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Supplementary Figure S2. The mpMRI-visible PCa tumors show marked differences between the malignant and non-malignant compartments. A, Box plots showing WD per mpMRI-invisible and -visible PCa tumors. P-values were calculated with the Mann-Whitney U test. B, Combination of WD plotted against the malignant cell density per mpMRI-visible and -invisible PCa tumors and patient relapse.

Supplementary Table S1. Clinicopathologic characteristics of each mpMRI-invisible and -visible PCa tumor.

Purpose:Multiparametric MRI (mpMRI) has become an indispensable radiographic tool in diagnosing prostate cancer. However, mpMRI fails to visualize approximately 15% of clinically significant prostate cancer (csPCa). The molecular, cellular, and spatial underpinnings of such radiographic heterogeneity in csPCa are unclear.Experimental Design:We examined tumor tissues from clinically matched patients with mpMRI-invisible and mpMRI-visible csPCa who underwent radical prostatectomy. Multiplex immunofluorescence single-cell spatial imaging and gene expression profiling were performed. Artificial intelligence–based analytic algorithms were developed to examine the tumor ecosystem and integrate with corresponding transcriptomics.Results:More complex and compact epithelial tumor architectures were found in mpMRI-visible than in mpMRI-invisible prostate cancer tumors. In contrast, similar stromal patterns were detected between mpMRI-invisible prostate cancer and normal prostate tissues. Furthermore, quantification of immune cell composition and tumor-immune interactions demonstrated a lack of immune cell infiltration in the malignant but not in the adjacent nonmalignant tissue compartments, irrespective of mpMRI visibility. No significant difference in immune profiles was detected between mpMRI-visible and mpMRI-invisible prostate cancer within our patient cohort, whereas expression profiling identified a 24-gene stromal signature enriched in mpMRI-invisible prostate cancer. Prostate cancer with strong stromal signature exhibited a favorable survival outcome within The Cancer Genome Atlas prostate cancer cohort. Notably, five recurrences in the 8 mpMRI-visible patients with csPCa and no recurrence in the 8 clinically matched patients with mpMRI-invisible csPCa occurred during the 5-year follow-up post-prostatectomy.Conclusions:Our study identified distinct molecular, cellular, and structural characteristics associated with mpMRI-visible csPCa, whereas mpMRI-invisible tumors were similar to normal prostate tissue, likely contributing to mpMRI invisibility.

Supplementary Data file with Methods and each Supplementary figure/table linked to legend

Severe and prolonged lymphopenia frequently occurs in patients with glioblastoma after standard chemoradiotherapy and has been associated with worse survival, but its underlying biological mechanism is not well understood. To address this, we performed a correlative study in which we collected and analyzed peripheral blood of patients with glioblastoma ( n = 20) receiving chemoradiotherapy using genomic and immune monitoring technologies. RNA sequencing analysis of the peripheral blood mononuclear cells (PBMC) showed an elevated concentration of myeloid-derived suppressor cell (MDSC) regulatory genes in patients with lymphopenia when compared with patients without lymphopenia after chemoradiotherapy. Additional analysis including flow cytometry and single-cell RNA sequencing further confirmed increased numbers of circulating MDSC in patients with lymphopenia when compared with patients without lymphopenia after chemoradiotherapy. Preclinical murine models were also established and demonstrated a causal relationship between radiation-induced MDSC and systemic lymphopenia using transfusion and depletion experiments. Pharmacological inhibition of MDSC using an arginase-1 inhibitor (CB1158) or phosphodiesterase-5 inhibitor (tadalafil) during radiation therapy (RT) successfully abrogated radiation-induced lymphopenia and improved survival in the preclinical models. CB1158 and tadalafil are promising drugs in reducing radiation-induced lymphopenia in patients with glioblastoma. These results demonstrate the promise of using these classes of drugs to reduce treatment-related lymphopenia and immunosuppression.

SUMMARYReference atlases, molecular and spatial maps of mammalian tissues, are critical resources for discovery efforts and translational research. Their utility is dependent on operationalizing the resulting data by identifying cell types, histological patterns, and predictive biomarkers underlying health and disease. The human lymph node (LN) offers a compelling use case because of its importance in immunity, structural and cellular diversity, and neoplastic involvement. One hematological malignancy, follicular lymphoma (FL), evolves from developmentally blocked germinal center B cells residing in and trafficking through these tissues. To promote survival and immune escape, tumor B cells undergo significant genetic changes and extensively remodel the lymphoid microenvironment. Here, we present an integrated portrait of healthy and FL LNs using multiple genomic and advanced imaging technologies. By leveraging the strengths of each platform, we identified several tumor-specific features and microenvironmental patterns enriched in individuals who experience early relapse, the most high-risk of FL patients.

Recent advances in immunotherapy demonstrate the need to further understand the characteristics of an individual cancer patient’s immune system and how it influences responses to cancer treatment. Here, we developed an immunoprofiling platform to evaluate the features in the blood of cancer patients to test the hypothesis that peripheral immune cell heterogeneity could be used to stratify these patients into different categories or immunotypes to monitor disease progression and treatment response. To that end, we established a unique diagnostic immunoprofiling assay and analytical framework based on the analysis of leukocytes in the peripheral blood using multiparameter flow cytometry. Supervised manual gating of flow cytometry data from a cohort of 50 healthy donors identified 415 cell types and immune activation states that were used to train and later independently validate machine learning models to automatically identify immune cell subsets from raw cytometry data. By applying this tool to peripheral blood samples from a mixed cohort of 299 healthy donors and 323 cancer patients, we developed a machine-learning classification model that can differentiate between these two groups with 93% accuracy. This model was further refined using spectral clustering with bootstrapping, revealing 5 clusters, or immunotypes, characterized by specific physiological immune profiles: (1) Myeloid-derived suppressor/NK cell, (2) Terminally-differentiated CD8+ T cells, (3) Mixed CD4+ T helper cells, (4) CD4+ Th1 & CD8+ T cell memory, and (5) Naive T and B lymphocytes. Interestingly, very few healthy donors could be found in clusters 1 and 2 but were assigned most frequently to cluster 5. Matched RNA-seq was used to further validate these profiles using the cellular deconvolution algorithm, Kassandra, and differential gene expression analysis revealed immunotype-specific signatures that are consistent with immune response potential. Patients in the terminally-differentiated CD8+ T cell cluster had a narrower range of HLA-types than the other clusters, and TCR repertoire analysis indicated significantly increased clonality and reduced clonotype diversity. Within this cluster there was a high degree of overlap between TCR sequences in the peripheral blood and the tumor, indicating a relationship between peripheral blood immunotype and tumor infiltration. Altogether, the establishment of these immunotypes using peripheral blood immunoprofiling represents a promising signature that can be used to identify and stratify cancer patients that will benefit from immune-based therapies. Citation Format: Daniiar Dyikanov, Iris Wang, Tatiana Vasileva, Polina Shpudeiko, Polina Turova, Arseniy A. Sokolov, Olga Golubeva, Evgenii Tikhonov, Anna Kamysheva, Ilya Krauz, Mary Abdou, Madison Chasse, Tori Conroy, Nicholas R. Merriam, Boris Shpak, Anastasia Radko, Anastasiia Kilina, Lira Nigmatullina, Linda Balabanian, Christopher J. Davitt, Alexander A. Ryabykh, Olga Kudryashova, Cagdas Tazearslan, Ravshan Ataullakhanov, Alexander Bagaev, Aleksandr Zaitsev, Nathan Fowler, Michael F. Goldberg. Comprehensive immunoprofiling of peripheral blood reveals five conserved immunotypes with implications for immunotherapy in cancer patients [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 6664.

Personalized cancer vaccines designed to target neoantigens represent a promising new treatment paradigm in oncology. In contrast to classical idiotype vaccines, we hypothesized that ‘polyvalent’ vaccines could be engineered for the personalized treatment of follicular lymphoma (FL) using neoantigen discovery by combined whole exome sequencing (WES) and RNA sequencing (RNA-Seq). Fifty-eight tumor samples from 57 patients with FL underwent WES and RNA-Seq. Somatic and B-cell clonotype neoantigens were predicted and filtered to identify high-quality neoantigens. B-cell clonality was determined by alignment of B-cell receptor (BCR) CDR3 regions from RNA-Seq data, grouping at the protein level, and comparison to the BCR repertoire of RNA-Seq data from healthy individuals. An average of 52 somatic mutations per patient (range: 2-172) were identified, and two or more (median: 15) high-quality neoantigens were predicted for 56 of 58 samples. The predicted neoantigen peptides were composed of missense mutations (76%), indels (9%), gene fusions (3%), and BCR sequences (11%). Building off of these preclinical analyses, we initiated a pilot clinical trial using personalized neoantigen vaccination combined with PD-1 blockade in patients with relapsed or refractory FL (#NCT03121677). Synthetic long peptide (SLP) vaccines were successfully synthesized for and administered to all four patients enrolled to date. Initial results demonstrate feasibility, safety, and potential immunologic and clinical responses. Our study suggests that a genomics-driven personalized cancer vaccine strategy is feasible for patients with FL, and this may overcome prior challenges in the field.

Human Ezrin Peptides (HEPs) are inhibitors of expression of IL-6 and other inflammatory cytokines, amplifiers of adaptive B cell and T cell immunity and enhancers of tissue repair. The mutation stable C-terminus of HIV gp120, mimics 69% of the “Hep-receptor”, a zipped α-helical structure in the middle of the α domain of human ezrin protein. Synthetic peptides homologous to the Hep-receptor of ezrin of five to fourteen amino acids, activate anti-viral immunity against a wide range of viruses (HIV, HCV, herpes, HPV, influenza and other human respiratory viruses). Human Ezrin Peptide One (HEP1) TEKKRRETVEREKE (brand name Gepon, registered for human use in Russia from 2001) is a successful treatment for opportunistic infections in HIV-infected patients. That treats HEP1and prevents mucosal candidiasis, herpes zoster outbreaks and infection-induced chronic diarrhea. There are clinical publications in Russian on the successful treatments of chronic recurrent vaginal candidiasis, acute and chronic enterocolitis and dysbacteriosis, which are accompanied by normalization of the mucosal microbiome, and the decline or disappearance of inflammation. HEP1 is also an effective treatment and prevention for recurrent inflammation and ulceration in the stomach, duodenum and colon. HEP1 and RepG3 GEKKRRETVEREGG (a derivative of HEP1) have been used successfully as an inhaled spray peptide solution to treat a small number of human volunteers with mild-to-moderate COVID, resulting from SARS-CoV-2 infection, based on earlier successes in treating acute viral respiratory disease with inflammatory complications. Ezrin peptides seem to correct a dysregulation of innate immune responses to SARS-CoV-2. They are also adjuvants of B cell adaptive immunity and increase antibody titres, resulting in protection from lethal virus infection of mice. In a clinical study in Moscow, orally administered HEP1 was shown to enhance antibody-titres produced in response to hepatitis-B vaccination. These very preliminary but promising results with ezrin peptide treatment of COVID must be replicated in large-scale randomised placebo controlled clinical studies, to be verified.

Intratumor heterogeneity (ITH) represents a major challenge for anticancer therapies. An integrated, multidimensional, multiregional approach dissecting ITH of the clear cell renal cell carcinoma (ccRCC) tumor microenvironment (TME) is employed at the single-cell level with mass cytometry (CyTOF), multiplex immunofluorescence (MxIF), and single-nucleus RNA sequencing (snRNA-seq) and at the bulk level with whole-exome sequencing (WES), RNA-seq, and methylation profiling. Multiregional analyses reveal unexpected conservation of immune composition within each individual patient, with profound differences among patients, presenting patient-specific tumor immune microenvironment signatures despite underlying genetic heterogeneity from clonal evolution. Spatial proteogenomic TME analysis using MxIF identifies 14 distinct cellular neighborhoods and, conversely, demonstrated architectural heterogeneity among different tumor regions. Tumor-expressed cytokines are identified as key determinants of the TME and correlate with clinical outcome. Overall, this work signifies that spatial ITH occurs in ccRCC, which may drive clinical heterogeneity and warrants further interrogation to improve patient outcomes.

Cellular deconvolution algorithms virtually reconstruct tissue composition by analyzing the gene expression of complex tissues. We present the decision tree machine learning algorithm, Kassandra, trained on a broad collection of9,400 tissue and blood sorted cell RNA profiles incorporated into millions of artificial transcriptomes to accurately reconstruct the tumor microenvironment (TME). Bioinformatics correction for technical and biological variability, aberrant cancer cell expression inclusion, and accurate quantification and normalization of transcript expression increased Kassandra stability and robustness. Performance was validated on 4,000 HE slides and 1,000 tissues by comparison with cytometric, immunohistochemical, or single-cell RNA-seq measurements. Kassandra accurately deconvolved TME elements, showing the role of these populations in tumor pathogenesis and other biological processes. Digital TME reconstruction revealed that the presence of PD-1-positive CD8

Despite the breast cancer microenvironment being recognized as a critical participant in tumor progression and therapeutic response, current technologies for tumor cell-to-cell interaction analyses are limited to qualitative and descriptive histological methods. Here, we utilized multiplex immunofluorescence (MxIF), RNA sequencing (RNA-seq), and microarray gene expression to quantitatively assess, using the BostonGene automated pipeline, the immune microenvironment composition of clinical stage 2 or 3 primary estrogen receptor-positive (ER+) HER2-negative (HER-) breast tumors (n = 29) from patients treated with neoadjuvant palbociclib in combination with anastrozole. Microenvironment characteristics of pretreatment tumor biopsies were associated with molecular subtype, cytokine signatures, and Ki67 response. MxIF analysis (21 markers, 2-13 ROIs per slide) of the tumor immune composition revealed that while the cellular composition was similar for each tumor region of the same patient, significant cellular heterogeneity was observed in tumor biopsies from different patients. The spatial heterogeneity was found in the distribution of both immune infiltrate cells and stromal components. T cells, CD68+ and CD163+ macrophages, and B cells were the predominant cell populations in the stromal component. Further MxIF analysis showed an increase in Ki67+ tumor cells in nonresponders (NR) versus responders (R). The number of immune cells was similar between Luminal A and B breast cancer molecular subtypes. A lower number of Ki67+ tumor cells was found in Luminal A compared to Luminal B tumors, as expected. MxIF analysis of cell-to-cell interactions revealed that distinct groups of cellular neighborhoods were significantly increased in NR compared with R: the immune inflammation cluster with a high density of CD4 T cells, Tregs, and CD8 T cells in the stromal component (p = 0.03); CD163+ macrophage-enriched cluster (p < 0.001); and tertiary lymphoid structures cluster (p < 0.001). Transcriptomic analysis showed that while CXCL1, CCL17, CCL13, CXCL9, and CCL5 expression correlated with the high density of CD4 and CD8 T cells in the stromal component cluster, VEGFA, IL1B, CXCL16, CCL3, and CCL7 were increased in the CD163+ and CD68+ macrophage-enriched clusters, indicating that differential cytokine expression is associated with the tumor architecture.Comprehensively characterizing, via MxIF, the immune microenvironment of ER+ HER2- breast cancer provided resolution of cellular architecture and elucidated spatial relationships among the immune and stromal cells of the tumor. Quantitatively assessing breast tumor cell-to-cell interactions has the potential to identify imaging markers of response, ultimately leading to the development of effective therapy options. Citation Format: Maria Bruttan, Souzan Sanati, Anna Belozerova, Ilia Galkin, Akshaya Ramachandran, Pavel Ovcharov, Grigory Perelman, Viktor Svekolkin, Ekaterina Postovalova, Alexander Bagaev, Krystle Nomie, Shana Thomas, Jeremy Hoog, Ravshan Ataullakhanov, James Hsieh, Cynthia Ma. Tumor microenvironment heterogeneity identifies potential biomarkers of response in ER-positive breast cancers treated with palbociclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6151.

Checkpoint immunotherapy (CPI) and its combination with tyrosine kinase inhibitor (TKI) therapy are widely used in the treatment of many cancers, including clear cell renal cell carcinoma (ccRCC). Although recent advances in these therapies demonstrate effectiveness, a subset of patients are unresponsive. In this study, using deep immune profiling by mass cytometry (CyTOF), we analyzed peripheral blood mononuclear cell (PBMC) samples from 86 ccRCC patients pre- and post-treatment. The cohort included patients treated with first-line CPI (n = 43), TKI (n = 15) and combinatorial CPI+TKI (n = 28), with PBMC samples collected pre- (n = 41) and post- (n = 69) therapy (n = 24 patients total with paired samples). Samples were characterized by CyTOF analysis of 30 different cell surface markers. Using t-distributed stochastic neighbor embedding (tSNE) and Phenograph clustering, the presence of immune populations and subpopulations were characterized and associated with therapeutic response as well as longitudinally tracked. While most cell types did not differ between pre- and post-treatment states, a decrease in CD8 and CD4 PD1+ T cell subsets was observed in patients treated with CPI and CPI+TKI (p < 0.001). Also, increased CD8 effector memory T cells were observed in post-treatment samples in those treated with CPI or CPI+TKI (p < 0.001).Subsequent analysis of pre-treatment samples in the context of clinical outcome revealed specific features associated with favorable response to immunotherapy. Responders (R) in comparison with non-responders (NR) were characterized by an increased number of naive CD4 T cells (p = 0.03), CD8 T cells (p = 0.038), immature NK cells (p = 0.005) and lower percentage of granulocytes (p = 0.012). Using unsupervised hierarchical clustering, 8 patient groups were identified with a unique immune profile enriched by a certain PBMC population. Three groups (G1-G3), which included a high ratio of R, were enriched by naive T cells, immature NK cells and non-classical monocytes, respectively. The G4 group, enriched with B cells, consisted of R and NR in equal numbers. The G5-G8 groups, enriched with granulocytes, classical monocytes, mature NK-cells and effector CD8 T cells, respectively, were primarily comprised of NR. Patients with favorable pre-treatment immune profiles also belonged to favorable groups post-treatment, with a similar trend observed with unfavorable profiles. In conclusion, this analysis revealed a remarkable association between the initial immune status or “portrait” of the patient before therapy and subsequent therapeutic outcome as well as the development of therapeutic resistance. Citation Format: Svetlana Shishkova, Dmitry Tabakov, Evgeny Egorov, Akshaya Ramachandran, Yang Lyu, Krystle Nomie, Jessica Brown, Vladimir Zyrin, Alexander Zaytcev, Natalia Miheecheva, Ekaterina Postovalova, Alexander Bagaev, Ravshan Ataullakhanov, James Hsieh. Deep immune profiling by mass cytometry revealed an association between the state of immune system before treatment and response to checkpoint inhibitor therapy in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2061.

ELISPOT allows detection of single IFN-γ-secreting cell, but the use of unseparated spleen cells removes the advantages of the method due to a high back ground of IFN-γ-production and uncertainty of the phenotype of secreting cells (CD4, CD8 or NK). We used high purity sorted CD4 and CD8 cells. For in vitro restimulation of CD8 T-cells, we used dendritic cells that present antigenic epitopes of M2, NP or of adenovirus in the context of MHC class I. In this instance, dendritic cells were transduced with Ad5-tet-M2, Ad5-tet-NP or Ad5-null, respectively. For restimulation of CD4 T-cells in vitro, we used dendritic cells that present antigenicM2 or NP epitopes in the context of MHC class II. For this purpose, dendritic cells were additionally activated by lipopolysaccharide E. coli (1 μg/ml) and were loaded with recombinant protein NP or M2e peptide..

Background: Immunomax® is a novel immunostimulant manufactured from potato sprouts. It consists of acidic peptidoglycans with a molecular mass of 1,000–40,000 kDa. In order to demonstrate its therapeutic effectiveness and safety, we here present data on the treatment of papilloma virus infections, and two pilot studies on prostatitis and prostate carcinoma treatment. Methods: Immunomax® was investigated using in vitro murine macrophage activation studies and by efficacy studies in human patients suffering from either recurrent anogenital warts caused by papilloma virus, or from chronic prostatitis type IIIa, as determined by the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI), or from prostate carcinoma. Results: Immunomax® stimulated nitric oxide (NO) production in murine bone marrow derived macrophages. In human patients, Immunomax® was effective for treating recurrent anogenital warts caused by papilloma virus. Immunomax® therapy also showed, in two pilot studies, a ...

The therapeutic agent OM-89 (Uro-Vaxom) contains lyophilized immunostimulating fractions from 18 Escherichia coil strains. It has been shown to provide protection against recurrent urinary tract infections in humans and against bacterial infections in mice. Here the immunostimulatory properties of OM-89 were investigated by in vitro and in vivo assays. In vitro the activation of murine spleen cells by the AlamarBlue assay was determined. OM-89 was effective in stimulating the metabolism of spleen cells within a concentration range of 0.625-2.5 mg/ml. The activation of murine bone marrow-derived macrophages by OM-89 was shown by the induction of NO production; OM-89 was a most effective stimulant at concentrations around 6 mg/ml. In the human system, the effect of OM-89 was tested in vitro:metabolic activity of peripheral blood lymphocytes (PBL) was stimulated starting at concentrations of approx. 250 microg/ml, and the spontaneous apoptosis of polymorphonuclear neutrophils (PMN) was reduced starting at OM-89 concentrations of approx. 100 microg/ml. Finally, in a mouse model, the in vivo protection of mice against infection with Salmonella typhimurium after the oral administration of OM-89 was tested (100 mg in a volume of 0.5 ml once a day for 10 consecutive days). The extract proved to be effective: 90% of the OM-89-treated animals survived compared to 58% of the untreated control group.

This report shows the therapeutic benefit of HEP1 (human ezrin peptide 324–337; TEKKRRETVEREKE) monotherapy of hepatitis C virus (HCV) infection in HIV infected patients in two clinical studies. In the Pilot Study I, 16 of 18 patients responded well to the treatment with significant reductions of HCV viral load and a normalization of serum liver enzymes. In 8 of 18 patients, HCV RNA became undetectable, and 3 of 8 interferon/ribavirin treatment failure patients showed undetectable HCV load following HEP1 treatment. In the second study, 8 of 10 patients responded well to the treatment with a pronounced reduction of the HCV viral load and a normalization of serum liver enzymes. Three of 15 patients (20 %) showed an undetectable viral load 30 days after the end of a 30-day course of HEP1 treatment. In both studies, all genotypes of HCV were sensitive to HEP1 treatment. Analysis of the combined data from both studies showed the overall efficacy of HEP1 therapy: in 37 HCV+HIV patients, HEP1 therapy gave the following results: 10 of 37 (27 %) HCV+HIV patients showed a reduction of viral load between –7 log (–10,000,000x) and –3 log (–1,000x); 4 of 37 (11 %) a reduction of –3 log (–1,000x); 6 of 37 (16 %) a reduction of –2 log (–100x); 11 of 37 (30 %) a reduction of –1 log (–10x); 6 of 37 (16 %) a reduction of less than –1 log (–10x); 0 of 37 (0 %) had an increase in viral load, and the average reduction in viral load for all 37 patients was –2 log (–100x). No adverse reactions or side effects were detected and the improving CD4/CD8 ratio showed that the therapy had no negative impact on the immunological status. Thus, oral HEP1 therapy matches the efficacy results for injectable peginterferon/oral ribavirin therapy with the advantages of more rapid action and less side effects. HEP1 therapy should be used in patients where either peginterferon/ribavirin therapy fails or is contraindicated.