José Antonio López-Guerrero

Histological heterogeneity of Ewing’s sarcoma/PNET: an immunohistochemical analysis of 415 genetically confirmed cases with clinical support

Ewing’s sarcoma (ES)/peripheral neuroectodermal tumor (PNET) are malignant neoplasms affecting children and young adults. We performed a study to typify the histological diversity and evaluate antibodies that may offer diagnostic/prognostic support. In total, 415 cases of genetically confirmed paraffin-embedded ES/PNET were analyzed on whole sections and in tissue microarrays. This study confirms the structural heterogeneity of ES/PNET, distinguishing three major subtypes: conventional ES (280 cases); PNET (53 cases); and atypical ES/PNET (80), including large cells, vascular-like patterns, spindle pattern, and adamantinoma-like configuration. All cases presented positivity for at least three of the four tested antibodies (CD99, FLI1, HNK1, and CAV1). CAV1 appeared as a diagnostic immunomarker of ES/PNET being positive in CD99-negative cases. Hence, the immunohistochemical analysis confirmed the diagnostic value of all four antibodies, which together cover more than 99% of the tumors, independently of the histological variety. The univariate analysis for survival revealed atypical ES as the only histological parameter apparently associated with less favorable clinical outcome, particularly in the subgroup of patients treated with surgery. In conclusion, the diagnosis of atypical ES is a challenge for the pathologist and needs support from molecular techniques to perform an optimal differential diagnosis with other small round cell tumors.

Array comparative genomic hybridization analysis of chromosomal imbalances and their target genes in gastrointestinal stromal tumors

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. The tumors characteristically harbor KIT or PDGFRA mutations, and mutant tumors respond to imatinib mesylate (Glivec™). Chromosomal imbalances resulting in altered gene dosage are known to have a role in the molecular pathogenesis of these tumors, but the target genes remain to be identified. The present study aimed to identify some of these genes. In total, 35 GIST samples were screened for chromosomal imbalances by array‐based comparative genomic hybridization. A cDNA array was used to define the minimal common overlapping areas of DNA copy number change. Eight confirmative, replicate hybridizations were performed using an oligonucleotide array. The most recurrent copy number losses were localized to 14q, 22q, and 1p. Gains were less common with 8q being the most recurrent. Two recurrent deleted regions of 14q were 14q11.2 harboring the PARP2, APEX1, and NDRG2 genes and 14q32.33 harboring SIVA. Additional target candidates were NF2 at chromosome 22, CDKN2A/2B at 9p, and ENO1 at 1p for copy number losses, and MYC at 8q for copy number gains. Array CGH proved to be an effective tool for the identification of chromosome regions involved in the development and progression of GISTs.

Molecular diagnosis of dermatofibrosarcoma protuberans: a comparison between reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization methodologies.

Dermatofibrosarcoma protuberans (DFSP) is characterized by the presence of the t(17;22)(q22;q13) that leads to the fusion of the COL1A1 and PDGFB genes. This translocation can be detected by multiplex reverse transcriptase‐polymerase chain reaction (RT‐PCR) or fluorescence in situ hybridization (FISH) techniques. We have evaluated the usefulness of a dual color dual fusion FISH probe strategy for COL1A1/PDGFB detection in a series of 103 archival DFSPs and compared the obtained results with RT‐PCR analyses. FISH and RT‐PCR were carried out on paraffin embedded tissue samples. Regarding the RT‐PCR approach, all COL1A1 exons and exon 2 of PDGFB were evaluated. Sensitivity, specificity, positive and negative predictive values were assessed considering the histological diagnosis as the gold standard. We also analyzed the relationship between the genetic findings and the clinicopathological variables of the tumors. The COL1A1/PDGFB translocation was detected in 93% of DFSP. Both techniques showed a similar specificity (100%), but FISH was more sensitive than RT‐PCR (90% vs. 72%). Regarding, clinicopathological features, a higher percentage of positive cells detected by FISH was significantly associated with the fibrosarcomatous DFSP variant (P < 0.001). Interestingly, all CD34 negative DFSP (n = 5) were positive for COL1A1/PDGFB translocation by both techniques. In conclusion, the majority of DFSP harbor the COL1A1/PDGFB translocation and FISH technique should be recommended as a routine diagnostic tool, especially in cases showing unusual histopathological subtypes and/or immunohistochemical features.

Molecular diagnosis of Ewing sarcoma family of tumors: a comparative analysis of 560 cases with FISH and RT-PCR.

Aims To compare the sensitivity and specificity of fluorescence in situ hybridization (FISH) with reverse transcription polymerase chain reaction (RT-PCR) in the diagnosis of Ewing sarcoma family of tumors (ESFTs) and other small round-cell tumors (SRCTs) in formalin-fixed paraffin-embedded tissue assembled in tissue microarrays (TMAs). The second objective is to confirm the value of molecular methods and immunohistochemical (IHC) assays, to perform a differential diagnosis between ESFTs and SRCTs with similar or overlapping morphology. Materials and Methods A total of 560 cases were selected for the present study out the 806 cases collected from the PROgnosis and THerapeutic Targets in the Ewings Family of TumorS project. Case selection bias included only the cases with enough material to enable the TMA construction, as FISH analysis and the majority of IHC studies were performed in TMAs. Histopathologic, IHC, and molecular assays were carried out. Results Of the 560 total cases, 411 (73.4%) were considered informative (with results by FISH and/or RT-PCR assays). From the informative cases, 382 (92.9%) were diagnosed as ESFT, 23 cases (5.6%) as non-ESFT but with specific diagnosis for another established entity, and 6 cases (1.5%) as small round cell tumors not otherwise specified. Sensitivity and specificity for the FISH assays was 96.3% and 95.2%, respectively, whereas RT-PCR presented a sensitivity of 97.5% and specificity of 92.9%. In concordant cases, both methods showed a sensitivity and specificity of 99.2% and 100%, respectively. Twenty-nine cases (7.1%) initially interpreted at morphologic level as atypical ESFTs were finally reclassified, with the support of molecular methods and IHC, as either non-ESFT with another specific histologic type or as small round cell tumors not otherwise specified. Conclusions FISH and RT-PCR are ancillary techniques possessing high sensitivity in the diagnosis of ESFT; nevertheless, FISH is more specific than RT-PCR in the diagnosis of formalin-fixed paraffin-embedded tissue. Both methods in combination displayed the highest sensitivity and specificity. The combination of histopathologic, IHC, and molecular findings is the method of choice for the diagnosis of ESFT, as well as for the differential diagnosis with other SRCTs.

High stability of microRNAs in tissue samples of compromised quality

Degradation of tissue samples limits performing RNA-based molecular studies, but little is known about the potential usefulness of samples of compromised quality for studies focused on miRNAs. In this work we analyze a series of cryopreserved tissue samples (n = 14), frozen samples that underwent a severe thawing process (n = 10), and their paired formalin-fixed paraffin-embedded (FFPE) tissue samples (n = 24) from patients with breast cancer obtained during primary surgical resection and collected in 2011. Quality and integrity analyses of the total and small fraction of RNA were carried out. Recovery of specific RNA molecules (miRNAs hsa-miR-21, hsa-miR-125b, and hsa-miR-191; snoRNA RNU6B; and mRNAs GAPDH and HPRT1) was also analyzed by quantitative RT-PCR. Our results suggest that visualisation of the small RNA electrophoretic profiles obtained using the Agilent 2100 bioanalyzer makes it possible to differentiate between the three groups of samples (optimally frozen, thawed, and FFPE). We demonstrate that specific miRNA molecules can be similarly recovered from different tissue sample sources, which supports their high degree of stability. We conclude that miRNAs are robustly detected irrespective of the quality of the tissue sample. In this regard, a word of caution should be raised before degraded samples are discarded: although prior quality assessment of the biological material to be analyzed is recommended, our work demonstrates that degraded tissue samples are also suitable for miRNA studies.

Use of reverse-transcriptase polymerase chain reaction (RT-PCR) for carcinoembryonic antigen, cytokeratin 19, and maspin in the detection of tumor cells in leukapheresis products from patients with breast cancer: comparison with immunocytochemistry.

This study evaluates the role of reverse-transcriptase polymerase chain reaction (RT-PCR) assay for carcinoembryonic antigen (CEA), cytokeratin 19 (CK19), and maspin transcripts to identify breast cancer cells (BCC) in leukapheresis products (LP) collected from breast cancer (BC) patients and compares these results with those obtained using immunocytochemistry (IC). Eighty-four LP obtained from 33 patients with stage II-III BC and control subjects without BC were screened for the presence of BCC by IC and CK19, CEA, and maspin expression using RT-PCR. CEA RT-PCR and IC were the only specific markers, as no false positives were detected in any patients without BC. CK19 RT-PCR gave 11% false positives, whereas maspin RT-PCR with 25% was the most unspecific marker. In LP from BC patients, positive results were observed in 70% and 63% for CK19 and CEA RT-PCR, respectively. For maspin RT-PCR, this percentage was 22%, and for IC it was 17%. There was a good correlation between the CEA and CK19 RT-PCR (p = 0.018). No correlation between CEA and CK19 RT-PCR and IC was found, and although 5 of the 6 IC+ samples were CEA+/CK19+, great discrepancies in the group of IC- samples were observed. Our data suggest that RT-PCR assays for CEA and, to a lesser extent, for CK19 have more sensitivity and specificity than IC to detect BCC in LP.

Emerging treatment strategies in recurrent platinum-sensitive ovarian cancer: Focus on trabectedin

Ovarian cancer (OC) is the leading cause of death from gynecological malignancies. In spite of high response rates to the standard front-line treatment for advanced disease with cytoreductive surgical debulking, followed by platinum/taxane-based chemotherapy, most patients eventually relapse developing drug-resistant disease. Owing to the molecular heterogeneity, genetic instability and mutagenicity of OC, increases in survival might be achieved by translating recent insights at the morpho-molecular levels to individual therapeutic strategies. Several emerging treatments have been shown to be active in platinum-sensitive (PS) recurrent OC (ROC), but an optimal strategy still has not been established. Based on the recent results, it is likely that the introduction of novel non-platinum based chemotherapies and molecular targeted therapies will have a major impact on the management of ROC. Some current strategies are focused on the extension of platinum-free interval (PFI) in patients with PS, particularly in those with partially PS disease. Apparently, the PFI extension by an effective non-platinum intervention, such as trabectedin plus pegylated liposomal doxorubicin (PLD), may reduce cumulative platinum-induced toxicities leading to longer survival after the reintroduction of subsequent platinum. The introduction of novel therapies, such as the antiangiogenic monoclonal antibody bevacizumab, opens a new field of targeted therapies in this indication. In this review, we aim to outline the therapeutic potential of new emerging approaches, particularly the role of non-platinum therapy with trabectedin in combination with PLD in patients with PS ROC.

High susceptibility of a human oligodendroglial cell line to herpes simplex type 1 infection

More than 20 infectious agents, ranging from retroviruses to mycobacteria, have been associated with multiple sclerosis onset or relapses in which oligodendrocytes, the myelin-forming cells of the central nervous system, are the initial target of the pathogenic status. In this work, the nature of the susceptibility of the human precursor oligodendroglial KG-1C cell line to herpes simplex virus type 1 (HSV-1) was investigated. Infection of KG-1C cells was characterized by a high level of virus production and a notable progression of the cytopathic effect. After infection, there was a significant shut-off of host mRNA translation, which was correlated with evident synthesis of viral proteins. An examination by electron microscopy of the infected cells revealed the presence of large clusters of mitochondria located in the proximity of intracellular HSV-1 particle groups. In addition, transmission electron microscopy and nuclear fluorescence analysis showed neither signs of chromatin condensation nor of apoptotic bodies. Furthermore, procaspase-3 remained uncleaved, suggesting that apoptosis does not take place, at least in this system. Finally, expression and localization of MAL2, a subpopulation of detergent-insoluble lipid raft protein, was studied. Detection of MAL2 significantly increased after infection and it was colocalized with HSV-1 proteins. From these findings the authors conclude that human oligodendrocyte-like cells are highly susceptible to HSV-1 infection. The implications of this for central nervous system viral infection are discussed.

Role of the small GTPase Rab27a during Herpes simplex virus infection of oligodendrocytic cells

BackgroundThe morphogenesis of herpes simplex virus type 1 (HSV-1) comprises several events, of which some are not completely understood. It has been shown that HSV-1 glycoproteins accumulate in the trans-Golgi network (TGN) and in TGN-derived vesicles. It is also accepted that HSV-1 acquires its final morphology through a secondary envelopment by budding into TGN-derived vesicles coated with viral glycoproteins and tegument proteins. Nevertheless, several aspects of this process remain elusive. The small GTPase Rab27a has been implicated in regulated exocytosis, and it seems to play a key role in certain membrane trafficking events. Rab27a also seems to be required for human cytomegalovirus assembly. However, despite the involvement of various Rab GTPases in HSV-1 envelopment, there is, to date, no data reported on the role of Rab27a in HSV-1 infection.ResultsHerein, we show that Rab27a colocalized with GHSV-UL46, a tegument-tagged green fluorescent protein-HSV-1, in the TGN. In fact, this small GTPase colocalized with viral glycoproteins gH and gD in that compartment. Functional analysis through Rab27a depletion showed a significant decrease in the number of infected cells and viral production in Rab27a-silenced cells.ConclusionsAltogether, our results indicate that Rab27a plays an important role in HSV-1 infection of oligodendrocytic cells.

HER2 amplification in recurrent breast cancer following breast-conserving therapy correlates with distant metastasis and poor survival

The authors analyzed the HER2 status in early‐stage nonrecurrent and recurrent breast cancer groups following breast‐conserving treatment. Retrospective analyses of a group of 36 invasive early breast cancer (IBC) patients who developed a local recurrence as a first event and of a random control group of 69 IBC patients were made. HER2 status was assessed by the HercepTest® (Dako Corp., Carpinteria, CA) and fluorescence in situ hybridization. The Kaplan–Meier proportional log‐rank test was used to study the impact of the biological factors on the metastasis‐free interval (MFI) and the overall survival (OS). The Cox proportional hazards model, using stepwise selection was performed to identify the independent predictors of poor outcome. The median time of follow‐up was 156 months (range: 22–230) for the nonrecurrent group of patients and 119 months (range: 36–228) for the recurrent group. No significant differences between either group were observed in terms of either patient or tumor characteristics, or of HER2 expression. However, a higher proportion of HER2 amplified cases were found in the recurrent group, in contrast to a higher proportion of hormonal receptor positive cases in the nonrecurrent group. After univariate and multivariate analyses, HER2 amplification was found to be an independent predictive factor for distant metastasis (HR = 10.75; p = 0.00008) and for survival (HR = 4.22; p = 0.004). In conclusion, HER2 amplification constitutes an independent poor prognostic factor for the MFI and OS in patients with recurrent breast cancer. The clinical implications are discussed.