Villitis of unknown etiology and chronic deciduitis are not associated with human papilloma virus and enterovirus infection.

Villitis of unknown etiology (VUE) and chronic deciduitis with plasma cells (CD) are supposed to be non infectious placental lesions caused by a pathologic immune reaction similar to a host versus graft mechanism. In some investigations, infection of human trophoblastic cells with human papilloma virus (HPV) has been described, and a relationship with miscarriage, preeclampsia, and chronic inflammatory placental lesions has been suspected. Infection with enterovirus, especially Coxsackievirus, has been observed in cases with spontaneous abortion and adverse perinatal outcome, respectively. We investigated 20 cases with VUE and 30 cases with chronic deciduitis with plasma cells. The placenta specimens were analyzed for expression of HPV capsid protein by immunohistochemistry, for presence of HPV DNA via polymerase chain reaction (PCR), and for presence of enterovirus mRNA using RT-PCR, respectively. VUE was associated with maternal diseases: atopic lesions in 21%, other autoimmune diseases in 15.5%, and obesity in 31.5%, respectively. Birth weight below the 10th percentile was detected in 63% of the cases with VUE. Chronic deciduitis was associated with preterm labor and preterm premature rupture of membranes (26%). Intrauterine fetal demise occurred in 5 cases with CD (18.5%). HPV DNA, HPV capsid protein, and enterovirus mRNA were not detected in all investigated VUE or CD cases. Our investigations show that a causal role for enterovirus and human papilloma virus in the development of VUE and CD is unlikely. Therefore, HPV vaccination is unlikely to reduce the incidence of VUE and CD in the future.

Complement receptor-associated CD163+/CD18+/CD11c+/CD206-/CD209- expression profile in chronic histiocytic intervillositis of the placenta.

INTRODUCTION: Chronic histiocytic intervillositis of unknown etiology (CIUE) is a non-infectious, most probably immunologic placenta lesion. CIUE is associated with recurrent miscarriage, intrauterine growth restriction and stillbirth. Among the pathologic-anatomic defined placental lesions this entity displays the highest risk of recurrence in following pregnancies (about 67-100%). The histiocytic cells accumulate in the placental blood space but do not infiltrate into the villi or decidua. Sparsely known is the expression profile of these intervillous cells regarding histiocytic markers. METHODS: We analysed 5-22 markers by immunohistochemistry in a total of 41 placenta samples and evaluated decidual, villous and intervillous histiocytic cells. RESULTS: In CIUE, intervillous CD163+ histiocytes over-express CD11c/CD18 and down-regulate CD206/CD209, while CD163+ decidual and Hofbauer cells show low CD11c/CD18 and higher CD206/CD209 protein expressions. DISCUSSION: CD163 expression indicates a M2-like polarisation. CD11c and CD18 form the complement receptor 4 which could be related to a complement mediated trigger for aberrant cell accumulation in CIUE.

Profile of fibrosis-related gene transcripts and megakaryocytic changes in the bone marrow of myelodysplastic syndromes with fibrosis.

Bone marrow fibrosis (MF) in myelodysplastic syndromes (MDS) is associated with an adverse prognosis. It is likely that molecular changes similar to those in primary myelofibrosis (PMF) lead to MDS-MF, but gene expression profiling has not yet been carried out. We analysed bone marrow biopsy samples by PCR, qPCR (45 transcripts per sample), and immunohistochemistry from MDS patients with fibrosis (n = 70/119; including 19/70 MF0 > MF follow-up cases), MDS without fibrosis (n = 49/119), and 33 controls. SRSF2 and JAK2 mutations were detectable in up to 13% including 3/19 follow-up cases with evidence of clonal evolution during MF progression. MDS-MF showed increased expression of thrombospondin 1 (THBS1), TIMP metallopeptidase inhibitor 1 (TIMP1), transforming growth factor beta 1 (TGFB1), matrix metallopeptidases 2 and 14 (MMP2, MMP14), SMAD family members 3 and 4 (SMAD3, SMAD4), and miR-146b. Paralleling MF progression, a subfraction of follow-up cases showed megakaryocytic changes with increased CD42b+ pro-platelet deposition in the bone marrow. In summary, fibrosis in MDS-MF and PMF shows many molecular and morphological similarities.

Molecular profile of inflammatory and megakaryocytic factors in pediatric myelodysplastic syndrome with acute myelofibrosis.

Pediatric fibrotic myelodysplastic syndromes (ped-MDS-MF) and pediatric primary myelofibrosis (ped-PMF) are rare, and the molecular changes which mediate fibrosis have never been investigated. Histology and gene expression profile of 119 fibrosis/angiogenesis/inflammation/megakaryopoiesis-related factors in bone marrow biopsies were performed (two ped-MDS-MF and one ped-PMF). In one progressive ped-MDS, comparison of MF grade 0 (no myelofibrosis) and MF grade 2 (dense network of reticulin fibres) after 4 months showed that expression of fibrosis-related transcripts increased and dysplastic megakaryocytes formed a dense net of CD42b+ proplatelets. These changes were not observed in another ped-MDS-MF, whereas ped-PMF showed a similar proplatelet pattern. These findings indicate that fibrotic changes in ped-MDS may involve proplatelet-related and unrelated pathways.

Molecular defects in BRAF wild-type ameloblastomas and craniopharyngiomas-differences in mutation profiles in epithelial-derived oropharyngeal neoplasms.

The aim of this study was to evaluate the mutation profile of BRAF wild-type craniopharyngiomas and ameloblastomas. Pre-screening by immunohistochemistry and pyrosequencing for identifying BRAF wild-type tumors was performed on archived specimens of ameloblastic tumors (n = 20) and craniopharyngiomas (n = 62). Subsequently, 19 BRAF wild-type tumors (nine ameloblastic tumors and ten craniopharyngiomas) were analyzed further using next-generation sequencing (NGS) targeting hot spot mutations of 22 cancer-related genes. Thereby, we found craniopharyngiomas mainly CTNNB1 mutated (8/10), including two FGFR3/CTNNB1-double mutated tumors. Ameloblastic tumors were often FGFR2 mutated (4/9; including one FGFR2/TP53/PTEN-triple mutated case) and rarely CTNNB1/TP53-double mutated (1/9) and KRAS-mutated (1/9). In the remaining samples, no mutation could be detected in the 22 genes under investigation. In conclusion, mutation profiles of BRAF wild-type craniopharyngiomas and ameloblastomas share mutations of FGFR genes and have additional mutations with potential for targeted therapy.

Cytogenetic and immunohistochemical biomarker profiling of therapy-relevant factors in salivary gland carcinomas.

OBJECTIVES: There is currently no established algorithm for the molecular profiling of therapy-relevant defects in salivary gland carcinomas (SGC). HER2 overexpression in a subfraction of SGC and low frequencies of EGFR mutations are known. Here, we established receptor and cell signalling profiles of 17 therapy-relevant factors and propose a molecular diagnostic algorithm for SGC. MATERIALS AND METHODS: Formalin-fixed and paraffin-embedded tissue samples from SGC (n = 38) were analysed with immunohistochemistry and fluorescence in situ hybridisation (FISH). RESULTS: Two or more expressed receptors and/or receptor gene amplification were detectable in eight of 38 (21%) tumours: HER2 3+/AR 1+, HER3 gene amplification/AR 1+/EGFR 1+, ER 3+/AR 1+, EGFR 2+/PR 1+ and EGFR 2+/PR 1+/AR 1+. No FGFR1-3, MET, ALK1, ROS1, RET, BRAF nor VEGFA defects were detectable, and ERCC1 was not overexpressed. No PD1+ tumour-infiltrating T cells were detectable. CONCLUSION: Personalised therapy of patients with salivary gland carcinomas should include HER2 and EGFR signalling testing and, in negative cases, evaluation of rare potential target molecules. ERCC1 and PD1 do not appear to be reliable markers for the decision for or against chemotherapy or immunotherapy, respectively.

Cytokine Expression Pattern in Bone Marrow Microenvironment after Allogeneic Stem Cell Transplantation in Primary Myelofibrosis.

The only curative therapy for primary myelofibrosis (PMF) is allogeneic stem cell transplantation (ASCT). However, although we know that patients can benefit from ASCT, we do not know the extent of the changes of the expression profile of cytokines and matrix modulation factors. In this first systematic analysis, we evaluated the expression profile of 103 factors before and after transplantation to identify potential biomarkers. The expression of fibrosis-, inflammation-, and angiogenesis-associated genes was analyzed in a total of 52 bone marrow biopsies: PMF patients (n = 14) before and after ASCT and, for control purposes, post-ASCT multiple myeloma patients (n = 14) and non-neoplastic hematopoiesis (n = 10). In post-ASCT PMF cases, decreased expression of tissue inhibitor of metalloproteinases (TIMP) and platelet-derived growth factor alpha (PDGFA) correlated with bone marrow remodeling and hematological remission. Expression of several other matrix factors remained at high levels and may contribute to post-ASCT remodeling. This is the first systematic analysis of cytokine expression in post-ASCT PMF bone marrow that shows that normalization of bone marrow microenvironment is paralleled by decreased expression of TIMP and PDGFA.

Expression of Toll-Like Receptors in Chronic Histiocytic Intervillositis of the Placenta.

Chronic histiocytic intervillositis of the placenta (CHI) shows monocytic/histiocytic infiltration of the intervillous space. Placental malaria has a CHI-like histopathology and induces an aberrant expression of Toll-like receptors (TLR) 3, 7-9. We hypothesized that, similar to placental malaria, CHI could be associated with increased TLR expression. TLR1-10 and other inflammation-associated factors were analyzed by real-time PCR and immunohistochemistry. A total of 31 formalin-fixed and paraffin-embedded placenta samples were evaluated: CHI (n = 9), and for control purposes, villitis of unknown etiology (VUE, n = 8) and placentas without inflammation (n = 14). CHI shows increased expression of monocytic TLR1, a receptor which is involved in bacterial lipopolysaccharide (LPS)-induced inflammation. This could indicate a TLR1-mediated immune mechanism in the placenta (e.g. triggered by transient, clinically inapparent maternal bacteraemia) which leads to massive monocytic/histiocytic accumulation in the intervillous space. The increased expression of TLR1 with no increased expression of TLR3 and TLR7-9 is different from that in malaria.

Aberrant expression of caspase 14 in salivary gland carcinomas.

OBJECTIVES: Caspase 14 is reduced in adenocarcinomas of the stomach and colon. In contrast, breast and lung adenocarcinomas frequently show an overexpression of caspase 14. Salivary gland adenocarcinomas have not been evaluated for potential aberrant caspase 14 expression. MATERIALS AND METHODS: Samples from salivary gland carcinomas (n = 43) were analysed by immunohistochemistry (caspase 14, filaggrin, GATA3 and Ki67) and fluorescence in situ hybridization. RESULTS: Caspase 14 is not expressed in normal salivary glands, while in a subfraction of carcinomas (32%) an aberrant expression was found. Filaggrin could not be detected. Caspase 14 staining was not associated with tumour dedifferentiation, GATA3 expression or amplification of gene locus 19p13. CONCLUSION: In summary, aberrant expression of caspase 14 can be found in a subfraction of salivary gland carcinomas but could not be used as a biomarker for a specific carcinoma subtype of the salivary gland.

Expression of cyclin-dependent kinase inhibitor 2A 16, tumour protein 53 and epidermal growth factor receptor in salivary gland carcinomas is not associated with oncogenic virus infection.

It is known that human papillomavirus (HPV) infection can cause squamous cell neoplasms at several sites, such as cervix uteri carcinoma and oral squamous carcinoma. There is little information on the expression of HPV and its predictive markers in tumours of the major and minor salivary glands of the head and neck. We therefore assessed oral salivary gland neoplasms to identify associations between HPV and infection-related epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitor 2A (CDKN2A/p16) and tumour protein p53 (TP53). Formalin-fixed, paraffin-embedded tissue samples from oral salivary gland carcinomas (n=51) and benign tumours (n=26) were analysed by polymerase chain reaction (PCR) analysis for several HPV species, including high-risk types 16 and 18. Evaluation of EGFR, CDKN2A, TP53 and cytomegalovirus (CMV) was performed by immunohistochemistry. Epstein-Barr virus (EBV) was evaluated by EBV-encoded RNA in situ hybridisation. We demonstrated that salivary gland tumours are not associated with HPV infection. The expression of EGFR, CDKN2A and TP53 may be associated with tumour pathology but is not induced by HPV. CMV and EBV were not detectable. In contrast to oral squamous cell carcinomas, HPV, CMV and EBV infections are not associated with malignant or benign neoplastic lesions of the salivary glands.

Expression of angiogenic factors is increased in metastasised renal cell carcinomas.

Clear cell renal cell carcinomas (ccRCC) have aberrant signalling pathways which affect vascular endothelial growth factor and are related to increased tumour angiogenesis. Little is known about other angiogenesis-associated genes in primary tumours and metastases. Quantitative PCR of 45 angiogenesis-associated gene transcripts was performed on formalin-fixed and paraffin-embedded tissues from primary ccRCC (n = 18) and their metastases (n = 17; in 8/17 cases the corresponding primary tumour could be analysed). In metastases, a significant increase was found in the expression of 15 pro-angiogenic (such as prostaglandin-endoperoxide synthase 1) and also anti-angiogenic (such as TIMP metallopeptidase inhibitor 2) factors. Comparison of a primary with its metastasis performed on eight cases showed that even without preceding anti-angiogenic therapy in metastases expression of angiogenic factors is increased. In ccRCC, the effects of anti-angiogenic factors are superimposed by pro-angiogenic factors. Increased expression of angiogenic factors in metastases might be related to development of resistance after anti-angiogenic therapy but might also be an inherent biological characteristic.