Association between parity and pregnancy-associated tumor features in high-grade serous ovarian cancer.

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 (TGFß1) 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 TGFß1. 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.

Clinical, pathological, and comprehensive molecular analysis of the uterine clear cell carcinoma: a retrospective national study from TMRG and GINECO network.

BACKGROUND: Uterine clear cell carcinomas (CCC) represent less than 5% of uterine cancers. Their biological characteristics and clinical management remain uncertain. A multicenter study to explore both clinical and molecular features of these rare tumors was conducted. METHODS: This multicenter retrospective national study was performed within the French TMRG (Rare Gynecologic Malignant Tumors) network. Clinical data and, when available, FFPE blocks were collected. Clinical features, treatments, and outcome (progression-free survival (PFS) and overall survival (OS)) were analyzed and correlated to the protein (tissue micro-array), RNA (Nanostring nCounter® technology), and DNA (array-Comparative Genomic hybridization and target-next generation sequencing) levels using the tumor samples available. RESULTS: Sixty-eight patients with uterine CCC were enrolled, 61 from endometrial localization and 5 with cervix localization. Median age at diagnosis was 68.9 years old (range 19-89.7). Most tumors were diagnosed at an early stage (78% FIGO stage I-II). Hysterectomy (performed in 90%) and lymph node dissection (80%) were the most frequent surgical treatment. More than 70% of patients received external beam radiotherapy and 57% received brachytherapy. Nearly half (46%) of the patients received chemotherapy. After a median follow-up of 24.7 months, median PFS was 64.8 months (95 CI [5.3-124.4]) and median OS was 79.7 (IC95 [31.0-128.4]). Low hormone receptor expression (13% estrogen-receptor positive), frequent PI3K pathway alterations (58% PTEN loss, 50% PIK3CA mutations), and P53 abnormalities (41%) were observed. Mismatch repair deficiency was identified in 20%. P16 expression was associated with shorter PFS (HR = 5.88, 95 CI [1.56-25], p = 0.009). Transcriptomic analyzes revealed a specific transcriptomic profile notably with a high expression of immune response-associated genes in uterine CCC displaying a very good overall prognosis. CONCLUSIONS: Uterine CCC reported to be potentially MSI high, hormone receptors negative, and sometimes TP53 mutated. However, some patients with immune response-associated features and better prognosis may be candidate to treatment de-escalation and immunotherapy.

Cytokeratin 20 expression is linked to stage progression and to poor prognosis in advanced (pT4) urothelial carcinoma of the bladder.

Cytokeratin 20 (CK20) expression is limited to umbrella cells in the normal urothelium. Since CK20 is often upregulated in neoplastic urothelial cells including dysplasia and carcinoma in situ, immunohistochemical CK20 analysis is often used for the assessment of bladder biopsies. CK20 expression is a feature of luminal bladder cancer subtype, but its prognostic relevance is disputed. In this study, we investigated CK20 on >2700 urothelial bladder carcinomas in a tissue microarray format by immunohistochemistry. Cytoplasmic and membranous CK20 staining was seen in 1319 (51.8%) cancers. The fraction of CK20 positive and especially strongly positive cases increased from pTaG2 low grade (44.5% strongly positive) and pTaG2 high grade (57.7%) to pTaG3 high grade (62.3%; p = 0.0006) but was lower in muscle-invasive (pT2-4) carcinomas (51.1% in all pTa vs. 29.6% in pT2-4; p < 0.0001). Within pT2-4 carcinomas, CK20 positivity was linked to nodal metastasis and lymphatic vessel invasion (p < 0.0001 each) and to venous invasion (p = 0.0177). CK20 staining was unrelated to overall patient survival if all 605 pT2-4 carcinomas were jointly analyzed but subgroup analyses revealed a significant association of CK20 positivity with favorable prognosis in 129 pT4 carcinomas (p = 0.0005). CK20 positivity was strongly linked to the expression of GATA3 (p < 0.0001), another feature of luminal bladder cancer. The combined analysis of both parameters showed best prognosis for luminal A (CK20+/GATA3+, CK20+/GATA3-) and worst outcome for luminal B (CK20-/GATA3+) and basal/squamous (CK20-/GATA3-) in pT4 urothelial carcinomas (p = 0.0005). In summary, the results of our study demonstrate a complex role of CK20 expression in urothelial neoplasms including neoexpression in pTa tumors, a subsequent loss of CK20 expression in a subset of tumors progressing to muscle-invasion, and a stage dependent prognostic role in muscle-invasive cancers.

Tissue Microarray Lipidomic Imaging Mass Spectrometry Method: Application to the Study of Alcohol-Related White Matter Neurodegeneration.

Central nervous system (CNS) white matter pathologies accompany many diseases across the lifespan, yet their biochemical bases, mechanisms, and consequences have remained poorly understood due to the complexity of myelin lipid-based research. However, recent advances in matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) have minimized or eliminated many technical challenges that previously limited progress in CNS disease-based lipidomic research. MALDI-IMS can be used for lipid identification, semi-quantification, and the refined interpretation of histopathology. The present work illustrates the use of tissue micro-arrays (TMAs) for MALDI-IMS analysis of frontal lobe white matter biochemical lipidomic pathology in an experimental rat model of chronic ethanol feeding. The use of TMAs combines workload efficiency with the robustness and uniformity of data acquisition. The methods described for generating TMAs enable simultaneous comparisons of lipid profiles across multiple samples under identical conditions. With the methods described, we demonstrate significant reductions in phosphatidylinositol and increases in phosphatidylcholine in the frontal white matter of chronic ethanol-fed rats. Together with the use of a novel rapid peak alignment protocol, this approach facilitates reliable inter- and intra-group comparisons of MALDI-IMS data from experimental models and could be extended to human disease states, including using archival specimens.

Analysis of More than 16,000 Human Tumor and Normal Tissues Identifies Uroplakin 3B as a Useful Diagnostic Marker for Mesothelioma and Normal Mesothelial Cells.

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.

Inhibin Alpha Expression in Human Tumors: A Tissue Microarray Study on 12,212 Tumors.

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.

Large-scale human tissue analysis identifies Uroplakin 1a as a putative diagnostic marker for urothelial cancer.

Uroplakin 1A (Upk1a) protein is relevant for stabilizing and strengthening urothelial cells and helps to prevent them from rupturing during bladder distension. Based on RNA expression data Upk1a is expressed in a limited number of normal tissues and tumors. To comprehensively evaluate the potential diagnostic and prognostic utility of Upk1a immunohistochemistry, a tissue microarray containing 6929 samples from 115 different tumor types and subtypes and 608 samples of 76 different normal tissue types was analyzed. Upk1a positivity was found in 34 (29.6 %) different tumor types including 9 (7.8 %) tumor types with at least one strongly positive case. The highest rates of Upk1a positivity were seen in various subtypes of urothelial neoplasms (42.6-98 %) including Brenner tumors of the ovary (64.9 %) followed by neoplasms of the thyroid (10.4-33.3 %). In urothelial tumors, Upk1a staining predominated at the cell membranes and staining intensity was often moderate to strong. In thyroidal neoplasms the staining was mostly purely cytoplasmic and of low to moderate intensity. Upk1a positivity was also seen in up to 15 % of cases in 25 additional tumor categories but the staining intensity was often cytoplasmic and the intensity was usually judged as weak and only rarely as moderate. Within non-invasive (pTa) tumors, the Upk1a positivity rate decreased from 94 % in pTa G2 (low grade) to 90.1 % in pTa G3 (p = 0.012) and was even lower in muscle-invasive carcinomas (41.5 %; p < 0.0001 vs pTaG3). Within muscle invasive carcinomas, Upk1a expression was unrelated to nodal metastasis (p > 0.05) and patient outcome (p > 0.05). In conclusion, Upk1a immunohistochemistry is a potentially useful and specific diagnostic marker for the distinction of urothelial carcinomas from other neoplasms. However, its sensitivity is less than 50 % in muscle-invasive cancers because Upk1a expression decreases during grade and stage progression.

Screening for Prognostic Biomarkers in Metastatic Adrenocortical Carcinoma by Tissue Micro Arrays Analysis Identifies P53 as an Independent Prognostic Marker of Overall Survival.

Advanced adrenocortical carcinoma (ACC) has poor but heterogeneous prognosis. Apart from Ki67 index, no prognostic or predictive biomarker has been validated in advanced ACC, so far. We aimed at analyzing expression of a large panel of proteins involved in known altered pathways in ACC (cell cycle, Wnt/ß-catenin, methylation) to identify and prioritize potential prognostic or predictive parameters metastatic ACC population. We conducted a retrospective multicentric study. Overall survival (OS) and partial response according to RECIST 1.1 were primary endpoints. TMA was set up and 16 markers were analyzed. Modified ENSAT and GRAS parameters were characterized for prognostic adjustment. Results: We included 66 patients with a mean age at metastatic diagnosis of 48.7 ± 15.5 years. Median survival was 27.8 months. After adjustment to mENSAT-GRAS parameters, p53 and PDxK were prognostic of OS. No potential biomarker has been identified as predictive factor of response. We identified for the first time P53 as an independent prognostic marker of metastatic adrenocortical carcinoma after mENSAT-GRAS parameter adjustment. Prognostic impact of Wnt/ß-catenin alterations was not confirmed in this cohort of metastatic ACC.

Cytokeratin 7 and cytokeratin 20 expression in cancer: A tissue microarray study on 15,424 cancers.

Combined analysis of cytokeratin 7 (CK7) and cytokeratin 20 (CK20) is often used for assessing the origin of metastatic cancer. To evaluate the diagnostic utility of CK7 and CK20, tissue microarrays containing 15,424 samples from 120 different tumor types and subtypes and 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry. CK7 positivity was seen in 52% (8.7% weak, 5.9% moderate, 37% strong) and CK20 positivity in 23% (5.1% weak, 3.4% moderate, 15% strong) of interpretable tumors. Of 8390 positive tumors, 1181 (14%) showed positivity for CK7 and CK20, 5380 (64%) showed positivity for CK7 alone, and 1829 (22%) showed positivity for CK20 alone. CK20 predominated in gastrointestinal tract, urothelial and Merkel cell carcinomas. CK7 was usually negative in prostate cancer and colorectal cancer. Combined evaluation of CK7/CK20 revealed the best diagnostic utility in CK20 positive tumors, where CK7 negativity is often linked to colorectal origin while CK7 positivity argues for urothelial origin or mucinous ovarian cancer. Associations with unfavorable tumor features were found for cytokeratin 7 loss in breast cancer of no special type, urothelial and renal cell carcinomas, for CK7 overexpression in high-grade serous ovarian and gastric cancer, and for CK20 overexpression in urothelial carcinoma. CK20 loss was linked to MSI in gastric (p = 0.0291) and colorectal adenocarcinoma (p < 0.0001). These analyses provide comprehensive data on the frequency of CK7 and CK20 immunostaining - alone or in combination - in human cancers. These data facilitate interpretation of CK7/CK20 immunostaining in cancers.

Large-Scale Tissue Microarray Evaluation Corroborates High Specificity of High-Level Arginase-1 Immunostaining for Hepatocellular Carcinoma.

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.

DOG1 expression is common in human tumors: A tissue microarray study on more than 15,000 tissue samples.

DOG1 (Discovered on GIST1) is a voltage-gated calcium-activated chloride and bicarbonate channel that is highly expressed in interstitial cells of Cajal and in gastrointestinal stromal tumors (GIST) derived from Cajal cells. To systematically determine in what tumor entities and normal tissue types DOG1 may be further expressed, a tissue microarray (TMA) containing 15,965 samples from 121 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. DOG1 immunostaining was found in 67 tumor types including GIST (95.7%), esophageal squamous cell carcinoma (31.9%), pancreatic ductal adenocarcinoma (33.6%), adenocarcinoma of the Papilla Vateri (20%), squamous cell carcinoma of the vulva (15.8%) and the oral cavity (15.3%), mucinous ovarian cancer (15.3%), esophageal adenocarcinoma (12.5%), endometrioid endometrial cancer (12.1%), neuroendocrine carcinoma of the colon (11.1%) and diffuse gastric adenocarcinoma (11%). Low level-DOG1 immunostaining was seen in 17 additional tumor entities. DOG1 expression was unrelated to histopathological parameters of tumor aggressiveness and/or patient prognosis in cancers of the breast (n = 1002), urinary bladder (975), ovary (469), endometrium (173), stomach (233), and thyroid gland (512). High DOG1 expression was linked to estrogen receptor expression in breast cancer (p < 0.0001) and absence of HPV infection in squamous cell carcinomas (p = 0.0008). In conclusion, our data identify several tumor entities that can show DOG1 expression levels at similar levels as in GIST. Although DOG1 is tightly linked to a diagnosis of GIST in spindle cell tumors, the differential diagnosis is much broader in DOG1 positive epithelioid neoplasms.

Absolute quantitative proteomics using the total protein approach to identify novel clinical immunohistochemical markers in renal neoplasms.

BACKGROUND: Renal neoplasms encompass a variety of malignant and benign tumors, including many with shared characteristics. The diagnosis of these renal neoplasms remains challenging with currently available tools. In this work, we demonstrate the total protein approach (TPA) based on high-resolution mass spectrometry (MS) as a tool to improve the accuracy of renal neoplasm diagnosis. METHODS: Frozen tissue biopsies of human renal tissues [clear cell renal cell carcinoma (n = 7), papillary renal cell carcinoma (n = 5), chromophobe renal cell carcinoma (n = 5), and renal oncocytoma (n = 5)] were collected for proteome analysis. Normal adjacent renal tissue (NAT, n = 5) was used as a control. Proteins were extracted and digested using trypsin, and the digested proteomes were analyzed by label-free high-resolution MS (nanoLC-ESI-HR-MS/MS). Quantitative analysis was performed by comparison between protein abundances of tumors and NAT specimens, and the label-free and standard-free TPA was used to obtain absolute protein concentrations. RESULTS: A total of 205 differentially expressed proteins with the potential to distinguish the renal neoplasms were found. Of these proteins, a TPA-based panel of 24, including known and new biomarkers, was selected as the best candidates to differentiate the neoplasms. As proof of concept, the diagnostic potential of PLIN2, TUBB3, LAMP1, and HK1 was validated using semi-quantitative immunohistochemistry with a total of 128 samples assessed on tissue micro-arrays. CONCLUSIONS: We demonstrate the utility of combining high-resolution MS and the TPA as potential new diagnostic tool in the pathology of renal neoplasms. A similar TPA approach may be implemented in any cancer study with solid biopsies.

DOG1 is commonly expressed in pancreatic adenocarcinoma but unrelated to cancer aggressiveness.

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.

Immunohistochemically detectable thyroglobulin expression in extrathyroidal cancer is 100% specific for thyroidal tumor origin.

Thyroglobulin is a secreted 660 kDa glycoprotein produced by thyroid follicular cells used in diagnostic pathology to secure or exclude a thyroidal origin of metastases of unknown primary tumors. This study was performed to estimate specificity of thyroglobulin immunohistochemistry. 9974 tumor samples from 109 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Thyroglobulin was strongly expressed in all normal thyroid samples but not in any other normal tissues. Thyroglobulin immunostaining was detected in 99.1% of 106 thyroid adenomas, 98.1% of 364 papillary, 95.2% of 147 follicular, and 7.5% of 40 anaplastic thyroid cancers. Twelve of 15 thyroid samples that were thyroglobulin negative on TMAs showed at least a weak focal thyroglobulin positivity in corresponding large sections, suggesting higher sensitivity of large section analysis. Thyroglobulin positivity in one diffuse large B-cell lymphoma of the thyroid, one chondrosarcoma metastasis to the thyroid, and 42.4% of 92 medullary thyroid cancers was considered to be caused by diffusion of thyroidal colloid from destroyed or even intact adjacent follicles. Thyroglobulin positivity was, however, not seen in 6403 extrathyroidal tumors from 104 different tumor types and subtypes. Our data demonstrate a complete specificity of positive thyroglobulin immunostaining for thyroid origin in tumor tissues obtained from extrathyroidal locations. However, for all tumors located within the thyroid, false positivity can occur as a result of tissue contamination by thyroglobulin rich thyroid colloid from adjacent normal tissue.

Napsin A Expression in Human Tumors and Normal Tissues.

Background: Novel aspartic proteinase of the pepsin family A (Napsin A, TAO1/TAO2) is a functional aspartic proteinase which is involved in the maturation of prosurfactant protein B in type II pneumocytes and the lysosomal protein catabolism in renal cells. Napsin A is highly expressed in adenocarcinomas of the lung and is thus commonly used to affirm this diagnosis. However, studies have shown that other tumors can also express Napsin A. Methods: To comprehensively determine Napsin A expression in normal and tumor tissue, 11,957 samples from 115 different tumor types and subtypes as well as 500 samples of 76 different normal tissue types were evaluable by immunohistochemistry on tissue microarrays. Results: Napsin A expression was present in 16 different tumor types. Adenocarcinoma of the lung (85.6%), clear cell adenocarcinoma of the ovary (71.7%), clear cell adenocarcinoma of the endometrium (42.8%), papillary renal cell carcinoma (40.2%), clear cell (tubulo) papillary renal cell carcinoma (16.7%), endometrial serous carcinoma (9.3%), papillary thyroid carcinoma (9.3%) and clear cell renal cell carcinoma (8.2%) were among the tumors with the highest prevalence of Napsin A positivity. In papillary and clear cell renal cell carcinoma, reduced Napsin A expression was linked to adverse clinic-pathological features (p ≤ 0.03). Conclusion: This methodical approach enabled us to identify a ranking order of tumors according to their relative prevalence of Napsin A expression. The data also show that loss of Napsin A is linked to tumor dedifferentiation in renal cell carcinomas.

MUC5AC expression is linked to mucinous/endometroid subtype, absence of nodal metastasis and mismatch repair deficiency in ovarian cancer.

Mucin 5AC (MUC5AC) is a secreted gel-forming mucin which is expressed by mucus producing cells of several organs but can also be found in cancer cells of the ovary, pancreas, and gastrointestinal tract. This study aimed to characterize the expression of MUC5AC and its potential prognostic implications in different ovarian cancer subtypes. MUC5AC expression was analyzed by immunohistochemistry on a tissue microarray containing 603 ovarian cancers. MUC5AC was commonly expressed in mucinous (27/36; 75%) and endometrioid (12/39; 31%) carcinomas, whereas malignant mixed Mullerian tumors (2/27; 7%), high-grade serous (20/373; 5%) and clear cell carcinomas (1/28; 4%) were only rarely MUC5AC positive and also showed lower expression levels. MUC5AC positive endometroid carcinomas and high-grade serous carcinomas lacked lymph node metastases (p = 0.0495 and p = 0.0216) suggesting a more favorable prognosis. Deficient mismatch repair (dMMR), associated with a favorable prognosis in different cancer types, was found in 4/39 (10%) MUC5AC positive cancers but in only 5/375 (1%) of MUC5AC negative cancers (p = 0.0052). In subgroup analyses MUC5AC positive endometroid carcinomas more frequently showed dMMR (4/10; 40%) as opposed to MUC5AC negative endometroid carcinomas (3/23; 13%; p = 0.0932). In summary, the results of our study show that MUC5AC expression is associated with mucinous and endometrioid ovarian carcinomas, lack of nodal metastases and dMMR. MUC5AC expressing ovarian cancers should be evaluated for dMMR.

Mesothelin Expression in Human Tumors: A Tissue Microarray Study on 12,679 Tumors.

Mesothelin (MSLN) represents an attractive molecule for targeted cancer therapies. To identify tumors that might benefit from such therapies, tissue microarrays including 15,050 tumors from 122 different tumor types and 76 healthy organs were analyzed for MSLN expression by immunohistochemistry. Sixty-six (54%) tumor types showed at least occasional weak staining, including 50 (41%) tumor types with at least one strongly positive sample. Highest prevalence of MSLN positivity had ovarian carcinomas (serous 97%, clear cell 83%, endometrioid 77%, mucinous 71%, carcinosarcoma 65%), pancreatic adenocarcinoma (ductal 75%, ampullary 81%), endometrial carcinomas (clear cell 71%, serous 57%, carcinosarcoma 50%, endometrioid 45%), malignant mesothelioma (69%), and adenocarcinoma of the lung (55%). MSLN was rare in cancers of the breast (7% of 1138), kidney (7% of 807), thyroid gland (1% of 638), soft tissues (0.3% of 931), and prostate (0 of 481). High expression was linked to advanced pathological tumor (pT) stage (p < 0.0001) and metastasis (p < 0.0001) in 1619 colorectal adenocarcinomas, but unrelated to parameters of malignancy in 1072 breast-, 386 ovarian-, 174 lung-, 757 kidney-, 171 endometrial-, 373 gastric-, and 925 bladder carcinomas. In summary, numerous important cancer types with high-level MSLN expression might benefit from future anti-MSLN therapies, but MSLN's prognostic relevance appears to be limited.

A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth.

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC50 at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.

Epithelial splicing regulatory protein 1 and 2 (ESRP1 and ESRP2) upregulation predicts poor prognosis in prostate cancer.

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.

Reduced KLK2 expression is a strong and independent predictor of poor prognosis in ERG-negative prostate cancer.

BACKGROUND: Kallikrein-related peptidase 2 (KLK2)-like KLK3 (prostate-specific antigen [PSA])-belongs to the highly conserved serine proteases of the glandular kallikrein protein family (KLK family). Studies suggested that measurement of KLK2 serum levels advanced the predictive accuracy of PSA testing in prostate cancer. METHODS: To clarify the potential utility of KLK2 as a prognostic tissue biomarker, KLK2 expression was analyzed by immunohistochemistry in more than 12 000 prostate cancers. RESULTS: Normal epithelium cells usually showed weak to moderate KLK2 immunostaining, whereas KLK2 was negative in 23%, weak in 38%, moderate in 35%, and strong in 4% of 9576 analyzable cancers. Lost or reduced KLK2 immunostaining was associated with advanced tumor stage, high Gleason score, lymph node metastasis, increased cell proliferation, positive resection margin, and early PSA recurrence (P < .0001). Comparison with previously analyzed molecular alterations revealed a strong association of KLK2 loss and presence of TMPRSS2:ERG fusion (P < .0001), most of all analyzed common deletions (9 of 11; P ≤ .03), and decreased PSA immunostaining (P < .0001 each). Cancers with combined negative or weak immunostaining of KLK2 and PSA showed worse prognosis than cancers with at least moderate staining of one or both proteins (P < .0001). Multivariate analyses including established preoperative and postoperative prognostic parameters showed a strong independent prognostic impact of KLK2 loss alone or in combination of PSA, especially in erythroblast transformation-specific-negative cancers (P ≤ .006). CONCLUSIONS: Loss of KLK2 expression is a potentially useful prognostic marker in prostate cancer. Analysis of KLK2 alone or in combination with PSA may be useful for estimating cancer aggressiveness at the time of biopsy.