Antonio Fernandez-Serra

Identification of miR-187 and miR-182 as Biomarkers of Early Diagnosis and Prognosis in Patients with Prostate Cancer Treated with Radical Prostatectomy

PURPOSE miRNAs are noncoding RNAs that negatively regulate target mRNA gene expression. Aberrant miRNA expression is associated with prostate cancer pathogenesis. We identified miRNAs as potential biomarkers for prostate cancer diagnosis and prognosis. MATERIALS AND METHODS Total RNA was obtained from 10 normal prostate and 50 prostate cancer samples, and analyzed using the GeneChip® miRNA 2.0 Array. At a median followup of 92 months (range 2 to 189) an independent cohort of 273 paraffin embedded prostate cancer samples was used for validation by quantitative reverse transcriptase-polymerase chain reaction. Another 92 urine samples from patients undergoing prostate biopsy were evaluated for these miRNAs. RESULTS miR-182 and 187, the miRNAs most differentially expressed between normal and tumor tissue, were selected for further validation. miR-187 inversely correlated with cT (p = 0.125) and pT (p = 0.0002) stages, Gleason score (p = 0.003) and TMPRSS2-ERG status (p = 0.003). The log rank test showed associations of miR-182 with biochemical (p = 0.026) and clinical (p = 0.043) progression-free survival, as also noted on multivariate analysis. A significant independent improvement in the definition of risk of progression was achieved by combining miR-182 expression with Gleason score (p <0.0001). miR-187 detection in urine provided an independent predictive value for positive biopsy. A prediction model including serum prostate specific antigen, urine PCA3 and miR-187 provided 88.6% sensitivity and 50% specificity (AUC 0.711, p = 0.001). CONCLUSIONS Results show that miR-182 and 187 are promising biomarkers for prostate cancer prognosis to identify patients at risk for progression and for diagnosis to improve the predictive capability of existing biomarkers.

Clinical Implications of TMPRSS2-ERG Gene Fusion Expression in Patients With Prostate Cancer Treated With Radical Prostatectomy

PURPOSE Molecular prognostic factors may be useful tools for prostate cancer that complement classic clinicopathological factors. Genetic rearrangements between TMPRSS2 and ETS have been described for prostate cancer but their clinical significance is still unclear. We analyzed the association of the TMPRSS2-ERG fusion gene with prostate cancer outcome in patients treated with radical prostatectomy. MATERIAL AND METHODS We analyzed prostate cancer samples from 226 patients treated with radical prostatectomy from 1996 to 2002 with a median followup of 84 months (range 9 to 153). TMPRSS2-ERG fusion gene expression was determined by reverse transcriptase-polymerase chain reaction. Clinicopathological and molecular variables were related to biochemical and clinical progression-free survival by the Kaplan-Meier proportional risk log rank test. A Cox proportional hazards model using stepwise selection was used to identify independent predictors of poor outcome. RESULTS TMPRSS2-ERG fusion was detected in 114 cases (50.4%). We noted no association between fusion gene status and prostate cancer clinicopathological characteristics. However, when patients were grouped by TMPRSS2-ERG fusion gene status, different clinicopathological prognostic factors defined each group for biochemical and clinical progression-free survival. Prostate specific antigen, specimen Gleason score and margin status were independent prognostic factors in patients with prostate cancer expressing the fusion gene. In the nonexpressing TMPRSS2-ERG group the prognostic factors were cT, Gleason score and margins. CONCLUSIONS TMPRSS2-ERG fusion gene status classifies patients with prostate cancer treated with radical prostatectomy into groups defined by different prognostic factors. This could be the basis for designing more refined treatment strategies.

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.

Fluorescence in situ hybridization for the differential diagnosis between Spitz naevus and spitzoid melanoma

Requena C, Rubio L, Traves V, Sanmartín O, Nagore E, Llombart B, Serra C, Fernández‐Serra A, Botella R & Guillén C 
(2012) Histopathology 61, 899–909

Resultados del uso expandido del PCA3 score en una población española con sospecha de cáncer de próstata

OBJECTIVES DD3(PCA3) (PCA3) gene expression is prostate cancer-specific. Routine use of this biomarker has resulted in a 35-67% reduction in the number of required biopsies. The aim of this study is to evaluate our outcomes in its routine use and to establish in which group of patients this is the most efficient, depending on the number of previous PCA3 biopsies. MATERIAL AND METHODS A total of 474 consecutive patients who had previously undergone a biopsy (group A, n=337) or not (group B, n=134) for whom a PCA3 was requested were analyzed. We subdivided group A into A(1) (a previous biopsy, n=182) and A(2) (<1 previous biopsy, n=155). The recommendation of whether to perform a biopsy or not was made independently by each of the 11 clinicians and guided by prostatic specific antigen (PSA) levels and digital rectal examination. RESULTS Median age was 65 years (range 38 to 84). PCA3 score had an informative ratio of 99.6%, with a median of 29 (range 1-3245). The percentage of biopsy sparing was 49% of the cases. ROC analysis demonstrated an AUC for PSA and PCA3 of 0.532 (P=.417) and 0.672 (P<.0001), respectively. Sensitivities of PSA≥ 4 and PCA3≥ 35 were 87% vs. 85%, with specificities of 12% vs. 33%, PPV 34% vs. 39% and NPV 63% vs. 81%, respectively. The PCA3 score showed direct correlation with the percentage of positive biopsies (P<.0001). CONCLUSIONS Routine use of PCA3, due to its high NPV, results in a significant reduction in the number of biopsies. PCA3 appears to be more efficient in biopsy-naive patients. Among patients already biopsied, the results are superior in those biopsied only once.

A de novo complete BRCA1 gene deletion identified in a Spanish woman with early bilateral breast cancer

BackgroundGermline mutations in either of the two tumor-suppressor genes, BRCA1 and BRCA2, account for a significant proportion of hereditary breast and ovarian cancer cases. Most of these mutations consist of deletions, insertions, nonsense mutations, and splice variants, however an increasing number of large genomic rearrangements have been identified in these genes.MethodsWe analysed BRCA1 and BRCA2 genes by direct sequencing and MLPA. We confirmed the results by an alternative MLPA kit and characterized the BRCA1 deletion by Array CGH.ResultsWe describe the first case of a patient with no strong family history of the disease who developed early-onset bilateral breast cancer with a de novo complete BRCA1 gene deletion in the germinal line. The detected deletion started from the region surrounding the VAT1locus to the beginning of NBR1 gene, including the RND2, ΨBRCA1, BRCA1 and NBR2 complete genes.ConclusionThis finding supports the large genomic rearrangement screening of BRCA genes in young breast cancer patients without family history, as well as in hereditary breast and ovarian cancer families previously tested negative for other variations.

Cáncer de próstata: la revolución de los genes de fusión

BACKGROUND TMPRSS2-ETS fusion gene rearrangements constitute a very common and specific alteration in prostate cancer cells. These genetic alterations lead the overexpression of ETS genes which encode the E26 family of transcription factors involved in cell proliferation. Of this family, the ERG oncogene is overexpressed in almost 50% of prostate cancer cases. EVIDENCE SYNTHESIS TMPRSS2-ERG overexpresses ERG through an androgen-mediated response. Structurally, the rearrangement is due to interstitial deletion and to a lesser extent to reciprocal translocation and plays a key role in cellular metabolism. Almost all fusion gene transcripts produce a truncated ERG protein and the presence of a specific isoform of this gene suggests the clonality of the tumor; hence, metastasis shares the fusion gene status of their primary lesion. Although the prognostic implications of TMPRSS2-ERG have not been fully elucidated, they constitutes a field of great diagnostic potential and, therefore, the development of techniques to identify and to analyze the presence and characteristics of this gene in a non-invasive fashion deserves great interest in this area. Currently, there is evidence supporting the hypothesis that the presence of fusion gene differentiates two molecular groups within prostate cancer with a differential behaviour making the fusion gene a potential therapeutic target. In this regard, the use of anti-HDAC (trichostatin), antagonists of estrogen receptor alpha and abiraterone acetate have shown promising results. CONCLUSIONS This review describes the great potential offered by the investigation of fusion genes in PC and the need for further studies.

MRP1 Overexpression Determines Poor Prognosis in Prospectively Treated Patients with Localized High-Risk Soft Tissue Sarcoma of Limbs and Trunk Wall: An ISG/GEIS Study

Patients with localized high-risk soft tissue sarcomas (STS) of the limbs and trunk wall still have a considerable metastatic recurrence rate of more than 50%, in spite of adjuvant chemotherapy. This drug-ceiling effect of chemotherapy in sarcoma setting could be explained, at least partially, by multidrug resistance (MDR) mechanisms. The aim of this study was to ascertain whether mRNA and protein expression of ABCB1 (P-glycoprotein), ABCC1 (MRP1), and GSTA1 (glutathione S-transferase pi) was prognostic in localized high-risk STS. Immunohistochemistry and reverse transcriptase-PCR studies were performed from biopsies at the time of diagnosis. Patients of this series were prospectively enrolled into a phase III trial that compared three versus five cycles of epirubicin plus ifosfamide. The series of 102 patients found 41 events of recurrence and 37 of death with a median follow-up of 68 months. In univariate analysis, variables with a statistically significant relationship with relapse-free survival (RFS) were: MRP1 expression (5-year RFS rate of 23% in positive cases and 63% in negative cases, P = 0.029), histology (5-year RFS rate of 74% in undifferentiated pleomorphic sarcoma and 43% in synovial sarcoma, P = 0.028), and ABCC1 expression (5-year RFS rate of 33% in overexpression and 65% in downregulation, P = 0.012). Combined ABCC1/MRP1 was the only independent prognostic factor for both RFS (HR = 2.704, P = 0.005) and overall survival (HR = 2.208, P = 0.029). ABCC1/MRP1 expression shows robust prognostic relevance in patients with localized high-risk STS treated with anthracycline-based chemotherapy, which is the standard front line treatment in STS. This finding deserves attention as it points to a new targetable protein in STS. Mol Cancer Ther; 13(1); 249–59. ©2013 AACR.

Clinico-pathological significance of the molecular alterations of the SPOP gene in prostate cancer

AIMS Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor recently described to be mutated in prostate cancer (PCa). Hence, studying the gene expression profile and the presence of SPOP mutations in PCa and understanding its clinico-pathological significance as prognostic and therapeutic biomarker are important to further understand its role in PCa development. PATIENTS AND METHODS A cohort of 265 paraffin-embedded PCa samples from patients with more than 5 years of follow-up and treated with radical prostatectomy were collected at our institution for SPOP evaluation. RT-qPCR analysis was performed for expression studies while mutations were assessed by next generation sequencing. Relationship with prognosis was analysed using log-rank analysis and multivariable Cox regression. RESULTS SPOP was found to be strongly down-regulated in PCa (median=0.24; range=0.04-9.98) and its expression was associated with both, biochemical (p=0.003) and clinical progression free survival (p=0.023), the very low SPOP expression levels being associated to the worst prognosis. Multivariate analysis demonstrated that low levels of SPOP independently predicted a worse prognosis for both, biochemical (Hazard ratio (HR)=0.5; confidence interval (CI) 95% [0.4-0.9], p=0.011) and clinical progression (HR=0.6; IC 95% [0.4-1], p=0.046). SPOP mutations were found in 10% of TMPRSS2-ERG (T2E)-negative cases. Log-rank tests showed that mutations were significantly associated with biochemical progression free survival (BPFS) (p=0.009) and also were significant in the multivariable analysis (HR=3.4; IC 95% [1.5-7.6], p=0.004). CONCLUSIONS The present study demonstrates that prognosis varies depending on SPOP expression and mutational status, hence, defining a new biotype of PCa associated with a worse prognosis.

A germline mutation of p14/ARF in a melanoma kindred.

CDKN2A is the most relevant gene involved in melanoma susceptibility found to date. Germline mutations at this gene have been reported to predispose to familial melanoma (FM) as well as to multiple primary melanoma [1,2]. CDKN2A is located at 9p21 and codifies for two distinct tumor suppressor proteins: p16/INK4A and p14/ ARF [3]. p16/INK4A, which participates in the cyclin D1/ CDK4/p16/pRB signaling pathway inhibiting proliferation of cells [4], is encoded by exons 1a, 2, and 3. In the same way, exon 1b splices to exons 2 and 3 of CDKN2A in a different reading frame of the sequence encoding p14/ ARF. The p14/ARF protein regulates both the p53 and pRb pathways by binding to and inhibiting the function of the proto-oncogene mdm2 and targeting the transcription factors E2F-1, E2F-2, and E2F-3 for degradation [5]. Linkage to 9p21 has been shown in approximately 50% of families with FM. Nevertheless, only a subgroup of these (from 20 to 40% of melanoma families) harbor known CDKN2A mutations [6], with more than 60 mutant alleles identified in human tumors and in individuals predisposed to melanoma [7,8]. Predominantly, these mutations target the shared p14/ARF and p16/INK4A exon 2, and to a lesser extent the p16/INK4A exon 1a. Approximately, 40% of the somatic INKA/ARF mutations identified in FM affect the common amino acid sequence of both genes [2,9–14]. In contrast, mutations exclusively affecting p14/ARF (specific exon 1b) in melanoma are rare, as also described for other tumors. Thus, the deletion of exon 1b has been reported in acute T-cell lymphoblastic leukemias [15], astrocytomas, and metastatic melanoma cell lines [16,17], and mutations have been identified in the HCT116 colon cancer cell line and in a primary colon carcinoma [18]. Concerning FM, just a few families have been described with specific genetic alterations in exon 1b: a germline deletion in a melanoma-neural system tumor family [19], a 16 base-pair germline insertion (60ins16) in an individual with multiple primary melanomas [20], a germline splice site mutation in a melanoma kindred [21], and up to five different variants at the splice donor site (g.192 A4T, g.193 G4C, g.193þ 1 G4A, g.193þ 2 T4C, g.193þ 3 A4G) [21,22].