RANK/OPG ratio of expression in primary clear-cell renal cell carcinoma is associated with bone metastasis and prognosis in patients treated with anti-VEGFR-TKIs.

BACKGROUND: Bone metastases (BMs) are associated with poor outcome in metastatic clear-cell renal carcinoma (m-ccRCC) treated with anti-vascular endothelial growth factor tyrosine kinase inhibitors (anti-VEGFR-TKIs). We aimed to investigate whether expression in the primary tumour of genes involved in the development of BM is associated with outcome in m-ccRCC patients treated with anti-VEGFR-TKIs. METHODS: Metastatic clear-cell renal cell carcinoma patients with available fresh-frozen tumour and treated with anti-VEGFR-TKIs. Quantitative real-time PCR (qRT-PCR) for receptor activator of NF-kB (RANK), RANK-ligand (RANKL), osteoprotegerin (OPG), the proto-oncogene SRC and DKK1 (Dickkopf WNT signalling pathway inhibitor-1). Time-to-event analysis by Kaplan-Meier estimates and Cox regression. RESULTS: We included 129 m-ccRCC patients treated between 2005 and 2013. An elevated RANK/OPG ratio was associated with shorter median time to metastasis (HR 0.50 (95% CI 0.29-0.87); P=0.014), shorter time to BM (HR 0.54 (95% CI 0.31-0.97); P=0.037), shorter median overall survival (mOS) since initial diagnosis (HR 2.27 (95% CI 1.44-3.60); P=0.0001), shorter median progression-free survival (HR 0.44 (95% CI 0.28-0.71); P=0.001) and mOS (HR 0.31 (95% CI 0.19-0.52); P<0.0001) on first-line anti-VEGFR-TKIs in the metastatic setting. Higher RANK expression was associated with shorter mOS on first-line anti-VEGFR-TKIs (HR 0.46 (95% CI 0.29-0.73); P=0.001). CONCLUSIONS: RANK/OPG ratio of expression in primary ccRCC is associated with BM and prognosis in patients treated with anti-VEGFR-TKIs. Prospective validation is warranted.

Overexpression and promoter mutation of the TERT gene in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a very aggressive tumor with no known curative treatment. Better knowledge of the molecular mechanisms of mesothelial carcinogenesis is required to develop new therapeutic strategies. MPM, like all cancer cells, needs to maintain telomere length to prevent senescence. Previous studies suggested that the telomere lengthening mechanism in MPM is based mainly on telomerase activity. For this reason, we focused on the key catalytic enzyme, TERT (telomerase reverse transcriptase), by analyzing its gene expression in MPM and by studying the mechanism underlying its upregulation. We used our large collection of MPM composed of 61 MPM in culture and 71 frozen MPM tumor samples. Evaluation of TERT mRNA expression by quantitative RT-PCR showed overexpression in MPM in culture compared with normal mesothelial cells, and in MPM tumor samples compared with normal pleura. We identified a 'hot spot' of mutations in the TERT gene core promoter in both MPM in culture and in MPM tumor samples with an overall frequency of 15%. Furthermore, data clearly identified mutation in the TERT promoter as a mechanism of TERT mRNA upregulation in MPM. In contrast, gene copy number amplification was not associated with TERT overexpression. Then, we analyzed the clinicopathological, etiological and genetic characteristics of MPM with mutations in the TERT promoter. TERT promoter mutations were more frequent in MPM with sarcomatoid histologic subtype (P<0.01), and they were frequently associated with CDKN2A gene inactivation (P=0.03). In conclusion, a subgroup of MPM presents TERT promoter mutations, which lead to TERT mRNA upregulation. This is the first recurrent gain-of-function oncogenic mutations identified in MPM.

Single-nucleotide polymorphisms associated with outcome in metastatic renal cell carcinoma treated with sunitinib.

BACKGROUND: There are no validated markers that predict response in metastatic renal cell cancer (RCC) patients treated with sunitinib. We aim to study the impact of single-nucleotide polymorphisms (SNPs) that have recently been proposed as predictors of outcome to anti-VEGF-targeted therapy in metastatic RCC in an independent cohort of patients. METHODS: We genotyped 16 key SNPs in 10 genes involved in sunitinib pharmacokinetics, pharmacodynamics and VEGF-independent angiogenesis in patients with metastatic clear-cell RCC treated with sunitinib as the first-line targeted therapy. Association between SNPs, progression-free survival (PFS) and overall survival (OS) were studied by multivariate Cox regression using relevant clinical factors associated with PFS and OS as covariates. RESULTS: In a series of 88 patients, both PFS and OS were associated significantly with SNP rs1128503 in ABCB1 (P=0.027 and P=0.025), rs4073054 in NR1/3 (P=0.025 and P=0.035) and rs307821 in VEGFR3 (P=0.032 and P=0.011). Progression-free survival alone was associated with rs2981582 in FGFR2 (P=0.031) and rs2276707 in NR1/2 (P=0.047), whereas OS alone was associated with rs2307424 in NR1/3 (P=0.048) and rs307826 in VEGFR3 (P=0.013). CONCLUSION: Our results confirm former communications regarding the association between SNPs in ABCB1, NR1/2, NR1/3 and VEGFR3 and sunitinib outcome in clear-cell RCC. Prospective validation of these SNPs is now required.

Negative impact of bone metastasis on outcome in clear-cell renal cell carcinoma treated with sunitinib.

BACKGROUND: The aim of our study was to determine whether the presence of bone metastases affects outcomes in patients with metastatic clear-cell renal cell carcinoma (m-ccRCC) receiving sunitinib. PATIENTS AND METHODS: We reviewed the charts of all patients in four academic centers in Belgium and France who started first-line sunitinib (50 mg/day; 4 weeks on and 2 weeks off) between January 2005 and December 2008. Data were collected on known prognostic factors for metastatic renal cell carcinoma and metastatic sites. Response and progression were evaluated by computed tomography scan (according to RECIST). RESULTS: Two hundred twenty-three patients were identified. With a median follow-up of 40 months, median progression-free survival (PFS) and median overall survival (OS) were significantly shorter in patients with bone metastases than in those without: respectively, 8.2 versus 19.1 months (P<0.0001) and 19.5 versus 38.5 months (P<0.0001). On multivariate analysis, taking on account platelet count, Eastern Cooperative Oncology Group performance status, number of metastatic sites, neutrophil count, corrected serum calcium, time from diagnosis to systemic treatment, and the presence of bone metastases, bone metastasis was the independent variable most significantly associated with poor PFS (P<0.0001) and OS (P=0.001). CONCLUSION: The presence of bone metastases in m-ccRCC patients has a significant and clinically relevant negative impact on outcome on sunitinib.

[Fundamental and translational research on hepatocellular carcinoma in 2008: forces and priorities]. / Recherche fondamentale et translationnelle sur le carcinome hépatocellulaire en 2008 : avancées récentes et perspectives.

Hepatocellular carcinogenesis is usually the result of a muti-step process. It begins with an exposure to various risk factors; followed by the development of a chronic hepatitis and cirrhosis that is a pre-neoplastic step; and finally after the occurrence of an hepatocellular carcinoma (HCC), different molecular events control aggressiveness of the tumors. The aim of this work was to identify in the international context, forces and priorities of the fundamental and translational HCC research.

Differential effects of inactivated Axin1 and activated beta-catenin mutations in human hepatocellular carcinomas.

Perturbations to the Wnt signaling pathway have been implicated in a large proportion of human hepatocellular carcinomas (HCCs). Activating beta-catenin mutations and loss of function mutations in Axin1 are thought to be functionally equivalent. We examined the Wnt pathway in HCC by comparing the expression of beta-catenin target genes and the level of beta-catenin-dependent transcriptional activation, in 45 HCC tumors and four cell lines. Among these samples, beta-catenin and AXIN1 were mutated in 20 and seven cases, respectively. We found a significant correlation between activated beta-catenin mutations and overexpression of mRNA for the target genes glutamine synthetase (GS), G-protein-coupled receptor (GPR)49 and glutamate transporter (GLT)-1 (P=0.0001), but not for the genes ornithine aminotransferase, LECT2, c-myc and cyclin D1. We also showed that GS is a good immunohistochemical marker of beta-catenin activation in HCC. However, we observed no induction of GS, GPR49 or GLT-1 in the five inactivated Axin1 tumors. Beta-catenin-dependent transcriptional activation in two Axin1-mutated HCC cell lines was much weaker than in beta-catenin-mutated cell lines. Our results strongly suggest that in HCC, contrary to expectation, the loss of function of Axin1 is not equivalent to the gain of function of beta-catenin. Our results also suggest that the tumor suppressor function of Axin1 in HCC may be related to another, non-Wnt pathway.

Genetics of hepatocellular tumors.

Numerous genetic alterations are accumulated during the process of hepatocarcinogenesis. These genetic alterations can be divided into two groups. The first set of genetic alterations is specific of hepatocellular tumor risk factors. It includes integration of hepatitis B virus (HBV) DNA, R249S TP53 (tumor protein p53) mutation in aflatoxin B1-exposed patients, KRAS mutations related to vinyl chloride exposure, hepatocyte nuclear factor 1alpha (HNF1alpha) mutations associated to hepatocellular adenomas and adenomatosis polyposis coli (APC) germline mutations predisposing to hepatoblastomas. The second set of genetic alterations are etiological nonspecific, it includes recurrent gains and losses of chromosomes, alteration of TP53 gene, activation of WNT/beta-catenin pathway through CTNNB1/beta-catenin and AXIN (axis inhibition protein) mutations, inactivation of retinoblastoma and IGF2R (insulin-like growth factor 2 receptor) pathways through inactivation of RB1 (retinoblastoma 1), P16 and IGF2R. Comprehensive analyses of these genetic alterations have defined two pathways of hepatocarcinogenesis according to the presence or the absence of chromosomal instability. Hepatitis B virus and poorly differentiated tumors are related to chromosome instable tumors associated with frequent TP53 mutations, whereas non-HBV and well-differentiated tumors are related to chromosomal stable samples that are frequently beta-catenin activated. These classifications have clinical relevance as genetic alterations may also be related to prognosis.

Identification of homozygous deletions at chromosome 16q23 in aflatoxin B1 exposed hepatocellular carcinoma.

Loss of heterozygosity (LOH) represents the most frequent genetic alteration observed in hepatocellular carcinoma (HCC). Chromosome 16q is of particular interest as it exhibits LOH in 29% of HCC tumors and is frequently lost in breast, prostate, ovarian and gastric carcinomas. We genotyped 157 HCC tumors for 17 microsatellite markers distributed on chromosome 16q and determined a common region of LOH localized between the markers D16S518 and D16S504. By refining the boundaries of two interstitial LOH and two homozygous deletions, the critical region was delimited to 180 kb between D16S3096 and D16S3029. This region is located in intron 8 of the WWOX/FOR gene, but a search for mutations in all coding exons of this gene in 27 HCC tumors and cell lines did not reveal any tumor somatic alterations. Furthermore, by RT-PCR, no abnormal transcripts of this WWOX/FOR gene was detected in nine HCC cell lines. Finally, analysis of the p53 gene mutations with the clinical parameters of all tumors revealed that the two homozygous deletions have occurred in tumors presenting a R249S mutation. Our data revealed a relationship between chromosome 16q homozygous deletions and R249S p53 mutations in tumors where the patient had been exposed to aflatoxin B1 (P=0.002). These results are consistent with a role of aflatoxin B1 in the instability of chromosome 16q at the fragile site FRA16D. However, the nature of the specific gene that is altered during hepatocarcinogenesis remains to be elucidated.

Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis.

BACKGROUND & AIMS: To evaluate how characterization of genetic alterations can help in the elucidation of liver carcinogenesis pathways, 137 tumors were analyzed. METHODS: High-density allelotype, p53, Axin1, and beta-catenin gene mutations were determined. Alterations were analyzed according to clinical parameters. RESULTS: Tumors could be divided into 2 groups according to chromosome stability status. In the first group, demonstrating a chromosome stability, beta-catenin mutation associated with chromosome 8p losses were frequently found as the single genetic alterations. beta-catenin mutations were associated with large tumor size and with negative hepatitis B virus status. In the second group, demonstrating a chromosome instability, the most frequent allelic losses were on chromosome 1p, 4q, 6q, 9p, 13q, 16p, 16q, and 17p; Axin1 and p53 were frequently mutated. All of these alterations, except losses on 6q and 9p, were associated with hepatitis B virus infection. P53 mutations, 17p, 13q losses, and a high value of the fractional allelic loss index were associated with poor differentiated tumors, independently of risk factors. Finally, in the whole series, chromosome 9p and 6q losses were associated with poor prognosis. CONCLUSIONS: Two main pathways defined by genetic alterations show different risk factors and clinical characteristics. Furthermore, loss of chromosome 9p or 6q is an independent prognostic indicator.

High resolution deletion analysis of constitutional DNA from neurofibromatosis type 2 (NF2) patients using microarray-CGH.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder whose hallmark is bilateral vestibular schwannoma. It displays a pronounced clinical heterogeneity with mild to severe forms. The NF2 tumor suppressor (merlin/schwannomin) has been cloned and extensively analyzed for mutations in patients with different clinical variants of the disease. Correlation between the type of the NF2 gene mutation and the patient phenotype has been suggested to exist. However, several independent studies have shown that a fraction of NF2 patients with various phenotypes have constitutional deletions that partly or entirely remove one copy of the NF2 gene. The purpose of this study was to examine a 7 Mb interval in the vicinity of the NF2 gene in a large series of NF2 patients in order to determine the frequency and extent of deletions. A total of 116 NF2 patients were analyzed using high-resolution array-comparative genomic hybridization (CGH) on an array covering at least 90% of this region of 22q around the NF2 locus. Deletions, which remove one copy of the entire gene or are predicted to truncate the schwannomin protein, were detected in 8 severe, 10 moderate and 6 mild patients. This result does not support the correlation between the type of mutation affecting the NF2 gene and the disease phenotype. This work also demonstrates the general usefulness of the array-CGH methodology for rapid and comprehensive detection of small (down to 40 kb) heterozygous and/or homozygous deletions occurring in constitutional or tumor-derived DNA.

Molecular characterization of germline NF2 gene rearrangements.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease that causes a predisposition to nervous system tumors. Deleterious point mutations have been found in about 55% of NF2 patients, and large genomic deletions account for approximately 33% of NF2 gene alterations. The majority of these deletions are larger than 50 kb, with a breakpoint usually lying outside the NF2 gene. We identified two cases of intragenic deletion with loss of 1.5 and 40 kb, respectively. In both cases, one boundary of the deletion was located in or at the proximity of an SVA sequence in NF2 intron 4. No sequence identity longer than 5 bases and no signal of specific recombination have been evidenced on either side of the deletion breakpoints. These observations are compatible with a nonhomologous recombination being responsible for the genomic deletions. In a third case, a paracentric inversion of chromosome 22 was found. This chromosomal rearrangement breaks the NF2 gene in two parts and carries the first NF2 exon in a juxta-centromeric position. The variability in position of the deletions and the observation of a new chromosomal rearrangement in the NF2 gene underscore the importance of FISH analysis in the molecular diagnosis of NF2.

Cryptic exons as a source of increased diversity of Ewing tumor-associated EWS-FLI1 chimeric products.

In the Ewing family of tumors (EFT), the EWS gene is rearranged with members of the ets oncogene family. Variability in genomic breakpoint locations is the source of significant heterogeneity in fusion product structure. As a consequence of variably included exon sequences from the two partner genes, a variable amount of additional peptide sequence is inserted in between the minimal transforming domains. Some of this molecular diversity has recently been correlated with disparate clinical outcome. Here we report on cryptic exons found in the chimeric RNA of three EFT with different EWS-FLI1 fusions. In two tumors, the emergence of a cryptic exon from FLI1 intron 5 in the chimeric RNA was apparently unrelated to the genomic rearrangement that occurred in FLI1 introns 4 and 5, respectively. In one case, a novel exon was generated through the creation of an artificial splice acceptor site in FLI1 intron 6 by the genomic rearrangement that occurred in EWS intron 8. These results further extend the spectrum of molecular diversity in EFT.

Beta-catenin mutations in hepatocellular carcinoma correlate with a low rate of loss of heterozygosity.

To determine the frequency of Wnt/Wingless beta catenin pathway alteration in human hepatocellular carcinoma, a beta catenin and APC gene mutation screening was performed in a series of 119 tumors. An activating beta catenin mutation in exon 3 was found in 18% of the cases. Among tumors lacking beta catenin mutation, no APC mutation has been evidenced in a subset of 30 cases tested. The correlation between beta catenin mutation status and chromosome segment deletions was studied on a set of 48 hyperploid tumors. Chromosome 1p, 4q and 16p deletions were significantly associated with the absence of beta catenin mutation (P<0.05). Furthermore the Fractional Allelic Loss was significantly smaller in the beta catenin mutated tumors than in the non-mutated tumors (0.12 versus 022). Taken together, these results suggest, the existence of two carcinogenesis mechanisms. The first mechanism implies a beta catenin activating mutation associated with a low rate of loss of heterozygosity. The second mechanism, operating in a context of chromosomal instability, would involve tumor suppressor genes.