In this project we introduced a spatial frequency-based objective function for optimization of dose distributions used in spatially fractionated radiotherapy (also known as grid therapy). Several studies indicate that tissues can tolerate larger mean doses of radiation if the dose is delivered heterogeneously or to a partial volume of the organ. The objective function rewards heterogeneous dose distributions in the collaterally irradiated healthy tissues and is based on the concept of a maximum stem-cell migration distance. The stem-cell depletion hypothesis stipulates that damaged tissues can be repopulated by nearby surviving stem-cells within a critical volume outlined by the maximum migration distance. Proton grid therapy dose distributions were calculated to study the viability of our spatial frequency-based objective function. These were computed analytically with a proton pencil beam dose kernel. A multi-slit collimator placed flush against the surface of a water phantom defined the entrance fluence. The collimator geometry was described by two free parameters: the slit width and the number of slits within a specified field width. Organs at risk (OARs) and a planning target volume (PTV) were defined. Two dose constraints were set on the PTV and objective function values were computed for the OARs. The objective function measures the high-frequency content of a masked dose distribution, where the distinction between low- and high frequencies was made based on a characteristic distance. Out of the feasible solutions, the irradiation geometry that produced the maximum objective function value was selected as the optimal solution. With the spatial frequency-based objective function we were able to find, by brute-force search, unique optimal solutions to the constrained optimization problem. The optimal solutions were found on the boundary of the solution space. The objective function can be applied directly to arbitrarily shaped regions of interest and to dose distributions produced by multiple field angles. The next step is to implement the objective function in an optimization environment of a commercial treatment planning system (TPS).

The dose of curative radiotherapy (RT) for cancer is commonly limited by adverse effects presenting years later. Late reacting normal tissues are, on average, more sensitive to the size of daily doses (fractions) than early reacting normal tissues and cancers. Clinical trials have shown breast cancers to be one exception to this rule, in that they are as sensitive to fraction size as the late reacting normal tissues. This has led to the adoption of hypofractionation (use of fractions >2.0 Gy) in the UK for the adjuvant therapy of women with early breast cancer. An understanding of the molecular basis of fraction size sensitivity is necessary to improve radiotherapy outcome. In this respect, it is relevant that late reacting normal tissues have lower proliferative indices than early reacting normal tissues and most cancers. Here, we test the hypothesis that tissue sensitivity to fraction size is determined by the DNA repair systems activated in response to DNA double strand breaks (DSB), and that these systems vary according to the proliferative status of the tissue. Clinical data suggest that sensitivity of epidermis to fraction size varies over a 5-week course of RT. It resembles a late reacting normal tissue in its sensitivity to fraction size in the first week of RT and loses fractionation sensitivity by weeks 4 & 5. We used this feature of human epidermis to test how fractionation sensitivity and DNA repair changed over 5 weeks of RT. Breast skin biopsies were collected 2 h after the 1st, 5th and last fractions from 30 breast cancer patients prescribed 50 Gy/25fractions/5weeks. Sections of epidermis were co-stained for Ki67, cyclin A, p21, RAD51, 53BP1 and β1-Integrin. After 5 weeks of radiotherapy, the mean basal Ki67 density increased from 5.72 to 15.46 cells per mm 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 in the basal epidermis. By week 5, there was a 4-fold increase (p=0.0003) in the proportion of Ki67-positive cells showing RAD51 foci, confirming an association between activation of homologous recombination (HR) and loss of tissue fractionation sensitivity. Subsequently, CHO cell lines deficient in specific DNA repair genes were used to test molecular pathways involved in sensitivity to fraction size. We irradiated AA8 (WT), irs-1SF (XRCC3-), V3-3 (DNA-PK-) and EM9 (XRCC1-) with 16 Gy gamma-rays in 1 Gy daily fractions over 3 weeks or 16 Gy in 4 Gy daily fractions over 4 days, and studied clonogenic survival, DNA double-strand break (DSB) repair kinetics (RAD51 & 53BP1 staining) and cell cycle analysis using flow cytometry. We found that wild-type and DNA repair defective cells acquire resistance to fractionated radiotherapy by accumulation in the late S/G2 phase of the cell cycle and increased use of HR. In contrast, the irs1SF cells, defective in HR, failed to acquire radioresistance and remained equally sensitive to ionizing radiation throughout the 3-week treatment. We also demonstrated that sensitivity to fraction size is associated with functional NHEJ. It was undetectable in V3-3 cells lacking NHEJ and thereby likely relying on HR. The high fidelity of HR, which is independent of induced DNA damage levels and hence, of fraction size, may explain the low fractionation sensitivity of cells using HR to repair radiation induced DSBs. We then wanted to investigate the modifying effects of small molecule inhibitors of DNA repair on fractionation responses. To this end we tested the effects of adding selected ATM, PARP, and DNAPK inhibitors to fractionated radiotherapy in WT CHO cells. Our results showed that the ATM inhibitor had a significant radiosensitising effect when combined with fractionated RT and resulted in loss of sparing effect of fractionation in wild type CHO cells, an observation that may be clinically relevant. We also examined DNA DSB repair kinetics (RAD51 & 53BP1 foci) with these drugs in the context of fractionated IR.

Investigations of DNA damage response (DDR) mechanisms in normal tissues have implications for both cancer prevention and treatments. The accumulating knowledge about protein function and molecular markers makes it possible to directly trace and interpret cellular DDR in a tissue context. Using immunohistochemical techniques and digital image analysis, we have examined several principal DDR events in epidermis from patients undergoing fractionated radiotherapy. Acquiring biopsies from different regions of the skin provides the possibility to determine in vivo dose response at clinically relevant dose levels throughout the treatment. A crucial event in cellular DDR is the repair of DNA double strand breaks (DSBs). These serious lesions can be directly visualised in cells by detecting foci forming markers such as γH2AX and 53BP1. Our results reveal that DSB-signalling foci can be detected and quantified in paraffin-embedded tissues. More importantly, epidermal DSB foci dose response reveals hypersensitivity, detected as elevated foci levels per dose unit, for doses below ~0.3Gy. The low-dose hypersensitive dose response is observed throughout the treatment course and also in between fractions: at 30 minutes, 3 hours and 24 hours following delivered fractions. The dose response at 24 hours further reveals that foci levels do not return to background levels between fractions. Furthermore, a low-dose hypersensitive dose response is also observed for these persistent foci. Investigations of end points further downstream in the DDR pathways confirmed that the low-dose hypersensitivity was preserved for: the checkpoint regulating p21 kinase inhibitor; mitosis suppression; apoptosis induction and basal keratinocyte reduction. Our results reveal preserved low-dose hypersensitivity both early and late in the DDR pathways. A possible link between the dose-response relationships is therefore suggested. The preserved low-dose hypersensitivity is a cause for re-evaluation of the risks associated with low-dose exposure and has implications for cancer treatments, diagnostics and radiation protection.

The structure, function and accessibility of epidermal skin provide aunique opportunity to study the DNA damage response (DDR) of a normaltissue. The in vivo response can be examined in detail, at a molecularlevel, and further associated to the structural changes, observed at atissue level. We collected an extensive skin biopsy material frompatients undergoing fractionated radiotherapy for 5 to 7 weeks. Several end-points inthe DDR pathways were examined before, during and after the treatment. Quantification of DNA double strand break (DSB) signalling focirevealed a hypersensitivity to doses below 0.3Gy. Furthermore, aconsiderable amount of foci persisted between fractions. The low dosehypersensitivity was observed throughout the treatment and was alsoobserved for several key parameters further downstream in the DDR-pathway, such as p21-associated checkpoint activation, apoptosisinduction and reduction in basal keratinocyte density (BKD).Furthermore, for dose fractions above 1.0 Gy, a distinct acceleration inDDR was observed half way into treatment. This was manifested as anaccelerated loss of basal keratinocytes, mirrored by a simultaneousincrease in DSBs and p21 expression. Quantifications of mitotic events revealed a pronounced suppression ofmitosis throughout the treatment which was clearly low dosehypersensitive. Thus, no evidence of accelerated repopulation could beobserved for fraction doses ranging from 0.05 to 2Gy. Our results suggest that the keratinocyte response primarily isdetermined by checkpoints, which leads to pre-mitotic cell elimination by permanent growth arrest and apoptosis. A comparison between the epidermal and dermal sub-compartments revealsa consistent up-regulation of the DDR response during treatment. Adifference was however observed in the recovery phase after treatment,where miR-34a and p21 remain up-regulated in dermis more persistentlythan in epidermis. Our observations suggest that the recovery phaseafter treatment can provide important clues to understand clinicalobservations such as the early and late effects observed in normaltissues during fractionated radiotherapy.