Rare cancers lack robust, evidence-based treatment options. We conduct a prospective, multi-histology phase 2 clinical trial of pembrolizumab in 154 patients (142 evaluable), observing an objective response rate of 14.8% and clinical benefit (CB) in 26.8%. Multi-modal profiling is performed on baseline, on-treatment, and progression samples. CB associates with high microsatellite instability (MSI-H)/high tumor mutation burden (TMB-H) (odds ratio [OR]: 13.9, p = 0.0013) and programmed cell death ligand 1 (PD-L1) combined positive score (CPS) ≥10 (p = 0.0285), although responses also occur in biomarker-negative tumors and vary by histology. CB tumors exhibit higher pre-treatment immune infiltration and T cell activation, whereas no CB (NCB) tumors show proliferative programs. In moderately infiltrated tumors, CB associates with increased immune content during treatment. Multiplex immunofluorescence confirms higher baseline T cell densities in CB and limited remodeling in NCB tumors. These findings suggest that tumor immune microenvironment features may serve as predictive markers beyond genomic assays and highlight immune cell recruitment during therapy in moderately infiltrated tumors.

Introduction: Immune checkpoint inhibitors have revolutionized cancer therapy; however, adverse events from an unchecked immune system such as immune checkpoint inhibitor mediated diarrhea and colitis (IMDC) can develop. Fecal microbiota transplantation (FMT) remains an option for patients with refractory colitis, but has not been tested in an upfront setting. Methods: From an open-label, phase I/II clinical trial (NCT0403861) starting June 2021, we report an analysis of adult patients with IMDC treated with upfront FMT. We performed fecal shotgun metagenomic sequencing, metabolomic, transcriptomic and immunofluorescence profiling pre-FMT and post-FMT and and plasma biomarkers of inflamamtion and immune response pre-FMT to predict response. Results: 13 patients were treated with FMT, of which 11 (84.6%) achieved clinical response with a median time to clinical improvement of 1(1-5) days. Among responders sequenced with baseline and follow-up (n = 8), 6 patients (75%) had an increase in alpha diversity post-FMT. Notably, in responders Lacrimispora amygdalina and Alistipes shahii increased independently at both 2 and 4 weeks post FMT. Interestingly, aspartic and propionic acid decreased post-FMT (p < 0.05). Using multiplex immunohistochemical immunofluorescence staining of samples obtained at presentation, we determined that total CK+ and CK+Ki67+ cell populations were reduced in non-responder patients. Additionally, there was a trend toward lower pre- and post-FMT CD20+, CD20+Ki67+, CD4+FOXP3+, and CD8+FOXP3+ cell populations in non-responder patients. Immune cell-type abundance scores showed increased plasma cells, neutrophils, (M1 and M2) macrophages, memory activated and resting memory CD4+ T cells , CD8+ T cells,T follicular helper (Tfh) cells, regulatory T cells (Treg), in pre-FMT samples which all decreased dramatically in post-FMT samples, likely related to response to FMT. Non-responders had lower baseline plasma GDF2, TLR3, CCL22, and FGF21 and higher baseline plasma CXCL14, OSM, IL-1β and IFNG. Conclusion: FMT is a promising front-line therapeutic option for patients who develop IMDC with high efficacy and favorable safety profile. Additional microbiome, blood and tissue analysis provides insights in future directions of potential mechanisms and targets for development of novel therapeutic intervention.

Despite the successes of immunotherapy in cancer treatment over recent decades, less than <10%-20% cancer cases have demonstrated durable responses from immune checkpoint blockade. To enhance the efficacy of immunotherapies, combination therapies suppressing multiple immune evasion mechanisms are increasingly contemplated. To better understand immune cell surveillance and diverse immune evasion responses in tumor tissues, we comprehensively characterized the immune landscape of more than 1,000 tumors across ten different cancers using CPTAC pan -cancer proteogenomic data. We identified seven distinct immune subtypes based on integrative learning of cell type compositions and pathway activities. We then thoroughly categorized unique genomic, epigenetic, transcriptomic, and proteomic changes associated with each subtype. Further leveraging the deep phosphoproteomic data, we studied kinase activities in different immune subtypes, which revealed potential subtype -specific therapeutic targets. Insights from this work will facilitate the development of future immunotherapy strategies and enhance precision targeting with existing agents.

The S1609 DART trial is designed to assess the clinical relevance of combination dual ipilimumab and nivolumab in rare tumors. We focused on immune profiling 2 cohorts comprised of subtypes of neuroendocrine carcinomas; cohort 23 (nonpancreatic neuroendocrine (NET) and cohort 52 (pan high-grade neuroendocrine). FFPE tissue collected at baseline and PBMCs and plasma collected at baseline and cycle 2 week 9 (C2W9) were utilized. Samples were subjected to multi-omics analyses including IHC, multiplex immunofluorescence (mIF), gene expression profiling, WES, TCR sequencing, CyTOF and Olink. Known viral TCRs were removed in silico from analysis. Hierarchical clustering based on an interacting distance of 25μm and minimum neighborhood size of 10 cells was used to identify neighborhoods of interacting cells in the TME from mIF. Biomarker data was dichotomized by median and Cox Proportional Hazard regression model used for multivariate survival analysis. For continuous biomarker data, we use Spearman's rank correlation, Mann-Whitney U test, or Kruskal-Wallis test. We dichotomized biomarker data and used Chi-Square test for response analysis. Benjamini & Hochbert method was used for multiple-testing adjustment of p-values. Immune cells clustered in the tumor regions of responding patients and in the stroma of non-responding patients. Gene expression profiling showed a B cell signature correlated with superior response (p=0.0037) and longer progression-free survival (PFS, p=0.0067) and a tertiary lymphoid signature (TLS) associated with better response (p=0.05). Lymphoid aggregates were confirmed using H&E and correlated with B cell and TLS gene signatures. Response was associated with a decrease in IL8, an increase in IFNg, and expansion of CD4+ T cell memory subsets and activation by C2W9 in peripheral blood. TCRseq revealed high richness and density correlation with improved PFS (p=0.00011) at C2W9 but not at baseline. With limited sample size fully acknowledged, multiomics analysis of NET cohorts revealed B cell/TLS signatures in tumor, diverse/dense T cells and expansion of CD4+ memory T cells in peripheral blood associate with improved outcome to ipi/nivo treatment.

BACKGROUND:The efficacy and feasibility of pembrolizumab combined with chemotherapy in frontline management of advanced high-grade epithelial ovarian cancer (EOC) is unknown. Additionally, modification of the tumor microenvironment following neoadjuvant therapy is not well understood. METHODS:In this single-arm phase 2 trial (this study was registered at ClinicalTrials.gov: NCT02520154), eligible patients received up to 4 cycles of neoadjuvant chemotherapy followed by interval cytoreduction, 3 cycles of adjuvant intravenous carboplatin/weekly paclitaxel/pembrolizumab, and finally maintenance pembrolizumab until progression or toxicity (maximum 20 cycles). The primary endpoint was progression-free survival (PFS). Secondary endpoints included feasibility, toxicity, and overall survival (OS). PD-L1 staining, multiplex immunofluorescence staining, RNA sequencing, reverse-phase protein array analyses were performed on pre- and post-chemotherapy samples. FINDINGS:Thirty-one eligible patients were enrolled. Median PFS and OS was 14.88 (95% CI 12.39-23.00) and 57.43 months (95% CI 30.88-not reached), respectively. Among those with PD-L1 combined positive score (CPS) ≥10, the median PFS and OS were not reached compared to those with CPS <10 (10.50 and 30.90 months, respectively). Feasibility was met, with all patients completing their planned adjuvant cycles. Treatment discontinuation due to immune-related toxicity occurred in 6 patients (20%). Chemotherapy resulted in an infiltration of anti-tumor immune cells in the tumor microenvironment. Samples of patients with the best PFS demonstrated increased expression of NF-κB, TGF-β, and β-catenin signaling. CONCLUSIONS:Pembrolizumab with chemotherapy was feasible and resulted in PFS within the historical range for this EOC population. Patients with CPS ≥10 may benefit more from this regimen, and future studies should investigate this potential biomarker. FUNDING:This investigator-initiated trial was funded by Merck.

Supplementary Figure 4. Neutrophil presence associated with exceptional response

BACKGROUND:Immune profiling has become an important tool for identifying predictive, prognostic and response biomarkers for immune checkpoint inhibitors from tumor microenvironment (TME). We aimed to build a multiplex immunofluorescence (mIF) panel to apply to formalin-fixed and paraffin-embedded tissues in mice tumors and to explore the programmed cell death protein 1/ programmed cell death 1 ligand 1 (PD-1/PD-L1) axis.RESULTS:An automated eight-color mIF panel was evaluated to study the TME using seven antibodies, including cytokeratin 19, CD3e, CD8a, CD4, PD-1, PD-L1, F4-80 and DAPI, then was applied in six mice lung adenocarcinoma samples. Cell phenotypes were quantified by software to explore the co-localization and spatial distribution between immune cells within the TME. This mice panel was successfully optimized and applied to a small cohort of mice lung adenocarcinoma cases. Image analysis showed a sparse degree of immune cell expression pattern in this cohort. From the spatial analysis we found that T cells and macrophages expressing PD-L1 were close to the malignant cells and other immune cells.CONCLUSIONS:Comprehensive immune profiling using mIF in translational studies improves our ability to correlate the PD-1/PD-L1 axis and spatial distribution of lymphocytes and macrophages in mouse lung cancer cells to provide new cues for immunotherapy, that can be translated to human tumors for cancer intervention.

Supplementary Table 5. Overall associations between cell phenotypes by compartment in both arms.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies 1 . Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8 + alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8 + cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

Supplementary Table 7. Comparation of median densities between patients who experienced exceptional responses and early progression/death according to compartment.

Introduction: Generating high levels of immunosuppressive adenosine in the tumor microenvironment contributes to cancer immune evasion. CD39 and CD73 hydrolyze adenosine triphosphate into adenosine; thus, efforts have been made to target this pathway for cancer immunotherapy. Our objective was optimizing a multiplex immunofluorescence (mIF) panel to explore the role of CD39 and CD73 within the tumor microenvironment.Materials and methods: In three-time points, a small cohort (n=8 ) of colorectal and pancreatic adenocarcinomas were automated staining using an mIF panel against CK, CD3, CD8, CD20, CD39, CD73 and CD68 to compare them with individual markers immunohistochemistry (IHC) for internal panel validation. Densities of immune cells and distances from different tumor-associated immune cells to tumor cells were exploratory assessment and compared with clinicopathologic variables and outcomes.Results: Comparing the three-time points and individual IHC staining results, we demonstrated high reproducibility of the mIF panel. CD39 and CD73 expression was low in malignant cells; the exploratory analysis showed higher densities of CD39 expression by various cells, predominantly stromal cells, followed by T cells, macrophages, and B cells. No expression of CD73 by B cells or macrophages was detected. Distance analysis revealed proximity of cytotoxic T cells, macrophages, and T cells expressing CD39 to malignant cells, suggesting a close regulatory signal driven by this adenosine marker.Conclusions: We optimized an mIF panel for detection of markers in the adenosine pathway, an emerging clinically relevant pathway. The densities and spatial distribution demonstrated that this pathway may modulate aspects of the tumor immune microenvironment.

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Supplementary Figure 5. Gene mutation profiles.

Background:Malignant pleural effusion (MPE),a frequent complication of advanced malignancies. This pilot study characterized and compared the immune landscapes of breast carcinoma (BC) and lung adenocarcinoma (LADC) primary tumors(PTs) and their corresponding MPEs and tested the incorporation of multiplexed image technology for the study of malignant fluids. Methods: We studied the immune contexture of 6 BC and 5 LADC PT samples and their MPEs using 3 multiplex immunofluorescence panels. We explored associations between sample characteristics and pleural effusion–free survival. Results: Although we found 3 out of 11 PTs PD-L1 positive more than 1% by malignant cells, no MPE samples reached positive expression by malignant cells. In addition, CD3+ T cells and CD3+CD8+ cytotoxic T cells predominated (median percentages for MPEs vs. PTs: 45.5% vs. 40.7% and 4.7% vs. 6.6%, respectively). In the BC samples, CD68+ macrophages predominated (median percentages for MPEs vs. PTs:61% vs. 57.1%). Generally, CD3+CD8+FOXP3+ T cells in PTs and the distances from the malignant cells to CD3+CD8+Ki67+ and CD3+PD-1+ T cells were correlated in the first event of MPE after diagnosis. Conclusions: The immune cell phenotypes in the MPEs and PTs were similar within each cancer type but were different for LADC vs. BC.MPE analysis could be used as a substitute for PT analysis, but an expanded study on this topic is essential.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin -embedded tissues is achieved when a specific fluorophore is annealed to an antibody -bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Supplementary material and method Supplementary Figure S1. Addition of a4-1BB does not lead CD8+ TIL into senescence. Supplementary Figure S2. Fold expansion of bulk and CD8+ PDAC TIL following the REP. Supplementary Figure S3. Level of MHC class I expression on PDAC tumor cell lines.

Supplementary figures and supplemental methods