Lara Navarro

Blattabacteria, the endosymbionts of cockroaches, have small genome sizes and high genome copy numbers

Blattabacteria are intracellular endosymbionts of cockroaches and primitive termites that belong to the class Flavobacteria and live only in specialized cells in the abdominal fat body of their hosts. In the present study we determined genome sizes as well as genome copy numbers for the endosymbionts of three cockroach species, Blattella germanica, Periplaneta americana and Blatta orientalis. The sole presence of blattabacteria in the fat body was demonstrated by rRNA-targeting techniques. The genome sizes of the three blattabacteria were determined by pulsed field gel electrophoresis. The resulting total genome sizes for the three symbionts were all approximately 650 +/- 15 kb. Comparison of the genome sizes with those of free-living Bacteroidetes shows extended reduction, as occurs in other obligatory insect endosymbionts. Genome copy numbers were determined based on cell counts and determination of DNA amounts via quantitative PCR. Values between 10.2 and 18.3 and between 323 and 353 were found for the symbionts of P. americana and B. orientalis respectively. Polyploidy in intracellular bacteria may play a significant role in the genome reduction process.

CCL27–CCR10 and CXCL12–CXCR4 chemokine ligand-receptor mRNA expression ratio: new predictive factors of tumor progression in cutaneous malignant melanoma

CXCR4, CCR7 and CCR10 chemokine receptors are known to be involved in melanoma metastasis. Our goal was to compare the relative intratumoral mRNA expression of these receptors with that of their corresponding chemokine ligands, CXCL12, CCL19, CCL21, and CCL27 across the full spectrum of human melanoma progression: thin and thick primary melanomas, as well as “in transit”, lymph node, and distant metastases. Expression was quantified by real-time RT-PCR in 103 melanoma samples: 51 primary tumors and 52 metastases. Particular emphasis was focused on chemokine ligand-receptor expression ratios. Immunohistochemistry was performed to identify the cell types expressing these molecules. CXCL12–CXCR4 and CCL27–CCR10 ratios were higher in thin than in thick primary melanomas, and all four chemokine-receptor ratios were higher in primary tumors than in melanoma metastases. CCL27–CCR10 and CXCL12–CXCR4 expression ratios in primary tumors were inversely associated with the development of distant metastases, and improved the predictive value of tumor thickness for distant metastasis, which is important since chemokine ligand-receptor ratios are not affected by the endogenous gene employed for normalizing mRNA expression. Both receptor and ligand immunolabeling were detected in neoplastic cells suggesting autocrine mechanisms. Our results support the concept that low CCL27/CCR10 and CXCL12/CXCR4 intratumoral mRNA ratios are associated with melanoma progression, and in combination with Breslow thickness, are the best predictive factors for the development of distant metastases in primary cutaneous melanoma.

Characterization of a New Human Cell Line (CH-3573) Derived from a Grade II Chondrosarcoma with Matrix Production

Chondrosarcomas are malignant cartilage-forming tumors that represent the third most common malignant solid tumor of bone. In patients with grades II and III, local recurrence, increasing tumor size and dedifferentiation have been associated with lower survival rates. These biologically poorly-understood neoplasms vary considerably in clinical presentation and biological behavior. Cytogenetic studies have shown that heterogeneity is related to karyotypic complexity; moreover, alterations in the 9p21 locus and TP53 gene are related to disease progression. Despite the relatively high frequency of chondrosarcoma only a limited number of cell lines exist in the scientific community, limiting the possibility to study hypothesis-derived research or primary drug interaction necessary for pre-clinical studies. We report a chondrosarcoma cell line, CH-3573, derived from a primary tumor that may serve as a useful tool for both in vitro and in vivo models to study the molecular pathogenesis. In addition, xenograft passages in nude mice were studied to characterize the genetic stability over the course of tumor progression. In contrary to other reported cell lines, an important feature of our established cell line was the retained matrix production, a characteristic feature of a conventional grade II chondrosarcoma. The cell line (CH-3573) was characterized by pathological, immunohistochemical and molecular genetic methods.

BRAF V600E Mutation in Two Distinct Meningeal Melanocytomas Associated With a Nevus of Ota

Introduction Meningeal melanocytomas are rare tumors of the CNS that develop from melanocytes that are present in leptomeninges, with differing pigmented appearance. They generally occur in the posterior fossa and the spinal cord. This lesion may manifest at any age, but most patients are in the fifth decade of life. Occasionally, these tumors appear in a complex neurocutaneous grouping with other melanocytic lesions. The nevus of Ota (oculodermal melanocytosis) is a blue hyperpigmented dermal lesion that affects the trigeminal dermatome. The association of a meningeal melanocytoma with an ipsilateral nevus of Ota is extremely rare; to our knowledge, only eight cases have been reported in the literature to date. In these cases, the melanocytomas were located in the supratentorial area. We present a patient with neurocutaneous melanosis showing a highly pigmented meningeal melanocytoma and a less pigmented meningeal melanocytoma in association with a meningeal melanocytosis and a congenital nevus of Ota. We have analyzed the histopathologic and molecular characteristics of these lesions. To our knowledge, this is the first report of melanocytomas with mutations in BRAF, PTEN, and NF2, genes that are involved in the melanomagenesis process.

Ewing-like sarcoma with CIC-DUX4 gene fusion in a patient with neurofibromatosis type 1. A hitherto unreported association

Sarcoma with CIC-DUX4 gene fusion is emerging as the most prevalent subset of Ewing-like undifferentiated small round cell sarcomas with around 50 cases published. We report hereby the case of a 40-year-old male who presented a CIC-DUX4 sarcoma in deep soft tissues in his thigh. He had been diagnosed with neurofibromatosis type 1 at age 19 and over the years underwent resection of multiple neural neoplasms, including two malignant peripheral nerve sheath tumors with classical spindle-cell histopathology. The CIC-DUX4 sarcoma was treated with surgical resection, radiation and chemotherapy, but lung and brain metastases developed and the patient died from the disease 14 months after diagnosis. This is the first case of sarcoma with CIC-DUX4 gene fusion reported in a patient with NF1. Whether this association is coincidental or CIC-DUX4 sarcomas could be related to NF1 remains to be clarified. Study of alternative molecular alterations in EWSR1-negative undifferentiated small round cell sarcomas is clinically relevant, since CIC-DUX4 sarcomas seem to be a very aggressive subset with poor response to the presently used therapeutic regimens.

Soft tissue myoepithelial carcinoma with rhabdoid-like features and EWSR1 rearrangement: Fine needle aspiration cytology with histologic correlation

A new case of soft tissue myoepithelial carcinoma (MEC) with rhabdoid‐like differentiation is presented including cytologic, histopathologic, immunohistochemical, and molecular biologic features. A 45‐year‐old woman was admitted to the Hospital with nodular mass involving the lower part of the abdominal wall. Fine‐needle aspiration cytology showed a round cell tumor with abundant cytoplasm in the myxoid background. The nuclei were uniform, round to ovoid, with finely distributed chromatin, nucleoli, and pale, vacuolated, or eosinophilic cytoplasm with rhabdoid‐like appearance resembling a soft tissue malignant rhabdoid tumor. The surgically removed tumor was poorly demarcated, yellow, soft, and myxoid. The histopathology revealed sheets of poorly differentiated round malignant cells with focal myxoid stroma and rhabdoid‐like morphology. Immunohistochemistry showed positivity for CK (AE1/AE3), EMA, S100, vimentin, CD99, and SMA; however desmin, CD34, and gliofibrilar acid protein (GFAP) were negative. Tumor cells revealed loss of INI1 expression. The EWSR1 gene rearrangement was detected by fluorescence in situ hybridization (FISH), but molecular biology failed to detect EWSR1/ETS, EWSR1/NR4A3, EWSR1/DDIT3, EWSR1/ATF1, EWSR1‐POU5F1, EWSR1/ZNF444, EWSR1‐PBX1 gene fusions. The final diagnosis was soft tissue malignant myoepithelioma with rhabdoid changes and EWSR1 gene rearrangement. The differential diagnosis included soft tissue malignant rhabdoid tumor, cellular extraskeletal myxoid chondrosarcoma, proximal epithelioid sarcoma, and other soft tissue tumor with EWSR1 rearrangement. To our knowledge, this is the first case of MEC with rhabdoid features and description of fine‐needle aspiration cytology. Diagn. Cytopathol. 2015;43:421–426.

Epigenetic changes in localized gastric cancer: the role of RUNX3 in tumor progression and the immune microenvironment.

Gastric cancer (GC) pathogenesis involves genetic, epigenetic and environmental factors. Epigenetic alterations, such as DNA methylation are considered pivotal in the inactivation of tumor-related genes. We assessed a methylation panel of 5 genes to study their association to GC progression and microsatellite instability (MSI), and studied the role of RUNX3 in GC pathogenesis and the tumor immune microenvironment. The methylation status of 47 promoter-CpG islands was studied through MALDI-TOF mass spectrometry analysis in 35 Microsatellite stable (MSS) GC, 26 MSI, and 18 cancer-free samples (CFS), and 6 MSS GC and 4 MSI GC cell lines. We also studied RUNX3 expression by immunohistochemistry (IHC) in 40 samples, and validated differences in methylation levels between tumor, normal, and immune tissue in 14 additional samples. Unsupervised hierarchical clustering of methylation levels revealed no distinct subgroups between MSI and MSS samples or cell lines. CFSs clustered together showing higher levels of RUNX3 methylation compared to GC samples. RUNX3 showed protein silencing in cancer and normal mucosa, compared to inflammatory peritumoural infiltrate in almost all cases, showing a non-lymphocytic predominant pattern and being correlated with epigenetic silencing. Our results show aberrant promoters methylation in APC, CDH1, CDKN2A, MLH1 and RUNX3 associated with GC, as well as a non-lymphocytic predominant infiltrate with high expression of RUNX3. Deep study of RUNX3 inflammation signaling could help in understanding inflammation and immune activation in the tumor microenvironment.

Association between epidermal growth factor receptor amplification and ADP-ribosylation factor 1 methylation in human glioblastoma.

PurposeGlioblastoma (GB) is the most frequent and most malignant primary brain tumor in adults. Previously, it has been found that both genetic and epigenetic factors may play critical roles in its etiology and prognosis. In addition, it has been found that the epidermal growth factor receptor gene (EGFR) is frequently over-expressed and amplified in primary GBs. Here, we assessed the promoter methylation status of 10 genes relevant to GB and explored associations between these findings and the EGFR gene amplification status.MethodsTumor samples were obtained from 36 patients with primary GBs. In addition, 6 control specimens were included from patients who were operated for diseases other than brain tumors. The amplification status of the EGFR gene, and its deletion mutant EGFRvIII, were evaluated using FISH and MLPA, respectively. The IDH1/2 gene mutation status was verified using Sanger sequencing. A commercial DNA methylation kit was used to assess the promoter methylation status of 10 pre-selected genes. Metabolic profiles were measured using HR-MAS NMR spectroscopy. The EGFR and ARF1 mRNA expression levels were quantified using qRT-PCR.ResultsOf the 10 genes analyzed, we found that only ARF1 promoter hypermethylation was significantly associated with EGFR gene amplification. ARF1 is a GTPase that is involved in vesicle trafficking and the Golgi apparatus. Subsequent tumor metabolism measurements revealed a positive association between EGFR amplification and different membrane precursors and methyl-donor metabolites. Finally, we found that EGFR gene amplifications were associated with distinct tumor infiltration patterns, thus representing a putative novel functional association between EGFR gene amplification and ARF1 gene promoter methylation in GB.ConclusionsThe results reported here provide a basis for a new hypotheses connecting EGFR gene amplification in GB cells with ARF1 gene promoter methylation, vesicle trafficking, membrane turnover and tumor metabolism. The mechanism(s) underlying these connections and their functional consequences remain to be established.

Epigenetic changes underlie the aggressiveness of histologically benign meningiomas that recur

Meningiomas are the most frequent primary brain tumor. Usually, they are curable by surgery, but even after seemingly complete resection, some low-grade lesions recur. Despite recent improvements, signatures having prognostic value in grade I tumors remain poorly characterized. The frequency and delicate location of these tumors suggest that the risk of recurrence might be more accurately predicted. Herein, we show an easy way to evaluate the methylation status of meningiomas and its correlation with the prognosis of the disease. A series of 120 meningiomas, including primary tumors and recurrences, were analyzed histopathologically, and 24 tumor suppressor genes (TSGs) were studied by methylation-specific multiple ligation probe amplification. Long-term follow-up was conducted to classify patients with grade I primary tumors according to their outcomes. We found that hypermethylation in at least one TSG is frequent. The number of hypermethylated TSG per case was significantly higher in recurrences than in primary tumors and in primary benign meningiomas that recurred than in tumors from patients who showed no evidence of disease during follow-up. Finally, hypermethylation in RASSF1A, MLH1, and CDKN2B was an independent prognostic factor associated with the time to recurrence of these benign tumors that were biologically aggressive. To our knowledge, this is one of the widest studies of primary grade I tumors of patients who developed a tumor recurrence. The frequency of epigenetic changes suggests that hypermethylation is an early event in meningiomas, whereas the accumulation of epigenetic changes is related to greater biological aggressiveness and may be a signature of potential clinical relevance.

Congenital undifferentiated sarcoma associated to BCOR-CCNB3 gene fusion

Small round cell sarcomas are aggressive bone and soft tissue tumors that predominantly affect children and young adults. A new group of sarcomas with a recurrent BCOR-CCNB3 gene fusion has been recently identified in previously unclassifiable small round cell sarcomas. BCOR-CCNB3 sarcomas share clinical and pathologic similarities with Ewing sarcoma, but show a stronger male predilection and less aggressiveness, as well as distinct gene expression profiling and pangenomic SNP array analyses. We report the unusual case of a congenital BCOR-CCNB3 retroperitoneal sarcoma in a female born at 34th gestational week, which was diagnosed in necropsy after 21hours of life. Immunohistochemical analysis showed diffuse expression of CD99 and CCNB3. SNPa showed two focal segmentary deletions at 5q34 and 22q11.23, the latter harboring among others the SMARCB1/INI1 tumor suppressor gene. Immunohistochemistry confirmed loss of INI1 in tumor cells, which has not been previously reported in this type of undifferentiated sarcomas.