Development of an automated gamma-H2AX immunocytochemistry assay.

gamma-H2AX is emerging as an important marker of ionizing radiation-induced double-strand breaks. Development of a significantly automated method to quantify gamma-H2AX would have broad application in assessing physiological responses to radiation exposure. PC-3 and DU145 prostate cancer cells grown on glass cover slips and 96-well plates were irradiated and assessed for gamma-H2AX focus formation by immunofluorescence analysis. The gamma-H2AX immunofluorescence staining was performed either manually or by using a preprogrammed automated robotic liquid handling system. A computer-controlled charge-coupled device camera acquired images serially throughout the thickness of each cell. Image analysis was performed manually and/or with automated image segmentation software. A robust relationship between radiation dose and gamma-H2AX focus numbers was demonstrated with both manual and automated image analysis methods, with excellent agreement observed between the two techniques. The r(2) correlation coefficients and Z factors exceeded 0.9 and 0.5, respectively, when gamma-H2AX focus formation was correlated with radiation dose using the automated technique. Inhibition of gamma-H2AX foci by drugs readily detected with this assay. Robotic specimen preparation with automated image acquisition and analysis can be used to quantify gamma-H2AX foci in irradiated cells, and the results agree well those obtained by manual counts. These data suggest that this assay has an excellent signal-to-noise ratio and is suitable for high-throughput applications.

Adjuvant radiotherapy and survival for patients with node-positive head and neck cancer: an analysis by primary site and nodal stage.

PURPOSE: Adjuvant radiotherapy (RT) is frequently recommended for node-positive head and neck squamous cell carcinoma (HNSCC) treated with primary surgery. The impact of RT on survival for various subgroups of node-positive HNSCC has not been clearly demonstrated. METHODS AND MATERIALS: Within the Surveillance, Epidemiology, and End Results (SEER) Database, we identified 5297 patients with node-positive (N1 to N3) HNSCC treated with definitive surgery with or without adjuvant RT between 1988 and 2001. The median follow-up was 4.4 years. RESULTS: Adjuvant RT significantly improved 5-year overall survival (46.3%: 95% confidence interval [CI], 44.7-48.0% for surgery + RT, vs. 35.2%: 95% CI, 32.0-38.5% for surgery alone, p < 0.001) and cancer-specific survival (54.8%: 95% CI, 53.2-56.4% for surgery + RT, vs. 46.2% for surgery alone 95% CI, 42.4-50.0%, p < 0.05). Use of adjuvant RT remained a significant predictor of survival on multivariable analysis (hazard ratio [HR], 0.75; 95% CI, 0.68-0.83; p < 0.001). Subset analyses demonstrated that adjuvant RT was associated with significantly improved survival for N1 (HR, 0.78; 95% CI; 0.67-0.90; p = 0.001), N2a (HR, 0.82; 95% CI, 0.67-0.99, p = 0.048) and N2b to N3 nodal disease (HR, 0.62; 95% CI, 0.51-0.75; p < 0.001). Adjuvant RT increased overall survival for node-positive patients with oropharynx (HR, 0.72; 95% CI, 0.57-0.90; p = 0.004), hypopharynx (HR, 0.66; 95% CI, 0.49 to 0.88; p = 0.004), larynx (HR, 0.66; 95% CI, 0.52-0.84; p = 0.001), and oral cavity (HR, 0.84; 95% CI, 0.73-0.98; p = 0.025) primary tumors. CONCLUSIONS: In a large population-based analysis, adjuvant RT significantly improves overall survival for patients with node-positive HNSCC. All nodal stages, including N1, appear to benefit from the addition of RT to definitive surgery.

(125)I monotherapy using D90 implant doses of 180 Gy or greater.

PURPOSE: The purpose of this study was to characterize the oncologic results and toxicity profile of patients treated with (125)I implants using the dose delivered to 90% of the gland from the dose-volume histogram (D90) of greater than 144 Gy. METHODS AND MATERIALS: From June 1995 to Feb 2005, a total of 643 patients were treated with (125)I monotherapy for T1-T2 prostate cancer with a D90 of 180 Gy or greater (median, 197 Gy; range, 180-267 Gy). Implantations were performed using a real-time ultrasound-guided seed-placement method and intraoperative dosimetry to optimize target coverage and homogeneity by using modified peripheral loading. We analyzed biochemical disease-free survival (bDFS) of 435 patients who had a minimum 2-year prostate-specific antigen follow-up (median follow-up, 6.7 years; range, 2.0-11.1 years). RESULTS: Five-year bDFS rates for the entire cohort using the American Society for Therapeutic Radiology and Oncology and Phoenix definitions were 96.9% and 96.5%, respectively. Using the Phoenix definition, 5-year bDFS rates were 97.3% for low-risk patients and 92.8% for intermediate/high-risk patients. The positive biopsy rate was 4.1%. The freedom rate from Grade 2 or higher rectal bleeding at 5 years was 88.5%. Acute urinary retention occurred in 10.7%, more commonly in patients with high pretreatment International Prostate Symptom Scores (p < 0.01). In patients who were potent before treatment, 73.4% remained potent at 5 years after implantation. CONCLUSIONS: Patients with a minimum D90 of 180 Gy had outstanding local control based on prostate-specific antigen control and biopsy data. Toxicity profiles, particularly for long-term urinary and sexual function, were excellent and showed that D90 doses of 180 Gy or greater performed using the technique described were feasible and tolerable.

Comparison study of intensity modulated arc therapy using single or multiple arcs to intensity modulated radiation therapy for high-risk prostate cancer.

PURPOSE: Intensity modulated arc therapy (IMAT) is a form of intensity modulated radiation therapy (IMRT) that delivers dose in single or multiple arcs. We compared IMRT plans versus single-arc field (1ARC) and multi-arc fields (3ARC) IMAT plans in high-risk prostate cancer. MATERIALS AND METHODS: Sixteen patients were studied. Prostate (PTV P ), right pelvic (PTV RtLN ) and left pelvic lymph nodes (PTV LtLN ), and organs at risk were contoured. PTV P , PTV RtLN , and PTV LtLN received 50.40 Gy followed by a boost to PTV B of 28.80 Gy. Three plans were per patient generated: IMRT, 1ARC, and 3ARC. We recorded the dose to the PTV, the mean dose (D MEAN ) to the organs at risk, and volume covered by the 50% isodose. Efficiency was evaluated by monitor units (MU) and beam on time (BOT). Conformity index (CI), Paddick gradient index, and homogeneity index (HI) were also calculated. RESULTS: Average Radiation Therapy Oncology Group CI was 1.17, 1.20, and 1.15 for IMRT, 1ARC, and 3ARC, respectively. The plans' HI were within 1% of each other. The D MEAN of bladder was within 2% of each other. The rectum D MEAN in IMRT plans was 10% lower dose than the arc plans (p < 0.0001). The GI of the 3ARC was superior to IMRT by 27.4% (p = 0.006). The average MU was highest in the IMRT plans (1686) versus 1ARC (575) versus 3ARC (1079). The average BOT was 6 minutes for IMRT compared to 1.3 and 2.9 for 1ARC and 3ARC IMAT (p < 0.05). CONCLUSION: For high-risk prostate cancer, IMAT may offer a favorable dose gradient profile, conformity, MU and BOT compared to IMRT.

Case series analysis of post-brachytherapy prostate edema and its relevance to post-implant dosimetry. Post-implant prostate edema and dosimetry.

PURPOSE: We evaluated the post-operative pattern of prostate volume (PV) changes following prostate brachytherapy (PB) and analyzed variables which affect swelling. MATERIAL AND METHODS: Twenty-nine patients treated with brachytherapy (14) or combined brachytherapy and external beam radiotherapy modality (15) underwent pre- and post-implant computed tomography (CT). Prostate volume measurements were done on post-operative days 1, 9, 30, and 60. An observer performed 139 prostate volume (PV) measurements. We analyzed the influence of pre-implant PV, number of needles and insertion attempts, number and activity of seeds, Gleason score, use of hormonal therapy and external beam radiation therapy on the extent of edema. We computed a volume correction factor (CF) to account for dosimetric changes between day 1 and day 30. Using the calculated CF, the dose received by 90% (D(90)) of the prostate on day 30 (D(90)Day30) was obtained by dividing day 1 (D(90)Day1) by the CF. RESULTS: The mean PV recorded on post-operative day 1 was 67.7 cm(3), 18.8 cm(3) greater than average pre-op value (SD 15.6 cm(3)). Swelling returned to pre-implant volume by day 30. Seed activity, treatment modality, and Gleason score were significant variables. The calculated CF was 0.76. After assessment using the CF, the mean difference between estimated and actual D(90)Day30 was not significant. CONCLUSIONS: We observed maximum prostate size on post-operative day 1, returning to pre-implant volume by day 30. This suggests that post-implant dosimetry should be obtained on or after post-operative day 30. If necessary, day 30 dosimetry can be estimated by dividing D(90)Day1 by a correction factor of 0.76.

The role of PET/CT in decreasing inter-observer variability in treatment planning and evaluation of response for cervical cancer.

We have previously introduced anatomic biologic contouring (ABC) with PET/CT, using a distinct "halo" to unify contouring methods in treatment planning for lung and head and neck cancers. The objective of this study is to assess the utility of PET/CT in planning and treatment response for cervical cancer. Forty-two patients with stages II-IIIB cervix cancer were planned for irradiation using PET/CT. A CT-based Gross Tumor Volume (GTV-CT) was delineated by two independent observers while the PET remained obscured. The Planning Target Volume (PTV) was obtained by adding a 1.5 cm margin around the GTV. The same volumes were recontoured using PET/CT data and termed GTV-ABC and PTV-ABC, respectively. The values of GTV-CT and GTV-ABC and the absolute differences between the two observers were analyzed. Additionally, 23 of these patients had PET/CT performed 3 months after treatment. The anatomic biologic value (ABV) was calculated using the product of maximum diameter and mean SUV of the cervical tumor. The pre- and post-treatment ABVs were compared. A "halo" was observed around areas of maximal SUV uptake. The mean halo SUV was 1.91 ± 0.56 (SD). The mean halo thickness was 2.12 ± 0.5 (SD) mm. Inter-observer GTV variability decreased from a mean volume difference of 55.36 cm(3) in CT-based planning to 12.29 cm(3) in PET/CT-based planning with a respective decrease in standard deviation (SD) from 55.78 to 10.24 (p <0.001). Comparison of mean pre-treatment and post-treatment ABV's revealed a decrease of ABV from 48.2 to 7.8 (p<0.001). PET/CT is a valuable tool in radiation therapy planning and evaluation of treatment response for cervical cancer. A clearly visualized "halo" was successfully implemented in GTV contouring in cervical cancer, resulting in decreased inter-observer variability in planning. PET/CT has the ability to quantify treatment response using anatomic biologic value.

Adjuvant radiotherapy improves overall survival for patients with lymph node-positive head and neck squamous cell carcinoma.

BACKGROUND: Although adjuvant radiotherapy (RT) is often recommended for locally advanced squamous cell carcinoma of the head and neck (HNSCC), its effect on overall or cancer-specific survival has not been clearly demonstrated. In the current study, the frequency and effect of adjuvant RT on overall survival was investigated in patients with resected lymph node-positive head and neck cancer. METHODS: Within the Surveillance, Epidemiology, and End Results (SEER) database, patients were selected with lymph node-positive HNSCC (American Joint Committee on Cancer and SEER stage 3/4) who were treated either with surgery alone or surgery and RT and were diagnosed between 1988 and 2001. A total of 8795 patients who met the inclusion criteria for analysis comprised the study population, with a median follow-up of 4.3 years for patients still alive at the time of last follow-up. RESULTS: Adjuvant RT was utilized in 84% of patients. Adjuvant RT improved the 5-year overall survival (43.2% [95% confidence interval (95% CI), 41.9-44.4%] for surgery + RT vs 33.4% [95% CI, 30.7-36.0%] for surgery alone; P < .001) and cancer-specific survival (50.9% for surgery + RT vs 42.1% for surgery) on univariate analysis. On multivariate analysis, adjuvant RT (hazards ratio [HR] of 0.78; 95% CI, 0.71-0.86 [P < .001]) remained a significant predictor of improved survival. The significant benefit of radiation on overall survival was noted for lymph node-positive patients with both primary tumors localized to the involved organ (HR of 0.81; 95% CI, 0.71-0.94 [P = .007]) and more locally invasive primary tumors (HR of 0.77; 95% CI, 0.68-0.87 [P < .001]). CONCLUSIONS: In what to the authors' knowledge is the largest reported analysis of adjuvant RT in patients with locally advanced HNSCC published to date, adjuvant RT resulted in an approximately 10% absolute increase in 5-year cancer-specific survival and overall survival for patients with lymph node-positive HNSCC compared with surgery alone. Despite combined surgery and adjuvant RT, outcomes in this high-risk population remain suboptimal, emphasizing the need for continued investigation of innovative treatment approaches.