TIGIT is an inhibitory immune checkpoint receptor and a putative target for novel immune therapies. Here, we analysed two different types of tissue microarrays of healthy lymphatic and various inflamed tissues, colorectal and lung cancers, as well as >1700 tumour samples from 86 different tumour entities for TIGIT and/or PD-1 by bright field and/or multiplex fluorescence immunohistochemistry. TIGIT was detected in CD8⁺ cytotoxic T cells, CD4⁺ T helper cells, FOXP3⁺ regulatory T cells, and NK cells, but not in CD11c⁺ dendritic cells, CD68⁺ macrophages, and CD20⁺ B lymphocytes. TIGIT expression paralleled that of PD-1. More than 70% of TIGIT⁺ cells were PD-1⁺, and more than 90% of the PD-1⁺ cells were TIGIT⁺. Expression varied between different tissue compartments. TIGIT expression in tonsil gradually increased from the interfollicular area over the marginal/mantle zone to the germinal centre in all T cell subtypes. In inflammatory diseases, the strongest expression of TIGIT/PD-1 was found in Hashimoto thyroiditis. TIGIT⁺ lymphocytes were seen in all 86 different tumour entities with considerable high variability of TIGIT positivity within and between different cancer entities. Particularly, high densities of TIGIT⁺ lymphocytes were, for example, seen in squamous cell cancers of various origins. In summary, the variable expression levels of TIGIT and PD-1 in cell types and tissue compartments illustrate the high complexity of immune microenvironments. The high frequency of TIGIT (and PD-1) expressing lymphocytes in cancers highlights considerable opportunities for cotargeting with checkpoint inhibitors.

The Ki‐67 labeling index (Ki‐67 LI) is a strong prognostic marker in prostate cancer, although its analysis requires cumbersome manual quantification of Ki‐67 immunostaining in 200–500 tumor cells. To enable automated Ki‐67 LI assessment in routine clinical practice, a framework for automated Ki‐67 LI quantification, which comprises three different artificial intelligence analysis steps and an algorithm for cell‐distance analysis of multiplex fluorescence immunohistochemistry (mfIHC) staining, was developed and validated in a cohort of 12,475 prostate cancers. The prognostic impact of the Ki‐67 LI was tested on a tissue microarray (TMA) containing one 0.6 mm sample per patient. A ‘heterogeneity TMA’ containing three to six samples from different tumor areas in each patient was used to model Ki‐67 analysis of multiple different biopsies, and 30 prostate biopsies were analyzed to compare a ‘classical’ bright field‐based Ki‐67 analysis with the mfIHC‐based framework. The Ki‐67 LI provided strong and independent prognostic information in 11,845 analyzed prostate cancers (p < 0.001 each), and excellent agreement was found between the framework for automated Ki‐67 LI assessment and the manual quantification in prostate biopsies from routine clinical practice (intraclass correlation coefficient: 0.94 [95% confidence interval: 0.87–0.97]). The analysis of the heterogeneity TMA revealed that the Ki‐67 LI of the sample with the highest Gleason score (area under the curve [AUC]: 0.68) was as prognostic as the mean Ki‐67 LI of all six foci (AUC: 0.71 [p = 0.24]). The combined analysis of the Ki‐67 LI and Gleason score obtained on identical tissue spots showed that the Ki‐67 LI added significant additional prognostic information in case of classical International Society of Urological Pathology grades (AUC: 0.82 [p = 0.002]) and quantitative Gleason score (AUC: 0.83 [p = 0.018]). The Ki‐67 LI is a powerful prognostic parameter in prostate cancer that is now applicable in routine clinical practice. In the case of multiple cancer‐positive biopsies, the sole automated analysis of the worst biopsy was sufficient. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Hodgkin’s lymphoma (HL) is characterized by a high background of inflammatory cells which play an important role for the pathogenesis of the disease. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory immune checkpoint receptor and a putative target for novel immunotherapies. To study patterns of TIGIT expression in the T cell background surrounding malignant cells including Hodgkin cells, Reed-Sternberg cells and histiocytic cells, a microenvironment (ME) tissue microarray (TMA) was constructed from tissue punches measuring 2 mm in diameter obtained from formalin-fixed tissue samples of Hodgkin’s lymphoma lymph nodes (n = 40) and normal human tonsil (n = 2). The ME-TMA was stained by brightfield and fluorescence multiplex immunohistochemistry (IHC) to evaluate expression levels of TIGIT and PD-1 as well as standard lymphocyte markers (CD3, CD8, CD4, FOXP3) in the lymphocytic background. All analyzed cases of HL contained 9–99% (median: 86%) of TIGIT+ lymphoid cells. In general, TIGIT localized to the same cells as PD-1. Strikingly, expression levels of TIGIT and PD-1 were highly variable among the analyzed samples. Highest levels of TIGIT and PD-1 were found in one sample of nodular lymphocytic-predominant HL (NLPHL). In conclusion, TIGIT expression is highly variable between patients with Hodgkin’s lymphoma. Our results encourage further studies evaluating the role of TIGIT as a target for immunotherapies in Hodgkin’s lymphoma.

Trichorhinophalangeal syndrome 1 (TRPS1) is a nuclear protein highly expressed in breast epithelial cells. TRPS1 immunohistochemistry (IHC) has been suggested as a breast cancer marker. To determine the diagnostic and prognostic utility of TRPS1 IHC, tissue microarrays containing 19,201 samples from 152 different tumor types and subtypes were analyzed. GATA3 IHC was performed in a previous study. TRPS1 staining was seen in 86 of 152 tumor categories with 36 containing at least one strongly positive case. TRPS1 staining predominated in various types of breast carcinomas (51%-100%), soft tissue tumors (up to 100%), salivary gland tumors (up to 46%), squamous cell carcinomas (up to 35%), and gynecological cancers (up to 40%). TRPS1 positivity occurred in 1.8% of 1083 urothelial neoplasms. In invasive breast carcinoma of no special type, low TRPS1 expression was linked to high grade (P = 0.0547), high pT (P < 0.0001), nodal metastasis (P = 0.0571), loss of estrogen receptor and progesterone receptor expression (P < 0.0001 each), and triple-negative status (P < 0.0001) but was unrelated to patient survival (P = 0.8016). In squamous cell carcinomas from 11 different sites, low TRPS1 expression was unrelated to tumor phenotype. Positivity for both TRPS1 and GATA3 occurred in 47.4% to 100% of breast cancers, up to 30% of salivary gland tumors, and 29 (0.3%) of 9835 tumors from 134 other cancer entities. TRPS1 IHC has high utility for the identification of cancers of breast (or salivary gland) origin, especially in combination with GATA3. The virtual absence of TRPS1 positivity in urothelial neoplasms is useful for the distinction of GATA3-positive urothelial carcinoma from breast cancer.

Quantity and the spatial relationship of specific immune cell types can provide prognostic information in bladder cancer. The objective of the study was to characterize the spatial interplay and prognostic role of different immune cell subpopulations in bladder cancer. A total of 2463 urothelial bladder carcinomas were immunostained with 21 antibodies using BLEACH&STAIN multiplex fluorescence immunohistochemistry in a tissue microarray format and analyzed using a framework of neuronal networks for an image analysis. Spatial immune parameters were compared with histopathological parameters and overall survival data. The identification of > 300 different immune cell subpopulations and the characterization of their spatial relationship resulted in numerous spatial interaction patterns. Thirty-nine immune parameters showed prognostic significance in univariate analyses, of which 16 were independent from pT, pN, and histological grade in muscle-invasive bladder cancer. Among all these parameters, the strongest association with prolonged overall survival was identified for intraepithelial CD8⁺ cytotoxic T cells (time-dependent area under receiver operating characteristic curve [AUC]: 0.70), while stromal CD8⁺ T cells were less relevant (AUC: 0.65). A favorable prognosis of inflamed cancers with high levels of "exhaustion markers" suggests that TIM3, PD-L1, PD-1, and CTLA-4 on immune cells do not hinder antitumoral immune response in tumors rich of tumor infiltrating immune cells. The density of intraepithelial CD8⁺ T cells was the strongest prognostic feature in muscle-invasive bladder cancer. Given that tumor cell killing by CD8⁺ cytotoxic T lymphocytes through direct cell-to-cell-contacts represents the "terminal end route" of antitumor immunity, the quantity of "tumor cell adjacent CD8⁺ T cells" may constitute a surrogate for the efficiency of cancer recognition by the immune system that can be measured straightaway in routine pathology as the CD8 labeling index. Quantification of intraepithelial CD8⁺ T cells, the strongest prognosticfeature identified in muscle-invasive bladder cancer, can easily be assessed by brightfield immunohistochemistry and is therefore "ready to use" for routine pathology.

Homozygous 9p21 deletions usually result in a complete loss of S‐methyl‐5′‐thioadenosine phosphorylase (MTAP) expression visualizable by immunohistochemistry (IHC). MTAP deficiency has been proposed as a marker for predicting targeted treatment response. A tissue microarray including 2,710 urothelial bladder carcinomas were analyzed for 9p21 deletion by fluorescence in situ hybridization and MTAP expression by IHC. Data were compared with data on tumor phenotype, patient survival, intratumoral lymphocyte subsets, and PD‐L1 expression. The 9p21 deletion rate increased from pTaG2 low (9.2% homozygous, 25.8% heterozygous) to pTaG2 high (32.6%, 20.9%; p < 0.0001) but was slightly lower in pTaG3 (16.7%, 16.7%) tumors. In pT2–4 carcinomas, 23.3% homozygous and 17.9% heterozygous deletions were found, and deletions were tied to advanced pT (p = 0.0014) and poor overall survival (p = 0.0461). Complete MTAP loss was seen in 98.4% of homozygous deleted while only 1.6% of MTAP negative tumors had retained 9p21 copies (p < 0.0001). MTAP loss was linked to advanced stage and poor overall survival in pT2–4 carcinomas (p < 0.05 each). The relationship between 9p21 deletions/MTAP loss and poor patient prognosis was independent of pT and pN (p < 0.05 each). The 9p21 deletions were associated with a noninflamed microenvironment (p < 0.05). Complete MTAP loss is strongly tied to homozygous 9p21 deletion, aggressive disease, and noninflamed microenvironment. Drugs targeting MTAP‐deficiency may be useful in urothelial bladder carcinoma. MTAP IHC is a near perfect surrogate for MTAP deficiency in this tumor type.