CDKN2A (p14(ARF)/p16(INK4a)) and ATM promoter methylation in patients with impalpable breast lesions.

Early detection of breast cancer increases the chances of cure, but the reliable identification of impalpable lesions is still a challenge. In spite of the advances in breast cancer detection, the molecular basis of impalpable lesions and the corresponding circulating biomarkers are not well understood. Impalpable lesions, classified by radiologists according to the Breast Imaging Reporting and Data System in the categories 3 and 4, can be either benign or malignant (slow growing or aggressive). In this article, we report the DNA methylation pattern in CDKN2A (p14(ARF)/p16(INK4a)) and in ATM gene promoters from 62 impalpable lesions, 39 peripheral blood samples, and 39 saliva samples, assessed by methylation-specific polymerase chain reaction method. ATM showed the greatest percentage of methylation in DNA from lesions (benign and malignant), blood (even with p16(INK4a)), and saliva, followed by p16(INK4a) and p14(ARF). Among the malignant cases, ATM promoter was the most hypermethylated in lesion DNA and in blood and saliva DNAs, and p14(ARF), the least. The highest percentage of p16(INK4a) methylation was found in the blood. Finally, our data are relevant because they were obtained using impalpable breast lesions from patients who were carefully recruited in 2 public hospitals of Rio de Janeiro.

Potential correlation between tumor aggressiveness and protein expression patterns of nipple aspirate fluid (NAF) revealed by gel-based proteomic analysis.

Breast cancer is the leading cause of cancer related deaths in women. Most breast cancers stem from mammary ductal cells that secrete nipple aspirate fluid (NAF), a biological sample that contains proteins associated with the tumor microenvironment. In this study, NAF samples from both breasts of 7 Brazilian patients with unilateral breast cancer were analyzed. These samples were systematically compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional fluorescence difference gel electrophoresis (2D-DIGE); substantial qualitative individual differences were observed. In general, when NAF samples were compared from both breasts within the same patient their electrophoretic patterns were very similar, regardless of their cancer status. A comparison of all patients identified 2 main NAF protein profiles. The HomEP, homogeneous expression profile, was characterized by typical SDS-PAGE and 2D-DIGE protein patterns that were observed in patients with a good breast cancer prognosis and were similar to previous Type I NAF classifications that used one-dimensional electrophoresis. The HetEP, heterogeneous expression profile, was characterized by distinct protein patterns that have not been reported in previous studies and have been primarily observed in breast cancer patients with a poor prognosis. The NAF samples were rich in metal-dependent proteolytic enzymes, as visualized by SDS-PAGE zymography. They varied qualitatively with respect to their gelatinolytic band distribution. However, there were no correlations between these characteristics and the pathologic features of these tumors. A comparative analysis of NAF samples taken from each breast in a single patient showed conserved zymographic patterns. In conclusion, the present study highlights important distinctions in the protein content of individual NAF samples and provides insight into the composition of the tumor microenvironment. These data reinforce breast cancer as a heterogeneous disease with a diverse natural history, which is becoming increasingly evident through other recent studies.

Genomic instability at the 13q31 locus and somatic mtDNA mutation in the D-loop site correlate with tumor aggressiveness in sporadic Brazilian breast cancer cases

OBJECTIVE: Genomic instability is a hallmark of malignant tissues. In this work, we aimed to characterize nuclear and mitochondrial instabilities by determining short tandem repeats and somatic mitochondrial mutations, respectively, in a cohort of Brazilian sporadic breast cancer cases. Furthermore, we performed an association analysis of the molecular findings and the clinical pathological data. METHODS: We analyzed 64 matched pairs of breast cancer and adjacent non-cancerous breast samples by genotyping 13 nuclear short tandem repeat loci (namely, D2S123, TPOX, D3S1358, D3S1611, FGA, D7S820, TH01, D13S317, D13S790, D16S539, D17S796, intron 12 BRCA1 and intron 1 TP53) that were amplified with the fluorescent AmpFlSTR Identifiler Genotyping system (Applied Biosystems, USA) and by silver nitrate staining following 6% denaturing polyacrylamide gel electrophoresis. Somatic mtDNA mutations in the D-loop site were assessed with direct sequencing of the hypervariable HVI and HVII mitochondrial regions. RESULTS: Half of the cancer tissues presented some nuclear instability. Interestingly, the D13S790 locus was the most frequently affected (36%), while the D2S123 locus presented no alterations. Forty-two percent of the cases showed somatic mitochondrial mutations, the majority at region 303-315 poly-C. We identified associations between Elston grade III, instabilities at 13q31 region (p = 0.0264) and mtDNA mutations (p = 0.0041). Furthermore, instabilities at 13q31 region were also associated with TP53 mutations in the invasive ductal carcinoma cases (p= 0.0207). CONCLUSION: Instabilities at 13q31 region and the presence of somatic mtDNA mutations in a D-loop site correlated with tumor aggressiveness.

Analysis of CYP1A1 exon 7 polymorphisms by PCR-SSCP in a Brazilian population and description of two novel gene variations.

CYP1A1 polymorphisms have been associated with a higher risk to develop lung cancer, particularly in Japanese. The type and the frequency of the polymorphisms can vary according to the ethnicity. In the present study, we aimed to determine the frequency of CYP1A1(*)2B and (*)4, and to look for other possible polymorphisms that may happen in exon 7 in individuals from Rio de Janeiro, an ethnic mixed population from Brazil. We developed a PCR-SSCP method for screening the genomic polymorphic region from 2289 to 2645 bp. Seven different migration patterns were found among 405 individuals, 130 healthy blood donors and 275 outpatients from Hospital Universitário Pedro Ernesto located in Rio de Janeiro. Five of the migration patterns corresponded to the genotypes: (*)1/(*)1 (the wild type); (*)1/(*)2B; (*)1/(*)4, (*)2B/(*)4 (heterozygous polymorphic) and (*)2B/(*)2B (homozygous polymorphic). Two other patterns corresponded to gene alterations not yet published: a C > T transition localized at the position 2461, and a C > T transition localized at position 2445. The genotype frequencies of the studied polymorphisms were: for CYP1A1(*)2B - 83.7% to (*)1/(*)1, 15.1% to (*)1/(*)2B and 1.2% to (*)2B/(*)2B; for CYP1A1(*)4 - 93.1% to (*)1/(*)1, 6.9% to (*)1/(*)4. The distribution of CYP1A1(*)2B and (*)4 genotypes combined were similar between white and non-white individuals. However, when the non-white individuals were stratified between blacks and mulattos, and then compared with white, black individuals showed a higher frequency of the wild type genotype (P = 0.008) and a lower frequency of genotype (*)1/(*)4 (P = 0.026). Additionally, when black and mulatto individuals were compared, blacks had a higher frequency of the wild type genotype (P = 0.008) and a lower frequency of the(*)1/(*)2B genotype (P = 0.0008), showing a different ethnic distribution of CYP1A1 polymorphisms.