Molecular alterations in retinoblastoma beyond RB1.

Retinoblastoma is the most common malignant ocular tumor in children. Although RB1 alterations are most frequently involved in the etiology of retinoblastoma, candidate driver events and somatic alterations leading to cell transformation, tumor onset and progression remain poorly understood. In this study, we identified novel genomic alterations in tumors with a panel of 160 genes. Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) were initially performed for identifying patients without apparent RB1 alterations in blood DNA. Subsequently, NGS analyses of 24 paired (blood/tumor) samples of these patients were carried out for identifying somatic mutations and copy number variation in RB1 and other 159 genes. One novel pathogenic RB1 mutation and seven novel VUS were identified as well as 90 novel pathogenic mutations in 61 other genes. Twenty-three genes appeared exclusively mutated in tumors without altered RB1 alleles and three frequently affected biological pathways while five other tumors did not show pathogenic RB1 alterations or SNV/indels in 159 other genes. Curiously, deletion of GATA2, AKT1, ARID1A, DNMT3A, MAP2K2, MEN1, MTOR, PTCH1 and SUFU (in homo- or heterozygosity) were exclusively found in these tumors when compared to those with any pathogenic alterations, probably indicating genes that might be essential for the development of retinoblastoma regardless of a functional RB1. Identification of genes associated with retinoblastoma will contribute to understanding presently unknown aspects of this malignancy, which might be essential for its initiation and progression, as well as providing valuable molecular markers.

The characterization of two novel neotropical primate papillomaviruses supports the ancient within-species diversity model.

Papillomaviruses (PVs) are non-enveloped icosahedral viruses with a circular double-stranded DNA genome of ∼8,000 base pairs (bp). More than 200 different PV types have been identified to date in humans, which are distributed in five genera, with several strains associated with cancer development. Although widely distributed in vertebrates, Neotropical Primates (NP) PV infection was described for the first time only in 2016. Currently, four complete genomes of NP PVs have been characterized, three from Saimiri sciureus (SscPV1 to SscPV3) and one from Alouatta guariba (AgPV1). In this work, we describe two novel PV strains infecting Callithrix penicillata (provisionally named CpenPV1 and CpenPV2), using anal swab samples from animals residing at the Brasilia Primatology Center and next generation sequencing. The genomes of CpenPV1 (7,288 bp; 41.5% guanine-cytosine content - GC) and CpenPV2 (7,250 bp; 40.7% GC) contain the characteristic open reading frames (ORFs) for the early (E6, E7, E1, E2, and E4) and late (L2 and L1) PV genes. The L1 ORFs, commonly used for phylogenetic identification, share 76 per cent similarity with each other and differ 32 per cent from any other known PV, indicating that these new strains meet the criteria for defining novel species. PV genes phylogenetic variance was analyzed and different degrees of saturation revealed similar levels of topological heterogeneity, ruling out saturation as primary etiological factor for this phenomenon. Interestingly, the two CpenPV strains form a monophyletic clade within the Gammapapillomavirus genus (provisionally named gammapapillomavirus 32). Unlike for other NP PV strains, which grouped into a new sister genus of Alphapapillomavirus, this is the first report of NP PV strains grouping into a genus previously considered to exclusively comprise Old World Primates (OWP) PVs, including human PVs. These findings confirm the existence of a common ancestor for Gammapapillomavirus already infecting primates before the split of OWP and NP at ∼40 million years ago. Finally, our findings are consistent with an ancient within-species diversity model and emphasize the importance of increasing sampling to help understanding the PV-primate codivergence dynamics and pathogenic potential.

Composite Analysis of the Virome and Bacteriome of HIV/HPV Co-Infected Women Reveals Proxies for Immunodeficiency.

The human cervical microbiome is complex, and its role in health and disease has just begun to be elucidated. In this study, 57 cervical swab samples from 19 HIV/HPV co-infected women were analyzed for both virome and bacteriome composition. Virome analysis focused on circular DNA viruses through rolling circle amplification followed by next-generation sequencing (NGS). Data were assigned to virus families and genera, and HPV types were identified. NGS data of bacterial 16S from a subset of 24 samples were assigned to operational taxonomic units and classified according to vaginal microbiome community state types (CSTs). Four viral families were found: Papillomaviridae, Anelloviridae, Genomoviridae, and Herpesviridae. Papillomavirus reads were more abundant in women with premalignant cervical lesions, which were also strongly associated with multiple (≥3) high-risk HPV infection. Anellovirus read abundance was negatively correlated with host CD4+ T-cell counts. The bacteriome revealed the presence of CST III and CST IV, and women with ≥1% frequency of genomovirus or herpesvirus reads displayed an increased risk of carrying CST IV. By characterizing the composition of the cervical circular DNA viruses and the bacteriome of HIV/HPV co-infected women, we identified putative interactions between these two microorganism communities and their associations with patients' clinical characteristics, notably immunodeficiency status.

Mutations, Differential Gene Expression, and Chimeric Transcripts in Esophageal Squamous Cell Carcinoma Show High Heterogeneity.

Esophageal squamous cell carcinoma (ESCC) is a frequent and lethal neoplasia. As recent advances in targeted therapy have not improved ESCC prognosis, characterization of molecular alterations associated to this tumor is of foremost relevance. In this study, we analyze, for the first time, the complete genomic profile of ESCC by RNA-seq. TP53 was the most frequently mutated gene in the investigation and validation sets (78.6% and 67.4%, respectively). Differential expression analysis between tumor and nontumor adjacent mucosa showed 6698 differentially expressed genes, most of which were overexpressed (74%). Enrichment analysis identified overrepresentation of Wnt pathway, with overexpressed activators and underexpressed inactivators, suggesting activation of canonical and noncanonical Wnt signaling pathways. Higher WNT7B expression was associated with poor prognosis. Twenty-one gene fusions were identified in 50% of tumors, none of which involving the same genes in different patients; 71% of fusions involved syntenic genes. Comparisons with TCGA data showed co-amplification of seven gene pairs involved in fusions in the present study (~33%), suggesting that these rearrangements might have been driven by chromoanagenesis. In conclusion, genomic alterations in ESCC are highly heterogeneous, impacting negatively in target therapy development.

The germline mutational landscape of BRCA1 and BRCA2 in Brazil.

The detection of germline mutations in BRCA1 and BRCA2 is essential to the formulation of clinical management strategies, and in Brazil, there is limited access to these services, mainly due to the costs/availability of genetic testing. Aiming at the identification of recurrent mutations that could be included in a low-cost mutation panel, used as a first screening approach, we compiled the testing reports of 649 probands with pathogenic/likely pathogenic variants referred to 28 public and private health care centers distributed across 11 Brazilian States. Overall, 126 and 103 distinct mutations were identified in BRCA1 and BRCA2, respectively. Twenty-six novel variants were reported from both genes, and BRCA2 showed higher mutational heterogeneity. Some recurrent mutations were reported exclusively in certain geographic regions, suggesting a founder effect. Our findings confirm that there is significant molecular heterogeneity in these genes among Brazilian carriers, while also suggesting that this heterogeneity precludes the use of screening protocols that include recurrent mutation testing only. This is the first study to show that profiles of recurrent mutations may be unique to different Brazilian regions. These data should be explored in larger regional cohorts to determine if screening with a panel of recurrent mutations would be effective.

Esophageal squamous cell carcinoma transcriptome reveals the effect of FOXM1 on patient outcome through novel PIK3R3 mediated activation of PI3K signaling pathway.

Esophageal squamous cell carcinoma (ESCC) presents poor prognosis, and patients diagnosed with this tumor currently lack target treatments. Therefore, in order to identify potential targets for ESCC treatment, we carried out a transcriptome analysis with ESCC and paired nonmalignant surrounding mucosa samples, followed by a master regulator analysis, and further explored the role of the identified central regulatory genes through in vivo and in vitro assays. Among the transcription factors deregulated/enriched in ESCC, we focused on FOXM1 because of its involvement in the regulation of critical biological processes. A new transcriptome analysis performed with ESCC cell lineage TE-1 showed that the modulation of FOXM1 expression resulted in PIK3R3 expression changes, whereas chromatin immunoprecipitation assay revealed that FOXM1 was capable of binding onto PIK3R3 promoter, thus demonstrating that PIK3R3 is a new FOXM1 target. Furthermore, FOXM1 overexpression resulted in the activation of PIK3/AKT signaling pathway through PIK3R3-mediated AKT phosphorylation. Finally, the analysis of the clinic-pathological data of ESCC patients revealed that overexpression of both FOXM1 and PIK3R3 was associated with poor prognosis, but only the latter was an independent prognosis factor for ESCC patients. In conclusion, our results show that FOXM1 seems to play a central role in ESCC carcinogenesis by upregulating many oncogenes found overexpressed in this tumor. Furthermore, PIK3R3 is a novel FOXM1 target that triggers the activation of the PI3K/AKT pathway in ESCC cells.

Investigation of major genetic alterations in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. This malignancy shows a wide spectrum of clinical outcome and its prognosis is conditioned by manifold biological and genetic factors. We investigated the tumor genetic profile and clinical data of 29 patients with NB by multiplex ligation-dependent probe amplification (MLPA) to assess therapeutic risk. In 18 of these tumors, MYCN status was assessed by fluorescence in situ hybridization (FISH). Copy number variation was also determined for confirming MLPA findings in two 6p loci. We found 2p, 7q and 17q gains, and 1p and 11q losses as the most frequent chromosome alterations in this cohort. FISH confirmed all cases of MYCN amplification detected by MLPA. In view of unexpected 6p imbalance, copy number variation of two 6p loci was assessed for validating MLPA findings. Based on clinical data and genetic profiles, patients were stratified in pretreatment risk groups according to international consensus. MLPA proved to be effective for detecting multiple genetic alterations in all chromosome regions as requested by the International Neuroblastoma Risk Group (INRG) for therapeutic stratification. Moreover, this technique proved to be cost effective, reliable, only requiring standard PCR equipment, and attractive for routine analysis. However, the observed 6p imbalances made PKHD1 and DCDC2 inadequate for control loci. This must be considered when designing commercial MLPA kits for NB. Finally, four patients showed a normal MLPA profile, suggesting that NB might have a more complex genetic pattern than the one assessed by presently available MLPA kits.

RPS6KA4/MIR1237 and AURKC promoter regions are differentially methylated in Wilms' tumor.

Wilms' tumor (WT) is the most frequent renal cancer in childhood, the occurrence of which is characterized by a relatively low frequency of associated mutations. While epigenetic alterations have been postulated to play a relevant role in the emergence of this tumor, the mechanisms involved in WT development remain largely unknown. In this study, the DNA methylation profile of WT was characterized with Beadchip array. Comparisons between WT with normal kidney identified 827 differentially methylated regions, most of which were attributable in hypermethylation in CpG islands. Among affected genes, WT1 and TP73 showed altered enhancers where hypermethylation was validaded by pyrosequencing. Thirty differentially methylated regions (DMRs) were identified in WT as compared to normal kidney, two of which were previously described. Two novel DMRs, located in RPS6KA4/MIR1237 and the AURKC promoter, were found to be hypermethylated in WT. Altogether, our data reinforced the relevance of alterations of DNA methylation in WT, highlighting the complex nature of these alterations that affect promoter regions as well as enhancers, UTRs and gene bodies.

The human retinoblastoma susceptibility gene (RB1): an evolutionary story in primates.

The tumor suppressor gene RB1 (Human Retinoblastoma Susceptibility Gene) plays a prominent role in normal development, gene transcription, DNA replication, repair, and mitosis. Its complete biallelic dysfunction in retinoblasts is the main cause of retinoblastoma in the human. Although this gene has been evolutionary conserved, comparisons between the reference and human RB1 coding region with its counterparts in 19 non-human primates showed 359 sites where nucleotide replacements took place during the radiation of these species. These resulted in missense substitutions in 97 codons, 91 of which by amino acids with radically different physicochemical properties. Several in frame deletions and two insertions were also observed in the N-terminal region of the pRB protein where the highest number of amino acid substitutions and radical amino changes were found. Fifty-six codons were inferred to be under negative selection and five under positive selection. Differences in codon usage showed evident phylogenetic signals, with hominids generally presenting higher indices of codon bias than other catarrhines. The lineage leading to platyrrhines and, within platyrrhines, the lineage leading to Saimiri boliviensis showed a high rate of nucleotide substitutions and amino acids. Finally, several RB1 alterations associated to retinoblastoma in the human were present in several non-human primates without an apparent pathological effect.

Identification of novel human papillomavirus lineages and sublineages in HIV/HPV-coinfected pregnant women by next-generation sequencing.

Infection by human papillomavirus (HPV) is a necessary condition for development of cervical cancer, and has also been associated with malignancies of other body anatomical sites. Specific HPV types have been associated with premalignant lesions and invasive carcinoma, but mounting evidence suggests that within-type lineages and sublineages also display distinct biological characteristics associated with persistent infections and evolution to cervical cancer. In the present study, we have assessed HPV multiple infection and variation from a cohort of highly susceptible, HIV(+) pregnant women using next-generation sequencing and an in-house pipeline for HPV full-length genome assembly. Seventy-two consensus sequences representing complete or near-complete (>97%) HPV genomes were assembled, spanning 28 different types. Genetic distance and phylogenetic analyses allowed us to propose the classification of novel HPV lineages and sublineages across nine HPV types, including two high-risk types. HPV diversity may be a hallmark of immunosuppressed patients upon HIV infection and AIDS progression.

Bonafide, type-specific human papillomavirus persistence among HIV-positive pregnant women: predictive value for cytological abnormalities, a longitudinal cohort study.

This study investigated the rate of human papillomavirus (HPV) persistence, associated risk factors, and predictors of cytological alteration outcomes in a cohort of human immunodeficiency virus-infected pregnant women over an 18-month period. HPV was typed through L1 gene sequencing in cervical smears collected during gestation and at 12 months after delivery. Outcomes were defined as nonpersistence (clearance of the HPV in the 2nd sample), re-infection (detection of different types of HPV in the 2 samples), and type-specific HPV persistence (the same HPV type found in both samples). An unfavourable cytological outcome was considered when the second exam showed progression to squamous intraepithelial lesion or high squamous intraepithelial lesion. Ninety patients were studied. HPV DNA persistence occurred in 50% of the cases composed of type-specific persistence (30%) or re-infection (20%). A low CD4+T-cell count at entry was a risk factor for type-specific, re-infection, or HPV DNA persistence. The odds ratio (OR) was almost three times higher in the type-specific group when compared with the re-infection group (OR = 2.8; 95% confidence interval: 0.43-22.79). Our findings show that bonafide (type-specific) HPV persistence is a stronger predictor for the development of cytological abnormalities, highlighting the need for HPV typing as opposed to HPV DNA testing in the clinical setting.

Bonafide, type-specific human papillomavirus persistence among HIV-positive pregnant women: predictive value for cytological abnormalities, a longitudinal cohort study

This study investigated the rate of human papillomavirus (HPV) persistence, associated risk factors, and predictors of cytological alteration outcomes in a cohort of human immunodeficiency virus-infected pregnant women over an 18-month period. HPV was typed through L1 gene sequencing in cervical smears collected during gestation and at 12 months after delivery. Outcomes were defined as nonpersistence (clearance of the HPV in the 2nd sample), re-infection (detection of different types of HPV in the 2 samples), and type-specific HPV persistence (the same HPV type found in both samples). An unfavourable cytological outcome was considered when the second exam showed progression to squamous intraepithelial lesion or high squamous intraepithelial lesion. Ninety patients were studied. HPV DNA persistence occurred in 50% of the cases composed of type-specific persistence (30%) or re-infection (20%). A low CD4+T-cell count at entry was a risk factor for type-specific, re-infection, or HPV DNA persistence. The odds ratio (OR) was almost three times higher in the type-specific group when compared with the re-infection group (OR = 2.8; 95% confidence interval: 0.43-22.79). Our findings show that bonafide (type-specific) HPV persistence is a stronger predictor for the development of cytological abnormalities, highlighting the need for HPV typing as opposed to HPV DNA testing in the clinical setting.

Array CGH Analysis of Paired Blood and Tumor Samples from Patients with Sporadic Wilms Tumor.

Wilms tumor (WT), the most common cancer of the kidney in infants and children, has a complex etiology that is still poorly understood. Identification of genomic copy number variants (CNV) in tumor genomes provides a better understanding of cancer development which may be useful for diagnosis and therapeutic targets. In paired blood and tumor DNA samples from 14 patients with sporadic WT, analyzed by aCGH, 22% of chromosome abnormalities were novel. All constitutional alterations identified in blood were segmental (in 28.6% of patients) and were also present in the paired tumor samples. Two segmental gains (2p21 and 20q13.3) and one loss (19q13.31) present in blood had not been previously described in WT. We also describe, for the first time, a small, constitutive partial gain of 3p22.1 comprising 2 exons of CTNNB1, a gene associated to WT. Among somatic alterations, novel structural chromosomal abnormalities were found, like gain of 19p13.3 and 20p12.3, and losses of 2p16.1-p15, 4q32.5-q35.1, 4q35.2-q28.1 and 19p13.3. Candidate genes included in these regions might be constitutively (SIX3, SALL4) or somatically (NEK1, PIAS4, BMP2) operational in the development and progression of WT. To our knowledge this is the first report of CNV in paired blood and tumor samples in sporadic WT.

The effect of human leukocyte antigen G alleles on human papillomavirus infection and persistence in a cohort of HIV-positive pregnant women from Brazil.

Patients with compromised immune systems have more severe intraepithelial lesions and more rapid disease progression, in addition to increased risk for cervical cancer. Persistent infection by the human papillomavirus (HPV) is a necessary step in that process. By inducing expression of inhibitory ligands of natural killer cells, like HLA-G, HPV avoids the elimination of infected cells. Recent studies have investigated polymorphisms in HLA-G that may be associated with susceptibility to HPV infection and persistence. One hundred-forty HIV(+) pregnant women from Brazil had a DNA fragment comprising HLA-G exons 2-4 PCR-amplified, cloned, sequenced and analyzed for allele determination. Altogether, 22 alleles comprising 52 different genotypes were found. Four novel HLA-G alleles were characterized. We have not observed association of specific HLA-G alleles with HPV infection, but found a protective effect of the G:01:01:02 allele against the occurrence of intraepithelial lesions. In addition to describing new HLA-G alleles and defining new reference sequences, our data provide a better understanding of the impact of HLA-G alleles on HPV-related disease.

MECP2, a gene associated with Rett syndrome in humans, shows conserved coding regions, independent Alu insertions, and a novel transcript across primate evolution.

BACKGROUND: The methyl-CpG Binding Protein two gene (MECP2) encodes a multifunctional protein comprising two isoforms involved in nuclear organization and regulation of splicing and mRNA template activity. This gene is normally expressed in all tissues, with a higher expression level in the brain during neuronal maturation. Loss of MECP2 function is the primary cause of Rett syndrome (RTT) in humans, a dominant, X-linked disorder dramatically affecting neural and motor development. RESULTS: We investigated the molecular evolution of MECP2 in several primate taxa including 36 species in 16 genera of neotropical (platyrrhine) primates. The coding region of the MECP2_e2 isoform showed a high level of evolutionary conservation among humans and other primates, with amino acid substitutions in 14 codons and one in-frame insertion of a single serine codon, between codons 357 and 358, in Ateles paniscus. Most substitutions occurred in noncritical regions of MECP2 and the majority of the algorithms used for analyzing selection did not provide evidence of positive selection. Conversely, we found 48 sites under negative selection in different regions, 23 of which were consistently found by three different algorithms. Similar to an inverted Alu insert found previously in a lesser ape at a parallel location, one Alu insertion of approximately 300 bp in Cebus and Sapajus was found in intron 3. Phylogenetic reconstruction of the intron 3 data provided a topology that was coincident with the consensus arrangement of the primate taxa. RNAseq data in the neotropical primate Callimico goeldii revealed a novel transcript consisting of a noncontinuous region of the human-homologous intron 2 in this species; this transcript accounted for two putative polypeptides. CONCLUSIONS: Despite the remarkable evolutionary conservation of MECP2, one in-frame codon insertion was observed in A. paniscus, and one region of intron 3 was affected by a trans-specific Alu retrotransposition in two neotropical primate genera. Moreover, identification of novel MECP2 transcripts in Callimico suggests that part of a homologous human intronic region might be expressed, and that the potential open reading frame in this region might be a subject of interest in RTT patients who carry an apparently normal MECP2 sequence.

Identification and characterization of highly divergent simian foamy viruses in a wide range of new world primates from Brazil.

Foamy viruses naturally infect a wide range of mammals, including Old World (OWP) and New World primates (NWP), which are collectively called simian foamy viruses (SFV). While NWP species in Central and South America are highly diverse, only SFV from captive marmoset, spider monkey, and squirrel monkey have been genetically characterized and the molecular epidemiology of SFV infection in NWPs remains unknown. We tested a large collection of genomic DNA (n = 332) comprising 14 genera of NWP species for the presence of SFV polymerase (pol) sequences using generic PCR primers. Further molecular characterization of positive samples was carried out by LTR-gag and larger pol sequence analysis. We identified novel SFVs infecting nine NWP genera. Prevalence rates varied between 14-30% in different species for which at least 10 specimens were tested. High SFV genetic diversity among NWP up to 50% in LTR-gag and 40% in pol was revealed by intragenus and intrafamilial comparisons. Two different SFV strains infecting two captive yellow-breasted capuchins did not group in species-specific lineages but rather clustered with SFVs from marmoset and spider monkeys, indicating independent cross-species transmission events. We describe the first SFV epidemiology study of NWP, and the first evidence of SFV infection in wild NWPs. We also document a wide distribution of distinct SFVs in 14 NWP genera, including two novel co-speciating SFVs in capuchins and howler monkeys, suggestive of an ancient evolutionary history in NWPs for at least 28 million years. A high SFV genetic diversity was seen among NWP, yet these viruses seem able to jump between NWP species and even genera. Our results raise concerns for the risk of zoonotic transmission of NWP SFV to humans as these primates are regularly hunted for food or kept as pets in forest regions of South America.

Screening of RB1 alterations in Brazilian patients with retinoblastoma and relatives with retinoma: phenotypic and genotypic associations.

PURPOSE: To identify constitutional alterations of the retinoblastoma 1 gene (RB1) in two cohorts of Brazilian patients with retinoblastoma and to analyze genotype-phenotype associations. METHODS: Molecular screening was carried out by direct sequencing of the 27 RB1 exons and flanking regions in blood DNA of 71 patients with retinoblastoma and 4 relatives with retinoma, and with multiplex ligation-dependent probe amplification (MLPA) in 21 patients. The presumed impact of nucleotide substitutions on the structure of the retinoblastoma protein (pRB) was predicted by Polymorphism Phenotyping-2 (PolyPhen-2). Kaplan-Meier and log-rank test were used for estimating 60-month survival rates. RESULTS: One hundred two nucleotide substitutions were detected, 92 substitutions in 59 patients with retinoblastoma and 10 substitutions in 4 individuals with retinoma. Eight substitutions were novel. The majority of substitutions were intronic (86.2%). More than one substitution was present in 37.3% of patients. Twenty-one duplications and 11 deletions were found in 12 patients; some of which with both types of alterations. Duplications/deletions were found in four patients lacking constitutional alterations when analyzed by sequencing, and in eight patients carrying one or more polymorphic intronic substitutions. The global 60-month survival rate in patients was 91.8% (Confidence Interval95% = 85.0 - 99.1). Significant, lower survival rates were found in extraocular presentation (81.0%) versus intraocular tumors (P = 0.014), first enucleation after 1 month following diagnosis (80.9%) versus earlier first enucleation (P = 0.020), and relapse (100.0%) versus absence of relapse (P = 0.0005). CONCLUSIONS: Fifteen substitutions (4 intronic and 11 exonic) were identified as probably or likely pathogenic. Four of these 11 exonic substitutions were novel. Survival rates, however, were not affected by presence of these probably or likely pathogenic alterations, most of which not found in patients with retinoblastoma from other Latin American countries. These differences might be related to the different ethnic composition of the Latin American cohorts. Portuguese Abstract.

HIV/HPV co-infection during pregnancy in southeastern Brazil: prevalence, HPV types, cytological abnormalities and risk factors.

OBJECTIVE: HIV(+) pregnant women are at a higher risk of HPV infection and development of cervical cancer. Our objectives were to assess the prevalence and HPV types in HIV(+) pregnant women and to identify risk factors for HPV infection and cytological abnormalities. METHODS: Cervicovaginal smears were collected during pregnancy from 140 women. Partial HPV L1 gene and the exon 4 of the human TP53 gene (containing codon 72) were PCR-amplified and sequenced. Amplified products indicating multiple HPV infection were further cloned and sequenced. The association of demographic, obstetric and HIV-related clinical variables with HPV infection and cervical lesions was tested by univariate analyses, and significant factors were subsequently tested by logistic regression multivariate analysis. RESULTS: HPV DNA tested positive for 118 patients and HPV types were identified in 104 samples. Twenty-eight different types were found, HPV-16 and HPV-58 being the most prevalent. High-risk types were present in 79.8% of samples and multiple infections in 16.3%. Abnormal cervical smears were found in 44 patients (31.4%). Absolute CD4(+) T-cell counts below 350 were associated with HPV infection. Younger age was associated with cervical abnormalities and higher CD4(+) T-cell count was an apparent protective factor. CONCLUSIONS: We found a high prevalence of HPV infection and high-risk types in this cohort. Our results highlighted the relevance of immune system integrity rather than TP53 variants for protecting this highly vulnerable population to HPV infection and carcinogenesis.

WT1, WTX and CTNNB1 mutation analysis in 43 patients with sporadic Wilms' tumor.

Wilms' tumor (WT) is a heterogeneous neoplasia characterized by a number of genetic abnormalities, involving tumor suppressor genes, oncogenes and genes related to the Wnt signaling pathway. Somatic biallelic inactivation of WT1 is observed in 5-10% of sporadic WT. Somatic mutations in exon 3 of CTNNB1, which encodes ß-catenin, were initially observed in 15% of WT. WTX encodes a protein that negatively regulates the Wnt/ß-catenin signaling pathway and mediates the binding of WT1. In this study, we screened germline and somatic mutations in selected regions of WT1, WTX and CTNNB1 in 43 WT patients. Mutation analysis of WT1 identified two single-nucleotide polymorphisms, one recurrent nonsense mutation (p.R458X) in a patient with proteinuria but without genitourinary findings of Denys-Drash syndrome (DDS) and one novel missense mutation, p.C428Y, in a patient with Denys-Drash syndrome phenotype. WT1 SNP rs16754A>G (R369R) was observed in 17/43 patients, and was not associated with significant difference in age at diagnosis distribution, or with 60-month overall survival rate. WTX mutation analysis identified five sequence variations, two synonymous substitutions (p.Q1019Q and p.D379D), a non-synonymous mutation (p.F159L), one frameshift mutation (p.157X) and a novel missense mutation, p.R560W. Two sequence variations in CTNNB1 were identified, p.T41A and p.S45C. Overall survival of bilateral cases was significantly lower (p=0.005). No difference was observed when survival was analyzed among patients with WT1 or with WTX mutations. On the other hand, the survival of two patients with the CTNNB1 p.T41A mutation was significantly lower (p=0.000517) than the average.