Early and late skin reactions to radiotherapy for breast cancer and their correlation with radiation-induced DNA damage in lymphocytes

INTRODUCTION Radiotherapy outcomes might be further improved by a greater understanding of the individual variations in normal tissue reactions that determine tolerance. Most published studies on radiation toxicity have been performed retrospectively. Our prospective study was launched in 1996 to measure the in vitro radiosensitivity of peripheral blood lymphocytes before treatment with radical radiotherapy in patients with breast cancer, and to assess the early and the late radiation skin side effects in the same group of patients. We prospectively recruited consecutive breast cancer patients receiving radiation therapy after breast surgery. To evaluate whether early and late side effects of radiotherapy can be predicted by the assay, a study was conducted of the association between the results of in vitro radiosensitivity tests and acute and late adverse radiation effects. METHODS Intrinsic molecular radiosensitivity was measured by using an initial radiation-induced DNA damage assay on lymphocytes obtained from breast cancer patients before radiotherapy. Acute reactions were assessed in 108 of these patients on the last treatment day. Late morbidity was assessed after 7 years of follow-up in some of these patients. The Radiation Therapy Oncology Group (RTOG) morbidity score system was used for both assessments. RESULTS Radiosensitivity values obtained using the in vitro test showed no relation with the acute or late adverse skin reactions observed. There was no evidence of a relation between acute and late normal tissue reactions assessed in the same patients. A positive relation was found between the treatment volume and both early and late side effects. CONCLUSION After radiation treatment, a number of cells containing major changes can have a long survival and disappear very slowly, becoming a chronic focus of immunological system stimulation. This stimulation can produce, in a stochastic manner, late radiation-related adverse effects of varying severity. Further research is warranted to identify the major determinants of normal tissue radiation response to make it possible to individualize treatments and improve the outcome of radiotherapy in cancer patients.

Optimized molecular resolution of cross-contamination alerts in clinical mycobacteriology laboratories

BACKGROUND. The phenomenon of misdiagnosing tuberculosis (TB) by laboratory cross-contamination when culturing Mycobacterium tuberculosis (MTB) has been widely reported and it has an obvious clinical, therapeutic and social impact. The final confirmation of a cross-contamination event requires the molecular identification of the same MTB strain cultured from both the potential source of the contamination and from the false-positive candidate. The molecular tool usually applied in this context is IS6110-RFLP which takes a long time to provide an answer, usually longer than is acceptable for microbiologists and clinicians to make decisions. Our purpose in this study is to evaluate a novel PCR-based method, MIRU-VNTR as an alternative to assure a rapid and optimized analysis of cross-contamination alerts. RESULTS. MIRU-VNTR was prospectively compared with IS6110-RFLP for clarifying 19 alerts of false positivity from other laboratories. MIRU-VNTR highly correlated with IS6110-RFLP, reduced the response time by 27 days and clarified six alerts unresolved by RFLP. Additionally, MIRU-VNTR revealed complex situations such as contamination events involving polyclonal isolates and a false-positive case due to the simultaneous cross-contamination from two independent sources. CONCLUSION. Unlike standard RFLP-based genotyping, MIRU-VNTR i) could help reduce the impact of a false positive diagnosis of TB, ii) increased the number of events that could be solved and iii) revealed the complexity of some cross-contamination events that could not be dissected by IS6110-RFLP.

Laboratory detection of respiratory viruses by automated techniques.

Advances in clinical virology for detecting respiratory viruses have been focused on nucleic acids amplification techniques, which have converted in the reference method for the diagnosis of acute respiratory infections of viral aetiology. Improvements of current commercial molecular assays to reduce hands-on-time rely on two strategies, a stepwise automation (semi-automation) and the complete automation of the whole procedure. Contributions to the former strategy have been the use of automated nucleic acids extractors, multiplex PCR, real-time PCR and/or DNA arrays for detection of amplicons. Commercial fully-automated molecular systems are now available for the detection of respiratory viruses. Some of them could convert in point-of-care methods substituting antigen tests for detection of respiratory syncytial virus and influenza A and B viruses. This article describes laboratory methods for detection of respiratory viruses. A cost-effective and rational diagnostic algorithm is proposed, considering technical aspects of the available assays, infrastructure possibilities of each laboratory and clinic-epidemiologic factors of the infection.

Human Papillomavirus (HPV) Type Distribution in Females with Abnormal Cervical Cytology. A Correlation with Histological Study

The aim of this study was to determine human papillomavirus (HPV) types distribution in cervical preneoplasic lesions in a Southern Spanish population and their relationship between HPV type and grade of histopathological abnormality. Finally, 232 cervical samples from 135 women with previous cytological abnormalities were included in this study. Colposcopy studies and biopsies were performed. Haematoxylin-eosin stained slides were observed and detection of HPV DNA in cervical swabs was carried out with use of a polymerase chain reaction and microarrays technology. The relationship between the presence of HPV infection and diagnostic variables was evaluated. HPV 16 was the most common type followed by HPV 58, 51, 33 and 31. However, the two HPV types targeted in the prophylactic vaccines such as HPV type 16 and 18 were detected in only 37 (21.2%) and 2 (1.1%) cases respectively. Thirty-three (18.9%) of samples were infected with multiple types, the majority of them with two types. In addition, during the follow-up of patients many changes in type distribution were observed. Several studies will be necessary in order to evaluate the HPV type distribution for therapeutically and prophylactic purposes such as vaccine treatment. Also, because of the differences obtained depending of use of various DNA technologies, the performance of some comparative studies of the different methods from detection of HPV would be advisable in a high population of patients and with the most homogeneous conditions possible.

Introduction of an automated system for the diagnosis and quantification of hepatitis B and hepatitis C viruses.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections pose major public health problems because of their prevalence worldwide. Consequently, screening for these infections is an important part of routine laboratory activity. Serological and molecular markers are key elements in diagnosis, prognosis and treatment monitoring for HBV and HCV infections. Today, automated chemiluminescence immunoassay (CLIA) analyzers are widely used for virological diagnosis, particularly in high-volume clinical laboratories. Molecular biology techniques are routinely used to detect and quantify viral genomes as well as to analyze their sequence; in order to determine their genotype and detect resistance to antiviral drugs. Real-time PCR, which provides high sensitivity and a broad dynamic range, has gradually replaced other signal and target amplification technologies for the quantification and detection of nucleic acid. The next-generation DNA sequencing techniques are still restricted to research laboratories.The serological and molecular marker methods available for HBV and HCV are discussed in this article, along with their utility and limitations for use in Chronic Hepatitis B (CHB) diagnosis and monitoring.

Evaluation of three automated systems for susceptibility testing of enterobacteria containing qnrB, qnrS, and/or aac(6')-Ib-cr.

The accuracy of the MicroScan WalkAway, BD Phoenix, and Vitek-2 systems for susceptibility testing of quinolones and aminoglycosides against 68 enterobacteria containing qnrB, qnrS, and/or aac(6 ')-Ib-cr was evaluated using reference microdilution. Overall, one very major error (0.09%), 6 major errors (0.52%), and 45 minor errors (3.89%) were noted.

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients.

BACKGROUND Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5' CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. METHODS Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). RESULTS Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. CONCLUSION Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient's resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.

Lack of correlation between the optimal glycaemic control and coronary micro vascular dysfunction in patients with diabetes mellitus: a cross sectional study.

Background Coronary microvascular dysfunction (CMD) is associated with cardiovascular events in type 2 diabetes mellitus (T2DM). Optimal glycaemic control does not always preclude future events. We sought to assess the effect of the current target of HBA1c level on the coronary microcirculatory function and identify predictive factors for CMD in T2DM patients. Methods We studied 100 patients with T2DM and 214 patients without T2DM. All of them with a history of chest pain, non-obstructive angiograms and a direct assessment of coronary blood flow increase in response to adenosine and acetylcholine coronary infusion, for evaluation of endothelial independent and dependent CMD. Patients with T2DM were categorized as having optimal (HbA1c < 7 %) vs. suboptimal (HbA1c ≥ 7 %) glycaemic control at the time of catheterization. Results Baseline characteristics and coronary endothelial function parameters differed significantly between T2DM patients and control group. The prevalence of endothelial independent CMD (29.8 vs. 39.6 %, p = 0.40) and dependent CMD (61.7 vs. 62.2 %, p = 1.00) were similar in patients with optimal vs. suboptimal glycaemic control. Age (OR 1.10; CI 95 % 1.04–1.18; p < 0.001) and female gender (OR 3.87; CI 95 % 1.45–11.4; p < 0.01) were significantly associated with endothelial independent CMD whereas glomerular filtrate (OR 0.97; CI 95 % 0.95–0.99; p < 0.05) was significantly associated with endothelial dependent CMD. The optimal glycaemic control was not associated with endothelial independent (OR 0.60, CI 95 % 0.23–1.46; p 0.26) or dependent CMD (OR 0.99, CI 95 % 0.43–2.24; p = 0.98). Conclusions The current target of HBA1c level does not predict a better coronary microcirculatory function in T2DM patients. The appropriate strategy for prevention of CMD in T2DM patients remains to be addressed. Keywords: Endothelial dysfunction; Diabetes mellitus; Coronary microcirculation