Tumour immune characterisation of primary triple-negative breast cancer using automated image quantification of immunohistochemistry-stained immune cells.

The tumour immune microenvironment (TIME) in breast cancer is acknowledged with an increasing role in treatment response and prognosis. With a growing number of immune markers analysed, digital image analysis may facilitate broader TIME understanding, even in single-plex IHC data. To facilitate analyses of the latter an open-source image analysis pipeline, Tissue microarray MArker Quantification (TMArQ), was developed and applied to single-plex stainings for p53, CD3, CD4, CD8, CD20, CD68, FOXP3, and PD-L1 (SP142 antibody) in a 218-patient triple negative breast cancer (TNBC) cohort with complementary pathology scorings, clinicopathological, whole genome sequencing, and RNA-sequencing data. TMArQ's cell counts for analysed immune markers were on par with results from alternative methods and consistent with both estimates from human pathology review, different quantifications and classifications derived from RNA-sequencing as well as known prognostic patterns of immune response in TNBC. The digital cell counts demonstrated how immune markers are coexpressed in the TIME when considering TNBC molecular subtypes and DNA repair deficiency, and how combination of immune status with DNA repair deficiency status can improve the prognostic stratification in chemotherapy treated patients. These results underscore the value and potential of integrating TIME and specific tumour intrinsic alterations/phenotypes for the molecular understanding of TNBC.

PAX8 expression in cancerous and non-neoplastic tissue: a tissue microarray study on more than 17,000 tumors from 149 different tumor entities.

PAX8 plays a role in development of the thyroid, kidney, and the Wolffian and Mullerian tract. In surgical pathology, PAX8 immunohistochemistry is used to determine tumors of renal and ovarian origin, but data on its expression in other tumors are conflicting. To evaluate PAX8 expression in normal and tumor tissues, a tissue microarray containing 17,386 samples from 149 different tumor types and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. PAX8 results were compared with previously collected data on cadherin 16 (CDH16). PAX8 positivity was found in 40 different tumor types. The highest rate of PAX8 positivity was found in thyroidal neoplasms of follicular origin (98.6-100%), gynecological carcinomas (up to 100%), renal tumors (82.6-97.8%), and urothelial neoplasms (2.3-23.7%). Important tumors with near complete absence of PAX8 staining (< 1%) included all subtypes of breast cancers, hepatocellular carcinomas, gastric, prostatic, pancreatic, and pulmonary adenocarcinomas, neuroendocrine neoplasms, small cell carcinomas of various sites, and lymphomas. High PAX8 expression was associated with low tumor grade in 365 non-invasive papillary urothelial carcinomas (p < 0.0001) but unrelated to patient outcome and/or tumor phenotype in clear cell renal cell carcinoma, high-grade serous ovarian cancer, and endometrioid endometrial carcinoma. For determining a renal tumor origin, sensitivity was 88.1% and specificity 87.2% for PAX8, while sensitivity was 85.3% and specificity 95.7% for CDH16. The combination of PAX8 and CDH16 increased specificity to 96.8%. In conclusion, PAX8 immunohistochemistry is a suitable diagnostic tool. The combination of PAX8 and CDH16 positivity has high specificity for renal cell carcinoma.

The Expression and Prognostic Value of Multiple Glucose Transporters in Cutaneous Melanoma.

BACKGROUND/AIM: Over-expression of glucose transporters (GLUTs), membrane proteins that facilitate glucose transport, has been implicated in cutaneous melanomas. Our prior studies have demonstrated increased expression of GLUT1 and GLUT3 in melanomas and their association with poorer prognosis. This study aimed to investigate the expression of GLUT isoforms 4 and 8 in melanocytic lesions, examine the co-expression status of multiple GLUTs, and evaluate their prognostic significance. MATERIALS AND METHODS: We analyzed 171 melanocytic lesions (97 primary melanomas, 19 metastatic melanomas, and 55 nevi) using a tissue microarray and immunohistochemistry using antibodies against GLUT4 and GLUT8. Membranous expression of GLUTs was scored using a semi-quantitative method. A combined GLUT total score was generated by summing scores from GLUT1, GLUT3, GLUT4, and GLUT8 (including data from previous studies). RESULTS: A significant up-regulation of GLUT4 and GLUT8 expression was found in melanomas compared to nevi (p<0.0001 for both). Concurrent over-expression of multiple GLUTs was more prevalent in melanomas compared to nevi (p<0.0001), and it was also more frequent in metastatic melanomas compared to primary melanomas (p=0.047). Importantly, high total GLUT expression scores were significantly correlated with negative prognostic factors, such as ulceration and mitoses (p=0.03 and p=0.008 respectively). Additionally, Kaplan-Meier survival curves revealed that patients with elevated GLUT total score in their melanomas had a lower disease-specific survival (p=0.006). Furthermore, analysis of multiple GLUTs improved diagnostic sensitivity. CONCLUSION: Similar to GLUT1 and GLUT3, melanoma exhibits up-regulation of GLUT 4 and GLUT8 compared to nevi. Evaluation of multiple GLUT isoforms improves diagnostic and prognostic values.

Expression Analysis of SSTR 1, 2 and 3 in Small-cell Lung Cancer Patients as Targets for Future Peptide Receptor Radionuclide Therapy.

BACKGROUND/AIM: Small-cell lung cancer (SCLC) is noted for its high proliferative rate, and while treatable, relapse is common. SCLC is known to potentially express somatostatin receptors (SSTRs). Somatostatin possesses antineoplastic activity through cell-cycle arrest and apoptosis, and angiogenesis inhibition. SSTRs, thus, serve as potential anticancer targets for somatostatin analogue therapies. The aim of the study was to determine the expression rate of SSTR subtypes 1, 2 and 3 in SCLC using immunohistochemistry, and potential predictors of such rates. MATERIALS AND METHODS: A total of 147 human, SCLC paraffin-embedded tissue microarrays with corresponding patient age, sex, TNM staging, and disease stage were utilized. Immunohistochemical analysis was performed using anti-SSTR 1, 2 and 3 antibodies, each calibrated using healthy human pancreatic islets as positive controls. Array slides were counterstained with hematoxylin and scored based on stain intensity and percentage of stained cells. RESULTS: No SCLC samples expressed SSTR 1, whereas SSTR 2 and 3 were expressed in 29.4% and 4.35% of cases respectively. While females had higher expression levels of SSTR 2/3, and there was a trend of decreased SSTR 2 expression with increased age, results were not statistically significant. No correlation between disease stage and SSTR expression was found. Co-expression of both SSTR2 and 3 occurred in 5.3% of patients. CONCLUSION: Immunohisto-chemistry is an efficient screening tool to reveal optimum SCLC patients for somatostatin analogue therapy. Whilst there currently exist no accepted norm or predictors of SSTR expression levels in SCLC patients, this study contributes insight into the receptors' varied expression.

Prevalence of S-methyl-5'-thioadenosine Phosphorylase (MTAP) Deficiency in Human Cancer: A Tissue Microarray Study on 13,067 Tumors From 149 Different Tumor Types.

Loss of S-methyl-5'-thioadenosine phosphorylase (MTAP) expression is a common event in cancer leading to a critical vulnerability of cancer cells towards anti-cancer drugs. Homozygous MTAP deletions result in a complete expression loss that can be detected by immunohistochemistry (IHC). In this study, a tissue microarray containing 17,078 samples from 149 different tumor entities was analyzed by IHC, and complete MTAP loss was validated by fluorescence in situ hybridization. MTAP loss was observed in 83 of 149 tumor categories, including neuroendocrine neoplasms (up to 80%), Hodgkin lymphoma (50.0%), mesothelioma (32.0% to 36.8%), gastro-intestinal adenocarcinoma (4.0% to 40.5%), urothelial neoplasms (10.5% to 36.7%), squamous cell carcinomas (up to 38%), and various types of sarcomas (up to 20%) and non-Hodgkin lymphomas (up to 14%). Homozygous MTAP deletion was found in 90% to 100% of cases with MTAP expression loss in most tumor categories. However, neuroendocrine tumors, Hodgkin lymphomas, and other lymphomas lacked MTAP deletions. MTAP deficiency was significantly linked to unfavorable tumor phenotype in selected tumor entities and the presence of PD-L1 expression on tumor cells, absence of PD-L1 expression on immune cells, and a low density of CD8 + lymphocytes. In summary, MTAP deficiency can occur in various tumor entities and is linked to unfavorable tumor phenotype and noninflamed tumor microenvironment, but is not always related to deletions. MTAP IHC is of considerable diagnostic value for the detection of neoplastic transformation in multiple different applications.

The utility of the lineage specific immunohistochemical stains SATB2, CDX2, and villin, and the mucin glycoproteins MUC2, MUC5AC, and MUC6 to distinguish pulmonary invasive mucinous adenocarcinoma from metastatic colorectal carcinoma.

CONTEXT: The lungs are a common site of tumor metastasis. While morphology and immunophenotype can help differentiate primary from metastatic tumors, distinguishing pulmonary invasive mucinous adenocarcinoma (PIMA) from metastatic colorectal adenocarcinoma (CRC) may occasionally be challenging due to overlapping morphological and immunohistochemical features. Lineage-specific markers such as CDX2, TTF-1, and napsin A are helpful with pulmonary non-mucinous adenocarcinoma (PNMA), however they are non-specific and insensitive when applied to PIMA. SATB2 is a newer marker that distinguishes CRC from upper gastrointestinal and pancreaticobiliary tumors; its utility in distinguishing CRC from PIMA has not been fully elucidated. OBJECTIVE: To evaluate the performance of lineage-specific and mucin glycoprotein immunostains in distinguishing PIMA and CRC. DESIGN: We stained tissue microarrays comprising 34 PNMA, 31 PIMA, and 32 CRC with CK7, CK20, SATB2, CDX2, villin, TTF-1, napsin A, and gel-forming mucins MUC2, MUC5AC, and MUC6. RESULTS: PIMA showed significant (>50% of cells) expression of SATB2 (6%), CDX2 (6%), villin (74%), TTF-1 (13%), and napsin A (23%). However, significant CK7 expression was seen in nearly all PIMA (30/31) and none of the metastatic CRC. CONCLUSION: Our results suggest that CK7 remains one of the most useful markers for distinguishing primary PIMA from metastatic CRC. Expression of the mucin glycoproteins MUC5AC and MUC6 and lack of expression of MUC2 favored a diagnosis of PIMA, but expression of these markers was too heterogeneous to be of clinical utility. To our knowledge this is the only study comparing the immunohistochemical profile of PIMA and metastatic CRC in lung metastasectomy specimens.

KLK7 expression in human tumors: a tissue microarray study on 13,447 tumors.

BACKGROUND: Kallikrein-related peptidase 7 (KLK7) is a chymotrypsin-like serine protease which is essential for the desquamation of corneocytes and thus plays a pivotal role in maintaining skin homeostasis. In cancer, KLK7 overexpression was suggested to represent a route for metastasis through cleavage of cell junction and extracellular matrix proteins of cancer cells. METHODS: To comprehensively determine KLK7 protein expression in normal and neoplastic tissues, a tissue microarray containing 13,447 samples from 147 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. RESULTS: KLK7 positivity was found in 64 of 147 tumor categories, including 17 tumor categories with at least one strongly positive case. The highest rate of KLK7 positivity was found in squamous cell carcinomas from various sites of origin (positive in 18.1%-63.8%), ovarian and endometrium cancers (4.8%-56.2%), salivary gland tumors (4.8%-13.7%), bilio-pancreatic adenocarcinomas (20.0%-40.4%), and adenocarcinomas of the upper gastrointestinal tract (3.3%-12.5%). KLK7 positivity was linked to nodal metastasis (p = 0.0005), blood vessel infiltration (p = 0.0037), and lymph vessel infiltration (p < 0.0001) in colorectal adenocarcinoma, nodal metastasis in hepatocellular carcinoma (p = 0.0382), advanced pathological tumor stage in papillary thyroid cancer (p = 0.0132), and low grade of malignancy in a cohort of 719 squamous cell carcinomas from 11 different sites of origin (p < 0.0001). CONCLUSIONS: These data provide a comprehensive overview on KLK7 expression in normal and neoplastic human tissues. The prognostic relevance of KLK7 expression and the possible role of KLK7 as a drug target need to be further investigated.

Unsupervised representation learning of chromatin images identifies changes in cell state and tissue organization in DCIS.

Ductal carcinoma in situ (DCIS) is a pre-invasive tumor that can progress to invasive breast cancer, a leading cause of cancer death. We generate a large-scale tissue microarray dataset of chromatin images, from 560 samples from 122 female patients in 3 disease stages and 11 phenotypic categories. Using representation learning on chromatin images alone, without multiplexed staining or high-throughput sequencing, we identify eight morphological cell states and tissue features marking DCIS. All cell states are observed in all disease stages with different proportions, indicating that cell states enriched in invasive cancer exist in small fractions in normal breast tissue. Tissue-level analysis reveals significant changes in the spatial organization of cell states across disease stages, which is predictive of disease stage and phenotypic category. Taken together, we show that chromatin imaging represents a powerful measure of cell state and disease stage of DCIS, providing a simple and effective tumor biomarker.

Tissue Microarray Immunohistochemical Staining for Androgen Receptor in Breast Cancer in a Ghanaian Cohort.

BACKGROUND: Despite the advancement in therapy, breast cancer still remains the most common malignancy in women globally due in part to its heterogeneity. Triple-negative breast cancer (TNBC) represents up to 20% of all breast cancer variants, an aggressive disease with poorer outcomes compared to other breast cancer subtypes. No targeted therapies are currently approved for TNBC, and newer treatment approaches are seriously needed. Androgen receptor (AR), another hormonal receptor, is often expressed in breast cancer, and its role depends on the relative levels of circulating estrogens and androgens. This study aimed to assess the expression of AR in breast cancer in a tertiary hospital in Ghana. METHODOLOGY: Immunohistochemical staining for AR was performed on tissue microarray (TMA) blocks, of which estrogen receptor, progesterone receptor, and Her-2/neu had already been done. 197 cases were suitable for the study. Results from the immunostaining were analyzed using the SPSS version 23 for descriptive statistics and correlations (χ2 and Pearson tests). RESULTS: 197 TMA cases were used. TNBCs constitute 61.9% of the cancers. The majority of these tumors were grade III, ductal carcinoma NST. The mean age was 49.86 ± 14.09, and the modal age group was 40-49 years. Our cases showed 23% AR expression in triple-negative cancers. The study also established that AR is more frequently expressed in low-grade tumors compared to high-grade ones. CONCLUSION: There is an appreciable level of AR expression in our cases; however, most are quadruple negative. However, AR is more frequently expressed in low-grade tumors than high-grade ones.

Résumé Contexte:Malgré les progrès thérapeutiques, le cancer du sein reste la tumeur maligne la plus courante chez les femmes dans le monde, en partie à cause de son hétérogénéité. Le cancer du sein triple négatif (CSTN) représente jusqu'à 20 % de toutes les variantes du cancer du sein, une maladie agressive dont les résultats sont moins bons que les autres sous-types de cancer du sein. Aucun traitement ciblé n'est actuellement approuvé pour le TNBC et de nouvelles approches thérapeutiques sont sérieusement nécessaires. Le récepteur aux androgènes (AR), un autre récepteur hormonal, est souvent exprimé dans le cancer du sein et son rôle dépend des niveaux relatifs d'œstrogènes et d'androgènes en circulation. Cette étude vise à évaluer l'expression de la RA dans le cancer du sein dans un hôpital tertiaire du Ghana.Méthodes:La coloration immunohistochimique pour AR a été réalisée sur des blocs de micropuces tissulaires (TMA) dont ER, PR, Her-2/neu avaient déjà été réalisés. 197 cas convenaient à l'étude. Les résultats de l'immunocoloration ont été analysés à l'aide de SPSS version 23 pour les statistiques descriptives et les corrélations (tests X2 et Pearson).Résultats:197 cas de TMA ont été utilisés. Les TNBC constituent 61,9 % des cancers. La majorité de ces tumeurs étaient des carcinomes canalaires NST de grade III. L'âge moyen était de 49,86 ± 14,09 et la tranche d'âge modale était celle des 40-49 ans. Nos cas ont montré une expression de 23 % de AR dans les cancers triples négatifs. L'étude a également établi que la RA est plus fréquemment exprimée dans les tumeurs de bas grade que dans celles de haut grade.Conclusion:Il existe un niveau appréciable d'expression des AR dans nos cas mais la plupart sont quadruple négatifs. Cependant, la RA est plus fréquemment exprimée dans les tumeurs de bas grade que dans celles de haut grade.

Advantages of automated immunostain analyses for complex membranous immunostains: An exemplar investigating loss of E-cadherin expression in colorectal cancer.

As pathology moves towards digitisation, biomarker profiling through automated image analysis provides potentially objective and time-efficient means of assessment. This study set out to determine how a complex membranous immunostain, E-cadherin, assessed using an automated digital platform fares in comparison to manual evaluation in terms of clinical correlations and prognostication. Tissue microarrays containing 1000 colorectal cancer samples, stained with clinical E-cadherin antibodies were assessed through both manual scoring and automated image analysis. Both manual and automated scores were correlated to clinicopathological and survival data. E-cadherin data generated through digital image analysis was superior to manual evaluation when investigating for clinicopathological correlations in colorectal cancer. Loss of membranous E-cadherin, assessed on automated platforms, correlated with: right sided tumours (p = <0.001), higher T-stage (p = <0.001), higher grade (p = <0.001), N2 nodal stage (p = <0.001), intramural lymphovascular invasion (p = 0.006), perineural invasion (p = 0.028), infiltrative tumour edge (p = 0.001) high tumour budding score (p = 0.038), distant metastasis (p = 0.035), and poorer 5-year (p= 0.042) survival status. Manual assessment was only correlated with higher grade tumours, though other correlations become apparent only when assessed for morphological expression pattern (circumferential, basolateral, parallel) irrespective of intensity. Digital assessment of E-cadherin is effective for prognostication of colorectal cancer and may potentially offer benefits of improved objectivity, accuracy, and economy of time. Incorporating tools to assess patterns of staining may further improve such digital assessment in the future.

Full-Automatic High-Efficiency Mueller Matrix Microscopy Imaging for Tissue Microarray Inspection.

This paper proposes a full-automatic high-efficiency Mueller matrix microscopic imaging (MMMI) system based on the tissue microarray (TMA) for cancer inspection for the first time. By performing a polar decomposition on the sample's Mueller matrix (MM) obtained by a transmissive MMMI system we established, the linear phase retardance equivalent waveplate fast-axis azimuth and the linear phase retardance are obtained for distinguishing the cancerous tissues from the normal ones based on the differences in their polarization characteristics, where three analyses methods including statistical analysis, the gray-level co-occurrence matrix analysis (GLCM) and the Tamura image processing method (TIPM) are used. Previous MMMI medical diagnostics typically utilized discrete slices for inspection under a high-magnification objective (20×-50×) with a small field of view, while we use the TMA under a low-magnification objective (5×) with a large field of view. Experimental results indicate that MMMI based on TMA can effectively analyze the pathological variations in biological tissues, inspect cancerous cervical tissues, and thus contribute to the diagnosis of postoperative cancer biopsies. Such an inspection method, using a large number of samples within a TMA, is beneficial for obtaining consistent findings and good reproducibility.

Correlation of RANK and RANKL with mammographic density in primary breast cancer patients.

PURPOSE: The receptor activator of nuclear factor kappa B (RANK) and its ligand (RANKL) have been shown to promote proliferation of the breast and breast carcinogenesis. The objective of this analysis was to investigate whether tumor-specific RANK and RANKL expression in patients with primary breast cancer is associated with high percentage mammographic density (PMD), which is a known breast cancer risk factor. METHODS: Immunohistochemical staining of RANK and RANKL was performed in tissue microarrays (TMAs) from primary breast cancer samples of the Bavarian Breast Cancer Cases and Controls (BBCC) study. For RANK and RANKL expression, histochemical scores (H scores) with a cut-off value of > 0 vs 0 were established. PMD was measured in the contralateral, non-diseased breast. Linear regression models with PMD as outcome were calculated using common predictors of PMD (age at breast cancer diagnosis, body mass index (BMI) and parity) and RANK and RANKL H scores. Additionally, Spearman rank correlations (ρ) between PMD and RANK and RANKL H score were performed. RESULTS: In the final cohort of 412 patients, breast cancer-specific RANK and RANKL expression was not associated with PMD (P = 0.68). There was no correlation between PMD and RANK H score (Spearman's ρ = 0.01, P = 0.87) or RANKL H score (Spearman's ρ = 0.04, P = 0.41). RANK expression was highest in triple-negative tumors, followed by HER2-positive, luminal B-like and luminal A-like tumors, while no subtype-specific expression of RANKL was found. CONCLUSION: Results do not provide evidence for an association of RANK and RANKL expression in primary breast cancer with PMD.

Detailed Profiling of the Tumor Microenvironment in Ethnic Breast Cancer, Using Tissue Microarrays and Multiplex Immunofluorescence.

Breast cancer poses a global health challenge, yet the influence of ethnicity on the tumor microenvironment (TME) remains understudied. In this investigation, we examined immune cell infiltration in 230 breast cancer samples, emphasizing diverse ethnic populations. Leveraging tissue microarrays (TMAs) and core samples, we applied multiplex immunofluorescence (mIF) to dissect immune cell subtypes across TME regions. Our analysis revealed distinct immune cell distribution patterns, particularly enriched in aggressive molecular subtypes triple-negative and HER2-positive tumors. We observed significant correlations between immune cell abundance and key clinicopathological parameters, including tumor size, lymph node involvement, and patient overall survival. Notably, immune cell location within different TME regions showed varying correlations with clinicopathologic parameters. Additionally, ethnicities exhibited diverse distributions of cells, with certain ethnicities showing higher abundance compared to others. In TMA samples, patients of Chinese and Caribbean origin displayed significantly lower numbers of B cells, TAMs, and FOXP3-positive cells. These findings highlight the intricate interplay between immune cells and breast cancer progression, with implications for personalized treatment strategies. Moving forward, integrating advanced imaging techniques, and exploring immune cell heterogeneity in diverse ethnic cohorts can uncover novel immune signatures and guide tailored immunotherapeutic interventions, ultimately improving breast cancer management.

Programmed death-ligand 1 expression in carcinoma of unknown primary.

We examined the expression of programmed death-ligand 1 (PD-L1) in carcinoma of unknown primary (CUP) and its potential implications. Tissue microarrays were constructed for 72 CUP cases (histologic subtypes: 22 adenocarcinoma, 15 poorly differentiated carcinoma, 19 squamous cell carcinoma, and 14 undifferentiated carcinoma; clinical subtype: favorable type 17 [23.6%], unfavorable type 55 [76.4%]), with immunohistochemical staining performed for PD-L1 (22C3, SP142, SP263, and 28 - 8), CK7, and CK20 to determine the association between staining results and clinicopathological parameters. In CUP, the PD-L1 positivity rate was 5.6-48.6% (tumor cells [TC] or tumor proportion score [TPS]: 5.6-36.1%, immune cell score [IC]: 8.3-48.6%, combined positive score [CPS]: 16.7%) using different cutoff values for 22C3 (TPS ≥ 1%, CPS ≥ 10), SP142 (TC ≥ 50%, IC ≥ 10%), SP263, and 28 - 8 (TC and IC ≥ 1%). PD-L1 SP142 TC and PD-L1 SP263 IC showed the lowest (5.6%) and highest (48.6%) positivity rates, respectively. The PD-L1 positivity rate did not significantly differ based on the histologic subtype, clinical subtype, or CK7/CK20 across clones. Considering TC κ ≥ 1%, TC κ ≥ 50%, IC κ ≥ 1%, and IC κ ≥ 10%, the PD-L1 positivity rate was TC = 4.2-36.1% and IC = 9.7-48.6%; the overall agreement between antibodies ranged from 69.4 to 93.1%, showing fair or better agreement (κ ≥ 0.21). In CUP, PD-L1 positivity varied depending on antibodies and scoring systems, with no difference observed according to histologic or clinical subtypes.

Temporal and spatial heterogeneity of HER2 status in metastatic colorectal cancer.

BACKGROUND: HER2-targeted therapies have recently emerged as an option in the management of metastatic colorectal cancer (mCRC) overexpressing HER2. However, data regarding HER2 status in primary CRC and its corresponding liver metastases are limited, potentially influencing clinical decisions. Therefore, the aim of this study was to compare the HER2 status in primary CRC and paired liver metastases. METHODS: Patients with mCRC who were operated from their primary colorectal cancer and their corresponding synchronous or metachronous liver metastases, in the digestive surgery department of Besançon University Hospital, between April 1999 and October 2021, were included. Tissue microarrays were constructed from matched primary CRC and liver metastastic tissue samples. HER2 status was assessed by immunohistochemistry and in situ hybridization according to Valtorta's criteria. RESULTS: A series of 108 paired primary CRC and liver metastases, including a series of multiple liver metastases originating from the same patients (n = 24), were assessed. Among the primary CRC, 89 (82.4%), 17 (15.8%) and 2 (1.8%) cases were scored 0, 1 + and 2 + respectively. In liver metastases, 99 (91.7%), 7 (6.5%) and 2 (1.8%) were scored 0, 1 + and 2, respectively. Overall, there was a 19% discrepancy rate in HER2 status between primary CRC and metastases, which increased to 21% in cases with multiple synchronous or metachronous liver metastases in a given patient. No significant difference was found between metachronous and synchronous metastases regarding the HER2 status (p = 0.237). CONCLUSIONS: Our study highlights the temporal and spatial heterogeneity of HER2 status between primary CRC and corresponding liver metastases. These findings raise the question of a sequential evaluation of the HER2 status during disease progression, to provide the most suitable treatment strategy.

EPM2AIP1 immunohistochemistry is inadequate as a surrogate marker for MLH1 promoter hypermethylation testing in colorectal cancer.

MLH1 promoter hypermethylation (MPH) analysis is an essential step in the universal tumor testing algorithm for Lynch syndrome, the most common inherited predisposition to colorectal cancer (CRC). MPH usually indicates sporadic CRC. EPM2AIP1 gene shares the same promoter as MLH1, therefore MPH should also silence EPM2AIP1 transcription leading to loss of protein expression on immunohistochemistry (IHC). It has been previously reported that EPM2AIP1 IHC can be used as a surrogate for MPH in endometrial cancer. Our goal was to evaluate the feasibility of EPM2AIP1 IHC as a surrogate for MPH in CRC. 101 microsatellite instable CRC cases were selected, including 19 cases from whole tumor sections and 82 cases from tissue microarrays. 74 cases were with MPH and 27 without MPH. All 74 cases with MPH showed absent MLH1 by IHC, but only 47 (64%) exhibited loss of expression of EPM2AIP1. Of the 27 cases without MPH, 9 (33%) cases had unexpected loss of EPM2AIP1 expression. Of note, 10 cases were MLH1-mutated Lynch syndrome without MPH, and 2 of these cases showed unexpected loss of EPM2AIP1 staining. Of the 6 cases with double somatic mutations of MLH1 gene (without MPH), only 4 cases demonstrated intact expression of EPM2AIP1 as expected. Taken together, EPM2AIP1 loss was 64% sensitive and 67% specific for MPH, with an accuracy of 64%. We conclude that, unless stain quality improves with different clones or platforms, EPM2AIP1 IHC will likely not be useful as a surrogate test for MPH in CRC.

The Prognostic Significance of Collagen VI in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and lack of biomarkers. A rich desmoplastic tumor stroma is considered a hallmark of PDAC and previous studies have indicated upregulated expression of collagen VI (COL6) in PDAC. COL6 is shown to associate with prognosis in many cancers but has been less extensively studied in PDAC. MATERIALS AND METHODS: The expression of COL6 was analyzed by immunohistochemistry in tissue microarrays containing resected tumor tissue samples from PDAC patients (n = 164). Significance of COL6 was estimated with Kaplan-Meier survival estimates and multivariable Cox regression analysis. COL6 protein and mRNA expression patterns were further investigated in publicly available datasets. RESULTS: There were no statistically significant ( P < 0.05) differences in survival when comparing high and low protein expression of any of the analyzed COL6 α-chains (α1(VI): hazard ratio [HR] 0.90, 95% confidence interval [CI] 0.64-1.28; α2(VI): HR 1.28, 95% CI 0.86-1.89; α3(VI): HR 0.91, 95% CI 0.64-1.29). Similar results were obtained when assessing public data from the Cancer Proteome Atlas, Clinical Proteomic Tumor Analysis Consortium, and The Cancer Genome Atlas. CONCLUSIONS: In contrast with previous studies and some other cancers, we did not find any association of COL6 tissue expression and PDAC survival.

Topographic analysis of pancreatic cancer by TMA and digital spatial profiling reveals biological complexity with potential therapeutic implications.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human malignancies. Tissue microarrays (TMA) are an established method of high throughput biomarker interrogation in tissues but may not capture histological features of cancer with potential biological relevance. Topographic TMAs (T-TMAs) representing pathophysiological hallmarks of cancer were constructed from representative, retrospective PDAC diagnostic material, including 72 individual core tissue samples. The T-TMA was interrogated with tissue hybridization-based experiments to confirm the accuracy of the topographic sampling, expression of pro-tumourigenic and immune mediators of cancer, totalling more than 750 individual biomarker analyses. A custom designed Next Generation Sequencing (NGS) panel and a spatial distribution-specific transcriptomic evaluation were also employed. The morphological choice of the pathophysiological hallmarks of cancer was confirmed by protein-specific expression. Quantitative analysis identified topography-specific patterns of expression in the IDO/TGF-ß axis; with a heterogeneous relationship of inflammation and desmoplasia across hallmark areas and a general but variable protein and gene expression of c-MET. NGS results highlighted underlying genetic heterogeneity within samples, which may have a confounding influence on the expression of a particular biomarker. T-TMAs, integrated with quantitative biomarker digital scoring, are useful tools to identify hallmark specific expression of biomarkers in pancreatic cancer.

CYP7B1 as a Biomarker for Prostate Cancer Risk and Progression: Metabolic and Oncogenic Signatures (Diagnostic Immunohistochemistry Analysis by Tissue Microarray in Prostate Cancer Patients-Diamond Study).

We aimed to analyze the association between CYP7B1 and prostate cancer, along with its association with proteins involved in cancer and metabolic processes. A retrospective analysis was performed on 390 patients with prostate cancer (PC) or benign prostatic hyperplasia (BPH). We investigated the interactions between CYP7B1 expression and proteins associated with PC and metabolic processes, followed by an analysis of the risk of biochemical recurrence based on CYP7B1 expression. Of the 139 patients with elevated CYP7B1 expression, 92.8% had prostate cancer. Overall, no increased risk of biochemical recurrence was associated with CYP7B1 expression. However, in a non-diabetic subgroup analysis, higher CYP7B1 expression indicated a higher risk of biochemical recurrence, with an HR of 1.78 (CI: 1.0-3.2, p = 0.05). PC is associated with elevated CYP7B1 expression. In a subgroup analysis of non-diabetic patients, elevated CYP7B1 expression was associated with an increased risk of biochemical recurrence, suggesting increased cancer aggressiveness.

Staining pattern of specific and cross-reacting Melan-A antibodies: A comparative study on 15,840 samples from 133 human tumor types.

The Melan-A (melanocyte antigen) protein, also termed 'melanoma antigen recognized by T cells 1' (MART-1) is a protein with unknown function whose expression is specific for the melanocyte lineage. Antibodies against Melan-A are thus used for identifying melanocytic tumors, but some Melan-A antibodies show an additional - diagnostically useful - cross-reactivity against an unspecified protein involved in corticosteroid hormone synthesis. To comprehensively compare the staining patterns of a specific and a cross-reactive Melan-A antibody in normal and neoplastic tissues, tissue microarrays containing 15,840 samples from 133 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry. For the Melan-A-specific antibody 'Melan-A specific' (MSVA-900M), Melan-A positivity was seen in 96.0% of 25 benign nevi, 93.0% of 40 primary and 86.7% of 75 metastatic melanomas, 82.4% of 85 renal angiomyolipomas as well as 96.4% of 84 neurofibromas, 2.2% of 46 granular cell tumors, 1.0% of 104 schwannomas, and 1.1% of 87 leiomyosarcomas. The cross-reactive antibody 'Melan-A+' (MSVA-901M+) stained 98.1% of the tumors stained by 'Melan-A specific'. In addition, high positivity rates were seen in sex-cord-stroma tumors of the ovary (35.3%-100%) and the testis (86.7%) as well as for adrenocortical neoplasms (76.3%-83.0%). Only nine further tumor groups showed Melan-A+ staining, including five different categories of urothelial carcinomas. Our data provide a comprehensive overview on the staining patterns of specific and cross-reactive Melan-A antibodies. The data demonstrate that both antibodies are highly useful for their specific purpose. It is important for pathologists to distinguish these two Melan-A antibody subtypes for their daily work.