T-cell Density Research Articles

Association between pathogenic germline mutations in BRCA2 and ATM and tumor-infiltrating lymphocytes in primary prostate cancer.

Pathogenic mutations in homologous recombination (HR) DNA repair genes may be associated with increased tumor mutational burden and numbers of tumor-infiltrating lymphocytes (TIL). Though HR-deficient prostate tumors have been anecdotally associated with improved responses to immunotherapy, it is unclear whether HR mutations or HR deficiency (HRD) scores predict for increased T-cell densities in this cancer. We evaluated 17 primary prostate tumors from patients with pathogenic germline BRCA2 mutations (gBRCA2) and 21 primary prostate tumors from patients with pathogenic germline ATM (gATM) mutations, which were compared to 19 control tumors lacking HR gene mutations, as well as the TCGA prostate cancer cohort. HRD score was estimated by targeted sequencing (gBRCA2 and gATM) or by SNP microarray (TCGA). Tumor-associated T-cell densities were assessed using validated automated digital image analysis of CD8 and FOXP3 immunostaining (gBRCA2 or gATM) or by methylCIBERSORT (TCGA). CD8 + and FOXP3 + T-cell densities were significantly correlated with each other in gBRCA2 and gATM cases. There was no significant difference between CD8 + or FOXP3 + TIL densities in gBRCA2 or gATM cases compared to controls. In the TCGA cohort, HRD score was associated with predicted CD8 + and FOXP3 + TILs. Associations were also seen for HRD score and TIL density among the germline-mutated cases. In contrast to mismatch repair-deficient primary prostate tumors, cancers from germline BRCA2 or ATM mutation carriers do not appear to be associated with elevated TIL density. However, measures of genomic scarring, such as HRD score, may be associated with increased tumor-infiltrating T-cells.

Prognostic Value of CD45Ro+ T-Cell Expression in Patients With Oral Squamous Cell Carcinoma.

The role of tumour-infiltrating CD45Ro+ T-cells in oral squamous cell carcinoma (OSCC) is unclear. This study aimed to evaluate prognostic biomarkers for OSCC. We determined the density of tumour-infiltrating CD45Ro+ T cells in the parenchyma and stroma at the tumour centre (TCe) and invasive front (IF) and examined the association between the density of these cells and histopathological status in 142 patients. Five-year overall survival (OS) and recurrence-free survival were favourable in patients with high CD45Ro+ T-cell density in the TCe stroma. OS was favourable in patients with high CD45Ro+ T-cell density in the IF stroma. Stepwise Cox regression model analysis indicated that CD45Ro+ T-cells in the stroma of the IF and TCe were an independent prognostic factor for OS. CD45Ro+ T-cells in the stroma of the IF and TCe play a role in cancer immune surveillance and may be a useful prognostic factor.

Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially dependent on IL‐8

Neutrophil extracellular traps (NETs) are webs of extracellular nuclear DNA extruded by dying neutrophils infiltrating tissue. NETs constitute a defence mechanism to entrap and kill fungi and bacteria. Tumours induce the formation of NETs to the advantage of the malignancy via a variety of mechanisms shown in mouse models. Here, we investigated the presence of NETs in a variety of human solid tumours and their association with IL-8 (CXCL8) protein expression and CD8+ T-cell density in the tumour microenvironment. Multiplex immunofluorescence panels were developed to identify NETs in human cancer tissues by co-staining with the granulocyte marker CD15, the neutrophil marker myeloperoxidase and citrullinated histone H3 (H3Cit), as well as IL-8 protein and CD8+ T cells. Three ELISA methods to detect and quantify circulating NETs in serum were optimised and utilised. Whole tumour sections and tissue microarrays from patients with non-small cell lung cancer (NSCLC; n= 14), bladder cancer (n= 14), melanoma (n= 11), breast cancer (n= 31), colorectal cancer (n= 20) and mesothelioma (n= 61) were studied. Also, serum samples collected retrospectively from patients with metastatic melanoma (n= 12) and NSCLC (n= 34) were ELISA assayed to quantify circulating NETs and IL-8. NETs were detected in six different human cancer types with wide individual variation in terms of tissue density and distribution. At least in NSCLC, bladder cancer and metastatic melanoma, NET density positively correlated with IL-8 protein expression and inversely correlated with CD8+ T-cell densities. In a series of serum samples from melanoma and NSCLC patients, a positive correlation between circulating NETs and IL-8 was found. In conclusion, NETs are detectable in formalin-fixed human biopsy samples from solid tumours and in the circulation of cancer patients with a considerable degree of individual variation. NETs show a positive association with IL-8 and a trend towards a negative association with CD8+ tumour-infiltrating lymphocytes. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Distinct Molecular Mechanisms of Altered HLA Class II Expression in Malignant Melanoma.

Simple SummaryThere exists limited knowledge about the underlying molecular processes controlling the expression of HLA class II APM components and their prognostic significance in melanoma. Therefore, this study analyzed the basal and regulated expression of HLA class II antigens and components in melanoma cell lines and patients’ lesions in conjunction to T-cell infiltration. The heterogeneous constitutive HLA class II APM expression was caused by distinct molecular mechanisms and was partially linked to immune cell infiltration and clinical parameters. These results contribute not only to a better understanding of the regulation of HLA class II expression in melanoma, but might have an impact on the design of novel (immuno)therapies for the treatment of this disease.Background: The human leukocyte antigen (HLA) class II molecules are constitutively expressed in some melanoma, but the underlying molecular mechanisms have not yet been characterized. Methods: The expression of HLA class II antigen processing machinery (APM) components was determined in melanoma samples by qPCR, Western blot, flow cytometry and immunohistochemistry. Immunohistochemical and TCGA datasets were used for correlation of HLA class II expression to tumor grading, T-cell infiltration and patients’ survival. Results: The heterogeneous HLA class II expression in melanoma samples allowed us to characterize four distinct phenotypes. Phenotype I totally lacks constitutive HLA class II surface expression, which is inducible by interferon-gamma (IFN-γ); phenotype II expresses low basal surface HLA class II that is further upregulated by IFN-γ; phenotype III lacks constitutive and IFN-γ controlled HLA class II expression, but could be induced by epigenetic drugs; and in phenotype IV, lack of HLA class II expression is not recovered by any drug tested. High levels of HLA class II APM component expression were associated with an increased intra-tumoral CD4+ T-cell density and increased patients’ survival. Conclusions: The heterogeneous basal expression of HLA class II antigens and/or APM components in melanoma cells is caused by distinct molecular mechanisms and has clinical relevance.

Mature tertiary lymphoid structures predict immune checkpoint inhibitor efficacy in solid tumors independently of PD-L1 expression.

Only a minority of patients derive long-term clinical benefit from anti-PD1/PD-L1 monoclonal antibodies. The presence of tertiary lymphoid structures (TLS) has been associated with improved survival in several tumor types. Here, using a large-scale retrospective analysis of three independent cohorts of cancer patients treated with anti-PD1/PD-L1 antibodies, we showed that the presence of mature TLS was associated with improved objective response rate, progression-free survival, and overall survival independently of PD-L1 expression status and CD8+ T-cell density. These results pave the way for using TLS detection to select patients who are more likely to benefit from immune checkpoint blockade.

Collagen Organization Does Not Influence T-Cell Distribution in Stroma of Human Pancreatic Cancer.

Simple SummaryThe excessive desmoplasia is the hallmark of human pancreatic cancer that influences the local T-cell-based immune response. In the present work, the stromal collagen organization in normal and malignant pancreatic tissues as well as its relationsship to T-cell distribution in pancreatic cancer were studied. It was found that differences in collagen organization do not change the spatial orientation of T-cell migration and do not influence the availability of tumor cells for T-cells. The results of the study do not support the concept of use of stroma collagen organization for improvement of spatial T-cell distribution in the tumor. The dominant intrastromal T-cell infiltration in pancreatic cancer is mainly caused by the contact guidance through the excessive desmoplastic reaction and could represent one of the obstacles to an effective immune response in this tumor type. This study analyzed the collagen organization in normal and malignant pancreatic tissues as well as its influence on T-cell distribution in pancreatic cancer. Human pancreatic tissue was analyzed using immunofluorescence staining and multiphoton and SHG microscopy supported by multistep image processing. The influence of collagen alignment on activated T-cells was studied using 3D matrices and time-lapse microscopy. It was found that the stroma of malignant and normal pancreatic tissues was characterized by complex individual organization. T-cells were heterogeneously distributed in pancreatic cancer and there was no relationship between T-cell distribution and collagen organization. There was a difference in the angular orientation of collagen alignment in the peritumoral and tumor-cell-distant stroma regions in the pancreatic ductal adenocarcinoma tissue, but there was no correlation in the T-cell densities between these regions. The grade of collagen alignment did not influence the directionality of T-cell migration in the 3D collagen matrix. It can be concluded that differences in collagen organization do not change the spatial orientation of T-cell migration or influence stromal T-cell distribution in human pancreatic cancer. The results of the present study do not support the rationale of remodeling of stroma collagen organization for improvement of T-cell–tumor cell contact in pancreatic ductal adenocarcinoma.

Relationship between PD-L1 expression, CD8+ T-cell infiltration and prognosis in intrahepatic cholangiocarcinoma patients

BackgroundProgrammed death- ligand 1 (PD-L1) seems to be associated with the immune escape of tumors, and immunotherapy may be a favorable treatment for PD-L1-positive patients. We evaluated intrahepatic cholangiocarcinoma (ICC) specimens for their expression of PD-L1, infiltration of CD8+ T cells, and the relationship between these factors and patient survival.MethodsIn total, 69 resections of ICC were stained by immunohistochemistry for PD-L1, programmed death factor-1 (PD-1), and CD8+ T cells. CD8+ T-cell densities were analyzed both within tumors and at the tumor-stromal interface. Patient survival was predicted based on the PD-L1 status and CD8+ T-cell density.ResultsThe expression rate of PD-L1 was 12% in cancer cells and 51% in interstitial cells. The expression rate of PD-1 was 30%, and the number of CD8+ T-cells increased with the increase of PD-L1 expression (p < 0.05). The expression of PD-L1 in the tumor was correlated with poor overall survival(OS) (p = 0.004), and the number of tumor and interstitial CD8+ T-cells was correlated with poor OS and disease-free survival (DFS) (All p < 0.001).ConclusionsThe expression of PD-L1 in the tumor is related to poor OS, and the number of tumor or interstitial CD8+ T-cells is related to poor OS and DFS. For patients who lose their chance of surgery, PD-L1 immunosuppressive therapy may be the focus of future research as a potential treatment.

Abstract 2733: Smoking and colorectal cancer Iincidence by tumor microenvironment in cancer tissue

Abstract Background: Biological evidence indicates that smoking influences immune responses through interacting with various immune cell populations. We therefore hypothesized that the association of smoking with colorectal cancer (CRC) incidence differs by T-cell, tumor-associated macrophage (TAM), and myeloid cell densities. Methods: We developed multiplexed immunofluorescence assays to identify immune cells, including T-cell subsets (CD3, CD4, CD8, CD45RO, and FOXP3), TAMs (overall, M1-like, M2-like), and myeloid cell subsets (CD14, CD15, HLA-DR, ARG) in tumor intraepithelial and stromal areas. Utilizing the Nurses' Health Study and the Health Professionals Follow-up Study, we examined the association of smoking with incidence of CRC subtypes classified by each immune cell subset. We applied inverse probability weighted Cox proportional hazards regression models to control for selection bias and potential confounders. Results: During follow-up of 131,144 participants (3,648,371 person-years), we documented 3,203 incident CRC cases including 871 CRC cases with available data on immune cell densities in tumor tissue. The association of pack-years smoked with colorectal cancer incidence was stronger for tumors with lower CD3+CD8+ T-cell densities (Pheterogeneity=0.074) and lower overall TAM densities (Pheterogeneity=0.004) in tumor stromal areas. Further, the association of pack-years smoked with colorectal cancer incidence was stronger for tumors with higher CD14+HLA-DR− immature monocytic myeloid cell densities (Pheterogeneity=0.001) in tumor intraepithelial areas. Conclusions: The association of smoking with CRC incidence differed by stromal CD3+CD8+ T-cell densities, stromal TAM densities, and intraepithelial CD14+HLA-DR− myeloid cell densities. Our findings suggest a complex interplay of T-cells, TAMs, and myeloid cells in smoking-related colorectal carcinogenesis. Cumulative Pack-years Smoked and Colorectal Cancer Incidence, Overall and by Immune Cell DensityCumulative Pack-years Smoked01-1920-39≥40P for trendP for heterogeneityAll colorectal cancer (N=871)361209149152--Multivariable HR (95% CI)1 (referent)1.06 (0.93-1.22)1.08 (0.92-1.25)1.25 (1.07-1.46)0.007-Stromal CD3+CD8+ cell density------Low (N=310)124685761--Multivariable HR (95% CI)1 (referent)1.07 (0.79-1.45)1.25 (0.91-1.73)1.46 (1.07-2.00)0.0120.074Intermediate (N=280)111764449--Multivariable HR (95% CI)1 (referent)1.24 (0.92-1.66)1.04 (0.73-1.47)1.38 (0.98-1.94)0.15-High (N=281)126664643--Multivariable HR (95% CI)1 (referent)0.91 (0.68-1.23)0.89 (0.64-1.26)0.98 (0.70-1.39)0.85-Stromal Macrophage density------Low (N=288)106715061--Multivariable HR (95% CI)1 (referent)1.31 (0.97-1.78)1.30 (0.92-1.84)1.70 (1.23-2.35)0.0030.004Intermediate (N=294)125605356--Multivariable HR (95% CI)1 (referent)0.82 (0.60-1.13)1.03 (0.75-1.43)1.36 (0.99-1.86)0.045-High (N=289)130784635--Multivariable HR (95% CI)1 (referent)1.12 (0.84-1.48)0.95 (0.67-1.33)0.80 (0.55-1.17)0.20-Intraepithelial CD14+HLA-DR− cell density-----Low (N=290)130763945--Multivariable HR (95% CI)1 (referent)1.08 (0.82-1.44)0.77 (0.54-1.11)0.99 (0.70-1.40)0.510.001Intermediate (N=285)120724350--Multivariable HR (95% CI)1 (referent)1.04 (0.77-1.40)0.89 (0.63-1.27)1.26 (0.90-1.77)0.36-High (N=279)98596656--Multivariable HR (95% CI)1 (referent)1.18 (0.85-1.62)1.85 (1.34-2.55)1.75 (1.26-2.44)&amp;lt;0.001- Citation Format: Tomotaka Ugai, Juha P. Väyrynen, Koichiro Haruki, Naohiko Akimoto, Mai Chan Lau, Rong Zhong, Sara A. Väyrynen, Melissa Zhao, Andressa Dias Costa, Jennifer Borowsky, Jennifer L. Guerriero, Charles S. Fuchs, Xuehong Zhang, Molin Wang, Marios Giannakis, Jeffrey A. Meyerhardt, Jonathan A. Nowak, Shuji Ogino. Smoking and colorectal cancer Iincidence by tumor microenvironment in cancer tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2733.

Abstract SY17-01: Analysis of immune contexture to refine prognosis in patients with colon cancer: Implications for immunotherapy

Abstract Current classification of colorectal cancer (CRC) is based on TNM staging which does not account for considerable within stage variability in clinical outcome. Currently, there is no immune-based classification of colon cancer although data suggest that intratumoral T-cell densities can provide prognostic information that is more robust than that has been provided by genomic or transcriptomic profiling. A subset of CRCs show microsatellite instability (MSI) that is a predictive biomarker for immune checkpoint inhibitors whose indication in CRC is limited to metastatic tumors. In a prior study, we compared the density and localization of CD3+ and cytotoxic CD8+ T-cell densities in non-metastatic colon cancers with deficient (dMMR) vs proficient (pMMR) DNA mismatch repair (MMR). CD3+ at the tumor invasive margin (IM) was most strongly associated with patient overall survival, and dichotomization of CD3+ prognostically stratified both dMMR and pMMR tumors. The ability to stratify dMMR tumors was a novel observation and revealed a poorer prognosis dMMR group. By multivariable (MV) analysis, CD3+ density and N (nodal) stage contributed the most to patient survival. In separate studies, expression of FOXP3+ Tregs in CRC has paradoxically been associated with more favorable survival. Interestingly, we found that colon cancers with low FOXP3+ and low CD8+ are a very poor prognostic subset. Immunoscore (IS; HalioDx) is a validated assay that examines CD3+ and CD8+ using digital pathology with conversion of densities into percentile scores using a software algorithm. In patients with stage III colon cancers from a completed adjuvant trial, recurrence rates were inversely related to IS score (0-4), and the dichotomized IS was associated with significantly better survival across all disease subgroups (Forest plot). Stage III colon cancers are categorized into low risk (T1-3N1) and high risk (T4 and/or N2) groups for guiding the duration of adjuvant chemotherapy (3 vs 6 months). IS was shown to prognostically stratify patients within both risk groups and was only a significant variable in low risk tumors by MV analysis. These data suggest the potential of IS to further risk stratify patients and to potentially inform treatment decisions. In patients with metastatic CRC, analysis of IS was shown to predict tumor metastatic burden which had prognostic value. Data also suggest that potential for IS to provide predictive information for chemotherapy (fluoropyrimidine + oxaliplatin) response in CRC, yet further studies are needed. A potential mechanism is immunogenic cell death induced by some anti-cancer drugs, including oxaliplatin. Regarding immune checkpoint inhibitors (ICPs), only 40-50% of patients with metastatic CRC respond to such therapy, and small studies in suggest that TIL densities may be a factor in observed variability in response to ICP inhibitors. To further explore this issue, we are examining tumor immunophenotypes in relationship to response and outcome in CRC patients treated with anti-PD-1 antibodies. We aim to develop a MV model, and to validate it in an independent dataset. Important and relevant issues include the fact that only a fraction of mutations result in neoantigens recognized by T-cells, the potential for poor presentation of neoantigens by MHC1, as well as activating or suppressive cytokines. Citation Format: Frank A. Sinicrope. Analysis of immune contexture to refine prognosis in patients with colon cancer: Implications for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY17-01.

First-line avelumab in a cohort of 116 patients with metastatic Merkel cell carcinoma (JAVELIN Merkel 200): primary and biomarker analyses of a phase II study

BackgroundAvelumab (anti-programmed death ligand 1 (PD-L1)) is approved in multiple countries for the treatment of metastatic Merkel cell carcinoma (mMCC), a rare and aggressive skin cancer. We report efficacy and...

Abstract 799: Reduced diversity of HLA class II alleles may contribute to the early manifestation of breast cancer in BRCA1 mutation carriers

Abstract Female carriers of germline BRCA1 mutation almost inevitably develop breast cancer (BC). However, the age of BRCA1-associated cancer onset varies widely. We hypothesize that age-related penetrance of BRCA1 mutations may depend on the inherited variability in the host immune system. We utilized high-throughput HLA-typing to describe 10 classical HLA class I/II loci for 90 young (&amp;lt;38 y.o.) and 73 elderly (&amp;gt;57 y.o.) BRCA1-mutated BC patients. 39% of young females were homozygous for two or more HLA-II loci; this was significantly higher than in the “elderly” cohort [OR = 3.23; p = 0.002]. In contrast, women who harboured identical alleles at HLA-B locus (class I) had increased chances to stay tumour-free to their older ages [OR = 3.02; p = 0.077]. Allele HLA-DBR1*11:03/04 was overtly overrepresented in elderly BRCA1-driven BC [11% vs. 3%, p = 0.006]. The frequencies of HLA-DRB1*01:01 and HLA-DQB1*05 were significantly higher in group of “young” patients [p = 0.020 and p = 0.011, correspondently]. The tumour immunogenicity was analyzed in early-onset and late-onset BC patients through the evaluation of the intratumoral density of cytotoxic T-lymphocytes (TILs) with anti-CD8 IHC staining. 5/10 carcinomas arisen in young BRCA1-carriers were classified as a “hot” phenotype with a high density of TILs across the tumour. Inversely, the BC developed in “elderly” women was characterized by the reduced levels of TILs. Conclusions: 1) the limited repertoire of HLA peptides caused by the homozygosity for HLA II loci may increase susceptibility to BC in BRCA1-carriers and manifest by early disease onset; 2) tumours from “elderly” BRCA1-associated patients have reduced levels of lymphocytic infiltration compared to “young” patients. The research is supported by RSF grant #19-15-00207 Citation Format: Ekaterina ShKuligina, Alexandr A. Romanko, Tatjana Jankevic, Aleksandr S. Martianov, Tatyana N. Sokolova, Alexandr O. Ivantsov, Evgeny N. Imyanitov. Reduced diversity of HLA class II alleles may contribute to the early manifestation of breast cancer in BRCA1 mutation carriers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 799.

A Subset of Localized Prostate Cancer Displays an Immunogenic Phenotype Associated with Losses of Key Tumor Suppressor Genes.

A subset of primary prostate cancer expresses programmed death-ligand 1 (PD-L1), but whether they have a unique tumor immune microenvironment or genomic features is unclear. We selected PD-L1-positive high-grade and/or high-risk primary prostate cancer, characterized tumor-infiltrating lymphocytes with multiplex immunofluorescence, and identified genomic alterations in immunogenic and nonimmunogenic tumor foci. One quarter of aggressive localized prostate cancer cases (29/115) had tumor PD-L1 expression more than 5%. This correlated with increased density of CD8+ T cells, a large fraction coexpressing PD-1, versus absent PD-1 expression on sparse CD8 T cells in unselected cases. Most CD8+PD-1+ cells did not express terminal exhaustion markers (TIM3 or LAG3), while a subset expressed TCF1. Consistent with these CD8+PD-1+TCF1+ cells being progenitors, they were found in antigen-presenting cell niches in close proximity to MHC-II+ cells. CD8 T-cell density in immunogenic prostate cancer and renal cell carcinoma (RCC) was nearly identical. Shallow RB1 and BRCA2 losses, and deep deletions of CHD1, were prevalent, the latter being strongly associated with a dendritic cell gene set in The Cancer Genome Atlas. Tumor mutation burden was variable; neither high microsatellite instability nor CDK12 alterations were present. A subset of localized prostate cancer is immunogenic, manifested by PD-L1 expression and CD8+ T-cell content comparable with RCC. The CD8+ T cells include effector cells and exhausted progenitor cells, which may be expanded by immune checkpoint inhibitors (ICI). Genomic losses of RB1, BRCA2, and CHD1 may be drivers of this phenotype. These findings indicate that immunotherapies may be effective in biomarker-selected subpopulations of patients with localized prostate cancer.

High T-cell infiltration in tumor tissue and younger age predict the response to pembrolizumab in recurrent urothelial cancer.

Targeting the programmed cell death-1 signaling pathway has been approved for the anti-cancer therapy in several cancers including urothelial cancer. To determine predictive factors of the responsiveness to pembrolizumab in urothelial cancer patients, a retrospective study that used clinical information and paraffin-embedded samples obtained from patients diagnosed with urothelial cancer between 2015 and 2020 were performed. Seventeen patients who underwent total cystectomy or nephroureterectomy of the primary lesion and were treated with pembrolizumab for chemo-resistant disease were enrolled, and immunohistochemical analysis was performed. A key difference in the characteristics between the non-responder group and the responder group was the age of the patients (74 vs. 63years, p = 0.0194). Although there was no statistically significant difference, the histological subtype with sarcomatoid and micropapillary components was only seen in the non-responder group, and squamous differentiation and lymph node metastasis were only seen in cases with a complete response. In the results of immunohistochemistry, the density of CD8-positive T-cells and Tregs was significantly increased in the responder group than in the non-responder group. In conclusion, younger age and a high number of tumor-infiltrating lymphocytes were predictive factors of a good response to immune checkpoint inhibitors, although further studies with more enrolled patients are necessary.

Low Concordance Between T-Cell Densities in Matched Primary Tumors and Liver Metastases in Microsatellite Stable Colorectal Cancer.

BackgroundThe subtype, density and location of tumor infiltrating T-cells are being explored as prognostic and predictive biomarkers in primary colorectal cancer (pCRC) and colorectal liver metastases (CLM). Very limited data exist comparing findings in pCRC and matched CLM.Patients and methodsFifty-eight patients with available pCRC and matched CLM (57/58 microsatellite stable) were included in this OSLO-COMET substudy. In immunohistochemically stained sections, total (Ttot), helper (TH), cytotoxic (CTL), and regulatory (Treg) T-cells were manually counted in hotspots from the invasive margin (IM), intratumor (IT), and tumor adjacent regions to determine T-cell densities.ResultsA striking accumulation of T-cells was found in IM of both pCRC and CLM with much lower densities in the IT region, exemplified by Ttot of 2838 versus 340 cells/mm2, respectively, in CLM. The correlation at the individual level between T-cell densities in pCRC and corresponding CLM was poor for all regions and T-cell subtypes; for instance, the correlation coefficient (R2) for IM Ttot was 0.07. The IT TH : CTL and Treg : TH ratios were 2.94 and 0.44, respectively, in pCRC, and 1.84 and 0.24, respectively, in CLM.ConclusionThe observed accumulation of T-cells in the IM regions of pCRC and CLM with low penetration to the IT regions, combined with high TH : CTL and Treg : TH ratios, point to the presence of an immune suppressive microenvironment. T-cell densities of CLM differed markedly from the matched pCRC, indicating that to evaluate T-cell biomarkers in metastasis, the commonly available pCRC cannot serve as a surrogate for the metastatic tumor.

Characterization of immune infiltration in sarcomatoid hepatocellular carcinoma.

Sarcomatoid hepatocellular carcinoma (sHCC) is a rare type of liver malignancy. Currently, the tumor immune features of sHCC are poorly understood. We recruited 31 patients with resected sHCC for whom tissue samples and complete clinicopathologic and follow-up data were available. To understand the immune infiltration of sHCC, immunohistochemical staining was performed on the resected sHCC samples to compare the expressions of programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), B7-H3, indoleamine 2,3-dioxygenase (IDO), lymphocyte-activation gene 3 (LAG-3), CD8, FOXP3, and CD68 in tumor and peritumoral tissues. Kaplan-Meier and Cox regression analyses were used to assess the predictive value of immune markers. Sarcomatoid components were characterized with significantly higher expression of PD-L1 and B7-H3 in tumor cells than in conventional HCC components, as well as in peritumoral tissue. Additionally, sarcomatoid components had a higher density of FOXP3+ and LAG-3+ cells and a lower density of CD8+ cells than conventional HCC components or peritumoral tissue. Higher expression of PD-L1 in tumor cells significantly correlated with higher densities of CD8+, PD-1+, and LAG-3+ cells. Increased tumor PD-L1 expression and decreased CD8+ T-cell density were associated with poor overall survival (OS) and disease-free survival (DFS) in patients of sHCC. These findings suggest further characterization on relative mechanism of sHCC immune infiltration may identify therapeutic targets for immunotherapy.

High density of cytotoxic T-lymphocytes is linked to tumoral PD-L1 expression regardless of the mismatch repair status in colorectal cancer

Background Immune checkpoint-inhibitors targeting the PD-1/PD-L1 system are FDA approved in microsatellite instable (MSI) or mismatch repair deficient (dMMR) colorectal cancer (CRC). PD-L1 expression is tightly linked to features connected to immune checkpoint inhibitor response, but studies on large subsets of cancers analyzing the correlation between different status of MSI/dMMR, tumor infiltrating lymphocytes and PD-L1 expression are still lacking. Methods More than 1800 CRC were analyzed for PD-L1 by immunohistochemistry in a tissue microarray format. Data were compared to MMR, the number of intratumoral CD8+ cytotoxic T-cells, and adverse clinico-pathological parameters. Different cutoff levels for defining PD-L1 positivity in tumor cells (1%, 5%, 10%, and 50%) yielded comparable results. Results At a cutoff level of 5%, PD-L1 positivity was seen in 5.1% of tumors. PD-L1 was more often positive in dMMR (18.6%) than in MMR proficient (pMMR) cancers (4.1%; p < 0.0001). The number of intratumoral CD8+ lymphocytes was strikingly higher in PD-L1 positive (939.5 ± 118.2) than in PD-L1 negative cancers (310.5 ± 24.8). A higher number of intratumoral CD8+ lymphocytes was found in dMMR CRC (PD-L1 positive: 1999.7 ± 322.0; PD-L1 negative: 398.6 ± 128.0; p < 0.0001) compared to pMMR CRC (PD-L1 positive: 793.2 ± 124.8; PD-L1 negative: 297.2 ± 24.2; p < 0.0001). In dMMR and pMMR CRC, PD-L1 expression in tumor cells was unrelated to tumor stage, lymph node status or lymphatic/venous invasion. PD-L1 positivity in tumor associated immune cells was seen in 47.5% of cases and was significantly linked to high numbers of tumor infiltrating CD8+, low tumor stage, and absence of lymph node metastasis and lymphatic/venous invasion (p < 0.0001 each). Conclusion The data support the previously suggested fact that PD-L1 expression in tumor cells is driven by extensive cytotoxic T-cell infiltration in highly immunogenic dMMR and pMMR CRC. Frequent and intense PD-L1 expression in tumor cells of dMMR CRC may contribute to the high response rates of dMMR CRC to immune checkpoint-inhibitors.

Favorable tumor immune microenvironment (TME) and robust chimeric antigen receptor (CAR) T-cell expansion may overcome tumor burden (TB) and promote durable efficacy with axicabtagene ciloleucel (axi-cel) in large B-cell lymphoma (LBCL).

7536 Background: Axi-cel is an autologous anti-CD19 CAR T-cell therapy approved for patients (pts) with relapsed/refractory LBCL after ≥2 prior systemic therapies. In the pivotal ZUMA-1 study, pts with high pretreatment (preTx) TB (estimated by sum of product diameters [SPD]) had lower peak CAR T-cell expansion normalized to TB and less frequent durable response rates vs pts with low TB ( &lt; 30% vs &gt; 60%, respectively; Blood Adv. 2020;4:3268). The number of CD8+ and CCR7+CD45RA+ product T cells infused and favorable immune contexture in preTx TME were also associated with axi-cel response ( Blood Adv. 2020;4:3268; Galon et al. ASCO 2020. #3022). As potential barriers to axi-cel efficacy are not fully elucidated, we systematically analyzed preTx TME characteristics, including myeloid-related biomarkers and product attributes, to identify such challenges in ZUMA-1 pts with high TB. Methods: Samples from evaluable pts in ZUMA-1 Phase (Ph) 1 and Ph2 Cohorts (C) 1–3 were analyzed (NCT02348216; Ph1 and Ph2 C1+2, ≥2-y follow-up; C3, ≥6-mo follow-up). PreTx immune TME was analyzed by multiplex immunohistochemistry (n = 18) and gene expression analysis (n = 30) as previously described (Rossi et al. AACR 2018. #LB-016; Galon et al. ASCO 2020. #3022). CAR T-cell product characteristics and other covariates were evaluated as previously described ( Blood Adv. 2020;4:3268). Correlative analyses of these covariates with clinical outcomes were performed by Wilcoxon or Kruskal-Wallis test. Median TB (by SPD) from ZUMA-1 Ph1 and Ph2 C1+2 was used as a cutoff for high ( &gt; 3721 mm2) vs low (≤3721 mm2) TB. Durable response refers to pts in ongoing response at time of data cutoff. Results: PreTx immune TME features related to suppressive myeloid-related activity, most notably ARG2, TREM2, and IL-8 gene expression, were elevated in pts who failed to respond or relapsed without documented loss of CD19 expression. ARG2 and TREM2 levels in preTx biopsies were negatively associated with CD8+ T-cell density. Pts with high TB who achieved durable response had low preTx ARG2 and TREM2 levels in TME and enhanced CAR T-cell expansion after axi-cel compared to pts with high TB who relapsed. High ratio of T-cell to suppressive myeloid cell markers (T/M ratio) in preTx biopsies associated positively with CAR T-cell expansion (peak and peak normalized to TB) and durable response in pts with high TB. Conclusions: Axi-cel may overcome high TB in pts with a favorable immune TME alongside robust CAR T-cell expansion. Favorable immune TME is characterized by reduced suppressive myeloid cell activity (low ARG2 and TREM2 expression) and increased T/M ratio. These data suggest possible actionable strategies to overcome high TB in the context of CAR T-cell therapy. [JC and VP contributed equally] Clinical trial information: NCT02348216.

Prognostic Impact of APOBEC3B Expression in Metastatic Urothelial Carcinoma and Its Association with Tumor-Infiltrating Cytotoxic T Cells.

APOBEC3B enzymes are endogenous carcinogenic mutagens. Metastatic urothelial carcinomas often harbor APOBEC3B-mediated mutations in which tCw to T or G substitution occurs. Here, we evaluated patient survival and CD8+ T-cell density according to APOBEC3B expression in patients with metastatic urothelial carcinoma who underwent cytotoxic chemotherapy. We performed a retrospective study on 94 patients with urothelial carcinoma who were treated with first line palliative chemotherapy. APOBEC3B expression and CD8+/CD3+ ratio of tumor-infiltrating lymphocytes were evaluated using immunohistochemistry. Kaplan–Meier survival curves were generated and the log-rank test was employed. The association between APOBEC3B expression and tumor-infiltrating lymphocytes was analyzed using Pearson’s chi-squared test. High APOBEC3B expression was detected in 71 of the 94 patients (75.5%). The median overall survival was longer in patients with high APOBEC3B expression (15 months) than in those with low expression (p = 0.045). The hazard ratio obtained based on the Cox regression analysis was 0.292 (95% confidence interval 0.118–0.723, p = 0.008). APOBEC3B expression was associated with the CD8+/CD3+ ratio (2.914, 95% confidence interval 1.030–8.249, p = 0.039). Collectively, APOBEC3B expression was an independent prognostic factor in patients with metastatic urothelial carcinoma treated with platinum-based chemotherapy. Tumor-infiltrating cytotoxic T cells were associated with APOBEC3B expression.

Synovial Immunohistological Biomarkers of the Classification of Undifferentiated Arthritis Evolving to Rheumatoid or Psoriatic Arthritis.

Background: Undifferentiated arthritis (UA) is defined as an inflammatory arthritis that does not fulfill criteria for a definite diagnosis. Delay in reaching a specific diagnostic and therapy may lead to impaired functional outcomes. Our aim was to identify synovial biomarkers associated with definitive diagnostic classification in patients with UA.Methods: DMARD-naïve UA patients with available initial synovial tissue (ST) and a final diagnosis of rheumatoid arthritis (RA) or psoriatic arthritis (PsA) during follow-up were included and compared with patients with well-defined disease (RA or PsA). Clinical, arthroscopic, and pathological data were compared between groups. Pathology included quantitative immunohistochemical (IHC) analysis of cell types and human interferon-regulated MxA. Principal component analysis (PCA) was performed to extract disease patterns.Results: One hundred and five patients were included: 31 patients with DMARD-naïve UA (19 evolving to RA and 12 to PsA during a median follow up of 7 years), 39 with established RA, and 35 with established PsA. ST from the UA group showed higher macrophage density compared with the established RA and PsA groups. Patients with UA evolving to RA (UA-RA) showed higher MxA expression and CD3+ T-cell density compared with established RA. UA patients evolving to PsA (UA-PsA) showed increased vascularity and lining synovial fibroblast density compared with established PsA. Synovitis of UA-PsA patients showed more mast cells and lining fibroblasts compared with UA-RA. No between-group differences in local or systemic inflammation markers were found.Conclusions: Our results show differences in the cellular composition of UA synovium compared with RA and PsA, with higher density of the cellular infiltrate in the UA groups. Initial expression of the interferon inducible gene MxA could be a biomarker of progression to RA, while higher mast cell and fibroblastic density may be associated with PsA progression.

Talimogene laherparepvec upregulates immune-cell populations in non-injected lesions: findings from a phase II, multicenter, open-label study in patients with stage IIIB–IVM1c melanoma

BackgroundTalimogene laherparepvec (T-VEC), an oncolytic virus, was designed to selectively replicate in and lyse tumor cells, releasing tumor-derived antigen to stimulate a tumor-specific immune response.MethodsIn this phase II study in...