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Purpose: High-grade serous ovarian cancer (HGSC) is the most common ovarian cancer subtype. Parity is an important risk-reducing factor, but the underlying mechanism behind the protective effect is unclear. Our aim was to study if the expression of hormones and proteins involved in pregnancy were affected by the woman's parity status, and if they may be associated with tumor stage and survival. Methods: We evaluated expression of progesterone receptor (PR), progesterone receptor membrane component 1 (PGRMC1), relaxin-2, and transforming growth factor beta 1 (TGFβ1) in tumor tissue from 92 women with HGSC parous (n = 73) and nulliparous (n = 19). Key findings were then evaluated in an independent expansion cohort of 49 patients. Survival rates by hormone/protein expression were illustrated using the Kaplan–Meier method. The independent prognostic value was tested by Cox regression, using models adjusted for established poor-prognostic factors (age at diagnosis, FIGO stage, type of surgery, and macroscopic residual tumor after surgery). Results: HGSC tumors from parous women were PR positive (≥ 1% PR expression in tumor cells) more often than tumors from nulliparous women (42% vs. 16%; p-value 0.04), and having more children was associated with developing PR positive tumors [i.e., ≥ 3 children versus nulliparity, adjusted for age at diagnosis and stage: OR 4.31 (95% CI 1.12–19.69)]. A similar result was seen in the expansion cohort. Parity status had no impact on expression of PGRMC1, relaxin-2 and TGFβ1. No associations were seen with tumor stage or survival. Conclusion: Tumors from parous women with HGSC expressed PR more often than tumors from nulliparous women, indicating that pregnancies might possibly have a long-lasting impact on ovarian cancer development. [ABSTRACT FROM AUTHOR]

Uroplakin 3B (Upk3b) is involved in stabilizing and strengthening the urothelial cell layer of the bladder. Based on RNA expression studies, Upk3b is expressed in a limited number of normal and tumor tissues. The potential use of Upk3b as a diagnostic or prognostic marker in tumor diagnosis has not yet been extensively investigated. A tissue microarray containing 17,693 samples from 151 different tumor types/subtypes and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. In normal tissues, Upk3b expression was largely limited to mesothelial cells, urothelial umbrella cells, and amnion cells. In tumor tissues, Upk3b was detectable in only 17 of 151 (11.3%) of tumor types. Upk3b expression was most frequent in mesotheliomas (82.1% of epithelioid and 30.8% of biphasic) and in urothelial tumors of the urinary bladder, where the positivity rate decreased from 61.9% in pTaG2 (low grade) to 58.0% in pTaG3 (high grade) and 14.6% in pT2-4 cancers. Among pT2-4 urothelial carcinomas, Upk3b staining was unrelated to tumor stage, lymph node status, and patient prognosis. Less commonly, Upk3b expression was also seen in Brenner tumors of the ovary (10.8%), as well as in four other subtypes of ovarian cancer (0.9–10.6%). Four additional tumor entities showed a weak to moderate Upk3b positivity in less than 5% of cases. In summary, Upk3b immunohistochemistry is a useful diagnostic tool for the distinction of mesotheliomas from other thoracic tumors and the visualization of normal mesothelial and umbrella cells. [ABSTRACT FROM AUTHOR]

As a result of its expression in corresponding normal cell types, inhibin alpha (INHA) is used as an immunohistochemical marker for adrenocortical neoplasms and testicular or ovarian sex cord stromal tumors. However, other tumors can also express INHA. To comprehensively determine INHA expression in cancer, a tissue microarray containing 15,012 samples from 134 different tumor types and subtypes was analyzed by immunohistochemistry. INHA positivity was found in 72 of 134 tumor categories, including 26 categories with ≥1 strongly positive case. A moderate to strong INHA positivity was found in 100% of 37 granulosa cell tumors of the ovary, 100% of 43 other sex cord stromal tumors of the ovary/testis, 100% of 31 granular cell tumors, 78.5% of 28 adenomas, 44% of 25 carcinomas of the adrenal cortex, and 46.7% of 15 pancreatic acinar cell carcinomas. At least a weak INHA positivity was seen in <33% of cases of 46 additional tumor entities. In summary, these data support the use of INHA antibodies for detecting sex cord stromal tumors, granular cell tumors, and adrenocortical neoplasms. Since INHA can also be found in other tumor entities, INHA immunohistochemistry should only be considered as a part of any panel for the distinction of tumor entities. [ABSTRACT FROM AUTHOR]

Background. DOG1 (ANO1; TMEM16A) is a voltage-gated calcium-activated chloride and bicarbonate channel. DOG1 is physiologically expressed in Cajal cells, where it plays an important role in regulating intestinal motility and its expression is a diagnostic hallmark of gastrointestinal stromal tumors (GIST). Data on a possible role of DOG1 in pancreatic cancer are rare and controversial. The aim of our study was to clarify the prevalence of DOG1 expression in pancreatic cancer and to study its association with parameters of cancer aggressiveness. Methods. DOG1 expression was analyzed by immunohistochemistry in 599 pancreatic cancers in a tissue microarray format and in 12 cases of pancreatitis on large tissue sections. Results. DOG1 expression was always absent in normal pancreas but a focal weak expression was seen in four of 12 cases of pancreatitis. DOG1 expression was, however, common in pancreatic cancer. Membranous and cytoplasmic DOG1 expression in tumor cells was highest in pancreatic ductal adenocarcinomas (61% of 444 interpretable cases), followed by cancers of the ampulla Vateri (43% of 51 interpretable cases), and absent in 6 acinus cell carcinomas. DOG1 expression in tumor associated stroma cells was seen in 76 of 444 (17%) pancreatic ductal adenocarcinomas and in seven of 51 (14%) cancers of the ampulla Vateri. Both tumoral and stromal DOG1 expression were unrelated to tumor stage, grade, lymph node and distant metastasis, mismatch repair protein deficiency and the density of CD8 positive cytotoxic T-lymphocytes in the subgroups of ductal adenocarcinomas and cancers of ampulla Vateri. Overall, the results of our study indicate that DOG1 may represent a potential biomarker for pancreatic cancer diagnosis and a putative therapeutic target in pancreatic cancer. However, DOG1 expression is unrelated to pancreatic cancer aggressiveness. [ABSTRACT FROM AUTHOR]

Background: Epithelial splicing regulatory protein 1 (ESRP1) and 2 (ESRP2) regulate alternative splicing events of various pre-mRNAs. Some of these targets play a role in cancer-associated processes, including cytoskeleton reorganization and DNA-repair processes. This study was undertaken to estimate the impact of ESRP1 and ESRP2 alterations on prostate cancer patient prognosis.Methods: A tissue microarray made from 17,747 individual cancer samples with comprehensive, pathological, clinical and molecular data was analyzed by immunohistochemistry for ESRP1 and ESRP2.Results: Nuclear staining for ESRP1 was seen in 38.6% (36.0% low, 2.6% high) of 12,140 interpretable cancers and in 41.9% (36.4% low, 5.3% high) of 12,962 interpretable cancers for ESRP2. Nuclear protein expression was linked to advanced tumor stage, high Gleason score, presence of lymph node metastasis, early biochemical recurrence, and ERG-positive cancers (p < 0.0001 each). Expression of ESRPs was significantly linked to 11 (ESRP1)/9 (ESRP2) of 11 analyzed deletions in all cancers and to 8 (ESRP1)/9 (ESRP2) of 11 deletions in ERG-negative cancers portending a link to genomic instability. Combined ESRPs expression analysis suggested an additive effect and showed the worst prognosis for cancers with high ESRP1 and ESRP2 expression. Multivariate analyses revealed that the prognostic impact of ESRP1, ESRP2 and combined ESRP1/ESRP2 expression was independent of all established pre- and postoperative prognostic features.Conclusions: Our data show a striking link between nuclear ESRP expression and adverse features in prostate cancer and identifies expression of ESRP1 and/or ESRP2 as independent prognostic markers with a potential for routine application. [ABSTRACT FROM AUTHOR]

SALL4 is a transcription factor originally identified as a homeotic gene essential for organ development. Early studies suggested that SALL4 is a useful marker to identify testicular and ovarian germ cell tumors. The aim of the study was to evaluate the diagnostic potential of SALL4 immunohistochemistry. Immunohistochemical staining was performed on a tissue microarray (TMA) with 3966 samples from 94 different tumor types and on a further TMA with 492 esophagus carcinomas. SALL4 immunostaining was by far most prevalent and most intensive in testicular tumors with a positivity rate of 93.1% in seminomas, 80% in mixed germ cell tumors (embryonic carcinomas/yolk sac tumors), and 18.5% in teratomas, respectively. However, SALL4 expression is not specific to germ cell tumors. We observed SALL4 positivity in non-germ cell tumors as carcinomas of the kidney (28.9% of chromophobe, 34.4% of clear cell carcinoma), in intestinal type adenocarcinoma of the stomach (10.9%), in adenocarcinoma (10.5%) and squamous cell carcinoma (7.2%) of the esophagus, and in malignant melanoma (8.1%) and invasive urothelial bladder carcinoma (20%). SALL4 expression was not found in lymphomas, in soft tissue tumors or breast tumors. At analysis of esophagus carcinoma TMA, no significant association was seen between SALL4 expression and overall survival in adenocarcinoma. However, SALL4 expression was strongly associated with worse overall survival in squamous cell carcinoma. SALL4 expression can be found at relevant frequencies in various tumors of different primary sites. SALL4 expression in squamous cell carcinoma of the esophagus may constitute a sign of dedifferentiation leading to poor patient prognosis. [ABSTRACT FROM AUTHOR]

A simple modification of the cell block technique for cultured cells grown in different conditions and suitable for the construction of tissue micro arrays (TMA) is described. The application of mechanical stirring during clot formation allows the uniform dispersion of cells in the fibrin mesh, thus increasing the final volume of the embedded material of evenly distributed cells. This technique is easily applied to spheres—obtained from cell lines cultured under appropriate conditions—that are enriched in stem cells. The possibility of constructing TMA using cell lines (grown in adherence and as spheres) and samples of the corresponding tumors or normal tissues may allow the direct comparison of original tumors with in vitro-expanded cell lines. [ABSTRACT FROM AUTHOR]

Tissue micro array ( TMA) is widely used in cancer research in search of new predictive and prognostic markers. Colon cancer is known to be heterogeneous and the present study addresses some methodological aspects using cores of different size and analysing markers with different cellular distribution. We selected 61 paraffin-embedded tissue blocks representing patients diagnosed with Dukes B colon cancer. Two 1 mm and two 2 mm cores were taken from both the centre and the invasive front of the tumour respectively. The immunostaining included MLH1, MSH2, PMS2, p53, COX-2, TIMP and Betacatenin. Twenty-five percent of the cores taken from paraffin blocks less than 0.5 cm was lost and the total loss was 8%. The homogeneous stains ( MLH1, MSH2 and PMS2) all showed high agreement between TMA and whole tissue stains (kappa = 0.96,1 and 1 respectively). The COX-2, p53 and Betacatenin illustrated moderate to high agreement (kappa = 0.54-0.9) whereas TIMP-1 had the lowest score (kappa 0.19-0.25). The application of TMA in Dukes B colon cancer has several pitfalls and depends substantially on the immunohistochemical marker in question. Therefore a validation study seems justified before applying large scale TMA in this setting. [ABSTRACT FROM AUTHOR]

Background: Formaldehyde (HCHO) is a gas (available as a 37% concentrated solution, stabilized with methanol). The 10% dilution (approximately 4% formaldehyde) has been used as a fixative since the end of the 19th century. Alternative fixatives are also commercially available or may be prepared in-house in laboratories. Statements by the IARC, along with other USA agencies (CalEPA, RoC/NTP) on the carcinogenicity of formaldehyde for humans renders its substitution in Pathology Departments necessary since the annual use of formalin may exceed 3,500 liters for a medium-large laboratory. To achieve a "formalin-free laboratory" we tested straightforward-to-make fixatives along with registered reagents offered as formalin substitutes. Methods: More than two hundreds specimens were fixed in parallel with in-laboratory made fixatives PAGA (Polyethylenglycol, ethyl Alcohol, Glycerol, Acetic acid), two zinc-based fixatives (ZBF, Z7), and commerciallyavailable alternatives (RCL2 and CellBlock). Tissue micro arrays were used for morphological and immunohistochemical comparison. Extraction of RNA was carried out to evaluate preservation of nucleic acids. Results: Differences compared to formalin fixation were evident in alcohol-based fixatives, mainly restricted to higher stain affinity and considerable tissue shrinkage. Conversely, nuclear detail was superior with these alcohol-based formulas compared to formalin or glyoxale-based recipes. RNA extraction was superior for Z7, PAGA and RCL2 with regard to concentration but relatively comparable regarding quality. Conclusions: Abolition of the human carcinogen formaldehyde from pathology laboratories is possible even in contexts whereby commercial alternatives to formalin are unavailable or are too expensive for routine use, and aspiration devices are lacking or not adequately serviced. The use of known formulations, possibly with simple and not-noxious ("alimentary grade") constituents, comparable with registered proprietary products, may expand the search for the ideal fixative combining satisfactory morphology with improved preservation of nucleic acids and proteins as well as being easy and safe to dispose of. [ABSTRACT FROM AUTHOR]

Arginase-1 catalyzes the conversion of arginine to ornithine and urea. Because of its predominant expression in hepatocytes, it serves as a marker for hepatocellular carcinoma, although other tumor entities can also express arginase-1. To comprehensively determine arginase-1 expression in normal and neoplastic tissues, tissue microarrays containing 14,912 samples from 117 different tumor types and 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry. In normal tissues, arginase-1 was expressed in the liver, the granular layer of the epidermis, and in granulocytes. Among tumors, a nuclear and cytoplasmic arginase-1 immunostaining was predominantly observed in hepatocellular carcinoma, where 96% of 49 cancers were at least moderately positive. Although 22 additional tumor categories showed occasional arginase immunostaining, strong staining was exceedingly rare in these entities. Staining of a few tumor cells was observed in squamous cell carcinomas of various sites. Staining typically involved maturing cells with the beginning of keratinization in these tumors and was significantly associated with a low grade in 635 squamous cell carcinomas of various sites (p = 0.003). Teratoma, urothelial carcinoma and pleomorphic adenomas sometimes also showed arginase expression in areas with squamous differentiation. In summary, arginase-1 immunohistochemistry is highly sensitive and specific for hepatocellular carcinoma if weak and focal staining is disregarded. [ABSTRACT FROM AUTHOR]

Background Targeting Her2 is highly established in cancers of the upper gastrointestinal tract, since clinical studies showed a survival benefit in Her2-positive gastric cancer. To date there is conflicting data concerning the impact of Her2 expression and gene amplification in esophageal adenocarcinoma (EAC), as most studies do not differ between cancers of the esophagus, gastroesophageal junction and stomach. The aim of this study was to analyze the expression and gene amplification of Her2 in EAC in correlation to clinical and pathological data to verify the prognostic impact. Methods We analyzed 506 patients with esophageal adenocarcinomas that underwent thoraco-abdominal esophagectomy between 1997 and 2013 using a tissue micro array (TMA) for the expression of Her2 by immunohistochemistry (IHC) according to the guidelines. Fluorescence-in-situ-hybridization (FISH) was performed for IHC score 2. The Her2-status was correlated with clinical and pathological data. Results Her2-positivity was found in 11.7% (n = 59) of all EAC (IHC score 3 + or IHC score 2 + with amplification) and demonstrated a significant better overall-survival (OS) in correlation to Her2-negative tumors (median OS 55.0 vs. 22.7 months, P  < 0.0001) (Figure below). Overexpression of Her2 was associated with lower tumor stages (pT1/pT2, P  = 0.038), absence of lymph node metastases (pN0/pN + , P  = 0.030) and was significantly associated with tubular and/or papillary tumor growth pattern (P  = 0.029). View large Download slide View large Download slide Conclusion We demonstrated a positive prognostic impact of Her2 expression and gene amplification in a large cohort of EAC, contrary to other solid malignancies including gastric cancer, but according to a large study of EAC from 2012. Her2-positive tumors significantly have a tubular and/or papillary tumor growth pattern, indicating a marker of differentiation in EAC. Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background Immunotherapy has grown to a rapidly advancing sphere of research on modern strategies for treatment of cancer. A well-known way of immunomodulation is the principle of immune checkpoints leading to a down-regulated immune response in tumor microenvironment. Recently, beside the PD1/PD-L1 pathway, several immunecheckpoint pathways have been found. However, almost nothing is known about the expression and the correlation as prognostic factor in esophageal adenocarcinoma. Methods Two tissue micro arrays (TMA) were construced, a multi-spot TMA with up to 8 spots from different tumor areas of 165 patients and a valdiation single-spot TMA of 620 patients with EAC. Immunehistochemistry analysis were performed for LAG3, Tim3, IDO, VISTA, MHC1, MHC2, CD3, CD8, CD20 and CD38 and correlated with clinico-pathological and survival data. Data were additionally correlated with molecular data like TP53 mutational and HER2-neu status. Results LAG3 was detectable in 13.5% of all tumors, VISTA in 40.2%, IDO in 57.9%, Tim3 in 36.3%. There was only minor intratumoral heterogeneity between the luminal and infiltration area of the tumor for all analyzed immunecheckpoints. LAG3 and VISTA expression on tumor infiltrating T-cells (TIL) was associated with superior overall-survival compared to LAG3 and VISTA negative tumors. In pT1/2 tumors, VISTA was found as excellent independent prognosticator identifiying long-term surivors in case of VISTA expression. High amount of CD3 positive TILs was also associated with a better OS than CD3 poor tumors. Conclusion We conducted an extensive analysis of the immunecheckpoint status and presecnce of TILs in EAC. To the best of our knowledge, this is the first report in a large patients cohort correlating the expression of LAG3, Tim3, IDO and VISTA in EAC. The prognostic impact of LAG3 and VISTA expression suggests potential promising targets for novel immune checkpoint inhibition therapies beyond the PD1/PD-L1 inhibition as it is already established in head and neck cancer and NSCLC. Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background: Intra-tumoral heterogeneity has been recently addressed in different types of cancer, including breast cancer. A concept describing the origin of intra-tumoral heterogeneity is the cancer stem-cell hypothesis, proposing the existence of cancer stem cells that can self-renew limitlessly and therefore lead to tumor progression. Clonal evolution in accumulated single cell genomic alterations is a further possible explanation in carcinogenesis. In this study, we addressed the question whether intra-tumoral heterogeneity can be reliably detected in tissue-micro-arrays in breast cancer by comparing expression levels of conventional predictive/prognostic tumor markers, tumor progression markers and stem cell markers between central and peripheral tumor areas.Methods: We analyzed immunohistochemical expression and/or gene amplification status of conventional prognostic tumor markers (ER, PR, HER2, CK5/6), tumor progression markers (PTEN, PIK3CA, p53, Ki-67) and stem cell markers (mTOR, SOX2, SOX9, SOX10, SLUG, CD44, CD24, TWIST) in 372 tissue-micro-array samples from 72 breast cancer patients. Expression levels were compared between central and peripheral tumor tissue areas and were correlated to histopathological grading. 15 selected cases additionally underwent RNA sequencing for transcriptome analysis.Results: No significant difference in any of the analyzed between central and peripheral tumor areas was seen with any of the analyzed methods/or results that showed difference. Except mTOR, PIK3CA and SOX9 (nuclear) protein expression, all markers correlated significantly (p < 0.05) with histopathological grading both in central and peripheral areas.Conclusion: Our results suggest that intra-tumoral heterogeneity of stem-cell and tumor-progression markers cannot be reliably addressed in tissue-micro-array samples in breast cancer. However, most markers correlated strongly with histopathological grading confirming prognostic information as expression profiles were independent on the site of the biopsy was taken. [ABSTRACT FROM AUTHOR]

Context/Background: Tissue micro array (TMA) is a method of harvesting small disks of tissue from a range of standard paraffin tissue blocks and placing them in an array on a recipient paraffin block such that hundreds of cases can be analyzed simultaneously by using only a few microliters of antibody used for immunohistochemistry in a single experiment. The TMA construction done with the help of automated tissue arrayer or commercially available rubber moulds are expensive. Aims: This study involved the fabrication of TMA using rubber-based additional silicone mould constructed in the Department and comparing it with two earlier methods of TMA technique. Materials and Methods: The TMA mould was fabricated using silicone material in the Department. The recipient blocks were prepared. The tissue core prepared from donor blocks were inserted into the recipient blocks. The sections taken from this were compared with the TMA using double-sided adhesive tape and TMA by punching out holes in prefabricated dummy paraffin recipient block for insertion of tissue core. Results: The TMA using mould made of silicone showed more advantages than other two methods. Conclusion: Fabricating TMA mould using silicone in the Department is inexpensive and yet efficient. [ABSTRACT FROM AUTHOR]