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Background: Aromatase (CYP19A1) regulates estrogen biosynthesis. Polymorphisms in CYP19A1 have been related to the pathogenesis of breast cancer (BC). Inhibition of aromatase with letrozole constitutes the best option for treating estrogen-dependent BC in postmenopausal women. We evaluate a series of polymorphisms of CYP19A1 and their effect on response to neoadjuvant letrozole in early BC.Methods: We analyzed 95 consecutive postmenopausal women with stage II-III ER/PgR [+] BC treated with neoadjuvant letrozole. Response to treatment was measured by radiology at 4th month by World Health Organization (WHO) criteria. Three polymorphisms of CYP19A1, one in exon 7 (rs700519) and two in the 3'-UTR region (rs10046 and rs4646) were evaluated on DNA obtained from peripheral blood.Results: Thirty-five women (36.8%) achieved a radiological response to letrozole. The histopathological and immunohistochemical parameters, including hormonal receptor status, were not associated with the response to letrozole. Only the genetic variants (AC/AA) of the rs4646 polymorphism were associated with poor response to letrozole (p = 0.03). Eighteen patients (18.9%) reported a progression of the disease. Those patients carrying the genetic variants (AC/AA) of rs4646 presented a lower progression-free survival than the patients homozygous for the reference variant (p = 0.0686). This effect was especially significant in the group of elderly patients not operated after letrozole induction (p = 0.009).Conclusions: Our study reveals that the rs4646 polymorphism identifies a subgroup of stage II-III ER/PgR [+] BC patients with poor response to neoadjuvant letrozole and poor prognosis. Testing for the rs4646 polymorphism could be a useful tool in order to orientate the treatment in elderly BC patients. [ABSTRACT FROM AUTHOR]

Purpose: The IMMUNOSARC trial combined an antiangiogenic agent (sunitinib) with a PD1 inhibitor (nivolumab) in advanced sarcomas. Here, we present the first correlative studies of the soft-tissue sarcoma cohort enrolled in this trial., Experimental Design: Formalin-fixed paraffin-embedded and peripheral blood samples were collected at baseline and week 13. Formalin-fixed paraffin-embedded samples were used for transcriptomics and multiplex immunofluorescence, whereas peripheral blood samples were used for multiplexed immunoassays. Flow cytometry and Luminex assays were performed to validate translational findings in tumor-isolated cells and peripheral blood mononuclear cells derived from patients., Results: The density of intratumoral CD8+ T cells, measured by multiplexed immunophenotyping, was significantly increased after treatment. This augment was accompanied by the dynamic significant increase in the gene expressions of CD86, CHI3L1, CXCL10, CXCL9, LAG3, and VCAM1 and the decrease in the expression levels of NR4A1. In peripheral blood, 12 proteins were significantly modulated by treatment at week 13. A score integrating the dynamic expression of the 7 genes and the 12 soluble factors separated 2 groups with distinct progression-free survival (PFS): 4.1 months [95% confidence interval, 3.5-not reached (NR)] versus 17 months (95% confidence interval, 12.0-NR), P = 0.014. This molecular score was predictive of PFS when applied to the normalized data determined in the baseline samples., Conclusions: Treatment with sunitinib and nivolumab inflamed the sarcoma microenvironment, increasing CD8+ T-cell density and the expression of several genes/proteins with relevance in the response to PD1 inhibitors. A molecular signature identified two groups of patients with distinct PFS for the combination of antiangiogenics plus PD1 inhibitor therapy., (©2024 American Association for Cancer Research.)

In this study, we identify USP1 as a transcriptional target of EWS::FLI1 and demonstrate the requisite function of USP1 in Ewing sarcoma (EWS) cell survival in response to endogenous replication stress. EWS::FLI1 oncogenic transcription factor drives most EWS, a pediatric bone cancer. EWS cells display elevated levels of R-loops and replication stress. The mechanism by which EWS cells override activation of apoptosis or cellular senescence in response to increased replication stress is not known. We show that USP1 is overexpressed in EWS and EWS::FLI1 regulates USP1 transcript levels. USP1 knockdown or inhibition arrests EWS cell growth and induces cell death by apoptosis. Mechanistically, USP1 regulates Survivin (BIRC5/API4) protein stability and the activation of caspase-9 and caspase-3/7 in response to endogenous replication stress. Notably, USP1 inhibition sensitizes cells to doxorubicin and etoposide treatment. Together, our study demonstrates that USP1 is regulated by EWS::FLI1, the USP1-Survivin axis promotes EWS cell survival, and USP1 inhibition sensitizes cells to standard of care chemotherapy., Implications: High USP1 and replication stress levels driven by EWS::FLI1 transcription factor in EWS are vulnerabilities that can be exploited to improve existing treatment avenues and overcome drug resistance., (©2023 The Authors; Published by the American Association for Cancer Research.)

Overexpression of the EPS15 Homology Domain containing 1 (EHD1) protein has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown. We establish that EHD1 is overexpressed in Ewing sarcoma (EWS), with high EHD1 mRNA expression specifying shorter patient survival. ShRNA-knockdown and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. RTK antibody arrays identified IGF-1R as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting., (© 2023. The Author(s).)

Background: Granulosa cell ovarian tumor (GCT) is characterized by a pathognomonic mutation in the FOXL2 gene (402 C > G) that leads to an overactivation of steroidogenesis. CYP17 is a key enzyme in such process and can be inhibited by ketoconazole., Methods: We designed a phase II clinical trial to assess the efficacy of ketoconazole in advanced GCT and conducted several in vitro studies to support the clinical findings., Results: From October 1st 2012 to January 31st 2014, six evaluable patients were recruited in ten hospitals of the Spanish Group for Transversal Oncology and Research in Orphan and Infrequent Tumors" (GETTHI). FOXL2 (402C > G) mutation was confirmed in three; two cases were wild type and it could not be assessed in one. No objective response by RECIST was observed, but five cases achieved stable disease longer than 12 months. Median progression-free survival was 14.06 months (CI 95% 5.43-22.69) for the whole study population (3.38 and 13.47 months for wild-type cases and 14.06, 20.67 and 26.51 for those with confirmed FOXL2 mutation). Median overall survival was 22·99 months (CI 95% 8.99-36.99). In vitro assays confirmed the activity of ketoconazole in this tumor and suggested potential synergisms with other hormone therapies., Conclusion: Ketoconazole has shown activity in advanced GCT in clinical and in vitro studies. Based on these data, an orphan designation was granted by the European Medicines Agency for ketoconazole in GCT (EU/3/17/1857)., Gov Identifier: NCT01584297., (© 2023. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

Pancreatic ductal adenocarcinoma (PDAC) prognoses and treatment responses remain devastatingly poor due partly to the highly heterogeneous, aggressive, and immunosuppressive nature of this tumor type. The intricate relationship between the stroma, inflammation, and immunity remains vaguely understood in the PDAC microenvironment. Here, we performed a meta-analysis of stroma-, and immune-related gene expression in the PDAC microenvironment to improve disease prognosis and therapeutic development. We selected 21 PDAC studies from the Gene Expression Omnibus and ArrayExpress databases, including 922 samples (320 controls and 602 cases). Differential gene enrichment analysis identified 1153 significant dysregulated genes in PDAC patients that contribute to a desmoplastic stroma and an immunosuppressive environment (the hallmarks of PDAC tumors). The results highlighted two gene signatures related to the immune and stromal environments that cluster PDAC patients into high- and low-risk groups, impacting patients' stratification and therapeutic decision making. Moreover, HCP5 , SLFN13 , IRF9 , IFIT2 , and IFI35 immune genes are related to the prognosis of PDAC patients for the first time.

Germline and tumor BRCA testing constitutes a valuable tool for clinical decision-making in the management of epithelial ovarian cancer (EOC) patients. Tissue testing is able to identify both germline (g) and somatic (s) BRCA variants, but tissue preservation methods and the widespread implementation of NGS represent pre-analytical and analytical challenges that need to be managed. This study was carried out on a multicenter prospective GEICO cohort of EOC patients with known gBRCA status in order to determine the inter-laboratory reproducibility of tissue sBRCA testing. The study consisted of two independent experimental approaches, a bilateral comparison between two reference laboratories (RLs) testing 82 formalin-paraffin-embedded (FFPE) EOC samples each, and a Ring Test Trial (RTT) with five participating clinical laboratories (CLs) evaluating the performance of tissue BRCA testing in a total of nine samples. Importantly, labs employed their own locally adopted next-generation sequencing (NGS) analytical approach. BRCA mutation frequency in the RL sub-study cohort was 23.17%: 12 (63.1%) germline and 6 (31.6%) somatic. Concordance between the two RLs with respect to BRCA status was 84.2% (g BRCA 100%). The RTT study distributed a total of nine samples (three commercial synthetic human FFPE references, three FFPE, and three OC DNA) among five CLs. The median concordance detection rate among them was 64.7% (range: 35.3-70.6%). Analytical discrepancies were mainly due to the minimum variant allele frequency thresholds, bioinformatic pipeline filters, and downstream variant interpretation, some of them with consequences of clinical relevance. Our study demonstrates a wide range of concordance in the identification and interpretation of BRCA sequencing data, highlighting the relevance of establishing standard criteria for detecting, interpreting, and reporting BRCA variants.

We hypothesized that the combination of olaparib and lurbinectedin maximizes DNA damage, thus increasing its efficacy. The POLA phase 1 trial established the recommended phase 2 dose of lurbinectedin as being 1.5 mg (day 1) and that of olaparib as being 250 mg/12 h (days 1-5) for a 21-day cycle. In phase 2, we explore the efficacy of the combination in terms of clinical response and its correlation with mutations in the HRR genes and the genomic instability (GI) parameters. Results: A total of 73 patients with high-grade ovarian ( n = 46), endometrial ( n = 26), and triple-negative breast cancer ( n = 1) were treated with lurbinectedin and olaparib. Most patients (62%) received ≥3 lines of prior therapy. The overall response rate (ORR) and disease control rate (DCR) were 9.6% and 72.6%, respectively. The median progression-free survival (PFS) was 4.54 months (95% CI 3.0-5.2). Twelve (16.4%) patients were considered long-term responders (LTR), with a median PFS of 13.3 months. No clinical benefit was observed for cases with HRR gene mutation. In ovarian LTRs, although a direct association with GI and a total loss of heterozygosity (LOH) events was observed, the association did not reach statistical significance ( p = 0.055). Globally, the total number of LOHs might be associated with the ORR ( p =0.074). The most common grade 3-4 toxicities were anemia and thrombocytopenia, in 6 (8.2%) and 3 (4.1%) patients, respectively. Conclusion: The POLA study provides evidence that the administration of lurbinectedin and olaparib is feasible and tolerable, with a DCR of 72.6%. Different GI parameters showed associations with better responses.

The poly (ADP-Ribose) polymerase (PARP) inhibitor olaparib has shown antitumor activity in patients with ovarian or breast cancer with or without BRCA1/2 mutations. Lurbinectedin is an ecteinascidin that generates DNA double-strand breaks. We hypothesized that the combination of olaparib and lurbinectedin maximizes the DNA damage increasing the efficacy. A 3 + 3 dose-escalation study examined olaparib tablets with lurbinectedin every 21 days. The purpose of this phase I study is to determine the dose-limiting toxicities (DLTs) of the combination, to investigate the maximum tolerated dose (MTD), the recommended phase II dose (RP2D), efficacy, pharmacokinetics, in addition to genotyping and translational studies. In total, 20 patients with ovarian and endometrial cancers were included. The most common adverse events were asthenia, nausea, vomiting, constipation, abdominal pain, neutropenia, anemia. DLT grade 4 neutropenia was observed in two patients in dose level (DL) 5, DL4 was defined as the MTD, and the RP2D was lurbinectedin 1.5 mg/m 2  + olaparib 250 mg twice a day (BID). Mutational analysis revealed a median of 2 mutations/case, 53% of patients with mutations in the homologous recombination (HR) pathway. None of the patients reached a complete or partial response; however, 60% of stable disease was achieved. In conclusion, olaparib in combination with lurbinectedin was well tolerated with a disease control rate of 60%. These results deserve further evaluation of the combination in a phase II trial.

A translational study was designed to analyze the expression of nucleotide excision repair (NER) and homologous recombination (HR) genes as potential predictive biomarkers for trabectedin in soft-tissue sarcoma (STS). This study is part of a randomized phase II trial comparing trabectedin plus doxorubicin versus doxorubicin in advanced STS. Gene expression levels were evaluated by qRT-PCR, while CUL4A protein levels were quantified by immunohistochemistry. Expression levels were correlated with patients' progression-free survival (PFS) and overall survival (OS). Gene expression was also evaluated in cell lines and correlated with trabectedin sensitivity. In doxorubicin arm and in the whole series, which includes samples from both arms, no significant differences in terms of PFS were observed amongst the analyzed genes. In the group treated with trabectedin plus doxorubicin, the median of PFS was significantly longer in cases with CUL4A , ERCC1, or ERCC5 overexpression, while BRCA1 expression did not correlated with PFS. Gene expression had no prognostic influence in OS. CUL4A protein levels correlated with worse PFS in doxorubicin arm and in the whole series. In cell lines, only overexpression of ERCC1 was significantly correlated with trabectedin sensitivity. In conclusion, CUL4A, ERCC5, and mainly ERCC1 acted as predictive factors for trabectedin efficacy in advanced STS.

Background: Solitary fibrous tumour is an ultra-rare sarcoma, which encompasses different clinicopathological subgroups. The dedifferentiated subgroup shows an aggressive course with resistance to pazopanib, whereas in the malignant subgroup, pazopanib shows higher activity than in previous studies with chemotherapy. We designed a trial to test pazopanib activity in two different cohorts of solitary fibrous tumour: the malignant-dedifferentiated cohort, which was previously published, and the typical cohort, which is presented here., Methods: In this single-arm, phase 2 trial, adult patients (aged ≥18 years) diagnosed with confirmed metastatic or unresectable typical solitary fibrous tumour of any location, who had progressed in the previous 6 months (by Choi criteria or Response Evaluation Criteria in Solid Tumors [RECIST]) and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 were enrolled at 11 tertiary hospitals in Italy, France, and Spain. Patients received pazopanib 800 mg once daily, taken orally, until progression, unacceptable toxicity, withdrawal of consent, non-compliance, or a delay in pazopanib administration of longer than 3 weeks. The primary endpoint was proportion of patients achieving an overall response measured by Choi criteria in patients who received at least 1 month of treatment with at least one radiological assessment. All patients who received at least one dose of the study drug were included in the safety analyses. This study is registered in ClinicalTrials.gov, NCT02066285, and with the European Clinical Trials Database, EudraCT 2013-005456-15., Findings: From June 26, 2014, to Dec 13, 2018, of 40 patients who were assessed, 34 patients were enrolled and 31 patients were included in the response analysis. Median follow-up was 18 months (IQR 14-34), and 18 (58%) of 31 patients had a partial response, 12 (39%) had stable disease, and one (3%) showed progressive disease according to Choi criteria and central review. The proportion of overall response based on Choi criteria was 58% (95% CI 34-69). There were no deaths caused by toxicity, and the most frequent adverse events were diarrhoea (18 [53%] of 34 patients), fatigue (17 [50%]), and hypertension (17 [50%])., Interpretation: To our knowledge, this is the first prospective trial of pazopanib for advanced typical solitary fibrous tumour. The manageable toxicity and activity shown by pazopanib in this cohort suggest that this drug could be considered as first-line treatment for advanced typical solitary fibrous tumour., Funding: Spanish Group for Research on Sarcomas (GEIS), Italian Sarcoma Group (ISG), French Sarcoma Group (FSG), GlaxoSmithKline, and Novartis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

CDKN2A germline mutations increase the risk of melanoma development and are present in 20 and 10% of familial and multiple melanoma cases, respectively. Pancreatic cancer has been associated with CDKN2A in some populations and, accordingly, its presence in first-degree or second-degree relatives of a melanoma patient is considered as a criterion for genetic testing. In this study, we show that in an area with low melanoma incidence, CDKN2A germline mutations in patients with melanoma and personal or family history of pancreatic cancer are mainly present in the setting of familial or multiple melanoma cases. In addition, a relatively young age (≤52 years) at pancreatic diagnosis is an additional single criterion that might also be considered.

Objectives: PCA3 performance as a single second line biomarker is compared to the European Randomised Study of Screening for Prostate Cancer risk calculator model 3 (ERSPC RC-3) in an opportunistic screening in prostate cancer (PCa)., Material and Methods: 5,199 men, aged 40-75y, underwent prostate-specific antigen (PSA) screening and digital rectal examination (DRE). Men with a normal DRE and PSA ≥3ng/ml had a PCA3 test done. All men with PCA3 ≥35 underwent an initial biopsy (IBx) -12 cores-. Men with PCA3 <35 were randomized 1:1 to either IBx or observation. We compared them to those obtained with ERSPC RC-3., Results: PCA3 test was performed on 838 men (16.1%). In PCA3(+) and PCA3(-) groups, global PCa detection rates were 40.9% and 14.7% with a median follow-up (FU) of 21.7 months (P<.001). In the PCA3(+) arm (n=301, 35.9%), PCa was identified in 115 men at IBx (38.2%). In the randomized arm, 256 underwent IBx and PCa was found in 46 (18.0%) (P<.001). The biopsy-sparing potential would have been 64.1% as opposed to 76.6% if we had used ERSPC RC-3. However, the estimated false negative cases for HGPCa would have been reduced by 37.1% (89 to 56 patients). Moreover, if we had applied PCA3-35 to avoid IBx, 14.7% PCa and 9.1% of clinical significant PCa patients would not have been diagnosed during this FU., Conclusions: When PCA3-35 is used as a second-line biomarker when PSA ≥3ng/ml and DRE is normal, IBx could be avoided in 12.5% less than if ERSPC RC-3 is used and would reduce the false negative cases by 36.2%. At a FU of 21.7 months, this dual protocol would miss 9.1% of clinically significant PCa, so strict FU is mandatory with established biopsy criteria based on PSA and DRE in cases with PCA3 <35., (Copyright © 2016 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.)

Background: Gastrointestinal stromal tumour (GIST) is the most common primary mesenchymal tumour of the gastrointestinal tract. Spindle cell monophasic synovial sarcoma (SS) can be morphologically similar. Angiogenesis is a major factor for tumour growth and metastasis. Our aim was to compare the angiogenic expression profiles of high-risk GIST and spindle cell monophasic SS by histological, immunohistochemical and molecular characterisation of the neovascularisation established between xenotransplanted tumours and the host during the initial phases of growth in nude mice., Methods: The angiogenic profile of two xenotransplanted human soft-tissue tumours were evaluated in 15 passages in nude mice using tissue microarrays (TMA). Tumour pieces were also implanted subcutaneously on the backs of 14 athymic Balb-c nude mice. The animals were sacrificed at 24, 48, and 96 h; and 7, 14, 21, and 28 days after implantation to perform histological, immunohistochemical, and molecular studies (neovascularisation experiments)., Results: Morphological similarities were apparent in the early stages of neoplastic growth of these two soft-tissue tumours throughout the passages in nude mice and in the two neovascularisation experiments. Immunohistochemistry demonstrated overexpression of pro-angiogenic factors between 24 h and 96 h after xenotransplantation in both tumours. Additionally, neoplastic cells coexpressed chemokines (CXCL9, CXCL10, GRO, and CXCL12) and their receptors in both tumours. Molecular studies showed two expression profiles, revealing an early and a late phase in the angiogenic process., Conclusion: This model could provide information on the early stages of the angiogenic process in monophasic spindle cell SS and high-risk GIST and offers an excellent way to study possible tumour response to antiangiogenic drugs.

Purpose: Doxorubicin and trabectedin are considered active drugs in soft tissue sarcoma (STS). The combination of both drugs was hypothesized to be advantageous and safe on the basis of preclinical evidence and a previous phase I trial, respectively. The aim of this study was to compare the clinical outcome of trabectedin plus doxorubicin with doxorubicin as first-line treatment of advanced STS patients., Patients and Methods: In this open-label randomized phase II trial, the main end point was progression-free survival (PFS). Trabectedin 1.1 mg/m(2) in a 3-hour infusion plus doxorubicin 60 mg/m(2) as the experimental arm and doxorubicin 75 mg/m(2) as the control arm were administered for up to six cycles. Translational research was planned to correlate the expression of apoptotic and DNA repair genes with clinical outcome., Results: In 115 randomly assigned patients, the median PFS was 5.5 months in the control arm and 5.7 months in the experimental arm (hazard ratio, 1.16; 95% CI, 0.79 to 1.71; P = .45) in the intent-to-treat analysis. The trial was stopped for futility after the interim analysis, because the results in the experimental arm showed the risk reduction for the main end point to be < 9.64%. The proportion of patients with grade 3 or 4 thrombocytopenia, asthenia, and liver toxicity was significantly higher in the experimental arm. FAS and p53 were shown to be prognostic factors for PFS (7.0 months if FAS+ and p53-; 3.4 months if FAS+/p53+ or FAS-/p53-; and 0.7 months if FAS- and p53+; P < .001) and for overall survival., Conclusion: Trabectedin plus doxorubicin did not show superiority over doxorubicin alone as first-line treatment of advanced STS. The prognostic role of apoptotic key genes, FAS and p53, was shown to be robust enough to continue this research line., (© 2016 by American Society of Clinical Oncology.)

Background: PCA3 has been included in a nomogram outperforming previous clinical models for the prediction of any prostate cancer (PCa) and high grade PCa (HGPCa) at the initial prostate biopsy (IBx). Our objective is to validate such IBx-specific PCA3-based nomogram. We also aim to optimize the use of this nomogram in clinical practice through the definition of risk groups., Methods: Independent external validation. Clinical and biopsy data from a contemporary cohort of 401 men with the same inclusion criteria to those used to build up the reference's nomogram in IBx. The predictive value of the nomogram was assessed by means of calibration curves and discrimination ability through the area under the curve (AUC). Clinical utility of the nomogram was analyzed by choosing thresholds points that minimize the overlapping between probability density functions (PDF) in PCa and no PCa and HGPCa and no HGPCa groups, and net benefit was assessed by decision curves., Results: We detect 28% of PCa and 11 % of HGPCa in IBx, contrasting to the 46 and 20% at the reference series. Due to this, there is an overestimation of the nomogram probabilities shown in the calibration curve for PCa. The AUC values are 0.736 for PCa (C.I.95%:0.68-0.79) and 0.786 for HGPCa (C.I.95%:0.71-0.87) showing an adequate discrimination ability. PDF show differences in the distributions of nomogram probabilities in PCa and not PCa patient groups. A minimization of the overlapping between these curves confirms the threshold probability of harboring PCa >30 % proposed by Hansen is useful to indicate a IBx, but a cut-off > 40% could be better in series of opportunistic screening like ours. Similar results appear in HGPCa analysis. The decision curve also shows a net benefit of 6.31% for the threshold probability of 40%., Conclusions: PCA3 is an useful tool to select patients for IBx. Patients with a calculated probability of having PCa over 40% should be counseled to undergo an IBx if opportunistic screening is required.

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Objectives: To reduce unnecessary biopsies (Bx) in an opportunistic screening programme of prostate cancer., Material and Methods: We perform a prospective evaluation of PCA3 as a second line biomarker in an opportunistic screening for prostate cancer (PCa). From September-2010 until September-2012, 2,366 men, aged 40-74 years and with >10 years life expectancy, were initially screened with PSA/digital rectal examination (DRE). Men with previous Bx or with recent urine infections were excluded. Men with abnormal DRE and/or PSA >3 ng/ml were submitted for PCA3. All men with PCA3 ≥ 35 underwent an initial biopsy (IBx) -12cores-. Men with PCA3 < 35 were randomized 1:1 to either IBx or observation. Re-biopsy(16-18 cores) criteria were PSA increase >.5 ng/ml at 4-6 months or PSAv > .75 ng/ml/year., Results: With median follow-up (FU) of 10.1 months, PCA3 was performed in 321/2366 men (13.57%), 289 at first visit and 32 during FU. All 110 PCA3+ men (34.3%) were biopsied and PCa was identified in 43 men in IBx (39.1%). In the randomized arm, 110 were observed and 101 underwent biopsy, finding 12 PCa (11.9%), showing a statistically significant reduction of PCa detection rate in this cohort (P<.001). Global PCa detection rates were 40.9% and 9.5% for the PCA3+ and PCA3- branches, respectively (P<.001). Area under the curve for PSA and PCA3 were .601 and .74, respectively. This is an ongoing prospective study limited by its short follow-up period and still limited enrolment., Conclusions: PCA3 as a second line biomarker within an opportunistic dual screening protocol, can potentially avoid 65.7% and 50.1% biopsies at first round and at median FU of 10.1 months, respectively, just missing around 3.2% of high grade PCa., (Copyright © 2013 AEU. Published by Elsevier Espana. All rights reserved.)

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., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

Background: Germline 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., Methods: We 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., Results: We 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 VAT1 locus to the beginning of NBR1 gene, including the RND2, ΨBRCA1, BRCA1 and NBR2 complete genes., Conclusion: This 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.

We determined whether ubiquitylation and sumoylation processes are involved in conventional renal cell carcinogenesis associated with chronic, long-term, persistent low doses of ionizing radiation (IR) in patients living for more than 20 years in cesium-137 ((137)Cs)-contaminated areas after the Chernobyl accident in Ukraine. To this end, we assessed the immunohistochemical expression of ubiquitin (Ub), SUMO1, SUMO E2 conjugating enzyme Ubc9, and the cell cycle regulators p53, mdm2, and p14(ARF) in 38 conventional renal cell carcinomas from Ukrainian patients with different degrees of radiation exposure after the Chernobyl accident. As control cases, 18 conventional renal carcinoma (cRCC) tissues from a Spanish cohort were analyzed. No significant differences between the Ukrainian and Spanish groups were found regarding Ub overexpression, although being higher in the Ukrainian cases. Furthermore, this expression was inversely associated with SUMO1 and Ubc9, with no correlation with tumor nuclear grade. There was also a direct relationship between Ubc9 and inflammatory response. These findings do not allow us to consider the immunohistochemical expression of ubiquitylation and sumoylation as valuable markers for discriminating the effects of long-term, low-dose IR exposure in cRCC carcinogenesis.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. The present review evaluates the most important parameters in GIST: epidemiology, principal clinical presentations, histopathological patterns of GIST with differential diagnosis and Fletcher's and Miettinen's risk classification, immunohistochemistry, prognostic factors, c-KIT and PDGFRalpha mutations and treatment of this tumor. The most frequent site for GISTs is the stomach, followed by the duodenum and small intestine; spindle cell morphology is described in 70% with CD17 positivity in around 95% of the cases; exon 11 of c-KIT is the most mutated, with Gleevec being the principal treatment. In GISTs, high mitotic rate, large tumor size, incomplete resection, extragastrointestinal site, duplication 502-503 in exon 9 of c-KIT, and secondary mutations are negative prognostic factors.

Aims: The role of IGF system in the pathogenesis of Ewing's sarcoma (EWS) is well-documented. However, still little information is available about the value of IGF system components as indicators of prognosis. Understanding the clinical role for IGF system in EWS patients may be important because different subtypes of patients have distinct outcome and may require different treatment protocol. We evaluated the expression of insulin-like growth factor (IGF)-receptor (IGF-IR), insulin receptor (IR), IGF-I and some major intracellular mediators (IRS1, p-ERK) in specimens from EWS patients with primary localised untreated tumours., Patients and Methods: 290 samples were used for immunohistochemistry studies; 57 samples for Real-Time PCR studies; and serum of 67 patients for ELISA., Results: IGF-IR and IR are expressed in virtually all EWS tumours. Usually both the receptors are present in the same tumour but when one receptor is lacking the other one is always present. Evaluation of IGF-IR, IR and IGF-I with quantitative methods may discriminate differential levels of expression with influences on the patients' outcome. High expression of IGF-IR, IR and IGF-I mRNAs is significantly associated with more favourable clinical outcomes. Higher circulating levels of IGF-I also correlated with lower risk to disease progression and death., Conclusions: Overall, our clinical data are in contrast with the assumption that higher amounts of IGF/IGF-IR are a surrogate for higher aggressiveness and indicate that in some cancers the transition to frank malignancy and poor treatment responsiveness seem to be associated with a reduction of IGF system activity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: To assess whether deletions involving codons 557 and/or 558 (critical deletions) of exon 11 of KIT are relevant in the prognosis of relapse-free survival (RFS) in gastrointestinal stromal tumor (GIST) patients with a long follow-up., Patients and Methods: A univariate and multivariate analysis for RFS were carried out on 162 localized GIST patients over the entire follow-up period and over the intervals 0-4 years and >4 years. Factors assessed among others were Fletcher/National Institutes of Health and Miettinen-Lasota/Armed Forces Institute of Pathology (M-L/AFIP) risk categories, critical deletions and non-deletion-type mutation (NDTM) within exon 11 of KIT., Results: Multivariate analyses revealed that M-L/AFIP [relative risk (RR) 11.45, confidence interval (CI) 4.40-29.76, for the high-risk subgroup and RR 5.97, CI 2.09-17.06, for the intermediate subgroup] and critical deletions (RR 3.05, CI 1.59-5.85) were independent prognostic factors for RFS for the first 4 years and for the entire follow-up period. Beyond 4 years, the high-risk M-L/AFIP subgroup (RR 8.12, CI 1.48-44.4) and NDTM (RR 6.42, CI 1.17-35.12) were independent prognostic factors for RFS. The median follow-up was 84 months., Conclusion: Critical deletions represent a time-dependent prognostic factor limited to the first 4 years after surgery, which could help identify a subset with higher and earlier risk for relapse in GIST patients.

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 Ewing's 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.

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.

Ewing sarcoma is a highly aggressive malignant bone tumor occurring preferentially in children and young adults. At present, only clinical features, such as patient age, presence of clinically evident metastases at diagnosis, and poor response to neoadjuvant chemotherapy, are widely accepted as prognostic indicators in Ewing sarcoma. In this study, we assessed the prognostic value of CCN3 (Nov), a matricellular protein that play crucial roles in bone formation. Polyclonal antibodies directed against each of the different CCN3 modules were used to identify variant CCN3 proteins in tumors and to draw potential relationships between the expression of these variants and the outcome of patients with Ewing sarcoma. Our results confirmed that expression of the full-length CCN3 in Ewing sarcoma is associated to a worse prognostic. Furthermore, we report a possible relationship between the expression of a CCN3 protein lacking an internal module (von Willebrand factor type C) and sensitivity to radiotherapy. We hypothesize that the increased level of variant CCN3 in the tumor cells reduces their tumorigenic potential and results in better outcome.

Background: Tissue microarrays (TMAs) are used to study genomics and proteomics in several tumour tissue samples. Cell lines (CC) are of great importance in the study of the genetic changes in tumours, and some reveal several aspects of tumour oncogenesis. There are few published reports on Ewing's tumours with TMAs including original tumours (OT) and corresponding CC., Methods: We have performed four TMAs, from 3 OT and the corresponding CC of successive in vivo and in vitro tumour passages. Xenotransplant CC in nude mice from OT (XT/OT) was made. Subsequently multiple XT were performed and in vitro XT cell line (CC/XT) was obtained. In vivo re-inoculation of CC/XT (XT/CC) was planned. TMAs with the successive tumour passages that grew in nude mice (XT/OT and XT/CC) were analyzed by morphologic pattern (Hematoxilin/eosin), immunohistochemical staining (CD99, FLI1, p16, p53, ki-67), fluorescent in situ hybridization-FISH-(EWSR1 break apart, p16 and p53 status) and gene fusion types., Results: Heterogeneous results of the p16, p53 and ki67 in OT, XT/OT, CC/XT and XT/CC were observed. The three cell lines revealed EWS/FLI1 rearrangements. p16 gene was deleted only in one case. The deletion was detected by FISH and confirmed by PCR assays. A p53 alteration was found in the second case with monosomy and subsequently polysomic status of chromosome 17 during the evolution of CC. The PCR study revealed p53 mutation. The third case showed hypermethylation in the promoter of p16. The growth of the tumour in nude mice was more accelerated when the inoculation was performed from the CC/XT, increasing progressively over the passages. The third case did not reveal tumour growth in nude mice after the re-inoculation of CC/XT., Conclusion: The study of several cores from original tumours and successive tumour passages in TMAs facilitated the analysis of the genetic alteration and protein expression in Ewing's tumours.

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 (Glivectrade mark). 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., ((c) 2007 Wiley-Liss, Inc.)

Developing a tissue bank database has become more than just logically arranging data in tables combined with a search engine. Current demand for high quality samples and data, and the ever-changing legal and ethical regulations mean that the application must reflect TuBaFrost rules and protocols for the collection, exchange and use of tissue. To ensure continuation and extension of the TuBaFrost European tissue bank, the custodianship of the samples, and hence the decision over whether to issue samples to requestors, remains with the local collecting centre. The database application described in this article has been developed to facilitate this open structure virtual tissue bank model serving a large group. It encompasses many key tasks, without the requirement for personnel, hence minimising operational costs. The Internet-accessible database application enables search, selection and request submission for requestors, whereas collectors can upload and edit their collection. Communication between requestor and involved collectors is started with automatically generated e-mails.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated Virtual Microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting bio-repositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

TuBaFrost is the consortium responsible for the creation of a virtual European human frozen tumour tissue bank: a collection of high quality frozen residual, accurately classified tumour tissue samples, which are stored in European cancer centres and universities. This virtual tissue bank, searchable on the internet, has rules for access and use, and a code of conduct to comply with the various legal and ethical regulations in European countries. The easy accessibility and the European scale of the bank will result in the availability of a large number of samples even of rarer tumour types. Standardisation of collection, storage and quality control throughout the network is achieved minimising inter-institutional variability. A website providing access to upload, search and request samples is a key tool of the tissue bank. The search engine makes use of virtual microscopy. An overview of the development of the European virtual frozen tissue bank infrastructure is described in this paper. The various key aspects are described in more detail in a series of articles to appear in this Journal.

When designing infrastructure for a networked virtual tumour bank (samples remain at the collector institutes and sample data are collected in a searchable central database), it is apparent that this can only function properly after developing an adequate set of rules for use and access. These rules must include sufficient incentives for the tissue sample collectors to remain active within the network and maintain sufficient sample levels in the local bank. These requirements resulted in a key TuBaFrost rule, stating that the custodianship of the samples remains under the authority of the local collector. As a consequence, the samples and the decision to issue the samples to a requestor are not transferred to a large organisation but instead remain with the collector, thus allowing autonomous negotiation between collector and requestor, potential co-authorship in publications or compensation for collection and processing costs. Furthermore, it realises a streamlined cost effective network, ensuring tissue visibility and accessibility thereby improving the availability of large amounts of samples of highly specific or rare tumour types as well as providing contact opportunities for collaboration between scientists with cutting edge technology and tissue collectors. With this general purpose in mind, the rules and responsibilities for collectors, requestors and central office were generated.

The regulatory regimes for research with residual tissue and accompanying data differ widely between countries in the European Union (EU): from specific consent to opt-out or even no consent at all. This could greatly hamper research where the exchange of tissue and accompanying data has become the gold standard, like in TubaFrost. Instead of adhering to international guidelines, which have a democratic deficit, or an attempt for a new set of possible harmonising rules, TubaFrost chose to create a coordinating rule: if tissue may legitimately be used for a certain kind of research in the country where it was taken and under whose jurisdiction the patient falls, it may also be used for such research in the country where it is sent to in the context of a scientific program even if in that other country other regulations would apply for research with residual tissue taken from patients under their jurisdiction. This coordinating rule has a sound basis in EU law in general and will solve the problems related to diverging national regulatory regimes in the case of cross national research with residual tissue.

Tumour Bank Networking presents a great challenge for oncological research as in order to carry out large-scale, multi-centre studies with minimal intrinsic bias, each tumour bank in the network must have some fundamental similarities and be using the same standardised and validated procedures. The European Human Frozen Tumour Tissue Bank (TuBaFrost) has responded to this need by the promotion of an integrated platform of tumour banks in Europe. The operational framework for TuBaFrost has drawn upon the best practice of standard workflows and operating procedures employed by members of the TuBaFrost project and key initiatives worldwide.

Glioblastoma multiforme (GBM) is characterized by intratumoral heterogeneity in both histomorphological and genetic changes, displaying a wide variety of numerical chromosome aberrations, the most common of which are trisomy 7 and monosomy 10. The amplification of the epidermal growth factor receptor (EGFR) gene is the most frequently reported genetic abnormality. The associations between these parameters and their implication in the tumoral progression are poorly understood. We performed simultaneous fluorescence in situ hybridization (FISH) with centromeric DNA probes for chromosomes 7 and 10 in smear preparations, and EGFR gene amplification by PCR from 25 cases of GBM. Trisomy/ polysomy for chromosome 7 was present in 76% of cases and monosomy 10 in 68%. Both alterations were associated in 56% of cases. The EGFR gene was amplified in 52% of tumors; in 44% associated with trisomy/ polysomy 7, and in 36% with monosomy 10. The three parameters were associated together in 28% of cases. Kaplan-Meier survival rate analysis demonstrated lower survival rates in patients with monosomy 10, trisomy 7, and monosomy associated with trisomy 7. The other combinations were not different in frequency in relation to survival. In the present study, trisomy/polysomy 7 and monosomy 10 have been found to be frequently associated. The combination of both anomalies is probably important in the tumorigenesis of glioblastoma. Moreover, this association is apparently independent of EGFR gene amplification, which could be a later event in this process.

Background: The aim of this study was to investigate new prognostic factors, by using a prognostic model, that could help to identify the patient group with the greatest probability of death., Patients and Methods: First, the clinicopathological variables were analyzed. Second, microvessels were immunohistochemically (IHC) stained with factor VIII-related antibody and then counted in the most intense vascularization area or hotspot, using an automatic image analyzer. In addition, biological angiogenesis-related factors such as vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase expression (iNOS) were also studied. Finally, we evaluated the IHC expression of p53 and p21WAF1 tumor supressor proteins., Results: The significant independent predictors were: tumor size (p=0.0063), angiogenesis (p=0.0271) and p21WAF1 (p=0.0478). Thus, the most important factor was tumor size 2.7462 [95% CI=1.3307-5.6673]. Finally, these variables were included in a risk model, in order to identify the group with the highest associated probability of death., Conclusion: The analysis of several prognostic factors could establish a more accurate patient risk profile.