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1.  Ectopic Activation of Germline and Placental Genes Identifies Aggressive Metastasis-Prone Lung Cancers 
Science translational medicine  2013;5(186):186ra66.
Activation of normally silent tissue-specific genes and the resulting cell “identity crisis” are the unexplored consequences of malignant epigenetic reprogramming. We designed a strategy for investigating this reprogramming, which consisted of identifying a large number of tissue-restricted genes that are epigenetically silenced in normal somatic cells and then detecting their expression in cancer. This approach led to the demonstration that large-scale “off-context” gene activations systematically occur in a variety of cancer types. In our series of 293 lung tumors, we identified an ectopic gene expression signature associated with a subset of highly aggressive tumors, which predicted poor prognosis independently of the TNM (tumor size, node positivity, and metastasis) stage or histological subtype. The ability to isolate these tumors allowed us to reveal their common molecular features characterized by the acquisition of embryonic stem cell/germ cell gene expression profiles and the down-regulation of immune response genes. The methodical recognition of ectopic gene activations in cancer cells could serve as a basis for gene signature–guided tumor stratification, as well as for the discovery of oncogenic mechanisms, and expand the understanding of the biology of very aggressive tumors.
PMCID: PMC4818008  PMID: 23698379
2.  Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil 
Genetics and Molecular Biology  2016;39(2):210-222.
In Brazil, breast cancer is a public health care problem due to its high incidence and mortality rates. In this study, we investigated the prevalence of hereditary breast cancer syndromes (HBCS) in a population-based cohort in Brazils southernmost capital, Porto Alegre. All participants answered a questionnaire about family history (FH) of breast, ovarian and colorectal cancer and those with a positive FH were invited for genetic cancer risk assessment (GCRA). If pedigree analysis was suggestive of HBCS, genetic testing of the BRCA1, BRCA2, TP53, and CHEK2 genes was offered. Of 902 women submitted to GCRA, 214 had pedigrees suggestive of HBCS. Fifty of them underwent genetic testing: 18 and 40 for BRCA1/BRCA2 and TP53 mutation screening, respectively, and 7 for CHEK2 1100delC testing. A deleterious BRCA2 mutation was identified in one of the HBOC probands and the CHEK2 1100delC mutation occurred in one of the HBCC families. No deleterious germline alterations were identified in BRCA1 or TP53. Although strict inclusion criteria and a comprehensive testing approach were used, the suspected genetic risk in these families remains unexplained. Further studies in a larger cohort are necessary to better understand the genetic component of hereditary breast cancer in Southern Brazil.
PMCID: PMC4910552  PMID: 27223485
Breast cancer predisposition syndrome; hereditary breast cancer; genetic cancer risk assessment
3.  Impact of G-quadruplex structures and intronic polymorphisms rs17878362 and rs1642785 on basal and ionizing radiation-induced expression of alternative p53 transcripts 
Carcinogenesis  2014;35(12):2706-2715.
This study demonstrates that the intronic TP53 polymorphisms rs1642785 and rs17878362 impact on the formation of G4 structures, mRNA splicing and stability, and thus on the differential expression of isoform-specific transcripts of the TP53 gene.
G-quadruplex (G4) structures in intron 3 of the p53 pre-mRNA modulate intron 2 splicing, altering the balance between the fully spliced p53 transcript (FSp53, encoding full-length p53) and an incompletely spliced transcript retaining intron 2 (p53I2 encoding the N-terminally truncated Δ40p53 isoform). The nucleotides forming G4s overlap the polymorphism rs17878362 (A1 wild-type allele, A2 16-base pair insertion) which is in linkage disequilibrium with rs1642785 in intron 2 (c.74+38 G>C). Biophysical and biochemical analyses show rs17878362 A2 alleles form similar G4 structures as A1 alleles although their position is shifted with respect to the intron 2 splice acceptor site. In addition basal FSp53 and p53I2 levels showed allele specific differences in both p53-null cells transfected with reporter constructs or lymphoblastoid cell lines. The highest FSp53 and p53I2 levels were associated with combined rs1642785-GG/rs17878362-A1A1 alleles, whereas the presence of rs1642785-C with either rs17878362 allele was associated with lower p53 pre-mRNA, total TP53, FSp53 and p53I2 levels, due to the lower stability of transcripts containing rs1642785-C. Treatment of lymphoblastoid cell with the G4 binding ligands 360A or PhenDC3 or with ionizing radiation increased FSp53 levels only in cells with rs17878362 A1 alleles, suggesting that under this G4 configuration full splicing is favoured. These results demonstrate the complex effects of intronic TP53 polymorphisms on G4 formation and identify a new role for rs1642785 on mRNA splicing and stability, and thus on the differential expression of isoform-specific transcripts of the TP53 gene.
PMCID: PMC4247521  PMID: 25269805
4.  Ancestry of the Brazilian TP53 c.1010G>A (p.Arg337His, R337H) Founder Mutation: Clues from Haplotyping of Short Tandem Repeats on Chromosome 17p 
PLoS ONE  2015;10(11):e0143262.
Rare germline mutations in TP53 (17p13.1) cause a highly penetrant predisposition to a specific spectrum of early cancers, defining the Li-Fraumeni Syndrome (LFS). A germline mutation at codon 337 (p.Arg337His, c1010G>A) is found in about 0.3% of the population of Southern Brazil. This mutation is associated with partially penetrant LFS traits and is found in the germline of patients with early cancers of the LFS spectrum unselected for familial history. To characterize the extended haplotypes carrying the mutation, we have genotyped 9 short tandem repeats on chromosome 17p in 12 trios of Brazilian p.Arg337His carriers. Results confirm that all share a common ancestor haplotype of Caucasian/Portuguese-Iberic origin, distant in about 72–84 generations (2000 years assuming a 25 years intergenerational distance) and thus pre-dating European migration to Brazil. So far, the founder p.Arg337His haplotype has not been detected outside Brazil, with the exception of two residents of Portugal, one of them of Brazilian origin. On the other hand, increased meiotic recombination in p.Arg337His carriers may account for higher than expected haplotype diversity. Further studies comparing haplotypes in populations of Brazil and of other areas of Portuguese migration are needed to understand the historical context of this mutation in Brazil.
PMCID: PMC4664269  PMID: 26618902
5.  Patterns of Benign and Malignant Lesions of the Thyroid in Two Wilayahs of Northeastern Algeria 
Journal of Cancer Epidemiology  2015;2015:849416.
The aim of this study is to compare histological patterns and to estimate the burden of thyroid cancers in the two Wilayahs (departments) of El-Taref and Guelma in northeast of Algeria (total population 0.9 million), locally reputed as having different rates of endemic thyroid diseases and cancer. A retrospective analysis of central pathology registers and clinical records of patients with thyroid diseases, covering the period 2008–2012, was conducted. A total of 145 cases of thyroid cancers with histological confirmation were registered in the two Wilayahs during the period, with a female to male ratio of 5.9 : 1. Estimates of crude incidence rates suggested that thyroid cancers were twice as frequent in the Wilayah of Guelma compared to El-Taref (p < 0.05) with a tendency to occur at a younger age in resident of the Wilayah of El-Taref. Diagnoses of thyroid adenoma were more frequent in the Wilayah of Guelma, whereas the prevalence of other thyroid lesions, including goitre, was similar in the two Wilayahs. This first descriptive study on geographic variations in thyroid cancer in Northern Africa suggests that significant differences may occur in relation with environmental and lifestyle exposures.
PMCID: PMC4670666  PMID: 26681943
6.  Osteopontin and Latent-TGF β Binding-Protein 2 as potential diagnostic markers for HBV-related Hepatocellular Carcinoma 
Chronic Hepatitis B (HB) is the main risk factor for chronic liver disease (CLD) and hepatocellular carcinoma (HCC) in many low-resource countries, where diagnosis is constrained by lack of clinical, histopathological and biomarker resources. We have used proteomics to detect plasma biomarkers that outperform α-Fetoprotein (AFP), the most widely used biomarker for HCC diagnosis in low-resource contexts. Deep plasma proteome analysis was performed in HCC patients, patients with chronic liver disease (CLD) and in HB-carrier controls from Thailand (South-East Asia) and The Gambia (West-Africa). Mass spectrometry profiling identified Latent-Transforming Growth Factor β Binding-Protein 2 (LTBP2) and Osteopontin (OPN) as being significantly elevated in HCC versus CLD and controls. These two proteins were further analysed by ELISA in a total of 684 plasma samples, including 183 HCC, 274 CLD and 227 asymptomatic controls. When combined, LTBP2 and OPN showed an area under the receiver operating curve (ROC) of 0.85 in distinguishing HCC from CLD in subjects with α-Fetoprotein (AFP) < 20 ng/mL. In a prospective cohort of 115 CLD patients from Korea, increased plasma levels of LTBP2 and/or OPN were detected in plasma collected over 2 years prior to diagnosis in 21 subjects who developed HCC. Thus, the combination of LTBP2 and OPN outperformed AFP for diagnosis and prediction of HCC and may therefore improve biomarker-based detection of HBV-related HCC.
PMCID: PMC4617682  PMID: 24803312
Biomarkers; chronic liver disease; hepatocellular carcinoma; LTBP2; OPN
8.  Molecular characteristics of Hepatitis B and chronic liver disease in a cohort of HB carriers from Bamako, Mali 
BMC Infectious Diseases  2015;15:180.
Hepatitis B (HB) infection is common in Mali. However, there is little information on molecular and biochemical characteristics of HB carriers.
A group of 1466 adult volunteers was recruited in the district of Bamako. Confirmed HB carriers were tested for HB viral load by quantitative PCR and HBV was genotyped by sequencing of HBS. Fibrosis and hepatitis activity were measured using the Fibrotest-Actitest. A mutation of TP53 at codon 249 (R249S), specific for exposure to aflatoxin, was detected in cell-free DNA extracted from plasma.
Overall, 276 subjects were HBsAg-positive (18.8%). Among 152 subjects tested for HBV load, 49 (32.2%) had over 104 copies/mL and 16 (10.5%) had levels below the limit of detection. The E genotype was found in 91.1% of carriers. Fibrotest scores ≥ F2 were observed in 52 subjects (35.4%). Actitest scores ≥ A2 were detected in 15 subjects (10.2%) and were correlated with Fibrotest scores (p = 0.0006). Among 105 subjects tested, 60% had detectable levels of R249S copies (>40 copies/mL plasma).
Chronic HB carriage in adults in Bamako district is well over epidemic threshold. About 1/3 of carriers have moderate to severe liver fibrosis and 60% have detectable aflatoxin-related TP53 R249S mutation. These results support introduction of anti-HB therapies to reduce the progression towards severe liver disease.
PMCID: PMC4403772  PMID: 25886382
HBV; Viral load; Chronic carriage; Fibrotest/Actitest; TP53 R249S mutation; Mali
9.  Age at cancer onset in germline TP53 mutation carriers: association with polymorphisms in predicted G-quadruplex structures 
Carcinogenesis  2013;35(4):807-815.
Germline TP53 mutations predispose to multiple cancers defining Li-Fraumeni/Li-Fraumeni-like syndrome (LFS/LFL), a disease with large individual disparities in cancer profiles and age of onset. G-quadruplexes (G4s) are secondary structural motifs occurring in guanine tracks, with regulatory effects on DNA and RNA. We analyzed 85 polymorphisms within or near five predicted G4s in TP53 in search of modifiers of penetrance of LFS/LFL in Brazilian cancer families with (n = 35) or without (n = 110) TP53 mutations. Statistical analyses stratified on family structure showed that cancer tended to occur ~15 years later in mutation carriers who also carried the variant alleles of two polymorphisms within predicted G4-forming regions, rs17878362 (TP53 PIN3, 16 bp duplication in intron 3; P = 0.082) and rs17880560 (6 bp duplication in 3′ flanking region; P = 0.067). Haplotype analysis showed that this inverse association was driven by the polymorphic status of the remaining wild-type (WT) haplotype in mutation carriers: in carriers with a WT haplotype containing at least one variant allele of rs17878362 or rs17880560, cancer occurred ~15 years later than in carriers with other WT haplotypes (P = 0.019). No effect on age of cancer onset was observed in subjects without a TP53 mutation. The G4 in intron 3 has been shown to regulate alternative p53 messenger RNA splicing, whereas the biological roles of predicted G4s in the 3′ flanking region remain to be elucidated. In conclusion, this study demonstrates that G4 polymorphisms in haplotypes of the WT TP53 allele have an impact on LFS/LFL penetrance in germline TP53 mutation carriers.
G-quadruplexes (G4) are regulatory secondary structures in guanine tracts in DNA or RNA. Polymorphisms in two G4-forming regions of TP53 modulate age at cancer onset in susceptible germline TP53 mutation carriers, suggesting that these G4 regulate p53 suppressor functions.
PMCID: PMC3977145  PMID: 24336192
10.  18F-FDG PET-CT for Surveillance of Brazilian Patients with Li-Fraumeni Syndrome 
Purpose: To evaluate the effectiveness of 18F-Fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) for detecting early cancer in carriers of germline TP53 mutation, the genetic defect underlying Li-Fraumeni and related syndromes, which predisposes to many forms of cancer throughout life.
Patients and methods: A total of 30 adult patients from six families with germline TP53 mutations were recruited. These patients did not have a diagnosis of cancer in the 24 months preceding the study. Anomalous concentrations from whole-body 18F-FDG-PET/CT were assessed by two independent experts. Suspicious lesions were excised and subjected to pathological examination.
Results: A total of 6/30 patients showed abnormal 18F-FDG-concentration. Confirmation studies revealed three cases of cancer, including one lung cancer, one ovarian cancer, and one disseminated breast cancer. Three patients had non-malignant lesions (one Bartholin’s cyst and two cases of reactive lymph nodes).
Conclusion: 18F-FDG-PET/CT is effective in detecting cancer in subjects who are asymptomatic according to current screening guidelines. These results further suggest that 18F-FDG-PET/CT is an appropriate method for surveillance of cancer risk in TP53 mutation carriers.
PMCID: PMC4333808  PMID: 25745605
Li-Fraumeni; PET-CT; screening; oncogenetic; p.R337H
11.  Association of prediagnostic physical activity with survival following breast cancer diagnosis: influence of TP53 mutation status 
Cancer causes & control : CCC  2013;24(12):10.1007/s10552-013-0294-x.
Physical activity both before and after breast cancer diagnosis has been associated with improved survival. However, it is not clear whether this association differs by molecular features of the tumor or by recency of the physical activity to the time of diagnosis.
We examined the association of prediagnostic physical activity with survival in a cohort of 1,170 women with primary, incident, and histologically confirmed breast cancer, examining tumor molecular subtypes. Cox regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI).
Mean follow-up time was 87.4 months after breast cancer diagnosis; there were 170 deaths identified. Compared with inactive patients (<3 hours/ week), women with higher average lifetime physical activity (>6 hours/ week) had reduced risk of all-cause mortality (adjusted HR = 0.61, 95% CI, 0.40-0.95; p trend = 0.04). There were no clear differences in the associations for lifetime and more recent physical activity. Lifetime physical activity was also weakly associated with decreased risk of breast cancer-specific mortality. Higher lifetime physical activity was associated with reduced risk of all-cause mortality among women with ER-positive tumors (HR = 0.52, 95% CI, 0.29-0.93), and mutant TP53 tumors (HR = 0.22, 95% CI, 0.06-0.72); however, no statistically significant interactions were observed for ER or TP53 status.
Our study further supports that prediagnostic physical activity improves overall survival following breast cancer, and suggests that the associations of prediagnostic physical activity with survival following breast cancer may vary by molecular features of the tumor, particularly ER and TP53 status.
PMCID: PMC3860883  PMID: 24068557
physical activity; breast cancer survival; TP53 mutation; epidemiology
12.  Prevalence of the TP53 p.R337H Mutation in Breast Cancer Patients in Brazil 
PLoS ONE  2014;9(6):e99893.
Germline TP53 mutations predispose individuals to multiple cancers and are associated with Li-Fraumeni/Li-Fraumeni-Like Syndromes (LFS/LFL). The founder mutation TP53 p.R337H is detected in 0.3% of the general population in southern Brazil. This mutation is associated with an increased risk of childhood adrenal cortical carcinoma (ACC) but is also common in Brazilian LFS/LFL families. Breast Cancer (BC) is one of the most common cancers diagnosed in TP53 mutation carriers. We have assessed the prevalence of p.R337H in two groups: (1) 59 BC affected women with a familial history (FH) suggestive of hereditary cancer syndrome but no LFS/LFL features; (2) 815 BC affected women unselected for cancer FH, diagnosed with BC at or before age 45 or at age 55 or older. Among group 1 and group 2 patients, 2/59 (3.4%, CI95%: 0.4%–11.7%) and 70/815 (8.6%, CI95%: 6.8%–10.7%), respectively, were p.R337H carriers in the germline. The prevalence of p.R337H was higher in women diagnosed with BC at or before age 45 (12.1%, CI95%: 9.1%–15.8%) than at age 55 or older (5.1%, CI95%: 3.2%–7.7%), p<0.001). The Brazilian founder p.R337H haplotype was detected in all carriers analysed. These results suggest that inheritance of p.R337H may significantly contribute to the high incidence of BC in Brazil, in addition to its recently demonstrated impact on the risk of childhood ACC.
PMCID: PMC4061038  PMID: 24936644
13.  The profile and contribution of rare germline copy number variants to cancer risk in Li-Fraumeni patients negative for TP53 mutations 
The Li-Fraumeni syndrome (LFS) is an inherited rare cancer predisposition syndrome characterized by a variety of early-onset tumors. Although germline mutations in the tumor suppressor gene TP53 account for over 50% of the families matching LFS criteria, the lack of TP53 mutation in a significant proportion of LFS families, suggests that other types of inherited alterations must contribute to their cancer susceptibility. Recently, increases in copy number variation (CNV) have been reported in LFS individuals, and are also postulated to contribute to LFS phenotypic variability.
Seventy probands from families fulfilling clinical criteria for either Li-Fraumeni or Li-Fraumeni-like (LFS/LFL) syndromes and negative for TP53 mutations were screened for germline CNVs.
We found a significantly increased number of rare CNVs, which were smaller in size and presented higher gene density compared to the control group. These data were similar to the findings we reported previously on a cohort of patients with germline TP53 mutations, showing that LFS/LFL patients, regardless of their TP53 status, also share similar CNV profiles.
These results, in conjunction with our previous analyses, suggest that both TP53-negative and positive LFS/LFL patients present a broad spectrum of germline genetic alterations affecting multiple loci, and that the genetic basis of LFS/LFL predisposition or penetrance in many cases might reside in germline transmission of CNVs.
PMCID: PMC4022048  PMID: 24775443
Li Fraumeni syndrome; Rare CNVs; Familial cancer
15.  Identification of Osteopontin as a Novel Marker for Early Hepatocellular Carcinoma 
Hepatology (Baltimore, Md.)  2011;55(2):483-490.
This study was to identify a biomarker that could improve α-fetoprotein (AFP) performance in hepatocellular carcinoma (HCC) surveillance among patients with cirrhosis. We performed proteomic profiling of plasma from patients with cirrhosis or HCC and validated selected candidate HCC biomarkers in two geographically distinct cohorts in order to include HCC of different etiologies. Mass spectrometry profiling of highly fractionated plasma from 18 cirrhosis and 17 HCC patients identified osteopontin (OPN) as significantly upregulated in HCC cases compared to cirrhosis controls. OPN levels were subsequently measured in 312 plasma samples collected from 131 HCC patients, 76 cirrhosis patients, 52 chronic hepatitis C (CHC) and B (CHB) patients and 53 healthy controls, in two independent cohorts. OPN plasma levels were significantly elevated in HCC patients compared to cirrhosis, CHC, CHB or healthy controls, in both cohorts. OPN alone or in combination with AFP had significantly better area under the receiver operating characteristic curve compared to AFP in comparing cirrhosis and HCC in both cohorts. OPN overall performance remained higher than AFP in comparing cirrhosis and the following HCC groups: HCV-related HCC, HBV-associated HCC and early HCC. OPN had also a good sensitivity in AFP negative HCC. In a pilot prospective study including 22 patients who developed HCC during follow-up, OPN was already elevated a year prior to diagnosis. Conclusion: OPN was more sensitive than AFP for the diagnosis of HCC in all studied HCC groups. In addition, OPN performance remained intact in samples collected a year prior to diagnosis.
PMCID: PMC3914762  PMID: 21953299
biomarker; HCC; early detection; OPN
16.  Transcriptional regulation of thymine DNA glycosylase (TDG) by the tumor suppressor protein p53 
Cell Cycle  2012;11(24):4570-4578.
Thymine DNA glycosylase (TDG) belongs to the superfamily of uracil DNA glycosylases (UDG) and is the first enzyme in the base-excision repair pathway (BER) that removes thymine from G:T mismatches at CpG sites. This glycosylase activity has also been found to be critical for active demethylation of genes involved in embryonic development. Here we show that wild-type p53 transcriptionally regulates TDG expression. Chromatin immunoprecipitation (ChIP) and luciferase assays indicate that wild-type p53 binds to a domain of TDG promoter containing two p53 consensus response elements (p53RE) and activates its transcription. Next, we have used a panel of cell lines with different p53 status to demonstrate that TDG mRNA and protein expression levels are induced in a p53-dependent manner under different conditions. This panel includes isogenic breast and colorectal cancer cell lines with wild-type or inactive p53, esophageal squamous cell carcinoma cell lines lacking p53 or expressing a temperature-sensitive p53 mutant and normal human bronchial epithelial cells. Induction of TDG mRNA expression is accompanied by accumulation of TDG protein in both nucleus and cytoplasm, with nuclear re-localization occurring upon DNA damage in p53-competent, but not -incompetent, cells. These observations suggest a role for p53 activity in TDG nuclear translocation. Overall, our results show that TDG expression is directly regulated by p53, suggesting that loss of p53 function may affect processes mediated by TDG, thus negatively impacting on genetic and epigenetic stability.
PMCID: PMC3562302  PMID: 23165212
TDG; p53; base-excision repair; glycosylase; transcriptional regulation; mutator phenotype; epigenetic stability
17.  Impact of Delay to Cryopreservation on RNA Integrity and Genome-Wide Expression Profiles in Resected Tumor Samples 
PLoS ONE  2013;8(11):e79826.
The quality of tissue samples and extracted mRNA is a major source of variability in tumor transcriptome analysis using genome-wide expression microarrays. During and immediately after surgical tumor resection, tissues are exposed to metabolic, biochemical and physical stresses characterized as “warm ischemia”. Current practice advocates cryopreservation of biosamples within 30 minutes of resection, but this recommendation has not been systematically validated by measurements of mRNA decay over time. Using Illumina HumanHT-12 v3 Expression BeadChips, providing a genome-wide coverage of over 24,000 genes, we have analyzed gene expression variation in samples of 3 hepatocellular carcinomas (HCC) and 3 lung carcinomas (LC) cryopreserved at times up to 2 hours after resection. RNA Integrity Numbers (RIN) revealed no significant deterioration of mRNA up to 2 hours after resection. Genome-wide transcriptome analysis detected non-significant gene expression variations of −3.5%/hr (95% CI: −7.0%/hr to 0.1%/hr; p = 0.054). In LC, no consistent gene expression pattern was detected in relation with warm ischemia. In HCC, a signature of 6 up-regulated genes (CYP2E1, IGLL1, CABYR, CLDN2, NQO1, SCL13A5) and 6 down-regulated genes (MT1G, MT1H, MT1E, MT1F, HABP2, SPINK1) was identified (FDR <0.05). Overall, our observations support current recommendation of time to cryopreservation of up to 30 minutes and emphasize the need for identifying tissue-specific genes deregulated following resection to avoid misinterpreting expression changes induced by warm ischemia as pathologically significant changes.
PMCID: PMC3835918  PMID: 24278187
18.  Hormonal Therapy and Risk of Breast Cancer in Mexican Women 
PLoS ONE  2013;8(11):e79695.
The use of hormonal therapies, including hormonal contraceptives (HC) and postmenopausal hormone replacement therapy (HRT) have been shown to influence breast cancer (BC) risk. However, the variations of these effects among populations and ethnic groups are not completely documented, especially among Hispanic women. We evaluated the association between HC and premenopausal BC risk, and between HRT and postmenopausal BC risk in Mexican women. Data from a Mexican multi-center population-based case–control study ofwomen aged 35 to 69 years were analysed. A total of 1000 cases and 1074 matched controls were recruited between 2004 and 2007. Information on hormonal therapy was collected through a structured questionnaire. Results were analysed using conditional logistic regression models. Overall, HC were used by 422/891 (47.3%) premenopausal women and HRT was used by 220/1117 (19.7%) postmenopausal women. For HC, odds ratios (ORs) for BC were 1.11 (95% confidence interval (CI): 0.82, 1.49) for current users and 1.68 (95% CI: 0.67, 4.21) for ever-users. No clear effect of duration of use was observed. For HRT, the OR for BC was significantly increased in ever users (OR: 1.45; 95% CI: 1.01, 2.08). A non-significant increased risk was observed for combined estrogen/progestin, (OR =  1.85; 95% CI: 0.84, 4.07) whereas no effect was observed for the use of estrogen alone (OR = 1.14; 95% CI: 0.68, 1.91). Our results indicate that, HC had a non-significant effect on the risk of pre-menopausal BC, but suggested that injected contraceptives may slightly increase the risk, whereas HRT had a significant effect on post-menopausal BC in this population. This study provides new information about the effects of HC and HRT on BC risk in a Mexican population, which may be of relevance for the population of Latin America as a whole.
PMCID: PMC3829873  PMID: 24260282
19.  Li-Fraumeni syndrome: report of a clinical research workshop and creation of a research consortium 
Cancer genetics  2012;205(10):479-487.
Li-Fraumeni syndrome (LFS) is a rare dominantly inherited cancer predisposition syndrome that was first described in 1969. In most families, it is caused by germline mutations in the TP53 gene and is characterized by early onset of multiple specific cancers and very high lifetime cumulative cancer risk. Despite significant progress in understanding the molecular biology of TP53, the optimal clinical management of this syndrome is poorly defined. We convened a workshop on November 2, 2010, at the National Institutes of Health in Bethesda, Maryland, bringing together clinicians and scientists, as well as individuals from families with LFS, to review the state of the science, address clinical management issues, stimulate collaborative research, and engage the LFS family community. This workshop also led to the creation of the Li-Fraumeni Exploration (LiFE) Research Consortium.
PMCID: PMC3593717  PMID: 22939227
Li-Fraumeni syndrome; hereditary cancer predisposition syndrome; TP53 mutations
20.  20-Years of Population-Based Cancer Registration in Hepatitis B and Liver Cancer Prevention in The Gambia, West Africa 
PLoS ONE  2013;8(9):e75775.
The Gambia Hepatitis Intervention Study (GHIS) was designed as a randomised control trial of infant hepatitis B vaccination applied to public health policy, with the main goal of preventing primary liver cancer later in adult life in The Gambia. To that effect, the National Cancer Registry of The Gambia (NCR), a population-based cancer registry (PBCR), was established in 1986 to actively collect data on all cancer diagnosis nation-wide. We extracted 20-years (1990-2009) of data to assess for the first time, the evolution of the most common cancers, also describe and demonstrate the role of the PBCR in a hepatitis B and liver cancer prevention programme in this population.
Methods and Findings
We estimated Age-Standardised Incidence Rates (ASR (W)) of the most common cancers registered during the period by gender. The registration period was divided into four 5-year intervals and incidence rates were estimated for each interval. The most common cancers in males were liver, prostate, lung plus bronchus, non-Hodgkin lymphoma (NHL) and stomach, accounting for 60%, 5%, 4%, 5% and 3%, respectively. Similarly, cancers of the cervix uteri, liver, breast and NHL, were the most common in females, accounting for 33%, 24%, 11% and 4% of the female cancers, respectively.
Cancer incidence has remained relatively stable over time, but as shown elsewhere in sub-Saharan Africa the disease is a threat in The Gambia. The infection related cancers which are mostly preventable (HBV in men and HPV/HIV in women) were the most common. At the moment the data is not enough to detect an effect of hepatitis B vaccination on liver cancer incidence in The Gambia. However, we observed that monitoring case occurrence through PBCR is a key public health pre-requisite for rational planning and implementation of targeted interventions for improving the health of the population.
PMCID: PMC3787012  PMID: 24098724
22.  Protein Profiling in Hepatocellular Carcinoma by Label-Free Quantitative Proteomics in Two West African Populations 
PLoS ONE  2013;8(7):e68381.
Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.
Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.
Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.
The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose definitive cut-offs and combinations for evaluation of performance.
PMCID: PMC3728326  PMID: 23935864
23.  TP53 R249S mutation, genetic variations in HBX and risk of hepatocellular carcinoma in The Gambia 
Carcinogenesis  2012;33(6):1219-1224.
In regions with high prevalence of chronic hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) exposure, hepatocellular carcinomas (HCCs) often contain TP53 mutation at codon 249 (R249S). Furthermore, a C-terminal truncated HBx protein expressed from hepatocyte integrated HBV is associated with HCC development. This study evaluates the association between R249S and HBX status in relation to HCC in West African population. HBX (complete or 3′-truncated) and HBS genes were assessed by PCR in cell-free DNA (CFDNA) from plasma of subjects recruited in a hospital-based case–control study (325 controls, 78 cirrhotic patients and 198 HCC cases) conducted in The Gambia. These samples had been previously analyzed for R249S and HBV serological status. Complete HBX sequence was frequently detected in CFDNA of HCC-R249S positive (77%, 43/56) compared with HCC-R249S-negative cases (44%, 22/50). Conversely, the proportion of 3′-truncated HBX gene was significantly higher in HCC-R249S negative than positive cases (34%, 17/50, compared with 12%, 7/56) (χ2 = 12.12; P = 0.002; distribution of R249S negative and positive according to HBX status). Occult HBV infection (detected by PCR) was present in 24% of HCC previously considered as negative by HBV serology. Moreover, HBV mutation analysis revealed that double mutation at nucleotides 1762T/1764A was associated with diagnosis of cirrhosis or HCC {cirrhosis: odds ratio (OR): 9.50 [95% confidence interval (CI) 1.50–60.11]; HCC: OR: 11.29 [95% CI 2.07–61.47]}. These findings suggest that in HCC from The Gambia, complete HBX sequences are often associated with the presence of TP53 R249S mutation.
PMCID: PMC3388490  PMID: 22759751
24.  Pooled Analysis of the Prognostic and Predictive Effects of KRAS Mutation Status and KRAS Mutation Subtype in Early-Stage Resected Non–Small-Cell Lung Cancer in Four Trials of Adjuvant Chemotherapy 
Journal of Clinical Oncology  2013;31(17):2173-2181.
We undertook this analysis of KRAS mutation in four trials of adjuvant chemotherapy (ACT) versus observation (OBS) to clarify the prognostic/predictive roles of KRAS in non–small-cell lung cancer (NSCLC).
KRAS mutation was determined in blinded fashion. Exploratory analyses were performed to characterize relationships between mutation status and subtype and survival outcomes using a multivariable Cox model.
Among 1,543 patients (763 OBS, 780 ACT), 300 had KRAS mutations (codon 12, n = 275; codon 13, n = 24; codon 14, n = 1). In OBS patients, there was no prognostic difference for overall survival for codon-12 (mutation v wild type [WT] hazard ratio [HR] = 1.04; 95% CI, 0.77 to 1.40) or codon-13 (HR = 1.01; 95% CI, 0.47 to 2.17) mutations. No significant benefit from ACT was observed for WT-KRAS (ACT v OBS HR = 0.89; 95% CI, 0.76 to 1.04; P = .15) or codon-12 mutations (HR = 0.95; 95% CI, 0.67 to 1.35; P = .77); with codon-13 mutations, ACT was deleterious (HR = 5.78; 95% CI, 2.06 to 16.2; P < .001; interaction P = .002). There was no prognostic effect for specific codon-12 amino acid substitution. The effect of ACT was variable among patients with codon-12 mutations: G12A or G12R (HR = 0.66; P = .48), G12C or G12V (HR = 0.94; P = .77) and G12D or G12S (HR = 1.39; P = .48; comparison of four HRs, including WT, interaction P = .76). OBS patients with KRAS-mutated tumors were more likely to develop second primary cancers (HR = 2.76, 95% CI, 1.34 to 5.70; P = .005) but not ACT patients (HR = 0.66; 95% CI, 0.25 to 1.75; P = .40; interaction, P = .02).
KRAS mutation status is not significantly prognostic. The potential interaction in patients with codon-13 mutations requires validation. At this time, KRAS status cannot be recommended to select patients with NSCLC for ACT.
PMCID: PMC4881333  PMID: 23630215
25.  TP53 p.R337H is a conditional cancer-predisposing mutation: further evidence from a homozygous patient 
BMC Cancer  2013;13:187.
Adrenocortical carcinomas (ACCs) are among the most common childhood cancers occurring in infants affected with the Li-Fraumeni and Li- Fraumeni-like (LFS/LFL) syndromes, which are caused by dominant germline mutations in the TP53 gene. In Brazil, a particular mutation, occurring in the tetramerisation domain of the gene, p.R337H, is exceedingly common due to a founder effect and is strongly associated with ACC. In this report, we describe the phenotype and long-term clinical follow-up of a female child diagnosed with ACC and homozygous for the TP53 p.R337H founder mutation.
Case presentation
At age 11 months, the patient was diagnosed with a virilising anaplastic adrenal cortical tumour, which was completely excised without disturbing the adrenal capsule. Family history was consistent with an LFL tumour pattern, and genotyping identified the TP53 p.R337H mutation in both alleles in genomic DNA from lymphocytes and fibroblasts. Haplotype analysis confirmed the occurrence of the mutation in the same founder haplotype previously described in other Brazilian patients. No other germline or somatic TP53 mutations or rearrangements were identified. At age 9 years, the child was asymptomatic and had no evidence of endocrine derangements. Full body and brain magnetic resonance imaging (MRI) failed to detect any suspicious proliferative lesions, and cardiopulmonary exercise testing results were within the normal reference for the child’s age, ruling out a major exercise capacity deficiency.
This is the first clinical and aerobic functional capacity documentation of a patient who carries two mutant TP53 alleles and no wild-type allele. Our results support the hypothesis that TP53 p.R337H, the most common TP53 mutation ever described in any population, is a conditional mutant. Furthermore, our observations over a long period of clinical follow-up suggest that TP53 p.R337H homozygotes do not have a more severe disease phenotype than do heterozygote carriers of the same mutation. Patients with the homozygous TP53 p.R337H genotype will require careful surveillance for lifetime cancer risk and for effects on metabolic capacity later in life.
PMCID: PMC3637265  PMID: 23570263

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