Edward Levine

Radiation-induced micronucleus induction in lymphocytes identifies a high frequency of radiosensitive cases among breast cancer patients: A test for predisposition?

Enhanced sensitivity to the chromosome-damaging effects of ionizing radiation is a feature of many cancer-predisposing conditions. We previously showed that 42% of an unselected series of breast cancer patients and 9% of healthy control subjects showed elevated chromosomal radiosensitivity of lymphocytes irradiated in the G2 phase of the cell cycle. We suggested that, in addition to the highly penetrant genes BRCA1 and BRCA2, which confer a very high risk of breast cancer and are carried by about 5% of all breast cancer patients, there are also low-penetrance predisposing genes carried by a much higher proportion of breast cancer patients, a view supported by recent epidemiological studies. Ideally, testing for the presence of these putative genes should involve the use of simpler methods than the G2 assay, which requires metaphase analysis of chromosome damage. Here we report on the use of a simple, rapid micronucleus assay in G0 lymphocytes exposed to high dose rate (HDR) or low dose rate gamma-irradiation, with delayed mitogenic stimulation. Good assay reproducibility was obtained, particularly with the HDR protocol, which identified 31% (12 out of 39) of breast cancer patients compared with 5% (2 out of 42) of healthy controls as having elevated radiation sensitivity. In the long term, such cytogenetic assays may have the potential for selecting women for intensive screening for breast cancer.

Apoptosis, intrinsic radiosensitivity and prediction of radiotherapy response in cervical carcinoma

Apoptosis is an important mechanism of cell death in tumours and it is seen both prior to and following radiotherapy. In this study patients with proven carcinoma of the cervix had measurement made of the percentage of apoptotic cells (apoptotic index or AI) in pre-therapy biopsies. Measurements of intrinsic radiosensitivity (SF2), already shown to be a predictor of outcome, had previously been made on the same pre-therapy biopsies. Mitotic index (MI) and Ki-67 antigen staining were also recorded as markers for proliferation. Patients were divided into those with an AI above or below the median and in general increasing apoptosis was associated with poor prognosis. The 5-year survival rate for tumours with an AI below the median was 79% and was significantly greater than the rate of 47% for those with an AI above the median (p = 0.003). There was also a significantly increased 5-year local recurrence-free rate for patients with an AI below the median compared with those with an AI above the median (79 versus 61%, p = 0.012). In addition, AI and SF2 acted as independent prognostic indicators. Patients with both an SF2 and AI value above the median did badly (25% 5-year survival, 46% local control) compared with those with an SF2 and AI below the median (80% 5-year survival, 100% local control). Apoptosis showed correlation with MI (n = 66, r = 0.34, p = 0.002) and cell staining for the Ki-67 antigen (n = 57, r = 0.25, p = 0.03), but neither MI nor Ki-67 were related to patient outcome. This suggests that while apoptosis may be a reflection of tumour proliferation this cannot in itself explain the ability of apoptosis to predict clinical outcome for this series of patients. The study raises the possibility of AI and SF2 being used together as predictors of tumour response to radiotherapy.

Association of transforming growth factor beta-1 single nucleotide polymorphisms with radiation-induced damage to normal tissues in breast cancer patients.

Purpose : To investigate whether transforming growth factor beta-1 (TGF β1) single nucleotide polymorphisms were associated with the susceptibility of breast cancer patients to severe radiationinduced normal tissue damage. Materials and methods : PCR-RFLP assays were performed for TGF β1 gene polymorphisms on DNA obtained from 103 breast cancer patients who received radiotherapy. The G −800A, C −509T, T+869C and G+915C polymorphic sites were examined, and genotype and allele frequencies of two subgroups of patients were calculated and compared. Results : The less prevalent −509T and +869C alleles were significantly associated with a subgroup of patients who developed severe radiation-induced normal tissue fibrosis (n =15) when compared with those who did not (n =88) (odds ratio=3.4, p =0.0036, and 2.37, p =0.035, respectively). Furthermore, patients with the −509TT or +869CC genotypes were between seven and 15 times more likely to develop severe fibrosis. Conclusions : These findings imply a role for the −509T and +869C alleles in the pathobiological mechanisms underlying susceptibility to radiation-induced fibrosis. Their predictive value would be limited to patients who are −509TT or +869CC, but if fibrosis-associated polymorphic sites in other genes could be identified, it may be possible to detect fibrosis prone individuals before radiotherapy with greater certainty.

Genetic predisposition in breast cancer

UI - 95057701LA - engRN - 0 (Genetic Markers)PT - LetterDA - 19941216IS - 0140-6736SB - AIMSB - IMCY - ENGLANDJC - L0S

Relationship between in vitro chromosomal radiosensitivity of peripheral blood lymphocytes and the expression of normal tissue damage following radiotherapy for breast cancer

BACKGROUND AND PURPOSEnThere is a need for rapid and reliable tests for the prediction of normal tissue responses to radiotherapy, as this could lead to individualization of patient radiotherapy schedules and thus improvements in the therapeutic ratio. Because the use of cultured fibroblasts is too slow to be practicable in a clinical setting, we evaluated the predictive role of assays of lymphocyte chromosomal radiosensitivity in patients having radiotherapy for breast cancer.nnnMATERIALS AND METHODSnRadiosensitivity was assessed using a micronucleus (MN) assay at high dose rate (HDR) and low dose rate (LDR) on lymphocytes irradiated in the G(0) phase of the cell cycle (Scott D, Barber JB, Levine EL, Burril W, Roberts SA. Radiation-induced micronucleus induction in lymphocytes identifies a frequency of radiosensitive cases among breast cancer patients: a test for predispostion? Br. J. Cancer 1998;77;614-620) and an assay of G(2) phase chromatid radiosensitivity (G(2) assay) (Scott D, Spreadborough A, Levine E, Roberts SA. Genetic predisposition in breast cancer. Lancet 1994; 344: 1444). In a study of acute reactions, blood samples were taken from breast cancer patients before the start of radiotherapy, and the skin reaction documented. 116 patients were tested with the HDR MN assay, 73 with the LDR MN assay and 123 with the G(2) assay. In a study of late reactions, samples were taken from a series of breast cancer patients 8-14 years after radiotherapy and the patients assessed for the severity of late effects according to theLENT SOMA scales. 47 were tested with the HDR assay, 26 with the LDR assay and 19 with the G(2) assay. For each clinical endpoint, patients were classified as being normal reactors or highly radiosensitive patients (HR patients (Burnet NG. Johansen J, Turesson I, Nyman J. Describing patients normal tissue reactions: Concerning the possiblity of individualising radiotherapy dose presciptions based on potential predictive assays of normal tissue radiosensitivity. Int. J. Cancer 1998;79:606-613)).nnnRESULTSnThe HR patients could be identified in some of the assays. For example, for acute skin reactions, 9/123 patients were judged as HR; they had significantly higher G(2) scores than normal reactors (P=0.004). For the late reactions, the mean HDR MN scores were higher for the 4/47 patients who had severe telangiectasia (P=0.042) and the 8/47 patients had severe fibrosis (P=0.055). However, there were no trends towards increased chromosomal radiosensitivity with the micronucleus scores at HDR or LDR, or with G(2) chromosomal radiosensitivity.nnnCONCLUSIONSnWhile these results support the concept of using lymphocytes to detect elevated sensitivity to radiotherapy (as an alternative to fibroblasts), these assays are unlikely to be of assistance for the prediction of normal tissue effects in the clinic in their present form.

TGF-β1 levels in pre-treatment plasma identify breast cancer patients at risk of developing post-radiotherapy fibrosis

A serious complication of radiotherapy in the treatment of cancer patients is the late onset of fibrosis in normal tissues. Transforming growth factor β (TGF‐β) is emerging as a key mediator of the fibrotic process through its effects on stimulation of fibroblast proliferation, migration and extracellular matrix (ECM) synthesis. The fact that radiation‐induced vascular injury tends to precede the development of fibrosis has led to the suggestion that vascular damage is crucial in its pathogenesis. CD105, the specific type III vascular receptor for TGF‐β1 and ‐β3, modulates cell proliferation and ECM production in response to TGF‐β in vitro. In this study, we have quantified the levels of TGF‐β1 and soluble CD105–TGF‐β1 complex in 91 pre‐radiotherapy plasma samples from early‐stage (T1 or T2) breast cancer patients utilising an enhanced chemiluminescence ELISA system. During the follow‐up period, 24 patients had developed moderate and one severe fibrosis of the breast. The mean TGF‐β1 level in these 25 patients was 203.2 ± 37.3 pg/ml, which was significantly elevated above the level for those with no fibrosis. Furthermore, a significantly lower CD105–TGF‐β1 complex level was observed in the former compared to the latter. Spearmans correlation analysis showed that TGF‐β1 was positively correlated and the CD105–TGFβ1 complex inversely correlated with the occurrence of breast fibrosis. Using a cut‐off value of 96 pg/ml, the sensitivity and specificity of TGF‐β1 levels in predicting breast fibrosis were 76% and 74%, respectively. Our results indicate that TGF‐β1 and the receptor–ligand complex appear to be of clinical value in identifying patients at risk of developing post‐radiotherapy fibrosis. Int. J. Cancer (Pred. Oncol.) 84:155–159, 1999.

Absence of mutations in the ATM gene in breast cancer patients with severe responses to radiotherapy

The effectiveness of cancer radiotherapy is compromised by the small proportion (approximately 5%) of patients who sustain severe normal tissue damage after standard radiotherapy treatments. Predictive tests are required to identify these highly radiosensitive cases. Patients with the rare, recessively inherited, cancer-prone syndrome ataxia-telangiectasia (A-T) sustain extremely severe normal tissue necrosis after radiotherapy and their cultured cells are also highly radiosensitive. Clinically normal carriers (heterozygotes) of the A-T gene have an increased risk of breast cancer, account for approximately 4% of all breast cancer cases and show a modest increase in cellular radiosensitivity in vitro. It has been suggested that a substantial proportion of highly radiosensitive (HR) breast cancer patients may be A-T heterozygotes, and that screening for mutations in the A-T gene could be used as a predictive test. We have tested this hypothesis in a group of cancer patients who showed adverse reactions to radiotherapy. Sixteen HR breast cancer patients showing mainly acute reactions (and seven HR patients with other cancers) were tested for ATM mutations using the restriction endonuclease fingerprinting assay. No mutations typical of those found in obligate A-T heterozygotes were detected. If the estimate that 4% of breast cancer cases are A-T gene carriers is correct, then ATM mutations do not confer clinical radiosensitivity. These early results suggest that screening for ATM mutations in cancer patients may not be of value in predicting adverse reactions.

Apoptosis as predictor of response to radiotherapy in cervical carcinoma

UI - 94335419LA - engPT - LetterDA - 19940915IS - 0140-6736SB - AIMSB - IMCY - ENGLANDJC - L0S

Aberrant CDKN1A transcriptional response associates with abnormal sensitivity to radiation treatment

Normal tissue reactions to radiation therapy vary in severity among patients and cannot be accurately predicted, limiting treatment doses. The existence of heritable radiosensitivity syndromes suggests that normal tissue reaction severity is determined, at least in part, by genetic factors and these may be revealed by differences in gene expression. To test this hypothesis, peripheral blood lymphocyte cultures from 22 breast cancer patients with either minimal (11) or very severe acute skin reactions (11) have been used to analyse gene expression. Basal and post-irradiation expression of four radiation-responsive genes (CDKN1A, GADD45A, CCNB1, and BBC3) was determined by quantitative real-time PCR in T-cell cultures established from the two patient groups before radiotherapy. Relative expression levels of BBC3, CCNB1, and GADD45A 2u2009h following 2u2009Gy X-rays did not discriminate between groups. However, post-irradiation expression response was significantly reduced for CDKN1A (P<0.002) in severe reactors compared to normal. Prediction of reaction severity of ∼91% of individuals sampled was achieved using this end point. Analysis of TP53 Arg72Pro and CDKN1A Ser31Arg single nucleotide polymorphisms did not show any significant association with reaction sensitivity. Although these results require confirmation and extension, this study demonstrates the possibility of predicting the severity of acute skin radiation toxicity in simple tests.

Irinotecan+5-fluorouracil with concomitant pre-operative radiotherapy in locally advanced non-resectable rectal cancer: a phase I/II study.

In the UK, 10% of patients diagnosed with rectal cancer have inoperable disease at presentation. This study ascertained whether the resectability rate of inoperable locally advanced rectal cancer was improved by administration of intravenous irinotecan, 5-fluorouracil (5-FU) and pelvic radiotherapy. During phase I of the trial (n=12), the dose of irinotecan was escalated in three-patient cohorts from 50u2009mgu2009m−2 to 60u2009mgu2009m−2 to 70u2009mgu2009m−2 to identify the maximum tolerated dose (60u2009mgu2009m−2). In phase II, 31 patients with non-resectable disease received 45u2009Gy radiotherapy and 5-FU infusions (200u2009mgu2009m−2 per day) for 5 weeks. Irinotecan (60u2009mgu2009m−2) was given on days 1, 8, 15 and 22. After treatment, patients were operated on if possible. Thirty patients completed the protocol, 28 underwent surgery. Before surgery, MRI restaging of 24 patients showed that 19 (79%) had a reduction in tumour stage after treatment (seven complete clinical response and 12 partial). Of 27 patients followed up after surgery, 22 (81%) had clear circumferential resection margins. Disease-free and overall survival estimates at 3 years were 65 and 90%, respectively. The regimen was well tolerated. Irinotecan, 5-FU and radiotherapy results in tumour downgrading, allowing resection of previously inoperable tumour with acceptable toxicity.