Raquel Catarino

Quantification of Free Circulating Tumor DNA as a Diagnostic Marker for Breast Cancer

AIM To determine whether the amounts of circulating DNA could discriminate between breast cancer patients and healthy individuals by using real-time PCR quantification methodology. METHODS Our standard protocol for quantification of cell-free plasma DNA involved 175 consecutive patients with breast cancer and 80 healthy controls. RESULTS We found increased levels of circulating DNA in breast cancer patients compared to control individuals (105.2 vs. 77.06 ng/mL, p < 0.001). We also found statistically significant differences in circulating DNA amounts in patients before and after breast surgery (105.2 vs. 59.0 ng/mL, p = 0.001). Increased plasma cell-free DNA concentration was a strong risk factor for breast cancer, conferring an increased risk for the presence of this disease (OR, 12.32; 95% CI, 2.09-52.28; p < 0.001). CONCLUSIONS Quantification of circulating DNA by real-time PCR may be a good and simple tool for detection of breast cancer with a potential to clinical applicability together with other current methods used for monitoring the disease.

Linkage of TP53 codon 72 pro/pro genotype as predictive factor for nasopharyngeal carcinoma development.

Genetic predisposition has been suggested as a cofactor for cancer aetiology and a polymorphism in TP53 codon 72 has been associated as a susceptibility factor for several cancers. Nasopharyngeal carcinoma is a rare neoplasia in western civilizations and genetic predisposition might play an important role in its development. We evaluated the linkage of the polymorphic variants (Arg/Pro) on TP53 codon 72 with nasopharyngeal cancer development in a case–control study with 392 individuals from a northern Portuguese population, including 107 patients with nasopharyngeal carcinoma and 285 healthy controls. This study revealed a three-fold risk for carriers of Pro/Pro genotype either against carriers of Arg/Arg (OR=2.62; 95% CI=1.10–6.30; P=0.016) or total Arg carriers (OR=2.67; 95% CI=1.21–5.90; P=0.012). Moreover, step-wise logistic regression analysis identified Pro/Pro genotype (OR=3.1; 95% CI=1.3–7.3; P=0.009), age >49 at diagnosis (OR=2.5; 95% CI=1.6–4.0; P<0.001) and male gender (OR=2.7; 95% CI=1.6–4.4; P<0.001) as predictive factors for the development of nasopharyngeal carcinoma. These results confirm the data from Asiatic populations suggesting that Pro/Pro genotype represents a stable risk factor for nasopharyngeal carcinoma development in Portugal and that TP53 codon 72 polymorphism can contribute as a genetic susceptibility marker, providing additional information to improve the knowledge about nasopharyngeal carcinoma aetiology.

Association of the A870G cyclin D1 gene polymorphism with genetic susceptibility to nasopharyngeal carcinoma.

Nasopharyngeal cancer (NPC) is multifactorial, and the genetic background may be a crucial etiologic factor. Cyclin D1 (CCND1) is a key regulator of the cell cycle, and its altered activity is associated with the development of cancer.

FcγRIIa polymorphism and clinical response to rituximab in non-Hodgkin lymphoma patients

Rituximab is a chimeric monoclonal antibody that specifically targets the CD20 surface marker expressed in neoplastic B-lymphoid cells. Combined with chemotherapy or alone, in maintenance/consolidation, it is used for the treatment of non-Hodgkin lymphoma (NHL). The role of a polymorphism in a specific Fc gamma receptor gene, FcgammaRIIa, in the clinical outcome of patients with NHL was investigated in this study. We characterized DNA samples from 64 non-Hodgkin lymphoma patients treated with rituximab using a polymerase chain reaction-restriction fragment length polymorphism method. The FcgammaRIIa HH genotype was significantly correlated with complete response to rituximab compared to the R allele (P=0.028). In terms of overall or event-free survival, no difference was found according to FcgammaRIIa alleles. We hypothesize that the HH genotype increases the affinity of the FcgammaRIIa receptor, not only for naturally occurring IgG2, but also to ameliorate connection with chimeric IgG1 rituximab, contributing to a genetic individual profile of great interest in clinical onco-hematology.

Genetic Risk Markers for Nasopharyngeal Carcinoma in Portugal: Tumor Necrosis Factor Alpha −308G >A Polymorphism

The tumor necrosis factor-alpha (TNF-α) is a strong proinflammatory cytokine produced by the activated macrophages in the immune response to viral infections. A common polymorphism on the promoter region of TNFA gene (-308G >A) has been associated with different susceptibilities to the development of several diseases including viral-associated neoplasias. Data suggest that the A allele has been associated with higher levels of TNF-α and, therefore, leads to increased risk of cancer development. We have performed a case-control study considering the role of the -308G >A polymorphism in 750 individuals from the northern region of Portugal, including 123 patients with nasopharyngeal carcinoma (NPC) and 627 healthy individuals. Our study revealed an increased frequency of the -308A TNFA allele in patients with NPC. The statistical analysis for recessive model revealed that -308AA genotype is associated with increased risk for the development of NPC (odds ratio = 2.46; 95% confidence interval, 0.98-6.17; p = 0.047); moreover, this effect was stronger in undifferentiated types, which are virtually 100% caused by the Epstein-Barr virus (odds ratio = 2.75; 95% confidence interval, 1.09-6.90; p = 0.025). These results reveal that in our population -308 TNFA AA genotype can represent a risk marker for NPC development and contributes for the definition of genetic susceptibility profiles for individuals at risk of development of a viral infection and associated neoplasia.

Circulating DNA: Diagnostic Tool and Predictive Marker for Overall Survival of NSCLC Patients

Purpose The purpose of our study was to determine whether the amounts of circulating DNA (cDNA) could discriminate between NSCLC patients and healthy individuals and assess its value as a prognostic marker of this disease. Methods We conducted a study of 309 individuals and the cDNA levels were assessed through real-time PCR methodology. Results We found increased cDNA levels in NSCLC patients compared to control individuals. We also found a decreased overall survival time in patients presenting high cDNA levels, when compared to lower cDNA concentrations. Conclusions Quantification of cDNA may be a good tool for NSCLC detection with potential for clinical applicability.

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.

Oncogenic virus-associated neoplasia: a role for cyclin D1 genotypes influencing the age of onset of disease?

Cyclin D1 (CCND1) is a key regulatory protein at the G1/S checkpoint of the cell cycle. The purpose of our study was to assess the role of CCND1 genotypes influencing the age of onset of oncogenic virus-associated neoplasia. We conducted a hospital-based case-control study of 581 individuals, including 247 controls and 334 cases (108 nasopharyngeal and 226 cervical cancer cases). The polymorphism analysis was performed in blood samples by PCR-RFLP methodology. Age-adjusted logistic regression analysis indicates that individuals carrying two G-alleles have an increased genetic susceptibility for the development of oncogenic virus-associated cancers (aOR=2.02, 95% CI 1.30-3.14, P=0.002). Moreover, our results indicate that the waiting time for onset of oncogenic virus-associated neoplasia in patients homozygous (GG) for CCND1 genotypes (52 years) was 12 years earlier in comparison with patients carrying AG or AA genotypes (60 years) (log-rank test: P=0.0003). Our results may be important in contributing to a more extensive knowledge of the mechanisms involved in oncogenic virus-associated carcinogenesis, as CCND1 may be an important target for the development of new strategies for cancer treatment and prevention.

CXCL12-3' A polymorphism and lung cancer metastases protection: new perspectives in immunotherapy?

Lung cancer remains a major worldwide health problem, being the most common form of cancer in the world, both in terms of incidence, 12.3% of all cancers (with an estimated 1.2 million new cases in 2000), and mortality (1.1 million deaths in 2000), representing 17.8% of the total number of deaths caused by cancer. More than half of the new cases occur in more developed countries (52%), with incidence rates lower in women (11.1 per 1,000,000), than in men (34.9 per 1,000,000) [17]. There are twomajor groups of lung carcinomas: smallcell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The latter includes epidermoid (squamous cell) carcinoma (which accounts for 25–40% of all lung cancers), adenocarcinoma and large cell carcinoma [5]. During the development of epidermoid carcinoma, oncogenic stimuli leads to a series of conversions of the normal bronchial epithelium, passing from premalignant lesions (hyperplastic, metaplastic, and dysplatic lesions) to carcinoma in situ that progresses to an overt carcinoma [19]. Adenocarcinomas are also thought to develop in part from premalignant precursor lesions such as atypical adenomatous hyperplasia [19]. Non-small cell lung cancer metastases to regional lymph nodes, liver, adrenal glands, contralateral lung, brain, and bone marrow are a key factor in the aggressiveness of this cancer [17] and experimental data have demonstrated that sites of metastases are determined both by the characteristics of neoplastic cells and the microenvironment of the specific organ [4]. Although tobacco smoking is pointed as the most important cause of lung cancers with 80–90% arising in cigarette smoker individuals [12], the biology of lung cancer is complex and poorly understood, and a variety of biomarkers have been implied in the virulence of this type of cancers [23]. Chemokines have recently emerged as an important family of proteins involved in tumourgenesis and organ-specific metastases [1, 13–15, 17, 21]. The chemokine receptor CXCR4 and its sole ligand SDF-1/ CXCL12 (Stromal Cell-Derived Factor-1) play important roles in inflammation and hematopoiesis, by acting as chemoattractant for leukocytes and stem cells [20]. Furthermore, it has been shown that in several types of cancer—including melanoma, ovarian, breast, and lung cancer—CXCL12 can stimulate the proliferation and/or survival of CXCR4-expressing cancer cells when they are grown under sub-optimal conditions, an adaptation that may allow tumour cells to grow in distant sites that would normally be less favourable [1]. The CXCR4-expressing metastatic cells might spread to many sites in the body, but only become established as metastatic locations where high levels of CXCL12 are found [1]. A single nucleotide polymorphism in the 3¢ untranslated region of the CXCL12 gene—CXCL12-3¢A—was reported as delaying AIDS progression to death [22, 24] and Zafiropoulos et al. associated the allelic frequency of the polymorphism with breast cancer and melanoma [25]. The aim of our study was to evaluate the genetic influence of CXCL12-3¢A polymorphism in the susceptibility to lung cancer development.

Prognostic Significance of Telomerase Polymorphism in Non-Small Cell Lung Cancer

Purpose: Lung cancer is the leading cause of death in oncologic patients of western countries, with very low survival rates. Telomerase main components are the catalytic subunit (hTERT) and the RNA template (hTR). A functional polymorphism in the hTERT gene was found in the promoter region (−1327T/C), and individuals homozygous for the −1327C/C genotype present shorter telomere length compared with T-carrier genotypes. Our purpose was to investigate the potential prognostic role of the hTERT functional genetic variant in non–small cell lung cancer (NSCLC) patients. Experimental Design: We prospectively conducted a study involving 226 patients with NSCLC treated with a first-line chemotherapeutic standard protocol. A follow-up study was undertaken (median follow-up time, 26 months) to evaluate treatment response and overall survival of NSCLC patients. The hTERT −1327T/C genetic variants were analyzed by allelic discrimination with real-time PCR. Results: Our results indicate an influence of the telomerase genetic variants in the overall survival of NSCLC patients. Cox regression analysis showed a significantly higher median estimated cumulative survival of 26.5 months in T-carrier patients, compared with that of 19.3 months in CC patients (hazard ratio, 0.52; 95% confidence interval, 0.35-0.77; P = 0.001). Conclusions: Telomerase functional polymorphism in the hTERT gene may contribute as a prognostic factor in NSCLC patients. Our findings indicate that hTERT genetic variants, by modulating telomere length, may confer an advantage in chemotherapy response. The assessment of telomerase genetic variants could supplement prognosis of survival in the course of NSCLC and may be a promising molecular marker of treatment response in these patients. Clin Cancer Res; 16(14); 3706–12. ©2010 AACR.