Deolinda Pereira

Importance of TP53 codon 72 and intron 3 duplication 16bp polymorphisms in prediction of susceptibility on breast cancer

BackgroundTP53 is one of major tumour suppressor genes being essential in preservation of genome integrity. Two very common polymorphisms have been demonstrated to contribute to cancer susceptibility and tumour behaviour. The purpose of this study was to evaluate the role of Arg72Pro and PIN3 Ins16bp polymorphisms in TP53 gene as genetic susceptibility and predictive markers to breast cancer.MethodsWe analysed DNA samples from 264 breast cancer patients and 440 controls, for TP53 Arg72Pro and PIN3 Ins16bp polymorphisms using PCR-RFLP.ResultsWe observed that women with A2A2 genotype have increased risk for developing breast cancer, either in women with or without familial history (FH) of the disease (OR = 4.40, 95% CI 1.60–12.0; p = 0.004; OR = 3.88, 95% CI 1.18–12.8; p = 0.026, respectively). In haplotype analysis, statistically significant differences were found between TP53 Arg-A2 haplotype frequencies and familial breast cancer cases and the respective control group (OR = 2.10, 95% CI 1.08–4.06; p = 0.028). Furthermore, both TP53 polymorphisms are associated with higher incidence of lymph node metastases.ConclusionOur findings suggest TP53 PIN3 Ins16bp polymorphism as a real risk modifier in breast cancer disease, either in sporadic and familial breast cancer. Furthermore, both TP53 polymorphisms are associated with higher incidence of lymph node metastases.

Platinum/paclitaxel-based chemotherapy in advanced ovarian carcinoma: glutathione S-transferase genetic polymorphisms as predictive biomarkers of disease outcome

AbstractBackground. The glutathione S-transferases (GSTs) are a group of multifunctional enzymes that catalyze the conjugation of glutathione with a variety of electrophilic compounds, including cytotoxic agents. A significant percentage of normal individuals exhibit genetic polymorphism with a homozygous deletion (null genotype) of the genes, leading to absence of the enzyme. Methods. In the present study we analyzed GSTM1 and GSTT1 polymorphisms in the genomic DNA isolated from peripheral blood of patients with ovarian cancer treated with chemotherapy (paclitaxel and cisplatinum) after cytoreductive surgery and assessed its correlation with the clinical outcome of these patients. The median follow-up for the patients was 30 months. Results. The estimated 3-year survival rate was 59.8% for all patients and 20.8% for carriers of GSTM1-wt/GSTT1-wt (wt indicates wild type) genotype combination (37.7% for GSTM1-wt alone) compared with 83.1% for non-GSTM1-wt/GSTT1-wt genotype carriers (100% for GSTM1-null). The mean survival time was significantly better in patients who are carriers of the GSTM1-null genotype (40.5 vs. 33.5; P = 0.006) or carriers of non-GSTM1-wt/GSTT1-wt genotypes (55.4 vs. 30.7; P = 0.009). The progression-free interval was more favorable for GSTM1-null carriers (41.9 vs. 27.4; P = 0.024). Conclusion. The study suggests that characterization of the drug-metabolizing genetic individual profile can be of great interest in clinical oncology. It can define the optimal chemotherapy for each patient, improve the efficiency, and reduce the incidence of drug toxicity and poor drug responses.

Analysis of Estrogen Receptor Polymorphism in Codon 325 by PCR‐SSCP in Breast Cancer: Association With Lymph Node Metastasis

Breast cancer is the most frequent neoplasm in women. Expression of the estrogen receptor (ER) has a key role in breast cancer; the ER gene is located at chromosome 6q24‐q27 and is made up of 8 exons with a total of 140 kb. The polymorphism in codon 325 of exon 4 (ER325) is a transition CCC→CCG. The objective of this study is to analyze the frequency of this polymorphism in breast cancer using the polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) technology. DNA was extracted from tumor cells of 70 breast cancer patients and from the peripheral blood of 69 individuals without any known pathology (control group). Amplification products of the ER gene were analyzed by SSCP. In breast cancer patients the ER325 polymorphism was detected in 42.8% of the cases. In contrast, in the control group, the frequency of the same polymorphism was 24.6. Statistical comparison of the frequency distributions revealed that they are significantly different (p = 0.023). There was also an association between ER325 polymorphism and the absence of lymph node metastases (p = 0.038). Our data suggest that there is a relationship between the ER325 polymorphism and susceptibility to breast cancer (OR = 2.3; 1.10 < OR < 5.1) and that it can also be related with the metastasization process.

The c.156_157insAlu BRCA2 rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal

We evaluated the contribution of an Alu insertion in BRCA2 exon 3 (c.156_157insAlu) to inherited predisposition to breast/ovarian cancer in 208 families originated mostly from northern/central Portugal. We identified the c.156_157insAlu BRCA2 mutation in 14 families and showed that it accounts for more that one-fourth of deleterious BRCA1/BRCA2 mutations in breast/ovarian cancer families originated from this part of the country. This mutation originates BRCA2 exon 3 skipping and we demonstrated its pathogenic effect by showing that the BRCA2 full length transcript is derived only from the wild type allele in carriers, that it is absent in 262 chromosomes from healthy blood donors, and that it co-segregates with the disease. Polymorphic microsatellite markers were used for haplotype analysis in three informative families. In two of the three families one haplotype was shared for all but two markers, whereas in the third family all markers telomeric to BRCA2 differed from that observed in the other two. Although the c.156_157insAlu BRCA2 mutation has so far only been identified in Portuguese breast/ovarian cancer families, screening of this rearrangement in other populations will allow evaluation of whether or not it is a population-specific founder mutation and a more accurate estimation of its distribution and age.

Highly sensitive detection of the MGB1 transcript (mammaglobin) in the peripheral blood of breast cancer patients.

We describe a new one‐step RT‐PCR assay for the detection of the mammaglobin (MGB1) gene transcript in the peripheral blood of breast cancer patients. With this approach, the MGB1 transcript could be detected in the peripheral blood of 22 of 54 (41%) breast cancer patients prior to any therapy. This method, using specific primers for cDNA synthesis, proved to be more sensitive (10−6 to 10−11, usually 10−7) than previously reported methodologies. This increased sensitivity was achieved without compromising specificity, as the MGB1 transcript was not detected in 38 blood samples of healthy donors and in only 1 of 18 blood samples of patients presenting with hematologic malignancies. A positive correlation was seen between MGB1 positivity and breast cancer stage: 0/3 (0%) in stage 0, 3/13 (23%) in stage I, 6/17 (35%) in stage II, 5/10 (50%) in stage III, 8/11 (73%) in stage IV (p = 0.003). The prognostic and therapeutic implications of MGB1 positivity by one‐step RT‐PCR in the peripheral blood of breast cancer patients, especially in clinically localized disease (stages I and II), should be evaluated after long‐term clinical follow‐up of these patients.

BRCA1 and BRCA2 germline mutational spectrum and evidence for genetic anticipation in Portuguese breast/ovarian cancer families

We present the first characterisation of the mutational spectrum of the entire coding sequences and exon–intron boundaries of the BRCA1 and BRCA2 genes as well as large BRCA1 rearrangements in Portuguese families with inherited predisposition to breast/ovarian cancer. Of the 100 probands studied, pathogenic mutations were identified in 22 (24.7%) of 89 breast and/or ovarian cancer families with more than one affected member (15 in BRCA1 and seven in BRCA2), but in none of the 11 patients without family history of cancer. One (6.7%) of the BRCA1 mutations is a large deletion involving exons 11–15. Seven pathogenic point mutations are novel: 2088C>T, 2156delinsCC, and 4255_4256delCT in BRCA1 and 4608_4609delTT, 5036delA, 5583_5584insT, and 8923C>T in BRCA2. The novel 2156delinsCC was identified in three probands from different families and probably represents a founder mutation in our population. We also found a previously reported 3450_3453del4 mutation in three unrelated patients. In addition to the 22 pathogenic mutations, we identified 19 missense mutations of uncertain pathogenic significance, three of them (5241G>C in BRCA1 and IVS6+13C>T and 3731T>C in BRCA2) previously undescribed. The percentage of cases with truncating mutations in BRCA1 and BRCA2 was higher in breast/ovarian cancer (37.0%, mostly BRCA1) and male breast cancer (40%, all BRCA2) families than in families with only female breast cancer (17.5%). Interestingly, we found evidence for genetic anticipation regarding age at diagnosis of both breast and ovarian cancer in those families presenting affected members in more than one generation. These findings should be taken into consideration while planning screening and prophylactic measures in families with inherited predisposition to breast and ovarian cancer.

No significant role for beta tubulin mutations and mismatch repair defects in ovarian cancer resistance to paclitaxel/cisplatin

BackgroundThe mechanisms of chemoresistance in ovarian cancer patients remain largely to be elucidated. Paclitaxel/cisplatin combination is the standard chemotherapeutic treatment for this disease, although some patients do not respond to therapy. Our goals were to investigate whether TUBB mutations and mismatch repair defects underlie paclitaxel and cisplatin resistance.MethodsThirty-four patients with primary ovarian carcinomas (26 serous and eight clear cell carcinomas) treated with paclitaxel/cisplatin were analysed. TUBB exon 4 was analysed by nested PCR after a first round PCR using intronic primers. Microsatellite analysis was performed with the quasimonomorphic markers BAT 26 and BAT 34.ResultsTwenty-two of the 34 ovarian cancers (64.7%) presented residual tumour after surgery, seven of which (7/22; 31.8%) were shown to be chemoresistant (five serous and two clear cell tumours). Sequence analysis did not find any mutation in TUBB exon 4. Microsatellite instability was not detected in any of the ovarian carcinomas.ConclusionWe conclude that TUBB exon 4 mutations and mismatch repair defects do not play a significant role in paclitaxel/cisplatin resistance.

Loss of β-catenin is associated with poor survival in ovarian carcinomas

The catenins (α-, β- and γ-) are cytoplasmic proteins that bind to the conserved tail of the epithelial cadherin molecule. The function of epithelial cadherin at the adherens junctions is dependent on the catenins for efficient cell-to-cell adhesion. Loss of catenin expression has been reported in several human cancers and associated with poor tumor differentiation, advanced tumor stage, and poor patient survival. In this study, we investigated the clinical relevance of α-, β-, and γ-catenin immunoexpression in 104 cases of primary ovarian carcinoma with respect to clinicopathological features and as predictors of disease recurrence and prognosis. The clinicopathological parameters studied were International Federation of Gynaecology and Obstetrics (FIGO) stage, histological type, tumor differentiation, peritoneal metastases, residual postoperative tumor, integrity of the tumor’s serosal surface, peritoneal cytology, and lymphatic/vascular invasion. Negative immunoreactivity of α-catenin, β-catenin, and γ-catenin was observed in 22 (21%), 15 (14%) and 23 (22%) cases, respectively. Immunoreactivity of α-catenin and γ-catenin did not correlate with any of the clinicopathological parameters tested. The immunoexpression pattern of β-catenin correlated with histological type (p = 0.026) and with a poorer overall survival in univariate analyses (p = 0.022). In the group of serous carcinomas, β-catenin-immunoexpression associated significantly with overall survival. Patients with β-catenin-negative serous carcinomas had a poorer overall survival than patients with β-catenin-positive serous carcinomas (p = 0.013). In the multivariate analysis, negative expression of β-catenin (p = 0.003) and the presence of residual tumor (p = 0.019) were the two most important independent prognostic factors predicting poorer overall survival. In conclusion, negative immunoreactivity of β-catenin in serous carcinomas and the presence of residual tumor seem to be useful markers in selecting patients likely to have an unfavorable course.

Ovarian cancer and genetic susceptibility: association of A61G polymorphism in the EGF gene.

Growth factors play an essential role in regulating cellular proliferation, and lack of control is characteristic of malignant development. The epidermal growth factor (EGF) gene codifies a growth factor that binds to the EGF receptor (EGFR), which is involved in activating pathways that promote cellular proliferation, survival, migration and differentiation. The purpose of this study was to appraise the association between EGF gene A61G polymorphism with ovarian cancer susceptibility. A total of 564 DNA samples were analysed from 175 women with ovarian cancer and 389 women without cancer, through PCR-RFLP. We found a decreased risk for developing ovarian cancer in GG carriers compared to AA carriers (OR = 0.46, CI = 0.25–0.83, P = 0.010). The seemingly protective role in GG carriers was observed in women under 53 years of age (OR = 0.38, CI = 0.16–0.86, P = 0.011) and in patients diagnosed with advanced stage disease (OR = 0.38, CI = 0.18–0.81, P = 0.012). Allelic comparison evidenced similar results, with decreased risk for G allele. We further observed a linear trend for G allele in cancer risk. Moreover, we analysed the influence of genotypes in the time to onset of the disease and observed that GG carriers had ovarian cancer later than AA carriers (P = 0.035). We hypothesize that this polymorphism confers protection for ovarian cancer development.

Frequent copy number gains at 1q21 and 1q32 are associated with overexpression of the ETS transcription factors ETV3 and ELF3 in breast cancer irrespective of molecular subtypes

Several ETS transcription factors are involved in the pathogenesis of human cancers by different mechanisms. As gene copy number gain/amplification is an alternative mechanism of oncogenic activation and 1q gain is the most common copy number change in breast carcinoma, we investigated how that genomic change impacts in the expression of the three 1q ETS family members ETV3, ELK4, and ELF3. We have first evaluated 141 breast carcinomas for genome-wide copy number changes by chromosomal CGH and showed that 1q21 and 1q32 were the two chromosome bands with most frequent genomic copy number gains. Second, we confirmed by FISH with locus-specific BAC clones that cases showing 1q gain/amplification by CGH showed copy number increase of the ETS genes ETV3 (located in 1q21~23), ELF3, and ELK4 (both in 1q32). Third, gene expression levels of the three 1q ETS genes, as well as their potential targets MYC and CRISP3, were evaluated by quantitative real-time PCR. We here show for the first time that the most common genomic copy number gains in breast cancer, 1q21 and 1q32, are associated with overexpression of the ETS transcription factors ETV3 and ELF3 (but not ELK4) at these loci irrespective of molecular subtypes. Among the three 1q ETS genes, ELF3 has a relevant role in breast carcinogenesis and is also the most likely target of the 1q copy number increase. The basal-like molecular subtype presented the worst prognosis regarding disease-specific survival, but no additional prognostic value was found for 1q copy number status or ELF3 expression. In addition, we show that there is a correlation between the expression of the oncogene MYC, irrespectively of copy number gain at its loci in 8q24, and the expression of both the transcriptional repressor ETV3 and the androgen respondent ELK4.