The impact of preoperative treatments on the immune environment of rectal cancer.

To improve local disease control, the use of preoperative radiotherapy either alone or combined with chemotherapy has become standard practice in rectal cancer, but it is unclear how these treatments modify the antitumoral immune response. We aimed to evaluate tumor histopathologic features and the prognostic effect of host immune response in rectal cancer with variable treatment modalities. Ninety-five rectal cancers with short-course radiotherapy (SRT), 97 with long-course chemoradiotherapy (CRT), and 154 without preoperative treatments, were evaluated for histopathologic features including Crohn's-like reaction (CLR). CD3+ and CD8+ immunohistochemistry and tumor cells were analyzed from tumor tissue microarray samples to calculate T-cell densities and G-cross function values to estimate cancer cell-T-cell co-localization (proximity score). We found that lymphocyte densities were diminished after SRT, but CLR was scarcer after CRT. Proximity score and CLR density were prognostic for survival in cancer without preoperative treatments and could be combined into an enhanced prognostic score (immune grade). In the irradiated tumors, CLR density remained prognostic while the impact of T-cell infiltration was insufficient alone. In multivariable analysis, the immune grade proved to be an independent prognostic factor for survival. In conclusion, the immune contexture of rectal cancer harbors prognostic significance even after preoperative radiotherapy.

Establishing Criteria for Tumor Necrosis as Prognostic Indicator in Colorectal Cancer.

Tumor necrosis has been reported to represent an independent prognostic factor in colorectal cancer, but its evaluation methods have not been described in sufficient detail to introduce tumor necrosis evaluation into clinical use. To study the potential of tumor necrosis as a prognostic indicator in colorectal cancer, criteria for 3 methods for its evaluation were defined: the average percentage method (tumor necrosis percentage of the whole tumor), the hotspot method (tumor necrosis percentage in a single hotspot), and the linear method (the diameter of the single largest necrotic focus). Cox regression models were used to calculate cancer-specific mortality hazard ratios (HRs) for tumor necrosis categories in 2 colorectal cancer cohorts with more than 1800 cases. For reproducibility assessment, 30 cases were evaluated by 9 investigators, and Spearman's rank correlation coefficients and Cohen's kappa coefficients were calculated. We found that all 3 methods predicted colorectal cancer-specific survival independent of other prognostic parameters, including disease stage, lymphovascular invasion, and tumor budding. The greatest multivariable HRs were observed for the average percentage method (cohort 1: HR for ≥ 40% vs. <3% 3.03, 95% CI, 1.93-4.78; cohort 2: HR for ≥ 40% vs. < 3% 2.97; 95% CI, 1.63-5.40). All 3 methods had high reproducibility, with the linear method showing the highest mean Spearman's correlation coefficient (0.91) and Cohen's kappa (0.70). In conclusion, detailed criteria for tumor necrosis evaluation were established. All 3 methods showed good reproducibility and predictive ability. The findings pave the way for the use of tumor necrosis as a prognostic factor in colorectal cancer.

Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors.

Background: Lynch syndrome (LS) is an autosomal dominant multi-organ cancer syndrome with a high lifetime risk of cancer. The number of cumulative colorectal adenomas in LS does not generally exceed ten, and removal of adenomas via routine screening minimizes the cancer burden. However, abnormal phenotypes may mislead initial diagnosis and subsequently cause suboptimal treatment. Aim: Currently, there is no standard guide for the care of multiple colorectal adenomas in LS individuals. We aimed to shed insight into the molecular features and reasons for multiplicity of adenomas in LS patients. Methods: We applied whole exome sequencing on nine adenomas (ten samples) and three assumed primary carcinomas (five samples) of an LS patient developing the tumors during a 21-year follow-up period. We compared the findings to the tumor profiles of two additional LS cases ascertained through colorectal tumor multiplicity, as well as to ten adenomas and 15 carcinomas from 23 unrelated LS patients with no elevated adenoma burden from the same population. As LS associated cancers can arise via several molecular pathways, we also profiled the tumors for CpG Island Methylator Phenotype (CIMP), and LINE-1 methylation. Results: All tumors were microsatellite unstable (MSI), and MSI was present in several samples derived from normal mucosa as well. Interestingly, frequent frameshift variants in RNF43 were shared among substantial number of the tumors of our primary case and the tumors of LS cases with multiple tumors but almost absent in our control LS cases. The RNF43 variants were completely absent in the normal tissue, indicating tumor-associated mutational hotspots. The RNF43 status correlated with the mutational signature SBS96. Contrary to LS tumors from the reference set with no elevated colorectal tumor burden, the somatic variants occurred significantly more frequently at C>T in the CpG context, irrespective of CIMP or LINE-1 status, potentially indicating other, yet unknown methylation-related mechanisms. There were no signs of somatic mosaicism affecting the MMR genes. Somatic variants in APC and CTNNB1 were unique to each tumor. Conclusion: Frequent somatic RNF43 hot spot variants combined with SBS96 signature and increased tendency to DNA methylation may contribute to tumor multiplicity in LS.

High immune cell infiltration predicts improved survival in cholangiocarcinoma.

Background: Antitumoral immune response has a crucial role in constraining cancer. However, previous studies on cholangiocarcinoma (CCA), a rare and aggressive cancer, have reported contradictory findings on the prognostic impact of tumor-infiltrating T-lymphocytes. We aimed to clarify the effect of tumor-infiltrating CD3+ and CD8+ lymphocytes and PD-1/PD-L1 expression on CCA prognosis. Methods: CD3+, CD8+, and PD-1+ lymphocyte densities, as well as PD-L1 expression rate were analyzed from stained tissue microarray samples from the tumor center and invasive margin of 47 cholangiocarcinomas. The association of CD3+ and CD8+ based Immune cell score (ICS) and its components with overall survival was evaluated, adjusting for age, sex, TNM stage, radicality of surgery, tumor location, and PD-L1 expression on immune cells. Results: Low ICS was a strong independent prognostic factor for worse overall survival (Hazard ratio 9.27, 95% confidence interval 2.72-31.64, P<0.001). Among the ICS components, high CD8+ lymphocyte infiltration at the tumor center had the most evident impact on patient outcome. PD-1 and PD-L1 expression on immune cells did not have a significant impact on overall survival alone; however, PD-L1 positivity seemed to impair survival for ICSlow subgroup. Conclusion: Identifying patient subgroups that could benefit from immunotherapy with PD-1/PD-L1 pathway blockade may help improve treatment strategies for this aggressive cancer. Our findings highlight the importance of evaluating the immune contexture in cholangiocarcinoma, as ICS serves as a strong independent prognostic and selective factor for patients who might benefit from immunotherapy.

CD3+ and CD8+ T cell-based immune cell score as a prognostic factor in clear-cell renal cell carcinoma.

BACKGROUND AND PURPOSE: Immunoscore® is a prognostic parameter based on densities of lymphocyte populations in the tumor center and invasive margin. Immunoscore® is validated in colorectal cancer as a high Immunoscore® is associated with longer survival. Previous studies have suggested that Immunoscore® may also predict oncological outcomes in clear-cell renal cell carcinoma (ccRCC). This study aims to assess the prognostic role of immune cell score in ccRCC. MATERIAL AND METHODS: All patients with ccRCC undergoing surgery between 2007 and 2020 in Central Finland Central Hospital were retrospectively identified. CD3+ and CD8+ cell densities were calculated from tissue samples to determine the immune cell score using Immunoscore® principles. Receiver-operating characteristic analysis, Kaplan-Meier survival curve, and Cox regression were used to evaluate the association between immune cell score and survival. RESULTS: A total of 203 patients (mean age 66.5 years) were identified. The median follow-up time was 6.2 years. Based on the immune cell score, the patients were divided into three groups: low, intermediate, and high. In Cox regression analysis, adjusted with age, sex, and Charlson Comorbidity Index, no significant differences in disease-specific mortality were observed among the three groups. The hazard ratios (HRs) for disease-specific mortality were 0.93 (95% confidence interval [CI] 0.48-1.79) and 1.12 (0.52-2.37) for intermediate- and high-immune cell score groups when compared to low-immune cell score group, respectively. INTERPRETATION: This study found no association between immune cell score and survival. These results indicate that immune cell score may not serve as a prognostic tool in ccRCC.

Quantitative Multiplexed Analysis of Indoleamine 2,3-Dioxygenase (IDO) and Arginase-1 (ARG1) Expression and Myeloid Cell Infiltration in Colorectal Cancer.

Indoleamine 2,3-dioxygenase (IDO) and arginase-1 (ARG1) are amino acid-metabolizing enzymes, frequently highly expressed in cancer. Their expression may deplete essential amino acids, lead to immunosuppression, and promote cancer growth. Still, their expression patterns, prognostic significance, and spatial localization in the colorectal cancer microenvironment are incompletely understood. Using a custom 10-plex immunohistochemistry assay and supervised machine learning-based digital image analysis, we characterized IDO and ARG1 expression in monocytic cells, granulocytes, mast cells, and tumor cells in 833 colorectal cancer patients. We evaluated the prognostic value and spatial arrangement of IDO- and ARG1-expressing myeloid and tumor cells. IDO was mainly expressed not only by monocytic cells but also by some tumor cells, whereas ARG1 was predominantly expressed by granulocytes. Higher density of IDO+ monocytic cells was an independent prognostic factor for improved cancer-specific survival both in the tumor center (Ptrend = .0002; hazard ratio [HR] for the highest ordinal category Q4 [vs Q1], 0.51; 95% CI, 0.33-0.79) and the invasive margin (Ptrend = .0015). Higher density of granulocytes was associated with prolonged cancer-specific survival in univariable models, and higher FCGR3+ARG1+ neutrophil density in the tumor center also in multivariable analysis (Ptrend = .0020). Granulocytes were, on average, located closer to tumor cells than monocytic cells. Furthermore, IDO+ monocytic cells and ARG1- granulocytes were closer than IDO- monocytic cells and ARG1+ granulocytes, respectively. The mRNA expression of the IDO1 gene was assessed in myeloid and tumor cells using publicly available single-cell RNA sequencing data for 62 colorectal cancers. IDO1 was mainly expressed in monocytes and dendritic cells, and high IDO1 activity in monocytes was associated with enriched immunostimulatory pathways. Our findings provided in-depth information about the infiltration patterns and prognostic value of cells expressing IDO and/or ARG1 in the colorectal cancer microenvironment, highlighting the significance of host immune response in tumor progression.

Multiplexed analysis of macrophage polarisation in pulmonary metastases of microsatellite stable colorectal cancer.

Tumour-associated macrophages (TAMs) express a continuum of phenotypes ranging from an anti-tumoural M1-like phenotype to a pro-tumoural M2-like phenotype. During cancer progression, TAMs may shift to a more M2-like polarisation state, but the role of TAMs in CRC metastases is unclear. We conducted a comprehensive spatial and prognostic analysis of TAMs in CRC pulmonary metastases and corresponding primary tumours using multiplexed immunohistochemistry and machine learning-based image analysis. We obtained data from 106 resected pulmonary metastases and 74 corresponding primary tumours. TAMs in the resected pulmonary metastases were located closer to the cancer cells and presented a more M2-like polarised state in comparison to the primary tumours. Higher stromal M2-like macrophage densities in the invasive margin of pulmonary metastases were associated with worse 5-year overall survival (HR 3.19, 95% CI 1.35-7.55, p = 0.008). The results of this study highlight the value of multiplexed analysis of macrophage polarisation in cancer metastases and might have clinical implications in future cancer therapy.

The prognostic and predictive role of tumor-infiltrating lymphocytes (FoxP3 + and CD8 +) and tumor-associated macrophages in early HER2 + breast cancer.

PURPOSE: In HER2-positive (HER2 +) breast cancer, tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) may influence the efficacy of the HER2-antibody trastuzumab and the patient's outcome. In this HER2 + patient cohort, our aim was to study the numbers of FoxP3 + regulatory TILs and CD8 + cytotoxic TILs, their correlations with CD68 + and CD163 + TAMs, and the prognostic and predictive value of the studied factors. METHODS: We evaluated 139 non-metastatic HER2 + breast cancer patients operated between 2001 and 2008. The FoxP3+TIL count (FoxP3+TILs) was assessed using the hotspot method, and the CD8 + TIL count (CD8+mTILs) utilizing a digital image analysis from invasive margin areas. The ratios between CD8+mTILs and FoxP3+TILs as well as CD8+mTILs and TAMs were calculated. RESULTS: FoxP3 + TILs and CD8 + mTILs correlated positively with each other (p<0.001). FoxP3+TILs had a positive correlation with CD68+and CD163+TAMs (p≤0.038), while CD8 + mTILs correlated only with CD68+TAMs (p<0.001). In the HER2 + and hormone receptor-positive Luminal B subgroup, high numbers of FoxP3+TILs were associated with shorter disease-free survival (DFS) (54% vs. 79%, p = 0.040). The benefit from adjuvant trastuzumab was extremely significant among patients with a high CD8 + mTILs/CD68 + TAMs ratio, with overall survival (OS) 84% vs. 33% (p = 0.003) and breast cancer-specific survival (BCSS) 88% vs. 48% (p = 0.009) among patients treated with or without trastuzumab, respectively. CONCLUSION: In the HER2 + Luminal B subgroup, high FoxP3 + TILs were associated with shorter DFS. A high CD8 + mTILs/CD68 + TAMs ratio seems to associate with impressive efficacy of trastuzumab.

Tertiary lymphoid structures in pulmonary metastases of microsatellite stable colorectal cancer.

Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates located at sites of chronic inflammation and recognized as prognosticators in several cancers. We aimed to analyse the prognostic effect of TLSs in colorectal cancer (CRC) pulmonary metastases and primary tumours, with a comparison to the CD3+ and CD8+ cell density-based immune cell score (ICS). For TLS density and TLS maximum diameter analysis, 67 pulmonary metastases and 63 primary tumours were stained with haematoxylin and eosin. For ICS scoring and analysis, CD3 and CD8 immunohistochemistry was performed. Excellent interobserver agreement was achieved in all TLS measurements. Of all patients, 36 patients had low TLS density (< 0.222 follicles/mm) and 31 patients had high TLS density (≥ 0.222 follicles/mm) in the first resected pulmonary metastases. TLS density (adjusted HR 0.91, 0.48-1.73) or maximum diameter (adjusted HR 0.78, 0.40-1.51) did not have prognostic value in pulmonary metastases. In primary tumours, higher TLS density (adjusted HR 0.39, 0.18-0.87) and maximum diameter (adjusted HR 0.28, 0.11-0.73) were associated with lower mortality. In the pulmonary metastases, ICS had superior prognostic value to TLSs; however, TLSs and ICS were significantly associated. In conclusion, TLSs in CRC pulmonary metastases had no prognostic value but correlated with the ICS. TLSs in primary tumours associated with favourable prognosis.

Immunological and prognostic significance of tumour necrosis in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) causes the second most cancer deaths worldwide, but the disease course varies according to tumour characteristics and immunological factors. Our objective was to examine the associations of tumour necrosis with tumour characteristics, immune cell infiltrates, serum cytokine concentrations, as well as prognosis in CRC. METHODS: Three independent CRC cohorts, including 1413 patients, were analysed. Associations of the areal percentage of tumour necrosis with clinicopathologic parameters, tumour infiltrating immune cells, cytokine concentrations in systemic and mesenteric vein blood, and survival were examined. RESULTS: Higher tumour necrosis percentage associated with shorter colorectal cancer-specific survival independent of tumour grade, T, N or M-class, mismatch repair status, BRAF status, and other possible confounding factors. In the largest cohort (N = 1100), the HR for high tumour necrosis percentage (≥40% vs. <3%) was 3.22 (95% CI 1.68-6.17, Ptrend < 0.0001). Tumour necrosis percentage positively correlated with peripheral serum levels of CXCL8, a proinflammatory chemokine, and negatively correlated with mesenteric serum levels of CXCL10 and mast cell densities in the invasive margin of the tumour. CONCLUSIONS: Our results support the value of tumour necrosis as a prognostic factor in colorectal cancer. CXCL8 may have a role in the systemic effects of tumour necrosis.

Spatially resolved multimarker evaluation of CD274 (PD-L1)/PDCD1 (PD-1) immune checkpoint expression and macrophage polarisation in colorectal cancer.

BACKGROUND: The CD274 (PD-L1)/PDCD1 (PD-1) immune checkpoint interaction may promote cancer progression, but the expression patterns and prognostic significance of PD-L1 and PD-1 in the colorectal cancer microenvironment are inadequately characterised. METHODS: We used a custom 9-plex immunohistochemistry assay to quantify the expression patterns of PD-L1 and PD-1 in macrophages, T cells, and tumour cells in 910 colorectal cancer patients. We evaluated cancer-specific mortality according to immune cell subset densities using multivariable Cox regression models. RESULTS: Compared to PD-L1- macrophages, PD-L1+ macrophages were more likely M1-polarised than M2-polarised and located closer to tumour cells. PD-L1+ macrophage density in the invasive margin associated with longer cancer-specific survival [Ptrend = 0.0004, HR for the highest vs. lowest quartile, 0.52; 95% CI: 0.34-0.78]. T cell densities associated with longer cancer-specific survival regardless of PD-1 expression (Ptrend < 0.005 for both PD-1+ and PD-1- subsets). Higher densities of PD-1+ T cell/PD-L1+ macrophage clusters associated with longer cancer-specific survival (Ptrend < 0.005). CONCLUSIONS: PD-L1+ macrophages show distinct polarisation profiles (more M1-like), spatial features (greater co-localisation with tumour cells and PD-1+ T cells), and associations with favourable clinical outcome. Our comprehensive multimarker assessment could enhance the understanding of immune checkpoints in the tumour microenvironment and promote the development of improved immunotherapies.

A pan-cancer analysis shows immunoevasive characteristics in NRF2 hyperactive squamous malignancies.

The NRF2 pathway is frequently activated in various cancer types, yet a comprehensive analysis of its effects across different malignancies is currently lacking. We developed a NRF2 activity metric and utilized it to conduct a pan-cancer analysis of oncogenic NRF2 signaling. We identified an immunoevasive phenotype where high NRF2 activity is associated with low interferon-gamma (IFNγ), HLA-I expression and T cell and macrophage infiltration in squamous malignancies of the lung, head and neck area, cervix and esophagus. Squamous NRF2 overactive tumors comprise a molecular phenotype with SOX2/TP63 amplification, TP53 mutation and CDKN2A loss. These immune cold NRF2 hyperactive diseases are associated with upregulation of immunomodulatory NAMPT, WNT5A, SPP1, SLC7A11, SLC2A1 and PD-L1. Based on our functional genomics analyses, these genes represent candidate NRF2 targets, suggesting direct modulation of the tumor immune milieu. Single-cell mRNA data shows that cancer cells of this subtype exhibit decreased expression of IFNγ responsive ligands, and increased expression of immunosuppressive ligands NAMPT, SPP1 and WNT5A that mediate signaling in intercellular crosstalk. In addition, we discovered that the negative relationship of NRF2 and immune cells are explained by stromal populations of lung squamous cell carcinoma, and this effect spans multiple squamous malignancies based on our molecular subtyping and deconvolution data.

The prognostic role of tumor budding and tumor-stroma ratio in pulmonary metastasis of colorectal carcinoma.

OBJECTIVE: To evaluate the prognostic value of tumor budding and tumor-stroma ratio (TSR) in resected pulmonary metastases of colorectal carcinoma (CRC). METHODS: In total, 106 pulmonary metastasectomies were performed to 74 patients in two study hospitals during 2000-2020. All relevant clinical data were retrospectively collected. Tumor budding based on the International Tumor Budding Consensus Conference recommendations and TSR in the first resected pulmonary metastases and primary tumors were evaluated from diagnostic hematoxylin-eosin-stained histopathological slides. RESULTS: 60 patients (85.7%) had low tumor budding (≤5 buds/field) and 10 patients (14.3%) had high tumor budding (>5 buds/field) in their first pulmonary metastases of CRC. 5-year overall survival rates of pulmonary metastasectomy in low and high total tumor budding were 28.3% and 37.3% (p = 0.387), respectively. 19 patients (27.1%) had low TSR and 51 patients (72.9%) had high TSR. The 5-year overall survival rates were 32.9% in low and 28.6% in high TSR of first pulmonary metastases (p = 0.746). Tumor budding and TSR did not provide prognostic value in Cox multivariate analysis. Tumor budding and TSR in resected pulmonary metastases were not associated with those of the primary tumor. CONCLUSION: Tumor budding and TSR in the resected pulmonary metastases of CRC showed no statistically significant prognostic value, however, additional well-powered confirmatory studies are needed.

Is HLA type a possible cancer risk modifier in Lynch syndrome?

Lynch syndrome (LS) is the most common inherited cancer syndrome. It is inherited via a monoallelic germline variant in one of the DNA mismatch repair (MMR) genes. LS carriers have a broad 30% to 80% risk of developing various malignancies, and more precise, individual risk estimations would be of high clinical value, allowing tailored cancer prevention and surveillance. Due to MMR deficiency, LS cancers are characterized by the accumulation of frameshift mutations leading to highly immunogenic frameshift peptides (FSPs). Thus, immune surveillance is proposed to inhibit the outgrowth of MMR-deficient cell clones. Recent studies have shown that immunoediting during the evolution of MMR-deficient cancers leads to a counter-selection of highly immunogenic antigens. The immunogenicity of FSPs is dependent on the antigen presentation. One crucial factor determining antigen presentation is the HLA genotype. Hence, a LS carrier's HLA genotype plays an important role in the presentation of FSP antigens to the immune system, and may influence the likelihood of progression from precancerous lesions to cancer. To address the challenge of clarifying this possibility including diverse populations with different HLA types, we have established the INDICATE initiative (Individual cancer risk by HLA type, http://indicate-lynch.org/), an international network aiming at a systematic evaluation of the HLA genotype as a possible cancer risk modifier in LS. Here we summarize the current knowledge on the role of HLA type in cancer risk and outline future research directions to delineate possible association in the scenario of LS with genetically defined risk population and highly immunogenic tumors.

Systemic circulating microRNA landscape in Lynch syndrome.

Circulating microRNAs (c-miRs) are small noncoding RNA molecules that migrate throughout the body and regulate gene expression. Global c-miR expression patterns (c-miRnomes) change with sporadic carcinogenesis and have predictive potential in early detection of cancers. However, there are no studies that have assessed whether c-miRnomes display similar potential in carriers of inherited pathogenic mismatch-repair gene variants (path_MMR), known as Lynch syndrome (LS), who are predisposed to highly increased cancer risk. Using high-throughput sequencing and bioinformatic approaches, we conducted an exploratory analysis to characterize systemic c-miRnomes of path_MMR carriers, sporadic rectal cancer patients and non-LS controls. We showed for the first time that cancer-free path_MMR carriers have a systemic c-miRnome of 40 differentially expressed c-miRs that can distinguish them from non-LS controls. The systemic c-miRnome of cancer-free path_MMR carriers also resembles the systemic c-miRnomes of cancer patients with or without path_MMR. Our pathway analysis linked the found differentially expressed c-miRs to carcinogenesis. A total of 508 putative target genes were identified for 32 out of 40 differentially expressed c-miRs, and 238 of them were enriched in cancer-related pathways. The most enriched c-miR-target genes include well-known oncogenes and tumor suppressor genes such as BCL2, AKT3, PIK3CA, KRAS, NRAS, CDKN1A and PIK3R1. Taken together, our findings suggest that LS and sporadic carcinogenesis share common biological pathways and alterations in these pathways can produce a c-miR signature which can track potential oncogenic stress in cancer-free path_MMR carriers. Therefore, c-miRs hold potential in monitoring the LS risk stratification patterns during clinical surveillance or cancer management.

Prognostic significance of spatial and density analysis of T lymphocytes in colorectal cancer.

BACKGROUND: Although high T cell density is a strong favourable prognostic factor in colorectal cancer, the significance of the spatial distribution of T cells is incompletely understood. We aimed to evaluate the prognostic significance of tumour cell-T cell co-localisation and T cell densities. METHODS: We analysed CD3 and CD8 immunohistochemistry in a study cohort of 983 colorectal cancer patients and a validation cohort (N = 246). Individual immune and tumour cells were identified to calculate T cell densities (to derive T cell density score) and G-cross function values, estimating the likelihood of tumour cells being co-located with T cells within 20 µm radius (to derive T cell proximity score). RESULTS: High T cell proximity score associated with longer cancer-specific survival in both the study cohort [adjusted HR for high (vs. low) 0.33, 95% CI 0.20-0.52, Ptrend < 0.0001] and the validation cohort [adjusted HR for high (vs. low) 0.15, 95% CI 0.05-0.45, Ptrend < 0.0001] and its prognostic value was independent of T cell density score. CONCLUSIONS: The spatial point pattern analysis of tumour cell-T cell co-localisation could provide detailed information on colorectal cancer prognosis, supporting the value of spatial measurement of T cell infiltrates as a novel, robust tumour-immune biomarker.

PD-L1 and PD-1 expression in thyroid follicular epithelial dysplasia: Hashimoto thyroiditis related atypia and potential papillary carcinoma precursor.

Programmed cell death ligand (PD-L1)/PD-1 expression has been studied in a variety of cancers and blockage of PD-L1/PD-1 pathway is a cornerstone of immunotherapy. We studied PD-L1/PD-1 immunohistochemical expression in 47 thyroid gland specimens in groups of (1) Hashimoto thyroiditis (HT) only; (2) HT and follicular epithelial dysplasia (FED); and (3) HT, FED, and papillary thyroid carcinoma (PTC). PD-1 positivity was found in immune cells, namely in lymphocytes, macrophages, and plasma cells with mean values for lymphocytes and macrophages 9% in HT group, 4% in FED group, and 4% in PTC group. PD-L1 positivity was identified in both immune cells and in the normal epithelial cells. In the HT group, mean PD-L1 staining on immune cells was 6%, in FED group 5%, and in PTC group 7%. The mean PD-L1 staining on the epithelial cells in the inflammatory parenchyma was 11.7% in HT, 13.4% in FED, and 8.3% in PTC group. The mean PD-L1 staining of FED foci was 47.2% in FED group and 33.6% in PTC group. The mean tumor proportion score (TPS) was 10.4%, and the mean combined positive score (CPS) was 15.5. At the moment, PTC is not a target of immunotherapy. However, understanding the complex issue of concurrent inflammation and autoimmunity can importantly influence the cancer treatment in future.

The Different Immune Profiles of Normal Colonic Mucosa in Cancer-Free Lynch Syndrome Carriers and Lynch Syndrome Colorectal Cancer Patients.

BACKGROUND & AIMS: Owing to the high load of immunogenic frameshift neoantigens, tumors arising in individuals with Lynch syndrome (LS), the most common inherited colorectal cancer (CRC) syndrome, are characterized by a pronounced immune infiltration. However, the immune status of normal colorectal mucosa in LS is not well characterized. We assessed the immune infiltrate in tumor-distant normal colorectal mucosa from LS CRC patients, sporadic microsatellite-unstable (MSI) and microsatellite-stable (MSS) CRC patients, and cancer-free LS carriers. METHODS: CD3-positive, FOXP3-positive, and CD8-positive T cells were quantified in, respectively, 219, 233, and 201 formalin-fixed paraffin-embedded (FFPE) normal colonic mucosa tissue sections from CRC patients and cancer-free LS carriers and 26, 22, and 19 LS CRCs. CD3-positive T cells were also quantified in an independent cohort of 97 FFPE normal rectal mucosa tissue sections from LS carriers enrolled in the CAPP2 clinical trial. The expression of 770 immune-relevant genes was analyzed in a subset of samples with the use of the NanoString nCounter platform. RESULTS: LS normal mucosa specimens showed significantly elevated CD3-, FOXP3-, and CD8-positive T-cell densities compared with non-LS control specimens. Gene expression profiling and cluster analysis revealed distinct immune profiles in LS carrier mucosa with and without cancer manifestation. Long-term follow-up of LS carriers within the CAPP2 trial found a correlation between mucosal T-cell infiltrate and time to subsequent tumor occurrence. CONCLUSIONS: LS carriers show elevated mucosal T-cell infiltration even in the absence of cancer. The normal mucosa immune profile may be a temporary or permanent tumor risk modifier in LS carriers.

CD3+ and CD8+ T-Cell-Based Immune Cell Score and PD-(L)1 Expression in Pulmonary Metastases of Microsatellite Stable Colorectal Cancer.

The objective of this study was to evaluate the prognostic value of CD3+ and CD8+ based immune cell score (ICS), programmed death -1 (PD-1) and programmed death ligand -1 (PD-L1) in pulmonary metastases of proficient mismatch repair colorectal cancer (CRC) patients. A total of 101 pulmonary metastases and 62 primary CRC tumours were stained for CD3+, CD8+, PD-1 and PD-L1 expression. The prognostic value of ICS, PD-1/PD-L1 expression in 67 first pulmonary metastases and 61 primary CRC tumour was analysed. Comparative analysis was also performed between primary tumours and pulmonary metastases, as well as between T-cell densities and PD-1/PD-L1 expression. The 5-year overall survival rates of low, intermediate, and high ICS in pulmonary metastases were 10.0%, 25.5% and 47.0% (p = 0.046), respectively. Patients with high vs. low ICS in pulmonary metastases had a significantly better 5-year survival (adjusted HR 0.25, 95% CI 0.09-0.75, p = 0.013). High tumour cell PD-L1 expression in the pulmonary metastases was associated with improved survival (p = 0.024). Primary tumour CD8+ expression was significantly correlated with all T-cell densities in pulmonary metastases. Conclusion: The ICS evaluated from the resected pulmonary metastases of CRC showed significant prognostic value. High PD-L1 expression in pulmonary metastases is associated with favourable prognosis.

Immunoprofiles and DNA Methylation of Inflammatory Marker Genes in Ulcerative Colitis-Associated Colorectal Tumorigenesis.

Immunological and epigenetic changes are interconnected and contribute to tumorigenesis. We determined the immunoprofiles and promoter methylation of inflammation-related genes for colitis-associated colorectal carcinomas (CA-CRC). The results were compared with Lynch syndrome (LS)-associated colorectal tumors, which are characterized by an active immune environment through inherited mismatch repair defects. CA-CRCs (n = 31) were immunohistochemically evaluated for immune cell scores (ICSs) and PDCD1 and CD274 expression. Seven inflammation-associated genes (CD274, NTSR1, PPARG, PTGS2, PYCARD, SOCS1, and SOCS2), the repair gene MGMT, and eight standard marker genes for the CpG Island Methylator Phenotype (CIMP) were investigated for promoter methylation in CA-CRCs, LS tumors (n = 29), and paired normal mucosae by multiplex ligation-dependent probe amplification. All but one CA-CRCs were microsatellite-stable and all LS tumors were microsatellite-unstable. Most CA-CRCs had a high ICS (55%) and a positive CD274 expression in immune cells (52%). NTSR1 revealed frequent tumor-specific hypermethylation in CA-CRC and LS. When compared to LS mucosae, normal mucosae from patients with CA-CRC showed significantly higher methylation of NTSR1 and most CIMP markers. In conclusion, CA-CRCs share a frequent ICShigh/CD274pos expression pattern with LS tumors. Elevated methylation in normal mucosa may indicate field cancerization as a feature of CA-CRC-associated tumorigenesis.