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Background Extrapulmonary small cell neuroendocrine carcinoma (EP-SC) is a rare malignancy with a poor prognosis. In our study, one hundred and thirty-eight EP-SCNC tissue samples underwent a complex analysis. Method Multicentric study included 138 high-grade small cell carcinomas exhibiting neuroendocrine morphology without known pulmonary involvement. Using TMA, we studied the possible benefit of TTF-1 (DAKO, clone 8G7G3/1, dilution 1:200), CDX-2 (DAKO, ready to use kit DAK-CDX2), PAX-8 (Cell Marque, polyclonal, dilution 1:50) and GATA-3 (Cell Marque, clone L50-823, 1:200) immunohistochemical analyses for determining the primary site of EP-SCNC origin. For statistical analyses, the Kaplan-Meier, the Cox regressions analyses, the Pearson chi-square test and the Fisher’s exact test were used. Results The median survival of our cohort was 11.5 months. Patients younger than 70 years had significantly better overall survival ( p  = 0.024). Across the full cohort, CDX-2 positivity was found in 19 tumors (13.9%), TTF-1 in 35 tumors (25.7%), PAX-8 in 63 tumors (46.3%) and GATA-3 in 8 tumors (6.5%), regardless of tumor origin. The expression of CDX-2, PAX-8 and GATA-3 did not differ significantly among different organ systems ( p  = 0.2; 0.3 and 0.12), respectively. The expression of TTF-1 differed significantly in tumors from various sites of origin ( p  = 0.009), expressed more often in breast, pancreatic, and genitourinary tumors. Interestingly, TTF-1 expression proved to have a negative prognostic impact relative to patient survival (age-adjusted Cox regression analysis, HR = 1.62, 95%CI:1.06–2.47, p  = 0.021). Conclusion As most patients with EP-SCNC had a metastatic presentation, finding the primary tumor origin, which is important for patient prognoses, is both challenging and important at the same time. Only TTF-1 immunohistochemical analysis proved to be helpful with this task. Moreover, a diagnosis of primary lung small cell carcinoma cannot be established based on TTF-1 positivity, since only 25.7% of EP-SCNC in our study displayed TTF-1 positivity.

Evaluation of fluid specimens involved by serous carcinoma might potentially include PAX8, GATA3, Uroplakin II, SOX2, and SALL4 antibodies. Those markers are commonly employed for diagnosing carcinomas of various types, including urothelial malignancies and germ cell tumors. There have been no comprehensive immunohistochemical studies, to our knowledge, for those markers on fluid specimens involved by serous carcinoma. To evaluate immunohistochemical markers PAX8, GATA3, SOX2, uroplakin II, and SALL4 in the diagnosis of high-grade serous carcinoma in fluid specimens. We examined 113 fluids (96 ascites specimens and 17 pleural fluid specimens) that were positive for carcinoma. Most (94 cases; 83.2%) consisted of high-grade serous carcinoma of Müllerian origin. Nineteen cases of non-high-grade serous carcinoma (including one case of low-grade serous carcinoma) of gynecologic origin were also included as anecdotal data. In 113 fluid specimens with positive results for carcinoma, including nonserous types, 99 (87.6%) had positive results for PAX8, 19 (16.8%) for GATA3; 19 (16.8%) for SOX2, 23 (20.4%) for uroplakin II, and 8 (7.1%) for SALL4. Of 94 fluids (83.2%) involved with high-grade serous carcinoma, 84 (89.4%) had positive results for PAX8, 18 (19.1%) for GATA3, 17 (18.1%) for SOX2, 22 (23.4%) for uroplakin II, and 8 (8.5%) for SALL4. Some of these specimens showed reactivity for more than one immunohistochemical marker. Most fluids involving high-grade serous carcinoma showed positive results for PAX8, and some cases expressed GATA3, SOX2, uroplakin II, and SALL4. Serous carcinoma in fluids may be positive for immunohistochemical markers not thought of traditionally as associated with gynecologic malignancy, an important consideration in avoiding misdiagnosis.

Background: The aim of this study is to elucidate the expression patterns of GATA transcription factors in neuroblastoma and the developing sympathetic nervous system (SNS). Methods: GATA-2, -3 and -4 and their cofactor friend-of-GATA (FOG)-2 were investigated in primary neuroblastoma by immunohistochemistry, real-time RT-PCR ( n =73) and microarray analysis ( n =251). In addition, GATA-2, -3 and FOG-2 expression was determined by northern-blot hybridisation. In the developing murine SNS, Gata-4 and Fog-2 were examined by immunohistochemistry. Results: Although Gata-2, -3 and Fog-2 are expressed in the developing nervous system, Gata-4 was not detected. In contrast, protein expression of all factors was observed in human neuroblastoma. Northern-blot hybridisation and real-time RT-PCR suggested specific expression patterns of the four genes in primary neuroblastoma, but did not show unequivocal results. In the large cohort examined by microarrays, a significant association of GATA-2 , -3 and FOG-2 expression with low-risk features was observed, whereas GATA-4 mRNA levels correlated with MYCN -amplification. Conclusion: The transcription factors GATA-2 and -3, which are essential for normal SNS development, and their cofactor FOG-2 are downregulated in aggressive but not in favourable neuroblastoma. In contrast, upregulation of GATA-4 appears to be a common feature of this malignancy and might contribute to neuroblastoma pathogenesis.

Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNF α and observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNF α without aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functions in vitro and in vivo . These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing.

Immunohistochemistry is an essential component of diagnostic breast pathology. The emergence of novel assays and applications is accompanied by new interpretation criteria and potential pitfalls. Immunohistochemistry assists in supporting breast origin for primary or metastatic carcinomas and identifying non-mammary metastases to the breast; however, no single immunostain is perfectly sensitive nor specific. GATA3 and Sox10 are particularly useful immunostains to identify triple negative breast carcinoma, which are often negative for other markers of mammary differentiation. Sox10 labeling is a major potential diagnostic pitfall, as Sox10 and S-100 label both triple negative breast carcinoma and metastatic melanoma; a pan-cytokeratin immunostain should always be included for this differential diagnosis. Novel immunohistochemistry serves as surrogates for the molecular alterations unique to several of special-type breast carcinomas, including the use of MYB in adenoid cystic carcinoma, pan-TRK in secretory carcinoma, and mutant IDH2 in tall cell carcinoma with reversed polarity (TCCRP). In addition, PD-L1 immunohistochemistry is an emerging, albeit imperfect, biomarker for breast cancer immunotherapy, with different assay parameters and scoring criteria in breast carcinoma compared to other tumor types. The expanding repertoire of novel immunohistochemistry provides additional diagnostic tools and biomarkers that improve diagnostic breast pathology and patient care.

Background: Modulation of Jak-STAT signalling may provide an effective therapeutic strategy in inflammatory arthritis. Objective: To document Jak-STAT expression in a cohort of patients with active rheumatoid arthritis (RA), spondyloarthritis (SpA), and osteoarthritis (OA) and compare these subsets with normal synovial tissue. Methods: Synovial tissue biopsy specimens from patients with RA, OA, and SpA and histologically normal tissue (n = 10 in each arthritis group) were examined for the presence of Jak3, STAT1, STAT4, and STAT6 expression using immunohistochemistry. Phenotyping was performed using immunohistochemistry and immunofluorescence. Clinical and serological characteristics of patients with RA expressing Jak3-STAT4 were assessed. Results: STAT1, STAT4, and Jak3 protein expression was generally increased in inflammatory arthritis. In contrast, STAT6 expression was relatively heterogeneous. A subpopulation of CD1a positive dendritic cells unique to seropositive patients with RA was detected. These cells showed intense protein expression for Jak3, STAT4, and STAT6. Conclusion: CD1a positive dendritic cells intensely express Jak3, STAT4, and STAT6 in seropositive RA tissue and may be an alternative marker for dendritic cells in their early stages of activation as well as providing a tool for identifying RA at the level of the synovium. Jak3 inhibition may be a potential therapeutic target to prevent dendritic cell maturation in RA. STAT1 expression is increased in inflammatory arthritis, suggesting that its pro-apoptotic and anti-inflammatory effects cannot effectively counteract inflammation. STAT6 expression is heterogeneous in synovium, suggesting a possible homoeostatic role in addition to any anti-inflammatory effects.

AimsThymic carcinoma and atypical thymoma (WHO type B3 thymoma) are unusual tumours the separation of which may be challenging in small biopsies. Both tumours consist of epithelioid tumour cells that share similar morphology and immunophenotype with conventional markers. Therefore, additional antibodies are needed to differentiate between these tumours.MethodsFor this purpose, a panel of immunohistochemical stains including PAX2, PAX5, PAX8 (all monoclonal) and CD70 was used on whole tumour sections of 30 thymic carcinomas and 30 atypical thymomas to determine the expression pattern of these antibodies. In addition, all tumours were stained with markers that are well known to be expressed in both tumours, including pancytokeratin and cytokeratin 5/6. The percentage of positive tumour cells as well as the intensity of staining were evaluated and scored.ResultsPAX5 stained close to 70% of thymic carcinomas while all atypical thymomas were negative for this marker. CD70 was expressed in 18 thymic carcinomas (60%) and in 1 case of atypical thymoma (3%). On the other hand, monoclonal PAX8 was negative in all cases while PAX2 was positive in a single thymic carcinoma. Of the established stains, pancytokeratin and cytokeratin 5/6 were equally positive in both tumours.ConclusionsAmong the markers explored, only PAX5 and CD70 appear to be differentially expressed and are predominantly restricted to thymic carcinomas. Therefore, in small biopsy specimens and in resections in which the morphological features remain equivocal, application of these particular stains may facilitate separation of thymic carcinoma and atypical thymoma.

Anaplastic thyroid carcinoma (ATC) is an aggressive malignant tumor composed of undifferentiated thyroid follicular cells. Pathological diagnosis of ATC can be challenging as the tumor may show morphological overlap with other neoplasms with anaplastic morphology. Immunohistochemical demonstration of thyroid origin facilitates the diagnosis of ATC. Previous studies using the polyclonal anti-PAX8 antibody 10336-1-AP suggested that PAX8 was the most sensitive marker, expressed in up to 80% of ATC. According to a 2018 NordiQC report, the monoclonal anti-PAX8 antibody MRQ-50 has become the most commonly used anti-PAX8 antibody worldwide. However, validation of this antibody in ATC is lacking. In this study, we recruited 182 ATC cases from seven institutions. Pathology slides were subjected to histology review. PAX8 immunohistochemistry using the MRQ-50 antibody was performed in whole tissue slides (n = 147) or tissue microarray sections (n = 35). We found PAX8 expression in 54.4% of the cases, which was significantly lower than those reported in prior studies with the polyclonal antibody. PAX8 expression was positively correlated with the presence of an epithelial pattern (63.6% vs 37.5%, p = 0.0008) and a coexisting differentiated thyroid carcinoma component (71.6% vs 44.3%, p = 0.0004), but was not associated with age, gender, specimen type, or presence of giant cell and sarcomatoid patterns. In conclusion, we demonstrated PAX8 expression using the monoclonal antibody MRQ-50 in only half of the cases in a large ATC series. Pathologists should be aware that PAX8 expression in ATC is less than those reported in early studies to avoid misdiagnosis.

Neuroendocrine tumors can present as liver metastases before discovery of the primary tumor. Islet 1 and PAX8 have recently been proposed as markers for neuroendocrine tumors of pancreatic origin. In this study, we compared the utility of Islet 1 and PAX8 in distinguishing pancreatic neuroendocrine tumors from neuroendocrine tumors of other sites and determined the usefulness of an immunohistochemical panel, including TTF1, CDX2, Islet 1 and/or PAX8, in identifying metastatic pancreatic neuroendocrine tumors. A total of 110 primary neuroendocrine tumors (33 pancreatic, 31 pulmonary, 23 ileal, 14 rectal, and 9 gastric) and 73 metastatic neuroendocrine tumors (28 pancreatic, 5 pulmonary, 37 ileal, 1 rectal, 1 colonic, and 1 duodenal) were studied. Islet 1 and PAX8 were positive in 27/33 (82%) and 29/33 (88%), respectively, of primary pancreatic neuroendocrine tumors, and in 19/28 (68%) and 15/28 (54%), respectively, of metastatic pancreatic neuroendocrine tumors. No cases of primary (0/23) or metastatic (0/37) ileal neuroendocrine tumors were positive with either Islet 1 or PAX8. There was Islet 1 positivity in 2/31 (6%) primary pulmonary, 12/14 (86%) primary rectal, and 1/1 metastatic rectal neuroendocrine tumors, and PAX8 positivity in 7/31 (23%) primary pulmonary, 11/14 (79%) primary rectal, and 2/9 (22%) primary gastric neuroendocrine tumors. ROC curve analysis incorporating sensitivity and specificity data of immunohistochemical panels for metastatic pancreatic neuroendocrine tumors showed that a four-stain panel, including Islet 1, PAX8, TTF1, and CDX2 significantly outperformed a three-stain panel composed of PAX8, TTF1, and CDX2 (P=0.019), and also showed a trend for better performance compared with a three-stain panel composed of Islet 1, TTF1, and CDX2 (P=0.072). Both Islet 1 and PAX8 are reliable immunohistochemical markers for pancreatic neuroendocrine tumors and would be useful adjuncts to other markers (TTF1, CDX2) currently used to work up a metastatic neuroendocrine tumor of unknown primary.

T lymphocytes are the most numerous immune cells in tumor-associated infiltrates and include several subpopulations of either anticancer or pro-tumorigenic functions. However, the associations between levels of different T cell subsets and breast cancer molecular subtypes as well as other prognostic factors have not been fully established yet. We performed immunohistochemistry for CD8 (cytotoxic T cells (CTL)), FOXP3 (regulatory T cells (Tregs)), and GATA3 (Th2 cells) in 106 formalin-fixed paraffin-embedded invasive breast cancer tissue samples and analyzed both the numbers and percentages of investigated cells in tumor-associated infiltrates. We observed that triple-negative breast cancer (TNBC) and HER2+ non-luminal breast tumors were associated with more numerous CTLs and Tregs and a higher Treg/Th2 cell ratio as compared with luminal A subtype. A higher Treg percentage was related to a decreased hormone receptor expression, an increase in the Ki67 level, a greater tumor size of luminal tumors, and the presence of lymph node metastases. Moreover, differences in the composition of T cell infiltrates were associated with HER2 status and histologic grade and type, and a distinct immune pattern was observed in tumors of different phenotypes regarding pT stage and nodal status. The results of our work show the diversity of T cell infiltrates in primary invasive breast cancers of different phenotypes and suggest that progression of luminal or non-luminal tumors is related to distinct tumor-associated T cell composition.