Navita Somaiah

Residual DNA and chromosomal damage in ex vivo irradiated blood lymphocytes correlated with late normal tissue response to breast radiotherapy

PURPOSE To test the association of DNA double-strand break (DSB) repair and chromosomal radiosensitivity in ex vivo irradiated blood lymphocytes with late-onset normal tissue responses following breast radiotherapy. METHODS Breast cancer patients with minimal (controls) or marked late radiotherapy changes (cases) were retrospectively selected. DSB were quantified by γH2AX/53BP1 immunofluorescence microscopy 0.5 and 24 h after exposure of unstimulated blood lymphocytes to 0.5 and 4 Gy X-rays, respectively. Chromosomal aberrations were scored in blood lymphocyte metaphases after 6 Gy X-rays. RESULTS Despite similar foci levels at 0.5 h in cases (n=7) and controls (n=7), foci levels 24 h after 4 Gy irradiation differed significantly between them (foci per cell were 12.8 in cases versus 10.2 in controls, p=0.004). Increased chromosomal radiosensitivity was also observed in cases (aberrations per cell were 5.84 in cases versus 3.79 in controls, p=0.001) with exchange and deletion type aberrations contributing equally to the difference between cases and controls. Residual foci correlated with formation of deletions (Spearmans R=0.589, p=0.027) but not exchanges (R=0.367, p=0.197) in blood lymphocytes from the same patients. CONCLUSIONS Higher levels of exchange type aberrations observed among radiosensitive breast cancer patients suggest a role for DSB misrepair, in addition to residual damage, as determinants of late normal tissue damage. Correlation of residual foci levels with deletion type aberration yields in the same cohort confirms their mechanistic linkage.

Randomised phase II trial of hyperbaric oxygen therapy in patients with chronic arm lymphoedema after radiotherapy for cancer.

BACKGROUND A non-randomised phase II study suggested a therapeutic effect of hyperbaric oxygen (HBO) therapy on arm lymphoedema following adjuvant radiotherapy for early breast cancer, justifying further investigation in a randomised trial. METHODS Fifty-eight patients with ≥ 15% increase in arm volume after supraclavicular ± axillary radiotherapy (axillary surgery in 52/58 patients) were randomised in a 2:1 ratio to HBO (n=38) or to best standard care (n=20). The HBO group breathed 100% oxygen at 2.4 atmospheres absolute for 100 min on 30 occasions over 6 weeks. Primary endpoint was ipsilateral limb volume expressed as a percentage of contralateral limb volume. Secondary endpoints included fractional removal rate of radioisotopic tracer from the arm, extracellular water content, patient self-assessments and UK SF-36 Health Survey Questionnaire. FINDINGS Of 53/58 (91.4%) patients with baseline assessments, 46 had 12-month assessments (86.8%). Median volume of ipsilateral limb (relative to contralateral) at baseline was 133.5% (IQR 126.0-152.3%) in the control group, and 135.5% (IQR 126.5-146.0%) in the treatment group. Twelve months after baseline the median (IQR) volume of the ipsilateral limb was 131.2% (IQR 122.7-151.5%) in the control group and 133.5% (IQR 122.3-144.9%) in the treatment group. Results for the secondary endpoints were similar between randomised groups. INTERPRETATION No evidence has been found of a beneficial effect of HBO in the treatment of arm lymphoedema following primary surgery and adjuvant radiotherapy for early breast cancer.

Acute skin toxicity associated with a 1-week schedule of whole breast radiotherapy compared with a standard 3-week regimen delivered in the UK FAST-Forward Trial

Background and purpose FAST-Forward is a phase 3 clinical trial testing a 1-week course of whole breast radiotherapy against the UK standard 3-week regimen after primary surgery for early breast cancer. Two acute skin toxicity substudies were undertaken to test the safety of the test schedules with respect to early skin reactions. Material and methods Patients were randomly allocated to 40 Gy/15 fractions (F)/3-weeks, 27 Gy/5F/1-week or 26 Gy/5F/1-week. Acute breast skin reactions were graded using RTOG (first substudy) and CTCAE criteria v4.03 (second substudy) weekly during treatment and for 4 weeks after treatment ended. Primary endpoint was the proportion of patients within each treatment group with grade ⩾3 toxicity (RTOG and CTCAE, respectively) at any time from the start of radiotherapy to 4 weeks after completion. Results 190 and 162 patients were recruited. In the first substudy, evaluable patients with grade 3 RTOG toxicity were: 40 Gy/15F 6/44 (13.6%); 27 Gy/5F 5/51 (9.8%); 26 Gy/5F 3/52 (5.8%). In the second substudy, evaluable patients with grade 3 CTCAE toxicity were: 40 Gy/15F 0/43; 27 Gy/5F 1/41 (2.4%); 26 Gy/5F 0/53. Conclusions Acute breast skin reactions with two 1-week schedules of whole breast radiotherapy under test in FAST-Forward were mild.

Homologous recombination mediates cellular resistance and fraction size sensitivity to radiation therapy

PURPOSE Cellular sensitivity to radiotherapy total dose and fraction size is strongly influenced by DNA double strand break (DSB) repair. Here, we investigate response to radiotherapy fraction size using CHO cell lines deficient in specific DNA repair pathways in response to radiation induced DNA double strand breaks (DSB). EXPERIMENTAL DESIGN We irradiated CHO cell lines, AA8 (WT), irs1SF (XRCC3-), V3-3 (DNA-PKcs-) and EM9 (XRCC1-) with 16 Gy in 1 Gy daily fractions over 3 weeks or 16 Gy in 4 Gy daily fractions over 4 days, and studied clonogenic survival, DNA DSB repair kinetics (RAD51 and 53BP1 foci staining) and cell cycle profiles (flow cytometry). RESULTS In response to fractionated radiotherapy, wild-type and DNA repair defective cells accumulated in late S/G2 phase. In cells proficient in homologous recombination (HR), accumulation in S/G2 resulted in reduced sensitivity to fraction size and increased cellular resistance (clonogenic survival). Sensitivity to fraction size was also lost in NHEJ-defective V3-3 cells, which likely rely on functional HR. By contrast, HR-defective irs1SF cells, with functional NHEJ, remained equally sensitive to fractionation throughout the 3-week treatment. CONCLUSIONS The high fidelity of HR, which is independent of induced DNA damage level, is postulated to explain the low fractionation sensitivity and cellular resistance of cells in S/G2 phase. In conclusion, our results suggest that HR mediates resistance to fractionated radiotherapy, an observation that may help future efforts to improve radiotherapy outcome.

Inter-individual and inter-cell type variation in residual DNA damage after in vivo irradiation of human skin

PURPOSE The aim of this study was to compare inter-individual and inter-cell type variation in DNA double-strand break (DSB) repair following in vivo irradiation of human skin. MATERIALS AND METHODS Duplicate 4mm core biopsies of irradiated and unirradiated skin were collected from 35 patients 24h after 4Gy exposure using 6MeV electrons. Residual DSB were quantified by scoring 53BP1 foci in dermal fibroblasts, endothelial cells, superficial keratinocytes and basal epidermal cells. RESULTS Coefficients of inter-individual variation for levels of residual foci 24h after in vivo irradiation of skin were 39.9% in dermal fibroblasts, 44.3% in endothelial cells, 32.9% in superficial keratinocytes and 46.4% in basal epidermal cells (p<0.001, ANOVA). In contrast, the coefficient of inter-cell type variation for residual foci levels was only 11.3% in human skin between the different epidermal and dermal cells (p=0.034, ANOVA). Foci levels between the different skin cell types were correlated (Pearsons R=0.855-0.955, p<0.001). CONCLUSIONS Patient-specific factors appear to be more important than cell type-specific factors in determining residual foci levels following in vivo irradiation of human skin.

The Relationship Between Homologous Recombination Repair and the Sensitivity of Human Epidermis to the Size of Daily Doses Over a 5-Week Course of Breast Radiotherapy

Purpose: A molecular understanding of tissue sensitivity to radiotherapy fraction size is missing. Here, we test the hypothesis that sensitivity to fraction size is influenced by the DNA repair system activated in response to DNA double-strand breaks (DSB). Human epidermis was used as a model in which proliferation and DNA repair were correlated over 5 weeks of radiotherapy. Experimental design: Radiotherapy (25 fractions of 2 Gy) was prescribed to the breast in 30 women with early breast cancer. Breast skin biopsies were collected 2 hours after the 1st and 25th fractions. Samples of contralateral breast skin served as controls. Sections were coimmunostained for Ki67, cyclin A, p21, RAD51, 53BP1, and β1-integrin. Results: After 5 weeks of radiotherapy, the mean basal Ki67 density increased from 5.72 to 15.46 cells per millimeter of basement membrane (P = 0.002), of which the majority were in S/G2 phase, as judged by cyclin A staining (P < 0.0003). The p21 index rose from 2.8% to 87.4% (P < 0.0001) after 25 fractions, indicating cell cycle arrest. By week 5, there was a 4-fold increase (P = 0.0003) in the proportion of Ki67-positive cells showing RAD51 foci, suggesting increasing activation of homologous recombination. Conclusions: Cell cycle arrest in S/G2 phase in the basal epidermis after a 5-week course of radiotherapy is associated with greater use of homologous recombination for repairing DSB. The high fidelity of homologous recombination, which is independent of DNA damage levels, may explain the low-fractionation sensitivity of tissues with high-proliferative indices, including self-renewing normal tissues and many cancers. Clin Cancer Res; 18(19); 5479–88. ©2012 AACR.

Phase II study of oral capecitabine in patients with hormone-refractory prostate cancer

Background:Currently available treatment for hormone refractory prostate cancer is limited in efficacy and associated with significant toxicity. This phase II study was performed to assess the efficacy of the oral fluoropyrimidine capecitabine in advanced prostate cancer.Patients and methods:Patients who had a rising prostate-specific antigen (PSA) despite androgen withdrawal, but who remained free from cancer-related symptoms. In total, 14 patients received oral capecitabine 1250 mg/m2 twice daily for two weeks of a three-week cycle. Tumour response was assessed using serum PSA measurement at 3-weekly intervals and, where present, imaging of soft tissue metastases.Results:One of 14 patients experienced a partial response as assessed by both PSA and imaging of liver metastases. In seven other patients (50%), treatment decreased the rate of PSA rise. The duration of PSA stabilisation was generally short, but in 5/14 patients (36%) was sustained beyond 18 weeks, and in one patient to 24 weeks. Toxicity was significant but manageable, the most common adverse events being nausea, mucositis and hand–foot syndrome, each occurring in 50% of patients. Other common side effects were diarrhoea and lymphopenia. All toxicities were grade 1 or 2, except for grade 3 hand–foot syndrome occurring in one patient, and no dose reduction was required because of toxicity.Conclusion:Capecitabine has limited activity as a single agent in prostate cancer, but appears to modulate tumour biology. Considering the added convenience of oral administration, these results support further evaluation of combinations containing capecitabine in hormone-refractory prostate cancer.

Hypofractionated radiotherapy in early breast cancer: Clinical, dosimetric and radio-genomic issues

Curative radiotherapy is enhanced by partitioning the total dose into daily dose increments, called fractions. Most human cancer types respond to total dose rather than to the size of daily fractions [1]. This is an important point of difference in comparison with the responses of normal tissues responsible for the most important late adverse effects, which are sensitive to fraction size as well as total dose. This difference underpins the historical use of ‘small’ fractions, classically 2.0 Gy, to deliver the highest possible tolerated total dose, thereby, ensuring the highest rate of tumour control. The a/b ratio is an empirical descriptor of fraction size sensitivity, early reacting normal tissues and most cancer types being insensitive (a/ b ratio 7e20 Gy) relative to the late reacting (dose limiting) normal tissues with low a/b ratios in the range 0.5e6 Gy [2]. This difference in fractionation sensitivity between cancers and late reacting normal tissues has been challenged in the last 20 years by randomised clinical trials offering high level evidence that breast cancer is an exception in showing comparable sensitivity to fraction size as the normal tissues of the breast and ribcage. The evidence base includes four randomised trials from Canada and the UK [3e7]. The results suggest that there is no disadvantage to hypofractionation

Where Do We Look for Markers of Radiotherapy Fraction Size Sensitivity

The response of human normal tissues to radiotherapy fraction size is often described in terms of cellular recovery, but the causal links between cellular and tissue responses to ionising radiation are not necessarily straightforward. This article reviews the evidence for a cellular basis to clinical fractionation sensitivity in normal tissues and discusses the significance of a long-established inverse association between fractionation sensitivity and proliferative indices. Molecular mechanisms of fractionation sensitivity involving DNA damage repair and cell cycle control are proposed that will probably require modification before being applicable to human cancer. The article concludes by discussing the kind of correlative research needed to test for and validate predictive biomarkers of tumour fractionation sensitivity.

FDXR is a biomarker of radiation exposure in vivo

Previous investigations in gene expression changes in blood after radiation exposure have highlighted its potential to provide biomarkers of exposure. Here, FDXR transcriptional changes in blood were investigated in humans undergoing a range of external radiation exposure procedures covering several orders of magnitude (cardiac fluoroscopy, diagnostic computed tomography (CT)) and treatments (total body and local radiotherapy). Moreover, a method was developed to assess the dose to the blood using physical exposure parameters. FDXR expression was significantly up-regulated 24 hr after radiotherapy in most patients and continuously during the fractionated treatment. Significance was reached even after diagnostic CT 2 hours post-exposure. We further showed that no significant differences in expression were found between ex vivo and in vivo samples from the same patients. Moreover, potential confounding factors such as gender, infection status and anti-oxidants only affect moderately FDXR transcription. Finally, we provided a first in vivo dose-response showing dose-dependency even for very low doses or partial body exposure showing good correlation between physically and biologically assessed doses. In conclusion, we report the remarkable responsiveness of FDXR to ionising radiation at the transcriptional level which, when measured in the right time window, provides accurate in vivo dose estimates.