Michele Biscuola

Micro-RNA signature of the epithelial–mesenchymal transition in endometrial carcinosarcoma†

Endometrial carcinosarcomas (ECSs) undergo a true epithelial‐mesenchymal transition (EMT). The molecular determinants of the EMT in vivo are unclear, although a role for some miRNAs, mainly involving the miR‐200 family, was recently suggested from in vitro cellular models. We analysed the microRNA (miRNA) signatures associated to EMT in human carcinosarcomas, and determined their relationships with EMT markers and repressors of E‐cadherin transcription. The expression of E‐, P‐ and N‐cadherin, cadherin‐11, p120, vimentin, SPARC, fascin and caveolin‐1 was studied in a group of 76 ECS by immunohistochemistry. In addition, real‐time PCR was used to measure the differences in the expression of 384 miRNAs, E‐cadherin, cadherin‐11, SPARC, SNAIL, ZEB1, ZEB2, TWIST‐1, TCF4, TGFβ1 and TGFβ2 between the epithelial and mesenchymal components of 23 ECSs. A loss of epithelial characteristics, including cadherin switching and the acquisition of a mesenchymal phenotype, was accompanied by changes in the profile of miRNA expression and the up‐regulation of all the E‐cadherin repressors analysed. A greater than five‐fold difference in the expression of 14 miRNAs between both neoplastic components was seen. Members of the miR‐200 family were down‐regulated in the mesenchymal part of the ECS. In addition, miR‐23b and miR‐29c, which are involved in the inhibition of mesenchymal markers, and miR‐203, which is involved in the inhibition of cell stemness, were also down‐regulated. Up‐regulated miRNAs included miR‐155, miR‐369‐5p, miR‐370, miR‐450a and miR‐542‐5p. These data suggest that in human ECS, the interplay between transcriptional repressors of E‐cadherin and miRNAs provides a link between EMT‐activation and the maintenance of stemness. Copyright

MicroRNA-200 Family Modulation in Distinct Breast Cancer Phenotypes

The epithelial to mesenchymal transition (EMT) contributes to tumor invasion and metastasis in a variety of cancer types. In human breast cancer, gene expression studies have determined that basal-B/claudin-low and metaplastic cancers exhibit EMT-related characteristics, but the molecular mechanisms underlying this observation are unknown. As the family of miR-200 microRNAs has been shown to regulate EMT in normal tissues and cancer, here we evaluated whether the expression of the miR-200 family (miR-200f) and their epigenetic state correlate with EMT features in human breast carcinomas. We analyzed by qRT-PCR the expression of miR-200f members and various EMT-transcriptional inducers in a series of 70 breast cancers comprising an array of phenotypic subtypes: estrogen receptor positive (ER+), HER2 positive (HER2+), and triple negative (TN), including a subset of metaplastic breast carcinomas (MBCs) with sarcomatous (homologous or heterologous) differentiation. No MBCs with squamous differentiation were included. The DNA methylation status of miR-200f loci in tumor samples were inspected using Sequenom MassArray® MALDI-TOF platform. We also used two non-tumorigenic breast basal cell lines that spontaneously undergo EMT to study the modulation of miR-200f expression during EMT in vitro. We demonstrate that miR-200f is strongly decreased in MBCs compared with other cancer types. TN and HER2+ breast cancers also exhibited lower miR-200f expression than ER+ tumors. Significantly, the decreased miR-200f expression found in MBCs is accompanied by an increase in the expression levels of EMT-transcriptional inducers, and hypermethylation of the miR-200c-141 locus. Similar to tumor samples, we demonstrated that downregulation of miR-200f and hypermethylation of the miR-200c-141 locus, together with upregulation of EMT-transcriptional inducers also occur in an in vitro cellular model of spontaneous EMT. Thus, the expression and methylation status of miR-200f could be used as hypothetical biomarkers to assess the occurrence of EMT in breast cancer.

ZEB1 overexpression associated with E-cadherin and microRNA-200 downregulation is characteristic of undifferentiated endometrial carcinoma

Undifferentiated endometrial carcinomas are very aggressive high-grade endometrial carcinomas that are frequently under-recognized. This study aimed to analyze the molecular alterations underlying the development of these endometrial carcinomas, focusing on those related to dedifferentiation. We assessed a series of 120 tumors: 57 grade 1 and 2 endometrioid endometrial carcinomas, 15 grade 3 endometrioid endometrial carcinomas, 27 endometrial serous carcinomas, and 21 undifferentiated endometrial carcinomas. We found a high frequency of DNA mismatch repair deficiency (38%) and moderate rate of p53 overexpression (∼33%) in undifferentiated carcinomas. In contrast to the characteristic endometrioid phenotype, there was a dramatic downregulation of E-cadherin expression in the undifferentiated subtype. Quantitative methylation studies dismissed CDH1 promoter hypermethylation as the mechanism responsible for this change in gene expression, while immunohistochemistry revealed that the E-cadherin repressor ZEB1 was frequently overexpressed (62%) in undifferentiated endometrial carcinomas. This finding was accompanied by a sharp downregulation in the expression of the miR-200 family of microRNAs, well-known targets of ZEB1. Furthermore, there was enhanced expression of epithelial-to-mesenchymal transition markers in undifferentiated endometrial carcinomas, such as N-cadherin, cytoplasmic p120, and osteonectin. In addition, HMGA2, a regulator of epithelial-to-mesenchymal transition that is expressed in aggressive endometrial tumors, such as endometrial serous carcinomas and carcinosarcomas, was expressed in >20% of undifferentiated carcinomas. These results suggest that ZEB1 overexpression, associated with E-cadherin and miR-200s downregulation, and the expression of mesenchymal markers might enhance the metastatic potential of undifferentiated endometrial carcinomas, leading to a poor prognosis. In addition, our observations suggest that the immnohistochemical analysis of E-cadherin and ZEB1 can aid in the differential diagnosis of the more agressive undifferentiated endometrial carcinomas from grade 3 endometrioid carcinomas.

Accurate identification of ALK positive lung carcinoma patients: Novel FDA-cleared automated fluorescence in situ hybridization scanning system and ultrasensitive immunohistochemistry

Background Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples. Methods Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioviews automated scanning system. Results All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. Conclusions The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations.

Characterization of human mesenchymal stem cells from ewing sarcoma patients. Pathogenetic implications.

Background Ewing Sarcoma (EWS) is a mesenchymal-derived tumor that generally arises in bone and soft tissue. Intensive research regarding the pathogenesis of EWS has been insufficient to pinpoint the early events of Ewing sarcomagenesis. However, the Mesenchymal Stem Cell (MSC) is currently accepted as the most probable cell of origin. Materials and Methods In an initial study regarding a deep characterization of MSC obtained specifically from EWS patients (MSC-P), we compared them with MSC derived from healthy donors (MSC-HD) and EWS cell lines. We evaluated the presence of the EWS-FLI1 gene fusion and EWSR1 gene rearrangements in MSC-P. The presence of the EWS transcript was confirmed by q-RT-PCR. In order to determine early events possibly involved in malignant transformation, we used a multiparameter quantitative strategy that included both MSC immunophenotypic negative/positive markers, and EWS intrinsic phenotypical features. Markers CD105, CD90, CD34 and CD45 were confirmed in EWS samples. Results We determined that MSC-P lack the most prevalent gene fusion, EWSR1-FLI1 as well as EWSR1 gene rearrangements. Our study also revealed that MSC-P are more alike to MSC-HD than to EWS cells. Nonetheless, we also observed that EWS cells had a few overlapping features with MSC. As a relevant example, also MSC showed CD99 expression, hallmark of EWS diagnosis. However, we observed that, in contrast to EWS cells, MSC were not sensitive to the inhibition of CD99. Conclusions In conclusion, our results suggest that MSC from EWS patients behave like MSC-HD and are phenotypically different from EWS cells, thus raising important questions regarding MSC role in sarcomagenesis.

Nuclear TAZ expression associates with the triple-negative phenotype in breast cancer

The Hippo signaling pathway, a conserved regulator of organ size, has emerged as an important regulatory pathway in cancer. The final transducer effectors of this pathway in mammals are the oncoproteins TAZ and YAP1, which are transcriptional coactivators of target genes involved in cell proliferation and survival. TAZ has been previously reported to play a role in tumorigenesis in breast cancer, but detailed analyses of the different breast cancer phenotypes have not been conducted thus far. We analyzed TAZ expression by immunohistochemistry in a retrospective series of 640 invasive breast carcinomas, comprising estrogen/progesterone receptor-positive (ER+/PR+), HER2-positive, and triple-negative (TN) tumors. We found a strong association of TAZ nuclear expression with the TN phenotype (60.5% TAZ-positive, P<0.001), which was strengthened when stratified into the basal-like subtype (70.8% TAZ-positive, P<0.001). Moreover, 90% of metaplastic breast carcinomas with morphological epithelial-mesenchymal transition features were TAZ-positive. We also investigated whether amplification or differential DNA methylation of the TAZ-encoding locus could account for the observed enhanced TAZ protein expression in the TN/basal phenotype. Amplification of the TAZ locus was analyzed by fluorescence in situ hybridization in 30 TN tumors, and we found gene amplification in some cases (6.45%). DNA methylation analysis was performed using the Sequenom MassArray MALDI-TOF platform, and we observed similar low methylation levels both in TN (n=25) and ER+/PR+ (n=26) tumors. These results were further confirmed using a panel of breast cancer cell lines and using the TCGA dataset. Finally, patients with strong TAZ expression showed poorer clinical outcomes with respect to both recurrence and overall survival.

Molecular events in endometrial carcinosarcomas and the role of high mobility group AT-hook 2 in endometrial carcinogenesis

The molecular events implicated in the development of endometrial carcinosarcoma remain poorly understood. Using complementary DNA microarrays, we analyzed a group of 15 endometrial carcinosarcomas and compared their gene expression profiles with those obtained from a group of 23 endometrioid endometrial carcinomas. We demonstrated changes in the expression of genes modulating processes such as the epithelial to mesenchymal transition, muscle differentiation, the expression of cancer/testis antigens, and immune response in endometrial carcinosarcomas. The high mobility group AT-hook 2 gene is an embryonic nuclear factor that mediates epithelial to mesenchymal transition in various tumor models, and it was among the genes overexpressed in endometrial carcinosarcomas. High mobility group AT-hook 2 overexpression was confirmed in 54% of endometrial carcinosarcomas by quantitative real time-polymerase chain reaction and immunohistochemistry. Moreover, we found a significant inverse correlation between the expression of high mobility group AT-hook 2 and let-7b, a member of the let-7 family of microRNAs that represses high mobility group AT-hook 2 expression. These changes were also associated with overexpression of Lin28B, a suppressor of microRNA biogenesis that is implicated in cancer progression and metastasis. Finally, high mobility group AT-hook 2 overexpression, which was detected in less than 3% of endometrioid endometrial carcinomas, was observed in many nonendometrioid carcinomas (46% of 28 samples). This pattern of expression, restricted to nonendometrioid carcinomas and endometrial carcinosarcomas, reflects a role for high mobility group AT-hook 2 in endometrial carcinogenesis that is associated with aggressive phenotypes and points to its potential use as a marker to distinguish between endometrioid and nonendometrioid tumors.

Oncogene alterations in endometrial carcinosarcomas

Endometrial carcinosarcomas are aggressive neoplasias composed of high-grade carcinomatous and sarcomatous elements. The pathogenesis and specific genetic alterations underlying these tumors are still not well known. We analyzed alterations in oncogenes involved in the pathogenesis of endometrial carcinomas that might represent predictive markers for specific therapies. Immunohistochemistry for HER2 (tyrosine kinase-type cell surface receptor HER2) and c-KIT (tyrosine-protein kinase Kit) and fluorescence in situ hybridization for EGFR (epidermal growth factor receptor) and ALK (anaplastic lymphoma receptor tyrosine kinase) were carried out for 76 endometrial carcinosarcoma samples on sequential tissue microarray sections. Analysis of 238 mutations across 19 common oncogenes was performed on 34 samples using the Sequenom OncoCarta Panel (Sequenom, Hamburg, Germany). We observed EGFR, HER2, and c-KIT expression in 71%, 1.5%, and 2.7% of tumors, respectively. EGFR amplification was detected in 11 of 76 endometrial carcinosarcomas (14.5%). Four samples showed both amplification and aneuploidy (5.2%). ALK amplification together with chromosome 2 polysomy was found in 1.3% of endometrial carcinosarcomas. In total, 23 mutations in 9 different oncogenes were detected in 15 (44.1%) of 34 endometrial carcinosarcomas. Five endometrial carcinosarcomas (14.7%) had 2 or more mutations. Eleven tumors (32.3%) had mutations affecting the PI3K (phosphoinositide-3-kinase)/AKT (v-akt murine thymoma viral oncogene homolog 1) (6 mutations in PIK3CA (PI3K catalytic alpha polypeptide) and 1 in AKT) and/or RAS/BRAF (serine/threonine-protein kinase B-raf) pathway (3 KRAS [kirsten RAS oncogene homolog], 2 NRAS [neuroblastoma RAS viral oncogene homolog], and 1 BRAF). Mutations in PDGFRA (platelet-derived growth factor receptor, alpha polypeptide) and/or KIT were found in 5 endometrial carcinosarcomas (14.7%). Finally, we found mutations in MET (met proto-oncogene [hepatocyte growth factor receptor]) in 2 tumors (5.9%) and in EGFR in one (2.9%). Our study evidences mutations in oncogenes in endometrial carcinosarcomas that are targets or modulators of response to specific therapies in other human cancers, with PI3K/AKT being the most frequently altered pathway.

Cutaneous Hodgkin-type lymphoproliferative lesion associated with immunomodulatory therapy for ulcerative colitis.

Immunomodulatory drugs have demonstrated efficacy in the therapy against autoimmune diseases such as rheumatoid arthritis, Crohns disease or ulcerative colitis. Tumor necrosis factor‐α (TNF‐α) represents a target molecule for the treatment of these entities. Use of monoclonal antibodies can block the proinflammatory function of TNF‐α. It has been shown that this action can reactivate quiescent chronic diseases as well as modify the immune response or potentiate carcinogens, thereby increasing the risk of secondary tumor development. In this context, different types of solid or hematological tumors have been documented. We present the case of a male with chronic ulcerative colitis who secondarily developed a cutaneous Hodgkin‐type lymphoproliferative lesion associated with immunodeficiency. This secondary tumor developed after 6 months of treatment with anti‐TNF‐α.

CK19 expression in breast tumours and lymph node metastasis after neoadjuvant therapy

Neoadjuvant therapy is used in many patients with breast cancer before surgery, with the aim of reducing the tumour size, allowing conservative resections. Sentinel node biopsy is a conservative procedure for handling the axilla in breast cancer; however, the use of this technique after neoadjuvant treatment is under discussion. For sentinel node assay, methods based on the detection of cytokeratin 19 (CK19) mRNA, such as one‐step nucleic acid amplification (OSNA), are available. However, if systemic therapy could alter protein expression, then CK19 would not be a good target for analysing these nodes. The aim of this study was to evaluate the immunohistochemical expression of CK19 within different cancer types, and to compare its expression in breast tumours and axillary nodes before and after treatment.