Piers N. Plowman

Prolonged expression of the γ-H2AX DNA repair biomarker correlates with excess acute and chronic toxicity from radiotherapy treatment.

The normal tissue tolerance levels to fractionated radiotherapy have been appreciated by a century of careful clinical observations and radiobiological studies in animals. During clinical fractionated radiotherapy, these normal tissue tolerance levels are respected, and severe sequelae of radiotherapy are avoided in the majority of patients. Notwithstanding, a minority of patients experience unexpectedly severe normal tissue reactions. The ability to predict which patients might form this minority would be important. We have conducted a study to develop a rapid and reliable diagnostic test to predict excessive normal tissue toxicity (NTT) in radiotherapy patients. A flow cytometric immunocytochemical assay was used to measure DNA damage in peripheral blood lymphocytes (PBL) from cancer patients exposed to 2‐Gy gamma radiation. DNA damage and repair was measured by induction of cellular γ‐H2AX in unirradiated and exposed cells at specific time points following exposure. In 12 cancer patients that experienced severe atypical NTT following radiotherapy, there was a failure to repair DNA double‐strand breaks (DSB) as measured by γ‐H2AX induction and persistence. In ten cancer patients that experienced little or no NTT and in seven normal (noncancer controls), efficient repair of DNA DSB was observed in the γ‐H2AX assay. We conclude that a flow cytometric assay based on γ‐H2AX induction in PBL of radiotherapy patients may represent a robust, rapid and reliable biomarker to predict NTT during radiotherapy. Further research is required with a larger patient cohort to validate this important study.

Ectopic intracranial retinoblastoma in childhood.

Twelve out of a series of 630 children with retinoblastoma, treated in the ocular oncology units at St Bartholomews and Moorfields Eye Hospitals during the past 30 years, have developed ectopic intracranial retinoblastoma. The ectopic tumour occurred in the pineal region in eight children and in the suprasellar region in four. Ten patients had bilateral retinoblastoma, one unilateral disease, and one child presented with an isolated suprasellar tumour but no evidence of retinal disease. The interval from the initial diagnosis of retinoblastoma to the development of ectopic intracranial disease ranged from 4 to 70 months, median 34 months. Methods of treatment for the ectopic tumour varied, but all 12 children died with a median survival of only 8 months following the diagnosis of ectopic retinoblastoma. Subsequent spread of tumour to other sites within the central nervous system proved to be the most frequent cause of death. Ectopic intracranial retinoblastoma is a potentially curable neoplasm, but it requires adequate therapy to the whole neuraxis as well as high dose equivalent radiotherapy to the primary tumour.

External beam radiotherapy for retinoblastoma: I. Whole eye technique.

A retrospective analysis has been performed of the results of external beam radiotherapy for retinoblastoma using a whole eye technique. Local tumour control has been assessed in a consecutive series of 175 eyes in 142 children all of whom received external beam radiotherapy as the primary treatment for retinoblastoma. Follow up ranged from 2 to 17 years (median 9 years). Tumour control rates have been analysed with respect to the Reese Ellsworth classification and the series includes eyes in groups I to V. Focal salvage therapy was given for persistent, recurrent, or new tumours after radiotherapy. Following whole eye radiotherapy alone, the overall ocular cure rate was 57%, though with salvage therapy 80% of eyes could be preserved.

Comparison of a micro-multileaf collimator with a 5-mm-leaf-width collimator for intracranial stereotactic radiotherapy

PURPOSE To dosimetrically compare a micro-multileaf collimator (minimum leaf width of 3 mm) with the 5-mm-leaf multileaf collimator (MLC) of a standard linear accelerator for stereotactic conformal radiotherapy treatment of intracranial lesions. MATERIALS AND METHODS Fourteen patients previously treated for a variety of irregularly shaped intracranial lesions using BrainLABs micro-MLC were retrospectively replanned using the Varian Millennium MLC (5 mm leaf width). All planning was performed with the BrainSCAN v 5.1 software. The same fixed, noncoplanar beam arrangement was used for both plans, and identical target coverage was achieved by adjusting the MLC shape around the planning target volume (PTV). The isodose distributions and dose-volume histograms (DVH) were computed and plans were compared in terms of conformity of the prescription isodose to the PTV and dose received by surrounding normal tissue. RESULTS Equivalent PTV coverage was achieved using the 5-mm collimator by adjusting the MLC shape around the target in every case. There was a statistically significant increase in the conformity index for the Varian MLC compared with the micro-MLC (p < 0.001), indicating a worse conformity of the prescription isodose to the PTV, but this parameter was within our (and Radiation Therapy Oncology Group) clinical criterion in all cases. There was no statistically significant difference in the maximum dose to critical structures, but DVH curves demonstrated an increased volume of normal tissue irradiated to the lower isodose levels. The mean increase in the volume of critical structure enclosed within the 50% and 70% isodose surfaces was 5.7% and 4.9%, respectively. CONCLUSIONS The micro-MLC consistently improves both PTV conformity and surrounding tissue sparing when compared to that of a standard linear accelerator. However, when viewed quantitatively, the improvements are small enough that individual centers may question their choice of equipment when outfitting a stereotactic radiotherapy service.

External beam radiotherapy for retinoblastoma: II. Lens sparing technique.

A retrospective analysis is presented of the results of external beam radiotherapy for retinoblastoma utilising an accurate lens sparing technique. Local tumour control has been assessed in a consecutive series of 67 eyes in 53 children all of whom received external beam radiotherapy as the primary treatment of retinoblastoma. Follow up ranged from 12 to 82 months (median 35 months) with 76% of the children followed for more than 2 years. Tumour control rates have been analysed with respect to the Reese-Ellsworth classification. The role of adjuvant and salvage focal therapy is emphasised. Following lens sparing radiotherapy with prior adjuvant treatment of anterior tumours, where appropriate, the overall ocular cure rate was 72%. With salvage therapy of persistent, recurrent, or new tumours, 93% of eyes could be preserved in this series which includes mainly eyes classified in Reese-Ellsworth groups I-III. These results compare favourably with those of whole eye external beam radiotherapy for comparable tumours, and with those of lens and anterior segment sparing using other techniques. They were achieved without the ocular morbidity associated with whole eye external beam radiotherapy.

Stereotactic Radiosurgery for Pituitary Adenomas

Pituitary adenoma is a radiosensitive disease and postoperative radiotherapy reduces the chance of relapse. Non-irradiated patients, followed in the modern era, suffer up to 20% five-year and up to 44% ten-year relapse. To some extent, predictors of relapse are available at the time of presentation or after surgery. Although conventionally fractionated radiotherapy has a very good track record with regard to controlling disease and safety in the modern age, there is considerable contemporary interest in the technique of radiosurgery (highly concentrated radiation therapy using stereotactic mapping). The usefulness of this technique in the treatment of pituitary adenoma is discussed in this review.

The effects of conventionally fractionated, extended portal radiotherapy on the human peripheral blood count

Conventionally fractionated, extended portal radiotherapy (CFEPRT) has been used to treat two diseases in which there was no marrow infiltration (viz. Hodgkins disease and medulloblastoma). Blood count indices have been monitored during therapy and in the recovery phase. The lymphocytes were the most sensitive and the monocytes the most refractory leucocytes to change; the monocyte count tended to recover during CFEPRT. The platelet count fell gradually and soon after the neutrophil count. The nadir counts for white cells and platelets occurred early or toward the middle of CFEPRT, after which levels were maintained. The hemoglobin slightly and progressively declined. The patterns of change were similar for the two portals analyzed. Absolute eosinophilia occurred in 9 of the 53 CFEPRT patients, often in the recovery period. All patients who maintained their early nadir levels throughout the rest of the CFEPRT demonstrated fast recovery of all indices following completion of radiotherapy; the lymphocyte count recovered fastest. Recent prior CFEPRT or standard MVPP (nitrogen mustard, vinblastine, procarbazine, prednisolone) chemotherapy rendered the blood count more liable to radiation induced cytopenia. A lapse of more than 3 months between MVPP and CFEPRT allowed greater tolerance to the radiotherapy. Recent MVPP may be less myelosuppressive than recent mantle radiotherapy with respect to subsequent tolerance to CFEPRT.

Stereotactic radiosurgery. XVI: Isodosimetric comparison of photon stereotactic radiosurgery techniques (gamma knife vs. micromultileaf collimator linear accelerator) for acoustic neuroma-and potential clinical importance

PURPOSE Two stereotactic photon radiation therapy methods are currently in practice for the treatment of acoustic neuroma. In the 1990s, our data and those of others demonstrated isodosimetric advantages for gamma knife technology over linear accelerator methodology. Since then, the introduction of micromultileaf collimator technology has improved the conformity of the linear accelerator method such that the isodosimetric differences between the two techniques have narrowed. MATERIALS AND METHODS In this study, modern gamma knife isodosimetry was compared to that of modern linac technology (conformal fixed fields and dynamic arcs) for the therapy of acoustic neuroma. This is an unusual target in that a special sensory nerve (holding the key to hearing preservation) frequently runs through the targeted volume, unlike the majority of other stereotactic radiation therapy targets. This was a single-dose prescription comparison; the perceived extra benefit of fractionation (a technique not routinely available to the gamma knife) was thereby abrogated. RESULTS Although the gamma knife technique maintained a slight, but statistically significant, advantage with regard to dose conformity (p < 0.02) (at the debatable cost of a lower minimum target dose), the much higher internal dose gradient (high maximum dose to prescription dose [MD:PD] ratio) could be interpreted as a disadvantage with respect to hearing preservation, although advantageous with regard to tumor ablation. Of the two linac methods, the dynamic arc method gave a statistically significant advantage over the fixed-field method as regards conformity (p < 0.05), at the expense of a slightly higher brainstem dose (an average of 12.4 Gy, cf. 11.7 Gy for fixed fields), but this result was not statistically significant. No significant difference was seen in the MD:PD ratio for the two single-isocenter linac techniques. CONCLUSIONS Gamma knife methodology remains well validated, with very good isodosimetry, but when hearing preservation is important, the improving linac technologies will compete with the gamma knife for optimal therapy. In these circumstances, the minor differences in isodosimetry between the two techniques will become important.

A novel splice variant of the DNA-PKcs gene is associated with clinical and cellular radiosensitivity in a patient with xeroderma pigmentosum

Background Radiotherapy-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions. Inherited defects in DNA DSB repair pathways lead to hypersensitivity to ionising radiation, immunodeficiency and increased cancer incidence. A patient with xeroderma pigmentosum complementation group C, with a scalp angiosarcoma, exhibited dramatic clinical radiosensitivity following radiotherapy, resulting in death. A fibroblast cell line from non-affected skin (XP14BRneo17) was hypersensitive to ionising radiation and defective in DNA DSB repair. Aim To determine the genetic defect causing cellular radiation hypersensitivity in XP14BRneo17 cells. Methods Functional genetic complementation whereby copies of human chromosomes containing genes involved in DNA DSB repair (chromosomes 2, 5, 8 10, 13 and 22) were individually transferred to XP14BRneo17 cells in an attempt to correct the radiation hypersensitivity. Clonogenic survival assays and γ-H2AX immunofluorescence were conducted to measure radiation sensitivity and repair of DNA DSBs. DNA sequencing of defective DNA repair genes was performed. Results Transfer of chromosome 8 (location of DNA-PKcs gene) and transfection of a mammalian expression construct containing the DNA-PKcs cDNA restored normal ionising radiation sensitivity and repair of DNA DSBs in XP14BRneo17 cells. DNA sequencing of the DNA-PKcs coding region revealed a 249-bp deletion (between base pairs 3656 and 3904) encompassing exon 31 of the gene. Conclusion We provide evidence of a novel splice variant of the DNA-PKcs gene associated with radiosensitivity in a patient with xeroderma pigmentosum and report the first double mutant in distinct DNA repair pathways being consistent with viability.

Multispectral imaging flow cytometry reveals distinct frequencies of γ-H2AX foci induction in DNA double strand break repair defective human cell lines

The measurement of γ‐H2AX foci induction in cells provides a sensitive and reliable method for the quantitation of DNA damage responses in a variety of cell types. Accurate and rapid methods to conduct such observations are desirable. In this study, we have employed the novel technique of multispectral imaging flow cytometry to compare the induction and repair of γ‐H2AX foci in three human cell types with different capacities for the repair of DNA double strand breaks (DSB). A repair normal fibroblast cell line MRC5‐SV1, a DSB repair defective ataxia telangiectasia (AT5BIVA) cell line, and a DNA‐PKcs deficient cell line XP14BRneo17 were exposed to 2 Gy gamma radiation from a 60Cobalt source. Thirty minutes following exposure, we observed a dramatic induction of foci in the nuclei of these cells. After 24 hrs, there was a predictable reduction on the number of foci in the MRC5‐SV1 cells, consistent with the repair of DNA DSB. In the AT5BIVA cells, persistence of the foci over a 24‐hr period was due to the failure in the repair of DNA DSB. However, in the DNA‐PKcs defective cells (XP14BRneo17), we observed an intermediate retention of foci in the nuclei indicative of partial repair of DNA DSB. In summary, the application of imaging flow cytometry has permitted an evaluation of foci in a large number of cells (20,000) for each cell line at each time point. This provides a novel method to determine differences in repair kinetics between different cell types. We propose that imaging flow cytometry provides an alternative platform for accurate automated high through‐put analysis of foci induction in a variety of cell types.