Analysis of radiosensitivity in South African cervical and breast cancer patients

Introduction: Ionising radiation can cause DNA double strand breaks (DSB), that result in chromosomal aberrations if un- or mis-repaired. Individuals with compromised DNA damage repair mechanisms display increased chromosomal radiosensitivity. The G0-micronucleus assay (MN assay) and the γ-H2AX assay are two assays used in radiobiology to study DNA DSB and repair. Breast cancer is the leading cancer amongst South African women, with a lifetime risk of 1 in 34. Since most cancer patients in South Africa present with late-stage disease, chemotherapy and radiotherapy are commonly-used treatments. Several international studies have shown breast cancer patients to be more chromosomally radiosensitive than healthy controls. These studies have not been confirmed on a cancer population living in South Africa. Cervical cancer is the second most common cancer in South Africa; however, it is the leading cancer amongst black women with a lifetime risk of 1/35 compared to 1/82 in white women. Studies show a genetic link to cervical cancer susceptibility and DNA damage repair genes. International studies on radiation-induced DNA damage in lymphocytes of cervical cancer patients remain inconclusive and have never been performed on a South African population. Cervical cancer is caused by infection with the Human Papilloma Virus (HPV). Human Immunodeficiency Virus (HIV), HPV and cervical cancer are epidemiologically linked. Due to the high rate of HIV in South Africa, a significant proportion of cervical cancer patients receiving radiotherapy treatment will be HIV-positive. Studies show an effect of HIV on chromosomal radiosensitivity, however this has not been confirmed on a cancer population. The MN assay on the biopsies and exfoliated cervical cells of cervical cancer patients could be used as a predictive test for response to radiotherapy. The overall aim was to study chromosomal radiosensitivity in South African cervical and breast cancer patients. Materials and methods: Chromosomal radiosensitivity of lymphocytes of cervical and breast cancer patients was examined using the MN assay with the Metafer 4 of Metasystems. Different scoring methods for the Metafer system were compared to each other. The effect of HIV, HPV, ethnicity, clinical parameters and age on micronuclei (MN) values in lymphocytes was investigated. The MN assay was attempted on cells from cervical biopsies and exfoliated cervical cells. The γ-H2AX was performed on the lymphocytes of a group of cervical cancer patients. Results: A new scoring method for the Metafer 4 system that is more reliable in patients with late-stage disease was introduced. Cervical cancer patients had significantly higher MN values with HIV patients having the highest values. HPV, clinical parameters and age had a limited effect on MN values. The MN assay was unsuccessful on biopsies and exfoliated cervical cells of cervical cancer patients. There was no difference in double strand break induction and repair between cervical cancer patients and controls. In breast cancer patients, ethnicity had an effect on MN values, with only white breast cancer patients having significantly higher MN counts. Conclusion: The study showed increased chromosomal radiosensitivity in cervical cancer and white breast cancer patients. Results highlight how such studies are important within the South African context, where factors like HIV, disease stage and ethnicity can have an effect on chromosomal radiosensitivity and where unique genes/polymorphisms may play a role in cancer risk

The effect of HIV and antiretroviral therapy on chromosomal radiosensitivity

Introduction: Antiretroviral Treatment (ART) has led to an improvement in survival of HIV infected individuals. Some of them will develop cancer during the course of their infection and will require radiation therapy. HIV positive cancer patients have presented with adverse side effects of radiotherapy and elevated chromosomal radiosensitivity. This study investigated if ART has an influence on chromosomal radiosensitivity of HIV positive individuals. Methods and Materials: Blood samples from 60 HIV positive individuals were in vitro exposed to doses of X-rays of 0, 2 and 4Gy and chromosomal radiosensitivity was assessed with the micronucleus assay. The micronucleus assay was also performed on lymphocytes of a group of non HIV-infected health care workers taking prophylactic post-exposure ART to measure the effect of these ART drugs on chromosomal radiosensitivity without HIV as a confounding factor. Results: All HIV patients (those on ART and without ART) had significantly higher radiation induced Micronuclei (MN) than healthy controls. The MN yields increased in the HIV patients taking ART compared to HIV patients not on treatment. The evaluation of chromosomal radiosensitivity of health care workers on ART revealed no effects of ART. Conclusions: HIV positive individuals show an increased chromosomal radiosensitivity. Antiretroviral treatment given to HIV positive individuals can lead to enhanced chromosomal radiosensitivity and therefore impose higher risks for radiotherapy side effects in these patients

Molecular characterisation of the Her2-Top2A amplicon in breast cancer

MSc (Med), Faculty of Health Sciences, University of the WitwatersrandThe HER2 gene is amplified in 20-30% of breast cancers, a common cancer amongst South African women. HER2 amplification is associated with a poor prognosis and predicts response to treatments such as Herceptin. The gold standard for HER2 testing is Fluorescent in situ Hybridisation (FISH) with dual colour probes for the HER2 gene and chromosome 17 centromere (CEP17) internal control. According to international guidelines, a HER2/CEP17 ratio >2.2 is considered positive. The HER2 FISH test is complicated by the emergence of ambiguous cases with increased CEP17 signals that cannot be accounted for by chromosome 17 polysomy (> 6 copies of CEP17) and that may hide true HER2 gene amplification. The aims of this study were to characterise the HER2 amplicon, in particular the copy number of genes in the vicinity of the HER2 gene, and to design an alternative control probe that could clarify the HER2 gene status in ambiguous cases. In addition, results on 1558 breast cancer specimens sent for routine testing were analysed to determine the trends of HER2 amplification amongst South African women. The rate of HER2 gene amplification was significantly higher (p < 0.05) in African patients (52%) than in Caucasian patients (43%). In Caucasian women, the rate of HER2 amplification in the younger group (68%) was significantly higher (p < 0.05) than in the general Caucasian group (43%), while the same was not seen in the African cohort. Nineteen ambiguous cases with more than 9 copies of CEP17 were further investigated. FISH assays with four different probe kits (PathVysion HER-2: Poseidon Repeat free TOP2A, HER2, CEP17: and Vysis PML-RARA respectively) were performed to determine the copy number of the HER2, TOP2A, RARA genes and CEP17. An in-house dual colour probe kit was designed using the ACTG1 gene as a control for HER2. Of the 19 ambiguous cases, 16 had centromeric amplification, showing that CEP17 is no longer an adequate internal control in FISH HER2 testing. The TOP2A gene was only amplified in HER2 positive cases and the RARA gene was only amplified when the TOP2A gene was also amplified. FISH with ACTG1 as v a control clearly revealed HER2 amplification in ambiguous cases on image analysis and gave HER2/ACTG1 ratios significantly higher than HER2/CEP17 ratios. However, screening of an additional 40 unambiguous cases showed an increased copy number, although limited ( 8), of the ACTG1 gene in four patients; this warrants further testing to assess the value of this gene as a control. Interestingly, a trend was observed for ACTG1 increased copy number in HER2 negative cases, this may point to the presence of a driver gene whose amplification tends to be mutually exclusive from HER2 amplification

Chromosomal radiosensitivity of human immunodeficiency virus positive/negative cervical cancer patients in South Africa

Cervical cancer is the second most common cancer amongst South African women and is the leading cause of cancer-associated mortality in this region. Several international studies on radiation-induced DNA damage in lymphocytes of cervical cancer patients have remained inconclusive. Despite the high incidence of cervical cancer in South Africa, and the extensive use of radiotherapy to treat it, the chromosomal radiosensitivity of South African cervical cancer patients has not been studied to date. Since a high number of these patients are human immunodeficiency virus (HIV)-positive, the effect of HIV infection on chromosomal radiosensitivity was also investigated. Blood samples from 35 cervical cancer patients (20 HIV-negative and 15 HIV-positive) and 20 healthy controls were exposed to X-rays at doses of 6 MV of 2 and 4 Gy in vitro. Chromosomal radiosensitivity was assessed using the micronucleus (MN) assay. MN scores were obtained using the Metafer 4 platform, an automated microscopic system. Three scoring methods of the MNScore module of Metafer were applied and compared. Cervical cancer patients had higher MN values than healthy controls, with HIV-positive patients having the highest MN values. Differences between groups were significant when using a scoring method that corrects for false positive and false negative MN. The present study suggested increased chromosomal radiosensitivity in HIV-positive South African cervical cancer patients

Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Chromosomal radiosensitivity of in vitro HIV infected cell lines

Purpose of the study: Compare the chromosomal radiosensitivity of HIV negative cell lines before and after in vitro infection with HIV. For this, chromosomal radiosensitivity is measured using the micronucleus (MN) assay in combination with a pan-centromeric probe. Brief description: As the prevalence of HIV infection is high in South Africa, many radiation workers and radiation therapy patients are HIV infected. Clinical observations show that the HIV positive cancer patients present with more adverse side effects to radiotherapy than HIV negative patients. Henceforth, HIV positive individuals also have shown a higher radiosensitivity on a chromosomal level than HIV negative individuals. An in vitro model to understand this radiosensitivity induced by HIV is developed. Different types of cells, derived from healthy HIV negative donors were infected with HIV in the laboratory. A non-infected culture of each cell line was used as control. Cells were exposed in vitro to 2Gy and 4Gy of 6 MV X-rays. After irradiations Cytochalasin B was added to inhibit cytokinesis. After 2 days the yield of binucleated cells and micronuclei were counted using the automated microscopy system of MetaSystems. Non-irradiated control samples from each donor were also analysed. A pan-centromeric FISH analysis was performed on non-irradiated samples to detect chromosomal instability induced by the HIV virus. Summary of the new, unpublished data: HIV infected cells showed higher MN values compared to their HIV negative counterparts. A high level of genomic instability was also observed in HIV infected cells. Statement of the conclusions: The HIV virus induces more chromosomal radiosensitivity in HIV infected cells compared to HIV negative cell lines. This change in sensitivity to ionizing radiation has significant consequences for both radiation workers and radiation therapy patient