in PFS or OS between patients who received different combinations. Patient summaryRenal cancer is one of the most frequent genitourinary tumors. Treatment is currently based on immunotherapy combinations or immunotherapy with tyrosine kinase inhibitors, but there are no comparisons between these.In this study, we have analyzed the clinical course of 930 patients from 58 centers in 20 countries around the world. We aimed to analyze the differences between the two main treatment strategies, combination of two immunotherapies versus immunotherapy + antiangiogenic therapy, and found in real-life data that intermediate-risk patients (approximately 60% of patients with metastatic renal cancer) seem to benefit more from the combination of immunotherapy + antiangiogenic therapy than from double immunotherapy. No such differences were found in poor-risk patients. This may have important implications in daily practice decision-making for these patients.

Discovery of BI 7446: A Potent Cyclic Dinucleotide STING Agonist with Broad-Spectrum Variant Activity for the Treatment of Cancer.

Activating the stimulator of interferon genes (STING) pathway with STING agonists is an attractive immune oncology concept to treat patients with tumors that are refractory to single-agent anti-PD-1 therapy. For best clinical translatability and broad application to cancer patients, STING agonists with potent cellular activation of all STING variants are desired. Novel cyclic dinucleotide (CDN)-based selective STING agonists were designed and synthesized comprising noncanonical nucleobase, ribose, and phosphorothioate moieties. This strategy led to the discovery of 2',3'-CDN 13 (BI 7446), which features unprecedented potency and activates all five STING variants in cellular assays. ADME profiling revealed that CDN 13 has attractive drug-like properties for development as an intratumoral agent. Injection of low doses of CDN 13 into tumors in mice induced long-lasting, tumor-specific immune-mediated tumor rejection. Based on its compelling preclinical profile, BI 7446 has been advanced to clinical trials (monotherapy and in combination with anti-PD-1 antibody).

Advances in Making Cancer Mouse Models More Accessible and Informative through Non-Germline Genetic Engineering.

Genetically engineered mouse models (GEMMs) allow for modeling of spontaneous tumorigenesis within its native microenvironment in mice and have provided invaluable insights into mechanisms of tumorigenesis and therapeutic strategies to treat human disease. However, as their generation requires germline manipulation and extensive animal breeding that is time-, labor-, and cost-intensive, traditional GEMMs are not accessible to most researchers, and fail to model the full breadth of cancer-associated genetic alterations and therapeutic targets. Recent advances in genome-editing technologies and their implementation in somatic tissues of mice have ushered in a new class of mouse models: non-germline GEMMs (nGEMMs). nGEMM approaches can be leveraged to generate somatic tumors de novo harboring virtually any individual or group of genetic alterations found in human cancer in a mouse through simple procedures that do not require breeding, greatly increasing the accessibility and speed and scale on which GEMMs can be produced. Here we describe the technologies and delivery systems used to create nGEMMs and highlight new biological insights derived from these models that have rapidly informed functional cancer genomics, precision medicine, and immune oncology.

Weight loss (WL) as a predictive factor for immune oncology (IO).

e14528 Background: WL is an adverse prognostic factor/symptom reported in ~40% cancer (ca) patients (pts) at diagnosis agnostic to site or histologic grade. The mechanism of WL in ca, however, remains elusive. The role of energetic waste in ca WL is doubtful (it neglects likely dietary compensation). On the other hand, lymphocytic activation, a clonal, energetically demanding process is accompanied with anorexia limiting caloric intake which leads frequently to WL if protracted over months as seen in lymphoma (B-symptom) or indecisive immune engagement (Eng) with tuberculosis. WL of &gt; 5% of body weight, was an adverse prognosticator and exclusion criterion in non-small cell lung cancer (NSCLC) chemotherapy (CT) trials, likely due to CT or steroids' abrogation of the putative, limited immune ca containment. We hypothesized that WL in ca reflects months of immune Eng, ineffective as it may be at containing the tumor, but nevertheless representing immune Eng with the ca. Such cases stand the best chance for IO benefit. Notably, IO impact in ca mirrors the 40% of WL in ca pts. Methods: Tumor registry retrieved data from all stage 4 cancers treated with monotherapy IO in our institution. We dichotomized pts to beneficiaries (B) or non-beneficiaries (NB). Bs survive longer than median survival for the respective stage/line of therapy in each ca derived from published trials. To account for missing WL data prior to IO, we reasoned that weight gain of ≥10 lbs (reliably charted over 1 year from IO infusions) is acceptable surrogate for WL and met our criteria. Results: WL in our cohort (n = 53) of Melanoma, NSCLC, renal cell carcinoma, and bladder ca was 43% of B; 10% in NB (ratio &gt; 4:1) rejecting the null hypothesis (p &lt; 0.05). PDL-1, Tumor Mutation Burden, both inconclusive in identifying durable benefit, were limited in our set precluding further analysis. Conclusions: Our results support, though do not prove, that WL identifies the appropriate IO pts. Improved outcome despite a historically-adverse prognosticator (WL); the fact that IO was the only ca therapy used, both support our interpretation. We hope to stimulate others to examine independent larger datasets to confirm this notion and to allow comprehensive subset analyses. For WL to be utilized properly the relevant clinical context is crucial, accounting for GI obstructing ca (esophageal ca), enzymatic anomalies (pancreatic ca), volume fluctuations (cirrhosis), catabolism (prolonged hospitalization), or dental issues, to name a few. Additionally, factors such as tumor bulk, perhaps discerned from the T of TNM staging, deserve examination since more vigorous immune activation against larger targets could produce more impact on appetite and caloric intake. Finally, available clinical trial datasets, not tumor registries, might provide a more crisp, reliable source as we continue this investigation. WL can, with confirmatory work, be clinically incorporated as a predictive factor for IO benefit.

Biomarkers predicting response to 5 immunotherapy arms in the neoadjuvant I-SPY2 trial for early-stage breast cancer (BC): Evaluation of immune subtyping in the response predictive subtypes (RPS).

102 Background: Previously, we showed that in our first PD1-inhibitor (PD1-inh) arm of I-SPY2, pCR associates with high STAT1/chemokine/dendritic signatures in TN and with high B-cell/low mast cell in HR+. From these results, we defined a research-grade Immune classifier incorporated into the RPS (PMID: 35623341), a schema designed to increase pCR if used to prioritize treatment. A clinical-grade version of the Immune (ImPrint) and other RPS biomarkers are now used in I-SPY2.2. Here we evaluate immune markers in 5 Immune-Oncology (IO) therapy arms (2 PD1-inh, 2 PD1-inh combinations, and 1 PDL1-inh combination). Methods: 343 patients with HER2-negative BC with information on pCR and mRNA in 5 IO arms (n 60-72 pts) plus controls (Ctr: 343) were considered. 32 continuous markers including 30 immune (7 checkpoint genes, 14 immune cell, 3 T/B-cell prognostic, 1 TGFB and 5 tumor-immune) and ESR1/PGR and proliferation signatures, were assessed for association with pCR using logistic regression. p-values were adjusted using the Benjamini-Hochberg method (BH p&lt;0.05). Correlations to multiplex immunofluorescence (mIF) data from our initial arm (immune cell and spatial proximity markers) were calculated. Performance of ImPrint, developed with Agendia Inc, was characterized overall and within HR subsets. Results: A larger number of the research-grade immune markers predict response to IO in HR+ than in TN, with the most for HR+ in combination-IO arms (27/32 biomarkers). Tumor-immune signatures dominated by chemokines/cytokines were most consistently associated with pCR across IO arms and across receptor status. Moreover, we found that these markers correlate to mIF spatial proximity measures reflecting high spatial co-localization of PD1+ immune and PDL1+ tumor cells, in TN especially (r=0.59; p=0.003). The ImPrint classifier was evaluated in the IO arms. In HR+, 28% were ImPrint+; and pCR rates were 76% in ImPrint+ vs. 16% in ImPrint-. In TN, 46% were ImPrint+; and pCR rates were 75% in ImPrint+ and 37% in ImPrint-. Overall (HR+ and TN, in all IO arms), pCR rates were 75% in ImPrint+ and 23% in ImPrint-. Performance varied by arm, with the highest pCR rates for HR+/ImPrint+ &gt;90%; and for TN/ImPrint+ &gt;81%. In contrast, pCR rates in the control arm were 34% for ImPrint+ (HR+:33%; TN: 34%) and 13% for ImPrint- (HR+: 21%; TN:8%). Conclusions: Tumor-immune signaling signatures predict response for IO drug class in both TN and HR+HER2-. The ImPrint single-sample classifier predicts response to a variety of IO regimens in both subsets and may inform prioritization of IO vs other treatments and best balance likely benefit vs risk of serious immune-related adverse events. Clinical trial information: NCT01042379 .

Distributional range of tumor mutational burden (TMB) across multiple cancer types emphasizing the importance of different cutoff values in different tumor types for clinical interpretation: A consensus statement from the SEQUOIA consortium.

2615 Background: The measurement of tumor mutational burden (TMB) is critical to select patients who are expected to respond to immune checkpoint inhibitors or immune-oncology (IO). The majority of the clinical guidelines on the interpretation &amp; utility of TMB values are based on studies performed on lung cancer, setting a cutoff of 10. Laboratories that are using comprehensive genomic profiling (CGP) for tumor testing are reporting TMB values &amp;/or interpretations (high/low) beyond lung cancer. Multiple studies have shown that the TMB signature is not universal, but rather tumor &amp;/or even patient-specific. However, the pan-cancer trial for IO (KEYNOTE-158) failed to consider setting different cutoffs per tumor type. Currently, no major guidelines or consensus statements address the issue of reporting TMB as a biomarker beyond lung cancer. In the current study, the membership of the SEQUOIA (Sequencing &amp; Oncology Informatics Academic Team) Consortium investigates the landscape of TMB values across multiple cancer types. Methods: We investigated TMB values in 11,670 patient samples (M = 6225; F= 5445) across 13 different tumor types &amp; histological subtypes, including cancers with lung, colorectal, gastrointestinal, brain, prostate, pancreatic, biliary, endometrioid, ovarian, skin, hematological (CLL myeloma) &amp; lymphoid origin. All samples were processed by a CGP panel of 523 genes. Multiple specimen types were included in the study consisting of tissue, blood, marrow, &amp; lymph node. Results: The range of TMB values varied significantly across different cancer types &amp; histological subtypes. The colorectal, melanoma &amp; endometrial cancers have a wider range of TMB values (2-400 mutations/MB) relative to prostate, breast, myeloma, &amp; serous cancers (0-30 mutations/MB). Along with the range, the mean, &amp; median TMB values are also significantly varied across these tumor types. We also identified that a subset of patients exhibits high TMB (&gt;30 mutations/MB) values across almost all types of cancer. Conclusions: TMB values &amp; their association with response to immune checkpoint inhibitors are not very well studied in all cancer types, although a TMB cut-off of 10 mutations/MB was defined as TMB-high for FDA-approved IO in patients with unresectable or metastatic solid tumors. The range, mean, median, cut-offs &amp; clinical interpretations of TMB-high &amp; low for IO based therapies for different tumor types has not been well established. Our data confirm the wide range, mean, &amp; median across different tumor types, &amp; thus laboratories &amp; oncologists need to be cautious in the clinical interpretation of TMB values for patient management. Future studies should assess responsiveness to IO in specific tumor types based on TMB value to determine tumor-specific TMB cutoff values.

FDA analysis of treatment-free survival in frontline advanced immuno-oncology–VEGF TKI renal cell carcinoma trials.

e13602 Background: Treatment-free survival (TFS) with and without (w/o) toxicity are outcome measures that describe health states where patients are experiencing clinically stable disease while off anti-cancer treatment, a clinically meaningful attribute seen with some immune-oncology (IO) regimens. We sought to characterize TFS with and w/o ongoing toxicity in addition to time on protocol therapy (combination and monotherapy) in trials of frontline IO-VEGF tyrosine kinase inhibitor (TKI) combinations in pts with advanced renal cell carcinoma (aRCC). Methods: We pooled randomized trials submitted to FDA evaluating IO-TKI combination in treatment-naïve aRCC with at least 30 months (mos) of median follow-up. OS was partitioned into health states, including TFS, defined as area between Kaplan-Meier curves for 2 time-to-event endpoints originating at randomization: 1) time to all protocol therapy cessation and 2) time to subsequent systemic therapy initiation or death. Health state areas under the curve were estimated by 30-mo restricted mean times. We pooled data by treatment arm. Results: Three trials (KEYNOTE-426, JAVELIN Renal 100, CheckMate 9ER) met criteria for analysis; in total, 1183 pts received IO-TKI vs. 1184 on control arms receiving sunitinib (SUN). IO-TKI and SUN groups spent 9% (2.7 mos [95% confidence interval (CI): 1.8, 3.5]) and 10% (2.9 mos [95% CI: 2.1, 3.8]) of the 30-mo period alive and treatment-free. Mean TFS with grade ≥3 toxicity were 1 and 0.6 mos in the IO-TKI and SUN groups, respectively. Mean TFS w/o grade ≥3 toxicity were 1.7 and 2.3 mos in IO-TKI and SUN groups, respectively. In IO-TKI group, the 30-mo mean time on combination therapy was 13.7 mos (46% of 30-mo period) and 3.9 mos on monotherapy (13% of 30-mo period). Following cessation of one drug in IO-TKI arm, monotherapy duration of TKI was longer than IO: 2.9 and 1 mos, respectively. Conclusions: In this descriptive, exploratory analysis, TFS and TFS w/o toxicity were similar in IO-TKI group compared to SUN group, in contrast to improved TFS in IO-IO vs. SUN seen in published literature. Among pts on IO-TKI, more time was spent on TKI monotherapy than IO monotherapy. These findings may reflect continuation of TKI until progression per protocol design in all trials and discontinuation of IO after 2 years in 2 trials, and may inform individual pt choice of IO-IO vs. IO-TKI. [Table: see text]

Molecular profiling and characterization of the tumor immune microenvironment (TME) in appendiceal carcinoma (AC).

3622 Background: The rarity of AC presents challenges in understanding disease pathogenesis. We previously showed that AC has higher rates of mutations in KRAS and GNAS and lower rates of TP53, APC, and PIK3CA than CRC. The appendix also has many lymphoid clusters and regulates IgA production in the large bowel, suggesting that AC may be subject to more lymphocytic regulation than CRC. We sought to characterize the molecular profile and TME across AC histopathological types. Methods: AC samples were analyzed by DNA sequencing (592 genes, NextSeq, or whole exome sequencing, NovaSeq), whole transcriptome sequencing (WTS, NovaSeq), and immunohistochemistry (IHC) for molecular profiling, including microsatellite instability (MSI), mismatch repair (MMR), PD-L1 (SP142), and tumor mutational burden (TMB). Microenvironment Cell Population-counter Quantiseq was used to quantify tumor immune contexture using WTS. AC histopathology was derived from pathology reports. Mann-Whitney U and ChiSquare tests were applied as appropriate, with P-values adjusted for multiple comparisons using Benjamini-Hochberg. Results: AC (N = 731) were grouped by histology: 5% goblet cell (GC), 6% high-grade adenocarcinoma (HGA), 14% low grade mucinous (LGM), 33% mucinous adenocarcinoma (MA), 1% pseudomyxoma peritonei (PMP), 6% signet ring cell carcinoma (SRC), and 35% adenocarcinoma not otherwise specified (NOS) (see Table). Age at collection and PD-L1+ (0-5.2%) did not differ by histopathology. Seven patients had dMMR (4 NOS and 3 MA). Median TMB was significantly higher in NOS vs. GC (4 mutations/megabase vs 2, q &lt; 0.001), NOS vs. LGM (4 vs 3, q = 0.048), MA vs. GC (4 vs 2, q &lt; 0.001), and MA vs. LGM (4 vs 3, q = 0.037). Distinct TME patterns were observed in NOS vs. MA (median cell fraction: dendritic cells 0.07 vs 0, q &lt; 0.01; M2 macrophages 0.055 vs 0.042, q = 0.030; natural killer cells 0.034 vs 0.031, q = 0.011; CD4 T cells 0.006 vs 0, q = 0.044) and GC vs. MA (M1 macrophages 0.039 vs 0.057, q = 0.021; dendritic cells 0.012 vs 0, q &lt; 0.01; B cells 0.061 vs 0.050, q = 0.013; M2 macrophages 0.074 vs 0.042, q &lt; 0.01; natural killer cells 0.041 vs 0.031, q = 0.01; and neutrophils 0.017 vs 0.038 q = 0.045). T cell inflamed signature (TIS) and immune-oncology (IO) marker gene expression were higher in HGA and lower in LGM and MA. Somatic mutation rates differed by histopathology (see Table). Conclusions: There is significant heterogeneity in TMB, TME, and mutational profiles across AC histologies. MA has a particularly immune-cold TME shown by lower infiltration of lymphocytes, TIS, and IO gene expression. These findings are critical to identify novel biomarkers and develop new therapeutic strategies for AC. [Table: see text]

Abstract 4385: Unprecedented tumor: immune interaction–clinical and drug-development implications

Abstract Background: Classic therapeutic (RX) oncology principles demand radiographic response or durable stable disease as surrogates of overall survival (OS) prolongation, the ultimate metric of Rx benefit. Progressive disease (PD), however, is an undisputed indication of treatment failure. Immune Oncology (IO) trials reporting individual patient outcomes using swimmers' plot (SP) suggest substantial OS subsequent to PD, unprecedented pre-IO. Hypothesis: Unrecognized phenomenon of OS benefit in the setting of radiographic PD, is tissue-agnostic and prevalent (&amp;gt;15%). Methods: We conducted a retrospective review of IO monotherapy trials in 3 cancers if SP were reported. Percentage of PD patients (Pts) surviving beyond defined time landmarks is reported (Table). Results: Realizing that 3rd or subsequent Rx lines rarely provide &amp;gt; 6 months OS duration in most cancers, our results indicate that 2/3rd of IO-treated pts, regardless of histology, or line of therapy are performing significantly better than expected; 1/3rd surviving &amp;gt; 1 year beyond PD. Discussion: In IO, radiographic bulk may increase despite undeniable OS benefit (in years). We likely underestimated this phenomenon since pts with PD at 1st assessment aren't included in the SP, and with longer follow up, OS of PD pts is likely to improve with longer time to manifest it. This phenomenon–we term 'Disguised Responders' (DRs)–challenges an oncology principal. DRs needs to be considered by clinicians and future drug developers to accurately identify putative novel drugs. A plausible mechanism of DRs posits that 'virulence' of a given subclone, regardless of size or bulk, dictates Rx outcome. Virulence may operate through cachexia-induction, propensity to metastasize or metabolic adaptability, to name a few. Novel experimental approaches are desperately awaited to probe and measure virulence of different tumor subclones in future drug evaluations. Table 1. Clinical Trials of IO Monotherapy Number and (%) of pts surviving beyond clinical PD (months) Tumor Rx Line 1st Author/Yr N PD (n) &amp;lt;6 6-11 12-23 24-35 36-47 &amp;gt; 48 NSCLC* 2nd + Topalian ''19 22 12 1 2 4 1 4 1st + Fredinandus ''21 45 2 1 1 1st + Kim ''22 139 22 13 5 3 N/A Bilger ''22 18 10 3 4 3 2nd Dart ''21 7 3 2 1 2nd + Tozuka (MR) ''20 11 11 4 6 1 2nd + Tozuka (PD) ''20 51 51 29 12 7 2 1 2nd + Waterhouse ''20 39 38 16 14 4 4 Subtotal 332 149 63 (42%) 44 (30%) 28 (18%) 4 (3%) 5 (3%) 4 (3%) Melanoma 1st + Warburton ''20 70 13 1 4 3 4 1 2nd + Topalian ''19 34 21 1 2 10 4 4 2nd + Weber ''15 38 4 2 1 1 2nd + Dimitriou ''21 125 9 3 4 2 Subtotal 267 47 3 (6%) 8 (17%) 10 (21%) 17 (36%) 5 (11%) 4 (8.5%) RCC** 2nd + Topalian 19'' 10 7 1 1 2 3 7 15% 15% 30% 42% Total 609 203 33% 25% 15% 10% 6% 5.4% *Non-small cell lung cancer. ** Renal Cell Carcinoma Citation Format: Farah Mazahreh, Liyan Mazahreh, Brad Fugere, Ahmad Mazin Safar. Unprecedented tumor: immune interaction–clinical and drug-development implications. [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 4385.

Abstract 4619: Spatial analysis of the immunosuppressive tumor microenvironment in brain cancer applying a novel multiplex immunofluorescence panel

Abstract Glioblastoma (GBM) and brain metastases (BM) account for &amp;gt;70% of all brain cancers. The annual incidence averages to 10-17 cases per 100.000 person (~ 3 cases for GBM and 7-14 for BM), resulting in 45.000-77.000 new cases in the European Union alone. The standard of care for GBM includes maximal surgical resection followed by radiation and chemotherapy with temozolomide, whilst whole brain irradiation or surgical removal with or without chemotherapy are used for BM. In spite of therapy, the median overall survival remains &amp;lt; 6 months for BM, and 16 months for GBM, suggesting brain cancers as one of the most therapy-resistant tumors. Despite the emergence of novel therapeutic strategies, such as anti-angiogenic therapy, targeting myeloid cells or checkpoint immunotherapy, no significant improvement in overall survival of brain cancer patients was observed. Several studies have suggested the resistance to therapy in brain cancers is largely attributed by the presence of an immunosuppressive brain tumor microenvironment. To investigate the spatial architecture of the tumor microenvironment in BM derived from lung and breast cancers, we employed the Lunaphore 13-plex immune-oncology (IO) Core Panel, consisting of clinically relevant IO biomarkers (CD3, CD4, CD8, CD45, FoxP3, PD1, PD-L1, CD11c, CD20, CD56, CD68, aSMA and Ki-67) for multiplex immunofluorescence (mIF) assays. These findings were then compared to isocitrate dehydrogenase-wildtype glioblastoma, which is a T-cell-poor cancer that is also characterized by an immunosuppressive tumor microenvironment. Further, the IO Core Panel was expanded with previously established antibodies targeting myeloid, blood-brain barrier and tumor cell markers in a proteomic workflow on COMET™, a fully automated, sequential immunofluorescence (seqIF™) platform. Comprehensive mapping of the GBM microenvironment and BM confirmed previous observations obtained by bulk and single cell RNA sequencing analysis that described profound differences in the tumor microenvironment of BM versus GBM. In contrast to GBM, anti-tumor immunity in BM was increased, whereas immunosuppressive cells remained unchanged between the two. Of note, T- and B-cells were not evenly distributed within the tumor tissue, providing an additional level of spatial information that would have been missed by bulk RNA or proteomic analyses. Our study is the first to demonstrate a successful application of Lunaphore’s IO core panel on GBM and BM samples. This highlights potential future use in the development of tools for personalized medicine for GBM and other tumor types. Citation Format: Yvonne Reiss, Jadranka Macas, François Rivest, Victor de Gautard, Saska Brajkovic, Bastian Nicolai, Tatjana Starzetz, Kathleen Sommer, Pinar Cakmak, Jonathan Schup, Jennifer H. Lun, Karl H. Plate. Spatial analysis of the immunosuppressive tumor microenvironment in brain cancer applying a novel multiplex immunofluorescence panel. [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 4619.

Abstract 5063: Optimizing benefit/risk in oncology: Review of post marketing dose optimization

Abstract Introduction: Molecularly targeted (MTAs) and immune-oncology (IO) agents are more selective with wider therapeutic window than cytotoxic agents resulting in maximal activity at doses &amp;lt; MTD. However, these agents commonly selected the MTD (or highest tested dose) which is assessed using dose-limiting toxicities in escalating doses in limited number of patients/dose. This led to several post-marketing requirement (PMR) studies to evaluate alternative dosing regimens to optimize the benefit-risk. Methods: Post-marketing dose optimization studies (required by FDA or sponsor initiated) for oncology MTAs/IO agents were reviewed. These studies/analyses were classified in to attempts to improve efficacy, safety, or ‘life cycle management’ (ie, fixed vs body size-based dosing, favorable route of administration, or extending the dosing interval). Approvals 1999-April/2022 were evaluated using FDA review documents and published literature. PMR studies for organ impairment, DDI, or food effect were excluded. Results: Overall, 27 (16 MTAs and 9 IO) out 126 FDA oncology approvals reviewed were identified to have conducted post-approval dose optimization studies (27/126, 21.4%). Dose optimization studies were either efficacy-driven (n = 4), safety-driven (n = 13), or to improve method of administration (ie, IV to SC route; body-weight to fixed dosing; less dosing frequency) (N = 14). Efficacy-driven PMRs evaluated a higher dose either for the overall population (1/4) or for a subgroup with lower exposure (3/4); the latter cases showed no clear benefit in this subgroup vs case-matched subgroup from the control arm. No changes of label dosing recommendations resulted from efficacy-driven PMRs. All safety-driven PMRs which evaluated lower doses were for drugs with a positive exposure-safety relationship and most had a flat exposure-efficacy relationship (9/13, 69%) suggesting that a lower dose might improve safety without compromising efficacy. Completed safety-driven PMR studies resulted in changing the approved dose in 2/7 cases (29%). Lower doses were commonly associated with better safety profiles, however, establishing efficacy non-inferiority vs approved doses was typically not successful except in 1 case where non-inferiority was met for the primary endpoint of overall survival; this did not lead to label change as secondary endpoints, eg, PFS, favored the higher dose.Attempts to improve method of administration tended to be feasible and resulted in a successful change of the label dose for drugs with relatively flat exposure-efficacy and safety profiles and with adequate efficacy/safety data at multiple dose levels. Conclusion: Our results suggest that safety or efficacy-driven post-marketing dose optimization studies were rarely successful in changing the label dose. Dose optimization could be more important for drugs with a steep exposure-safety relationship and a flat exposure-efficacy relationship. Citation Format: Pooneh Soltantabar, Hoi-Kei Lon, Kourosh Parivar, Diane Wang, Mohamed Elmeliegy. Optimizing benefit/risk in oncology: Review of post marketing dose optimization. [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 5063.

Abstract 1806: Modulation of natural killer cell immune response to tumor with novel synthetic tumor -immune cell agonist, NK-TICA(r)

Abstract The tumor specific activation of natural killer (NK) cells with Bicycles is an area of active investigation in immune oncology. NK cells are highly responsive immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Bicycles are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle platform technology to discover and evaluate a new class of fully synthetic molecules termed NK tumor immune cell agonists (NK-TICA®). The NK-TICA® consists of chemically coupled Bicycles® that bind specifically to the key activating receptor, NKp46, and to tumor antigens, that results in highly potent, antigen-dependent receptor activation and NK cell activation. We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA® compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner. In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle NK-TICA® as a multifunctional immune cell engager will promote the modulation of NK cells, as well as the infiltration and anti-tumor activity of NK cells in solid tumors. Citation Format: Fay Dufort, Christopher J. Leitheiser, Kathleen Ho, Tucker Ezell, Alexandra Rezvaya, Peter Brown, Liuhong Chen, Philip Brandish, Kevin McDonnell, Michael Skynner, Nicholas Keen. Modulation of natural killer cell immune response to tumor with novel synthetic tumor -immune cell agonist, NK-TICA(r) [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 1806.

Abstract 3471: A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment

Abstract Tumor-associated macrophages (TAMs) are important players to maintain immunosuppression within the tumor microenvironment (TME) and attenuate the function of effector immune cells to promote tumor survival. High density of TAMs is well-recognized as a feature of tumor progression and poor prognostic factor across various tumor types. Therefore, therapeutic strategies to dampen TAMs’ suppressive potential in TME and reprogram them towards a pro-inflammatory phenotype are being increasingly appreciated as being paramount for an effective immunotherapy. Nevertheless, the key mediators of TAM-induced immunosuppression remain largely unknown, identification of which can lead to the development of novel immunotherapeutic strategies aimed at re-educating the immunosuppressive TAMs to an anti-tumor phenotype. To systematically interrogate the genes responsible for the immune-suppressive phenotype of TAMs, we developed a functional, high-throughput genetic screening platform (iOTarg™) based on human primary monocyte-derived M2-like macrophages. Using CRISPR, we knocked out a proprietary library of well-expressed and druggable genes (1400 genes) in two independent donor-derived M2-like macrophages and cocultured them with activated autologous effector T cells (TCs). We achieved nearly complete knockout (KO) efficacy over a range of control genes. Functional impact of individual KO on macrophage viability and phenotype, as well as on TC activity, was measured using multiparametric assay readouts. Initial hits were reconfirmed in three additional donors in a secondary screen that integrated an additional tumor lysis readout. As expected, knockout of CSF1R, which is essential for macrophage maintenance, resulted in a dramatic loss of macrophage cell viability, whereas KO of established TAM markers, TREM2 and Clever-1, induced a change in macrophage phenotype. Furthermore, KO of immune-inhibitory receptor LILRB2 reduced the M2-like phenotype and restored TC activity. Inhibition of M2 activation of macrophages and subsequent increase in TC activation culminated in strong tumor cell killing for a subset of genes, highlighting an untapped repertoire of novel TAM-associated immune-checkpoint targets. Taken together, we report for the first time a highly sophisticated target discovery platform that addresses the functional role of any TAM-expressed gene in regulating macrophage viability, phenotype, T cell activity and even its gross impact on tumor cell lysis, all done in a high-throughput format employing CRISPR-edited primary human immune cells and multi-parametric functional immunological assays. As a result, we could confirm well-known targets in clinical testing, as well as identify additional novel targets that could lead to first-in-class, TME-based therapeutics and expansion of treatment options in immune oncology. Citation Format: Kritika Sudan, Tillmann Michels, Carmen Amerhauser, Claudia Tschulik, Leonie Majunke, Lucille Albert, Valentina Volpin, Adriana Turqueti-Neves, Ronny Milde, Nisit Khandelwal. A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment [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 3471.

Abstract 4444: Targeting PI3K isoforms to improve the effectiveness of T cell mediated immunotherapy

Abstract Hyperactivation of the PI3K pathway has been reported to correlate with resistance to immune checkpoint blockade therapy (ICB) in melanoma, highlighting the therapeutic potential of combining PI3K inhibition (PI3Ki) with ICB. To maximize the clinical benefit of PI3Ki-based immune oncology (IO) combination, we characterized the role of PI3K isoforms in tumor and T cells and determined the immunological impacts of PI3Ki alone or in combination with ICB. Inhibitions of PI3K were achieved by either genetic knockdown (KD) or the bioactive compound in PTEN-present (B16/MC38), PTEN-absent (BP/D4M) tumor cell lines, and CD8+ T cells (Pmel-1). Following PI3Ki, we determined the activation status of the PI3K pathway (p-AKT level), transcriptional profile, and cellular function of these cells. We found both in vitro KD and pharmacological inhibition of either PI3Kα or PI3Kβ displayed a dramatic reduction of the PI3K pathway in tumor cells but moderate or no reduction in T cells, whereas the PI3K pathway significantly decreased in T cells with PI3Kγ or PI3Kδ inhibition. KD of PI3Kα or β isoforms drastically sensitized both D4M and MC38 tumors to αPD1 in vivo. We also observed that only PI3Kγ or PI3Kδ inhibition profoundly suppressed cytokine production and cytotoxicity of CD8+ T cell, suggesting that PI3Kα or PI3Kβ isoform inhibition can achieve tumor specific PI3Ki with limited impacts on T cell function. Furthermore, we used multiple syngeneic melanoma models to determine whether PI3K isoform inhibition can synergize the antitumor activity of ICB in vivo. In PTEN-present tumors, BYL719 (BYL, a PI3Kα inhibitor) synergized with αPD1 to delay tumor growth and extend survival (median survival of MC38-bearing mice in control (Ctrl), BYL, αPD1, and combination (Comb) groups: 30, 36, 33, and &amp;gt;45 respectively; p&amp;lt;0.05: Ctrl/BYL/αPD1 vs Comb). However, a limited combinatorial effect between GSK2636771(a PI3Kβ inhibitor) and αPD1 was observed in PTEN-present tumor models. Moreover, the combination of BYL and αPD1 exhibits superior antitumor activity in a spontaneous Braf-mutant, PTEN-loss melanoma model when compared with either reagent. Mechanistically, the combination of BYL and αPD1 improved CD8+ T cells tumor infiltration (14 days treatment, mean CD8+ number/mg of the tumor, Ctrl:1392.9, BYL:2073.9, αPD1:1545.2, Comb:4691.8; p&amp;lt;0.01: Ctrl/BYL/αPD1 vs Comb) and reduced MDSCs in MC38 tumors (p&amp;lt;0.05: Ctrl vs Comb). Multi-omics profiling of tumor cells with in vitro and in vivo PI3K isoform inhibition is ongoing. Collectively, our results demonstrate that PI3Kα inhibitor can potentiate T cell-mediated antitumor immune responses regardless of PTEN status, providing a strong rationale for the clinical development of the BYL-based IO combination. In collaboration with Novartis, MD Anderson Cancer Center will launch a Phase I/II trial of the FDA-approved BYL in combination with αPD1 in advanced melanoma and breast cancer patients. Citation Format: Ritu Bohat, Xiaofang Liang, Chunyu Xu, Yitao Tang, Jiakai Hou, Nicholas A. Egan, Leilei Shi, Ashley Guerrero, Roshni Jaffery, Elizabeth M. Burton, Han Liang, Hussein Tawbi, Michael A. Davies, Weiyi Peng. Targeting PI3K isoforms to improve the effectiveness of T cell mediated immunotherapy. [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 4444.

Abstract 2269: Bexmarilimab induces B-cell activation and autoantibody production

Abstract Introduction: Bexmarilimab, a Clever-1 targeting humanized antibody, is a macrophage checkpoint inhibitor promoting antigen presentation and pro-inflammatory cytokine secretion. Data from the first-in-human clinical Phase I/II study (MATINS; NCT03733990) demonstrate that single agent bexmarilimab is able to ignite an interferon response with survival benefit in 30% of patients with advanced gastric cancer, cutaneous melanoma and cholangiocarcinoma (ASCO 2022). Since Clever-1 knockout mice have been reported to have enhanced T-cell antibody production and bexmarilimab increases peripheral B-cell populations, we investigated B-cell phenotype, clonality and autoantibody formation in bexmarilimab treatmented MATINS patients. Methods: We first performed comprehensive phenotyping of peripheral B-cells from a colorectal carcinoma patient showing partial response (per RECIST v1.1). Autoantibody reactivities were measured in 80 serum samples of bexmarilimab treated patients (37 pre-treatment and 43 post-treatment samples from cycles 3 and 4) and compared to serums from 53 healthy individuals using Oncimmune's SeroTag multiplex technology with an immune-oncology (IO) specific protein array comprised of 1162 antigens. Results: Single-cell sequencing together with BCR sequencing of the responder’s B-cells during cycle 4 revealed an induced and activated B-cell population consisting of four transcriptionally similar clusters expressing IGHM, IGHD, CD23 but not CD43 and one cluster expressing CD79B, PLD4 and MZB1, which was distant from the naïve CCR7 expressing B-cells and IGHA1 and IGHG1 expressing plasma blasts. The expansion of B-cells was not due to clonal expansion as no overlapping BCR clones were identified between B-cells at pre-dose and cycle 4. Autoantibody profiling revealed great inter-individual heterogeneity in the number and targets of induced antibodies among patients treated with bexmarilimab reflecting individual patterns in self- and tumor antigens. However, shared autoantibody changes against targets such as cancer testis antigens (GAGE2), typical autoimmune disease antigens (SNRPC, TPO, TOP1) and antigens related to the induction of innate immunity and interferon responses, e.g. TRIM21 were observed. Importantly, we identified a set of pre-treatment autoantibodies that were associated with longer time to disease progression and were predictive of clinical response (disease control rate [DCR], consisting of CR, PR, SD) and progression-free survival (PFS) on bexmarilimab therapy. Conclusions: Our data implies that bexmarilimab can induce activation and secondary Ig rearrangements in mature B-cells, which has been reported to occur in germinal centres during T-cell dependent antibody responses to increase B-cell diversity and affinity of antigen receptors. B cell diversity and activation was reflected in patient autoantibody production and may enhance cancer immune recognition. Citation Format: Elisa M. Vuorinen, Mari L. Björkman, Reetta Virtakoivu, Juho Jalkanen, Sofia Aakko, Akira Takeda, Petra Budde, Hans-Dieter Zucht, Manuel Brautigam, Behnaz Ahangarianabhari, Petri Bono, Maija Hollmén. Bexmarilimab induces B-cell activation and autoantibody production [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 2269.

Abstract 6375: Transcriptomic signatures in melanoma lymph node metastasis that relate to immune oncology treatment

Abstract Background: Frequently, patients with primary cutaneous melanoma develop regional lymph node metastasis (LNM). The outcomes in melanoma patients with LNM can be poor, depending on clinicopathological features including the size and the number of the LNM. Newer immunotherapies including immune checkpoint inhibitors (ICI) have significantly improved outcomes in metastatic melanoma patients, and when tumors respond to ICI the durable responses are noteworthy. The aim of the current study is to identify transcriptomics changes in immune-related genes in LNM that correlate to outcomes in melanoma patients receiving adjuvant ICI therapy. Methods: The analysis were performed in formalin-fixed paraffin-embedded (FFPE) sections from LNM positive (LNM(+); n=17) and LNM negative (LNM(-); n=8) of patients who received surgery at SJHC. For each tissue, a single section was processed and analyzed for 2,002 genes using the direct (mRNA extraction-free) transcriptomic assay of the HTG EdgeSeq AutoImmune Panel (HTG-AI). The HTG-AI transcriptomic normalized data were compared between both groups. For verification of gene signatures, publicly available RNA-seq datasets were obtained from patients with clinical outcomes receiving anti-PD-1. All biostatistical analysis were performed using HTG Reveal and/or GraphPad Prism software. Results: Comparisons between melanoma LNM(+) and LNM(-) samples showed 229 DEGs (Log2fold-change ←2; &amp;gt;2 FDR&amp;lt;0.01), with 213 genes upregulated and 16 downregulated. Pathway analyses using Reactome indicated that the DEGs in LNM(+) were associated with cytokine, interferon (IFN), and chemokine signaling, as well as signaling by receptor tyrosine kinases (FDR&amp;lt;0.0001). The chemokines CXCL8, CXCL2, and CXCL10 were significantly upregulated in LNM(+) (p&amp;lt;0.001). LNM(+) showed significantly higher levels of IFN signaling mediators (STAT1, STAT2, IFI6, IFI30, IFI44) compared to LNM(-) specimens (p&amp;lt;0.001). Also, LNM(+) showed a significant increase in CD276 (B7-H3) levels (p&amp;lt;0.001). Then, we correlated the mRNA levels of IFN signaling (STAT1, STAT2, IFI6, IFI30, and IFI44) obtained from samples before anti-PD-1 therapy (Nivolumab or Pembrolizumab) to overall survival (OS). Of clinical importance, patients with elevated levels of the IFN signaling genes showed better OS for patients receiving adjuvant anti-PD-1 (HR=0.6, CI=0.42-0.86, Log-rank test, p=0.0044). Conclusions: We identified a distinctive transcriptomic profile in LNM(+). Several genes were upregulated and associated with specific pathways. Using publicly available data, we demonstrated that STAT1, STAT2, IFI6, IFI30, and IFI44 gene signature correlated with improved OS for patients receiving adjuvant ICI treatment. The STAT1, STAT2, IFI6, IFI30, and IFI44 genes may represent new biomarkers to assess in biopsies of patients that will receive adjuvant or neoadjuvant ICI therapies. Citation Format: Matias A. Bustos, Shodai Mizuno, Suyeon Ryu, Dave S. Hoon. Transcriptomic signatures in melanoma lymph node metastasis that relate to immune oncology treatment [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 6375.

Abstract 1018: Multiplex immunofluorescence and image analysis to investigate the role of the immune contexture and fibroblast activation for tumor cell budding in colorectal cancer

Abstract The advent of immune oncology had a significant impact on the stratification of cancer patients in the past few years. Furthermore, immune cell phenotyping of the tumor microenvironment is becoming a tool not only for the identification of novel predictive biomarkers for cancer immunotherapy, but also for prognostic markers that may help to understand several mechanisms like invasion and epithelial-mesenchymal transition (EMT). We demonstrate how multiplexed immunofluorescence (mIF) assays and digital image analysis helped to investigate tumor budding in colorectal cancer by evaluating the tumor microenvironment and activated fibroblasts. Akoya Bioscience’s Opal detection system and fluorophores were optimized on LEICA Bond RX for use with human normal tonsil control tissues and FFPE colorectal cancer specimens. The 6-plex mIF panel included CD4 (clone SP35), CD8 (clone C8/144B), CD68 (clone PG-M1), FoxP3 (clone SP97), PD-L1 (clone SP263) and pan-Cytokeratin (clones AE1/AE3). In addition, FAP-alpha single-plex IHC was used to investigate reactivity of the stroma surrounding tumor cell buds. Each single marker of the mIF panel was independently validated for specificity regarding stripping efficacy and epitope stability, and for accuracy compared to single plex bright field immunohistochemistry. Automated image analysis and cellular phenotyping followed a workflow of custom Visiopharm apps. The tissue was segmented into regions of interest, such as areas with and without tumor cell budding, and bud microenvironment. Additional parameters like MSI (Microsatellite Instability) status and TNM classification were also taken into consideration for data analysis. The applied mIF immune cell panel and automated image analysis workflow identified a broad number of different immune cell phenotypes, including rare double- or triple-positive cell subtypes, as well as their spatial relation to tumor cell buds within the activated stroma region. This revealed new insights into the complexity of the tumor microenvironment in colorectal cancer around tumor cell buds, suggesting a potential effect of both the immune system and the stromal cells on tumor cell invasion into the surrounding tissue. Citation Format: Dirk Zielinski, Rebekka Vogtmann, Anne Siemon, Christina Koppel, Christina Schipper, Anastasiia Tereshchenko, Suso Platero, Hans-Ulrich Schildhaus. Multiplex immunofluorescence and image analysis to investigate the role of the immune contexture and fibroblast activation for tumor cell budding in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1018.