João D. Barros-Silva

FLI1 is a novel ETS transcription factor involved in gene fusions in prostate cancer.

To characterize the pattern of ETS rearrangements and to uncover novel ETS fusion genes, we analyzed 200 prostate carcinomas (PCa) with TaqMan low‐density arrays (TLDAs), followed by selective analyses with fluorescence in situ hybridization (FISH), RT‐PCR, and sequencing. Besides confirming the recurrent presence of ERG, ETV1, ETV4, and ETV5 rearrangements, we here report FLI1 as the fifth ETS transcription factor involved in fusion genes in prostate cancer. Outlier expression of the FLI1 gene was detected by TLDAs in one PCa that showed relative overexpression of FLI1 exons 4:5 as compared with FLI1 exons 2:3. A structural rearrangement was found using FISH probes flanking the FLI1 gene and RT‐PCR and sequencing analyses showed fusion of SLC45A3 exon 1 with FLI1 exon 3. Interestingly, we found four cases with two different ETS rearrangements in the index tumor, thus revealing intratumor genetic heterogeneity. Correlation analysis with clinico‐pathological data showed association of ERG rearrangements with locally advanced disease (pT3, P = 0.007) and MYC overexpression (P = 0.001), and association of ETV1 rearrangements with PTEN downregulation (P = 0.015). We report that FLI1 is a novel ETS transcription factor involved in gene fusions in prostate cancer and that intratumor genetic heterogeneity of ETS rearrangements can occasionally be found in index primary tumors.

Cysteine-Rich Secretory Protein-3 (CRISP3) Is Strongly Up-Regulated in Prostate Carcinomas with the TMPRSS2-ERG Fusion Gene

A large percentage of prostate cancers harbor TMPRSS2-ERG gene fusions, leading to aberrant overexpression of the transcription factor ERG. The target genes deregulated by this rearrangement, however, remain mostly unknown. To address this subject we performed genome-wide mRNA expression analysis on 6 non-malignant prostate samples and 24 prostate carcinomas with (n = 16) and without (n = 8) TMPRSS2-ERG fusion as determined by FISH. The top-most differentially expressed genes and their associations with ERG over-expression were technically validated by quantitative real-time PCR and biologically validated in an independent series of 200 prostate carcinomas. Several genes encoding metabolic enzymes or extracellular/transmembrane proteins involved in cell adhesion, matrix remodeling and signal transduction pathways were found to be co-expressed with ERG. Within those significantly over-expressed in fusion-positive carcinomas, CRISP3 showed more than a 50-fold increase when compared to fusion-negative carcinomas, whose expression levels were in turn similar to that of non-malignant samples. In the independent validation series, ERG and CRISP3 mRNA levels were strongly correlated (r(s) = 0.65, p<0.001) and both were associated with pT3 disease staging. Furthermore, immunohistochemistry results showed CRISP3 protein overexpression in 63% of the carcinomas and chromatin immunoprecipitation with an anti-ERG antibody showed that CRISP3 is a direct target of the transcription factor ERG. We conclude that ERG rearrangement is associated with significant expression alterations in genes involved in critical cellular pathways that define a subset of locally advanced PCa. In particular, we show that CRISP3 is a direct target of ERG that is strongly overexpressed in PCa with the TMPRSS2-ERG fusion gene.

Altered expression of MGMT in high-grade gliomas results from the combined effect of epigenetic and genetic aberrations.

MGMT downregulation in high-grade gliomas (HGG) has been mostly attributed to aberrant promoter methylation and is associated with increased sensitivity to alkylating agent-based chemotherapy. However, HGG harboring 10q deletions also benefit from treatment with alkylating agents. Because the MGMT gene is mapped at 10q26, we hypothesized that both epigenetic and genetic alterations might affect its expression and predict response to chemotherapy. To test this hypothesis, promoter methylation and mRNA levels of MGMT were determined by quantitative methylation-specific PCR (qMSP) or methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) and quantitative RT-PCR, respectively, in a retrospective series of 61 HGG. MGMT/chromosome 10 copy number variations were determined by FISH or MS-MLPA analysis. Molecular findings were correlated with clinical parameters to assess their predictive value. Overall, MGMT methylation ratios assessed by qMSP and MS-MLPA were inversely correlated with mRNA expression levels (best coefficient value obtained with MS-MLPA). By FISH analysis in 68.3% of the cases there was loss of 10q26.1 and in 15% of the cases polysomy was demonstrated; the latter displayed the highest levels of transcript. When genetic and epigenetic data were combined, cases with MGMT promoter methylation and MGMT loss depicted the lowest transcript levels, although an impact in response to alkylating agent chemotherapy was not apparent. Cooperation between epigenetic (promoter methylation) and genetic (monosomy, locus deletion) changes affecting MGMT in HGG is required for effective MGMT silencing. Hence, evaluation of copy number alterations might add relevant prognostic and predictive information concerning response to alkylating agent-based chemotherapy.

Relative 8q gain predicts disease-specific survival irrespective of the TMPRSS2-ERG fusion status in diagnostic biopsies of prostate cancer

Screening tools have greatly improved prostate cancer (PCa) detection, but the disease course is heterogeneous, and standard clinicopathological parameters do not fully discriminate aggressive from indolent tumors. To evaluate the prognostic value of the TMPRSS2‐ERG fusion gene combined with chromosome arm 8q relative gain in diagnostic biopsies of PCa, we studied a consecutive series of 200 diagnostic needle biopsies from patients with 10‐year disease‐specific survival data. TMPRSS2‐ERG fusion gene status and relative 8q gain were assessed by fluorescent in situ hybridization in whole formalin fixed paraffin‐embedded biopsies. The TMPRSS2‐ERG fusion gene was detected in 43.5% of PCa and was associated with lower Gleason score (P = 0.045), whereas relative 8q gain was present in 48% of PCa and was associated in high‐Gleason score (P < 0.001). ERG rearrangement alone was not associated with clinical outcome, whereas relative 8q gain predicted worse disease‐specific survival in PCa patients both with and without the TMPRSS2‐ERG fusion gene (P < 0.001), independently of Gleason score, clinical stage, and treatment modality. We conclude that relative 8q gain in diagnostic needle biopsies is a poor prognostic factor independent of the TMPRSS2‐ERG fusion gene status and of standard clinicopathological parameters.

Feasibility of differential diagnosis of kidney tumors by comparative genomic hybridization of fine needle aspiration biopsies.

The association of a genetic analysis that could improve the diagnostic accuracy of renal cell tumors in biopsy samples would allow better‐informed therapeutic decisions. We performed comparative genomic hybridization (CGH) on an ex vivo fine‐needle aspiration (FNA) biopsy and a tumor fragment obtained from 75 patients consecutively diagnosed with renal tumors and subjected to radical nephrectomy. The pattern of genomic changes by CGH was used blindly to classify the renal tumors and the genetic findings were subsequently compared with the histopathologic diagnosis. In particular cases, including in two carcinomas with morphologically distinct tumor areas, we performed FISH with several locus‐specific probes, and looked for VHL point mutations, exonic rearrangements, or promoter methylation. CGH was successful in 82.7% FNA biopsies and in 96% tumor fragments, with the former allowing genetic diagnosis in 75% of renal cell tumors. The genetic and the initial histological classification differed in two renal neoplasias, but the genetic diagnosis was confirmed after review. The genetic pattern correctly diagnosed 93.5% of clear cell renal cell carcinomas (RCC), 61.5% of chromophobe RCC, 100% of papillary RCC, and 14.3% of oncocytomas, with the negative predictive value being 93.9, 90.7, 100, and 90.2%, respectively. The positive predictive value and specificity of copy number profiles was 100%. We demonstrate that genetic diagnosis by CGH on FNA biopsies can improve differential diagnosis in patients with kidney tumors.

Genetic and clinical characterization of 45 acute leukemia patients with MLL gene rearrangements from a single institution

Chromosomal rearrangements affecting the MLL gene are associated with high‐risk pediatric, adult and therapy‐associated acute leukemia. In this study, conventional cytogenetic, fluorescence in situ hybridization, and molecular genetic studies were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL‐related leukemia treated in a single institution between 1998 and 2011. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL–AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL–MLLT3 (27%), a MLL–MLLT1 (20%), or MLL–MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloid leukemia was the MLL–MLLT3 fusion (42%), followed by MLL–MLLT10 (23%), MLL–MLLT1 (8%), MLL–ELL (8%), MLL–MLLT4 (4%), and MLL–MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL–MLLT4 fusion variant.

Potential downstream target genes of aberrant ETS transcription factors are differentially affected in Ewing's sarcoma and prostate carcinoma.

FLI1 and ERG, the major ETS transcription factors involved in rearrangements in the Ewing’s sarcoma family of tumors (ESFT) and in prostate carcinomas (PCa), respectively, belong to the same subfamily, having 98% sequence identity in the DNA binding domain. We therefore decided to investigate whether the aberrant transcription factors in both malignancies have some common downstream targets. We crossed a publicly available list of all putative EWSR1-FLI1 target genes in ESFT with our microarray expression data on 24 PCa and 6 non-malignant prostate tissues (NPT) and choose four genes among the top-most differentially expressed between PCa with (PCa ERG+) and without (PCa ETS-) ETS fusion genes (HIST1H4L, KCNN2, ECRG4 and LDOC1), as well as four well-validated direct targets of the EWSR1-FLI1 chimeric protein in ESFT (NR0B1, CAV1, IGFBP3 and TGFBR2). Using quantitative expression analysis in 16 ESFT and seven alveolar rhabdomyosarcomas (ARMS), we were able to validate the four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein, showing overexpression of CAV1 and NR0B1 and underexpression of IGFBP3 and TGFBR2 in ESFT as compared to ARMS. Although none of these four genes showed significant expression differences between PCa ERG+ and PCa ETS-, CAV1, IGFBP3 and TGFBR2 were less expressed in PCa in an independent series of 56 PCa and 15 NPT, as also observed for ECRG4 and LDOC1, suggesting a role in prostate carcinogenesis in general. On the other hand, we demonstrate for the first time that both HIST1H4L and KCNN2 are significantly overexpressed in PCa ERG+ and that ERG binds to the promoter of these genes. Conversely, KCNN2 was found underexpressed in ESFT relative to ARMS, suggesting that the EWSR1-ETS oncoprotein may have the opposite effect of ERG rearrangements in PCa. We conclude that aberrant ETS transcription factors modulate target genes differently in ESFT and PCa.

Prostate Cancer Prognosis Defined by the Combined Analysis of 8q, PTEN and ERG

Overtreatment is a major concern in men diagnosed with prostate cancer. The aim of this study was to evaluate the combined prognostic role of three frequent molecular alterations in prostate cancer, namely relative 8q gain, ERG overexpression, and loss of PTEN expression, in a series of 136 patients with prostate cancer treated with prostatectomy and with a long follow-up. Fluorescent in situ hybridization was used to detect the relative copy number of 8q and immunohistochemistry was used for quantitative assessment of ERG and PTEN expression. During a median follow-up period of 117.8 months, 66 (49%) patients had disease recurrence. Relative 8q gain, ERG overexpression, and loss of PTEN expression were observed in 18%, 56%, and 33% of the cases, respectively. No association with patient recurrence-free survival was found for relative 8q gain or ERG overexpression on their own, whereas loss of PTEN expression was associated with worse recurrence-free survival (P = .006). Interestingly, in the subgroup of patients with normal PTEN expression, we found that the combined relative 8q gain/ERG overexpression is associated with high risk of recurrence (P = .008), suggesting that alternative mechanisms exist for progression into clinically aggressive disease. Additionally, in intermediate-risk patients with normal PTEN expression in their tumors, the combination of 8q gain/ERG overexpression was associated with a poor recurrence-free survival (P < .001), thus indicating independent prognostic value. This study shows that the combined analysis of 8q, ERG and PTEN contributes to an improved clinical outcome stratification of prostate cancer patients treated with radical prostatectomy.

Deregulation of PAX2 expression in renal cell tumours: mechanisms and potential use in differential diagnosis

Expression of PAX2 (Paired‐box 2) is suppressed through promoter methylation at the later stages of embryonic development, but eventually reactivated during carcinogenesis. Pax‐2 is commonly expressed in the most prevalent renal cell tumour (RCT) subtypes—clear cell RCC (ccRCC), papillary RCC (pRCC) and oncocytoma—but not in chromophobe RCC (chrRCC), which frequently displays chromosome 10 loss (to which PAX2 is mapped). Herein, we assessed the epigenetic and/or genetic alterations affecting PAX2 expression in RCTs and evaluated its potential as biomarker. We tested 120 RCTs (30 of each main subtype) and four normal kidney tissues. Pax‐2 expression was assessed by immunohistochemistry and PAX2 mRNA expression levels were determined by quantitative RT‐PCR. PAX2 promoter methylation status was assessed by methylation‐specific PCR and bisulfite sequencing. Chromosome 10 and PAX2 copy number alterations were determined by FISH. Pax‐2 immunoexpression was significantly lower in chrRCC compared to other RCT subtypes. Using a 10% immunoexpression cut‐off, Pax‐2 immunoreactivity discriminated chrRCC from oncocytoma with 67% sensitivity and 90% specificity. PAX2 mRNA expression was significantly lower in chrRCC, compared to ccRCC, pRCC and oncocytoma, and transcript levels correlated with immunoexpression. Whereas no promoter methylation was found in RCTs or normal kidney, 69% of chrRCC displayed chromosome 10 monosomy, correlating with Pax‐2 immunoexpression. We concluded that Pax‐2 expression might be used as an ancillary tool to discriminate chrRCC from oncocytomas with overlapping morphological features. The biological rationale lies on the causal relation between Pax‐2 expression and chromosome 10 monosomy, but not PAX2 promoter methylation, in chrRCC.

NCOA2 is a candidate target gene of 8q gain associated with clinically aggressive prostate cancer

Prostate carcinomas harboring 8q gains are associated with poor clinical outcome, but the target genes of this genomic alteration remain to be unveiled. In this study, we aimed to identify potential 8q target genes associated with clinically aggressive prostate cancer (PCa) using fluorescence in situ hybridization (FISH), genome‐wide mRNA expression, and protein expression analyses. Using FISH, we first characterized the relative copy number of 8q (assessed with MYC flanking probes) of a series of 50 radical prostatectomy specimens, with available global gene expression data and typed for E26 transformation specific (ETS) rearrangements, and then compared the gene expression profile of PCa subsets with and without 8q24 gain using Significance Analysis of Microarrays. In the subset of tumors with ERG fusion genes (ERG+), five genes were identified as significantly overexpressed (false discovery rate [FDR], ≤5%) in tumors with relative 8q24 gain, namely VN1R1, ZNF417, CDON, IKZF2, and NCOA2. Of these, only NCOA2 is located in 8q (8q13.3), showing a statistically higher mRNA expression in the subgroup with relative 8q gain, both in the ERG+ subgroup and in the whole series (P = 0.000152 and P = 0.008, respectively). Combining all the cases with NCOA2 overexpression, either at the mRNA or at the protein level, we identified a group of tumors with NCOA2 copy‐number increase, independently of ETS status and relative 8q24 gain. Furthermore, for the first time, we detected a structural rearrangement involving NCOA2 in PCa. These findings warrant further studies with larger series to evaluate if NCOA2 relative copy‐number gain presents prognostic value independently of the well‐established poor prognosis associated with MYC relative copy‐number gain.