A Novel Panel of 80 RNA Biomarkers with Differential Expression in Multiple Human Solid Tumors against Healthy Blood Samples.

The aim of this study was to identify genes with higher expression in solid tumor cells by comparing human tumor biopsies with healthy blood samples using both in silico statistical analysis and experimental validations. This approach resulted in a novel panel of 80 RNA biomarkers with high discrimination power to detect circulating tumor cells in blood samples. To identify the 80 RNA biomarkers, Affymetrix HG-U133 plus 2.0 microarrays datasets were used to compare breast tumor tissue biopsies and breast cancer cell lines with blood samples from patients with conditions other than cancer. A total of 859 samples were analyzed at the discovery stage, consisting of 417 mammary tumors, 41 breast lines, and 401 control samples. To confirm this discovery, external datasets of eight types of tumors were used, and experimental validation studies (NanoString n-counter gene expression assay) were performed, totaling 5028 samples analyzed. In these analyses, the 80 biomarkers showed higher expression in all solid tumors analyzed relative to healthy blood samples. Experimental validation studies using NanoString assay confirmed the results were not dependent of the gene expression platform. A panel of 80 RNA biomarkers was described here, with the potential to detect solid tumor cells present in the blood of multiple tumor types.

Mutation profiling in the PIK3CA, TP53, and CDKN2A genes in circulating free DNA and impalpable breast lesions.

Breast impalpable lesions have become a clinical dilemma because they are small, presenting a heterogeneous cellular phenotype. The aim of this study was to evaluate the mutational profile of the PIK3CA, TP53, and CDKN2A genes, comparing the mammary tissue with the respective circulating free DNA (cfDNA). The PIK3CA, TP53, and CDKN2A genes were sequenced (PCR-Sanger) in 58 women with impalpable lesions (49 malignant and 9 benign) with the respective cfDNA. The chi-square or Fisher's exact test was used to evaluate statistical significance between the clinical variables and mutational profile. A total of 51 out of 58 samples generated successful mutation profiles in both breast lesion and cfDNA. Of the 37 mutations detected, 10 (27%) and 16 (43%) mutations were detected in benign and malignant breast lesions, respectively, while 2 (5%) and 9 (24%) were found in cfDNA of women with benign and malignant lesions, respectively. The lymph node involvement with mutations in the PIK3CA in malignant lesions (P = 0.001), and the relationship between mutations in PIK3CA, comparing ductal tumors with benign lesions (P = 0.05), were statistically significant. This study detected different mutations in PIK3CA, TP53, and CDKN2A genes, which represent, in part, the heterogeneity of impalpable lesions. The results confirm that more studies should be conducted on the functional role of cfDNA in the impalpable lesions.

Low Levels of Vitamin D in a Cohort of Women with Impalpable Breast Lesions from Rio de Janeiro/Brazil

Background: Low levels of vitamin D have been described as a risk factor for the development of breast cancer. The aim of this study was to evaluate the serum levels of vitamin D (25OHD) in patients with impalpable breast lesions comparing with a control group. Methods: Vitamin D quantification (25OHD) was assessed in the plasma of 65 patients with impalpable breast lesions and from 20 health controls using a chemiluminescent microparticle immunoassay. Pearson's chi-square test and nonparametric t-Student were used to evaluate statistical significance between the clinical variables and the means of quantification of vitamin D. The receiver operating characteristic (ROC) curve was used to evaluate the correlation between age and vitamin sufficiency for the cases and the controls. Results: The prevalence of vitamin D deficiency and/or insufficiency in women with malignant lesions was 84% and 60% for the control group. Using the chi-square or Fisher's exact test, the relationship between vitamin D levels and age presented significant association only for the control group (P=0.002). Using ROC curve, the plot area (0.778) for the control group defined a cut-off value of 45 years to age, with specificity and sensitivity of 60% and 50%, respectively. Thus, the odds ratio for vitamin D insufficiency in women over 45 years was 1.37 (P=0.011). For the case group, clinical characteristics, histological grade, and lymph node involvement did not show any significant association. Conclusion: The prevalence of vitamin D deficiency/insufficiency is high in women with impalpable breast lesions, as well as in the control group, even in a tropical city. According to the results the age advancement may be involved with the decrease in vitamin D levels in plasma, but there was no statistical association between low levels of Vitamin D and breast cancer.

Clotrimazole disrupts glycolysis in human breast cancer without affecting non-tumoral tissues.

Human breast cancer tissues, as well as normal tissues from the same patients, were treated with clotrimazole (CTZ) and have their capacities for glucose consumption and lactate production evaluated. This treatment strongly decreased the lactate production rate by tumor tissues (85% inhibition) without affecting the other measurements made, i.e. lactate production by control tissues or glucose consumption by both, control and tumor tissues. This result directly correlates with the inhibition promoted by CTZ on the activity of the major regulatory glycolytic enzyme 6-phosphofructo-1-kinase (PFK) that was observed in tumor tissues (84% inhibition) but not in control tissues. Fractionation of the tissues revealed that this inhibition does not occur in the soluble fraction of the enzyme, but is exclusive of a particulate fraction. It has been previously shown that the particulate fraction of PFK activity in tumors is associated to actin filaments (f-actin). Thus, we investigated whether CTZ would affect the association between PFK and f-actin and we found that the drug directly induces the dissociation of the two proteins in the same extent that it inhibits lactate production, total PFK activity and the particulate PFK activity. We concluded that CTZ disrupts glycolysis on human breast tumor tissues, inhibiting PFK activity by dissociating the enzyme from f-actin.

Quimioterapia primária em câncer de mama localmente avançado: estudo comparativo emtre dois esquemas terapêupticos com intensificação de doses

Realizou-se estudo clínico prospectivo, fase lII, multicêntrico, aberto, aleatório e comparativo. Foram avaliadas 60 pacientes portadoras de câncer de mama localmente avançado, estádio IIIA, divididas em dois grupos, que foram submetidas à quimioterapia primária, com ou sem intensificação de dose, por quatro ciclos, e à cirurgia. Utilizou-se protocolo FEC 50 no grupo A (5-FU 500 mg/m2, epirubicina 50 mg/m2 e ciclofosfamida 500 mg/m2) e FEC 100 no grupo B (5-FU 500 mg/m2, epirubicina 100 mg/m2 e ciclofosfamida 500 mg/m2). Foram analisados, durante a quimioterapia, o estado geral, variação ponderal, alopécia, alterações digestivas, hematológicas e cardiotoxicidade. Após a quimioterapia avaliou-se a resposta tumoral clínica e, na peça cirúrgica, a resposta anatomopatológica. O estado geral alterou-se em 22 pacientes (36%), sendo que 5 (8%) no grupo A e 17 (28%) no grupo B. A maioria, 38 pacientes (64%), manteve seu estado geral inicial inalterado . Verificou-se um maior ganho ponderal no grupo B, porém dentro de valores médios. Evidenciou-se uma diminuição progressiva dos valores hematológicos médios. No grupo A não houve necessidade de redução ou adiamento das doses. No grupo B a mielossupressão foi mais intensa e determinou redução das doses em 34 ciclos (28%) de 16 pacientes, porém não levou a situações clínicas de risco de vida. Náuseas e vômitos ocorreram em 68,7% dos ciclos. No grupo B houve um maior percentual de formas severas. A diarréia foi manifestação digestiva pouco freqüente, comprometendo apenas 6 ciclos e sempre de forma leve. Houve redução nos níveis da fração de ejeção ventricular esquerda (FEVE) em 90% dos casos, sendo 80% no grupo A e 100% no grupo B. Em apenas 8 casos (13%) a FEVE esteve abaixo do normal, sendo 2 (6%) no grupo A e 6 (20%) no grupo B. Nas outras pacientes as reduções da FEVE foram menores que 20%. Houve alterações inespecíficas no eletrocardiograma (ECO). As mais freqüentes foram alterações da repolarização ventricular, taquicardia e extra-sístoles. As alterações da FEVE e ECO foram mais freqüentes e intensas no grupo B, porém não tiveram manifestações clínicas. A resposta clínica objetiva (resposta completa e resposta parcial) nos grupos A e B foi 930/0 e 96%, respectivamente. Os resultados foram similares, porém no grupo B houve um percentual maior de respostas clínicas completas. Avaliou-se a doença residual no sítio primário e nos linfonodos axilares. No grupo A houve 4 (13%) casos de resposta anatomopatológica completa, 12 (40%) de tumor residual microscópico e 14 (47%) de tumor macroscópico. No grupo B ocorreram 10 (33%) casos de resposta anatomopatológica completa, 7 (23%) de tumor residual microscópico e 13 (440/0) de tumor macroscópico. Concluiu-se que a quimioterapia primária proporcionou resposta clínica e anatomopatológica maior no grupo com intensificação de dose. A toxicidade, tolerável e reversível, foi mais acentuada no grupo de altas doses.

A prospective clinical trial, phase III, multicenter, open, comparative and randomized, was performed. Sixty patients with locally advanced breast cancer, stage IlIA, were divided in two groups and submitted to primary chemotherapy, with and without intensification of doses, for 4 cycles and surgery. It has been used regimen FEC 50 in ann A (5-FU 500 mg/m2, epirubicin 50 mg/m2 and cyclophosphamide 500 mg/m2) and FEC 100 in arm B (5-FU 500 mg/m2, epirubicin 100 mg/m2 and cyclophosphamide 500 mg/m2). It has been analised cyclically: the performance status, weight change, hair loss, digestive and hematologic disorders and cardiotoxicity. After the primary chemotherapy, the clinical objective response rate and pathological response in the surgical specimens, were evaluated. The performance status has changed in 22 (360/0) patients, 5 (8%) in arm A and 17 (28%) in ann B. Most of the patients, 38 (64%) preserved the initial performance status. There was an increased in the weight gain in ann B, but at normal values. There was a progressive decrease in the mean haematological values. In arm A there was no need of reduction or postponement of the doses. In arm B the myelosupression was intense and determined reduction in 34 cycles (280/0) of 16 patients, but there was no clinical situation of risks. There were nausea and vomiting in 68,7% of the cycles. In ann B there was a high percentual of severe forms. Diarrhoea was less frequent, only in six cycles. The left ventricular ejection fraction (L VEF) has decreased in 90% of the cases, 800/0 in arm A and 100% in arm B. Only 8 cases (13%) were below the normal range, 2 (60/0) in ann A and 6 (20%) in arm B. There were inespecific changes in the eletrocardiograms of 22 (360/0) patients. There was no clinical manifestation of cardiac abnormalities. The clinical response (complete + partial response) was 93% in arm A and 96% in arm B. There was a high proportion of complete response in ann B. The residual tumor was analised in the surgical speciments. In arm A there was 4 (13%) complete pathological responses, 12 (40%) microscopic residual tumor and 14 (47%) macroscopic. In arm B there were 10 (33%) complete pathological responses, 7 (23%) microscopic residual tumor and 13 (44%) macroscopic. It has been concluded that the primary chemotherapy with intensification of doses promoted higher clinical objective and pathological response rates. The toxicity, reversible and tolerable, was more intense in the arm of high doses.