K.K. Ang

Apoptosis in irradiated murine tumors

Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors.

DNA repair biomarker profiling of head and neck cancer: Ku80 expression predicts locoregional failure and death following radiotherapy

Purpose: Radiotherapy plays an integral role in the treatment of head and neck squamous cell carcinoma (HNSCC). Although proteins involved in DNA repair may predict HNSCC response to radiotherapy, none has been validated in this context. We examined whether differential expression of double-strand DNA break (DSB) repair proteins in HNSCC, the chief mediators of DNA repair following irradiation, predict for treatment outcomes. Experimental Design: Archival HNSCC tumor specimens (n = 89) were assembled onto a tissue microarray and stained with antibodies raised against 38 biomarkers. The biomarker set was enriched for proteins involved in DSB repair, in addition to established mechanistic markers of radioresistance. Staining was correlated with treatment response and survival alongside established clinical and pathologic covariates. Results were validated in an independent intramural cohort (n = 34). Results: Ku80, a key mediator of DSB repair, correlated most closely with clinical outcomes. Ku80 was overexpressed in half of all tumors, and its expression was independent of all other covariates examined. Ku80 overexpression was an independent predictor for both locoregional failure and mortality following radiotherapy (P < 0.01). The predictive power of Ku80 overexpression was confined largely to HPV-negative HNSCC, where it conferred a nine-fold greater risk of death at two years. Conclusions: Ku80 overexpression is a common feature of HNSCC, and is a candidate DNA repair-related biomarker for radiation treatment failure and death, particularly in patients with high-risk HPV-negative disease. It is a promising, mechanistically rational biomarker to select individual HPV-negative HNSCC patients for strategies to intensify treatment. Clin Cancer Res; 17(7); 2035–43. ©2011 AACR.

Response of parotid gland organ culture to radiation

Organ cultures of rhesus parotid tissue in medium enriched with homologous serum and supplemented with low levels of isoproterenol were irradiated with single photon doses of 2.5, 5.0, 7.5, 10.0, 12.5, or 15.0 Gy. Following irradiation, the tissue was incubated while being agitated for 24 h; then it was fixed in formalin. Microscopically, death of serous acinar cells was seen in areas unaltered by autolysis. Based on the numbers of nuclear aberrations, the dose response did not differ significantly from that observed at 24 h in parotid gland irradiated in vivo. The similar rapid response under the two conditions shows that apoptosis of irradiated parotid serous cells is a direct expression of interphase cell death.

An International Collaboration to Harmonize the Quantitative Plasma Epstein-Barr Virus DNA Assay for Future Biomarker-Guided Trials in Nasopharyngeal Carcinoma

Purpose: Persistently elevated posttreatment plasma EBV DNA is a robust predictor of relapse in nasopharyngeal carcinoma (NPC). However, assay standardization is necessary for use in biomarker-driven trials. We conducted a study to harmonize the method between four centers with expertise in EBV DNA quantitation. Experimental Design: Plasma samples of 40 patients with NPC were distributed to four centers. DNA was extracted and EBV DNA copy number was determined by real-time quantitative PCR (BamHI-W primer/probe). Centers used the same protocol but generated their own calibrators. A harmonization study was then conducted using the same calibrators and PCR master mix and validated with ten pooled samples. Results: The initial intraclass correlations (ICC) for the first 40 samples between each center and the index center were 0.62 [95% confidence interval (CI): 0.39–0.78], 0.70 (0.50–0.83), and 0.59 (0.35–0.76). The largest variability was the use of different PCR master mixes and calibrators. Standardization improved ICC to 0.83 (0.5–0.95), 0.95 (0.83–0.99) and 0.96 (0.86–0.99), respectively, for ten archival frozen samples. For fresh plasma with spiked-in EBV DNA, correlations were more than 0.99 between the centers. At 5 EBV DNA copies per reaction or above, the coefficient of variance (CV) was less than 10% for the cycle threshold (Ct) among all centers, suggesting this concentration can be reliably used as a cutoff for defining the presence of detectable EBV DNA. Conclusions: Quantitative PCR assays, even when conducted in experienced clinical labs, can yield large variability in plasma EBV DNA copy numbers without harmonization. The use of common calibrators and PCR master mix can help to reduce variability. Clin Cancer Res; 19(8); 2208–15. ©2013 AACR.

The relationship between apoptosis and atrophy in the irradiated lacrimal gland

Atrophy is generally considered to be a true late effect of radiation. However, in serous glands, atrophy was thought to be a consequential late effect because serous cells die within hours of irradiation and the apparent effects of atrophy are observed contemporaneously with radiation treatment. Therefore, to determine the pathogenesis of atrophy in serous glands, it is necessary to differentiate between parenchymal loss as a result of direct radiation death of serous cells and parenchymal loss as a result of serous cell death that is secondary to fibrosis, vascular damage, or precursor cell death. The lacrimal glands of 62 rhesus monkeys have been irradiated to single doses of 2.5 to 20 Gy and examined at intervals of 4 hr to 112 days postirradiation. Serous cells (nuclei) and acini were counted in at least 30 high power fields per (dose, time) point. At each dose and time of sacrifice, the average number of nuclei per acinus and the average number of acini per high power field were calculated. Also at each dose and time, the distribution of the number of nuclei per acinus was examined to determine how the frequency of acinar sizes changed as a function of irradiation. The number of cells per acinus appears to rise initially, but this is likely a result of the degranulated cells being physically smaller, yielding an artificially higher count. Within 4 days after 12.5 Gy, the average number of nuclei per acinus approaches control values and remains within the range of controls for at least 112 days. The number of acini per high power field decreases steadily for 30 days after 12.5 Gy. From 30 to 112 days, there is some recovery of this number, but it remains well below control values. At 24 hr, the number of nuclei per acinus shows a distinct dose response up to 20 Gy. However, at 30 days there is no evidence of a dose response for this parameter. These results indicate that even though serous cells die in significant numbers within hours of irradiation, the atrophy of the lacrimal gland (and by extension, the parotid gland) is a result of the death of the serous stem cell or precursor. Consequently, protection of serous cells from radiation apoptosis will not diminish serous gland atrophy.

Quality-Adjusted Survival Analysis of Radiation Therapy Oncology Group (RTOG) 90-03: Phase III Randomized Study Comparing Altered Fractionation to Standard Fractionation Radiotherapy for Locally Advanced Head and Neck Squamous Cell Carcinoma

To evaluate quality‐adjusted survival (QAS) of patients with locally advanced squamous cell carcinoma of the head and neck treated with 4 different radiation fractionation schedules.

Geographic variation in human papillomavirus–related oropharyngeal cancer: Data from 4 multinational randomized trials

There are variations in the proportions of head and neck cancers caused by the human papillomavirus (HPV) between countries and regions. It is unclear if these are true variations or due to different study designs and assays.

Role of Epidermal Growth Factor Receptor and Its Inhibition in Radiotherapy

Growth factors are substances that regulate cell growth and proliferation, and maintain architectural and functional homeostasis in normal tissues. They bind to specific cell membrane receptors setting in motion a highly regulated network of cellular events, signal transduction, gene activation, transcription, etc., which then regulate cell cycle checkpoints. Growth factors act locally by autocrine or paracrine functions or on distant tissues via endocrine activities. Over a hundred different growth factors have been identified, many of which interact with each other rendering complimentary or opposing effects on cell growth. Compared to normal tissues, growth factor signaling pathways in tumors are commonly subverted to result in inordinate division and function of cells. Tumors, which are composed of both malignant cells and many types of normal cells that infiltrate tumors, including endothelial cells, fibroblasts and lymphoid cells, secrete a variety of growth factors, which regulate tumor growth and dissemination. Comparable to their action in normal tissues, these factors can be autocrine, paracrine or affect cells at a distance from their production site and may have complementary or opposing effects on tumor cell growth.

Role of Cyclooxygenase-2 (COX-2) and Its Inhibition in Tumor Biology and Radiotherapy

In recent years our understanding of the fundamental biology of cancer has greatly increased. It has become clear that many molecular processes and signaling pathways that normally regulate growth, survival and function of cells become dysregulated in cancer cells, contributing to the more aggressive behavior of malignant tumors. In parallel, the knowledge of molecular, biochemical and cellular effects of ionizing radiation in both tumors and normal tissues has broadened, allowing design and implementation of more effective therapy. Increasing evidence shows that dysregulation in epidermal growth factor receptor (EGFR) signaling, presence of cyclooxygenase-2 (COX-2) enzyme, mutated ras, and secretion of proangiogenic molecules by tumor cells are associated with tumor resistance to cytotoxic therapies, including radiotherapy. Targeting these molecules or their signaling pathways has increasingly been explored as a therapeutic strategy, and newly developed agents directed against these molecules are already reaching the clinic. In preclinical studies, many of these novel agents have been particularly effective when combined with chemotherapeutic drugs or radiation, where they rendered tumor cells more susceptible to the cytotoxic action of chemotherapy and radiation. Targeting dysregulated molecular processes should, in theory, affect only malignant cells and thus is unlikely to sensitize normal tissues to injury by cytotoxic drugs or radiation.

Tissue Repair and Repopulation in the Tumor Bed Effect

These experiments were designed to study the kinetics and magnitude of cell repair and repopulation in tissues whose damage results in the tumor bed effect. The right hind thighs of mice were irradiated with single doses or two equal gamma-ray fractions. Interfraction intervals ranging from 30 min to 24 h (to measure the kinetics of repair from sublethal damage) and 6 and 12 weeks (to determine the extent of repopulation) were used. One day after the second radiation dose 5 X 10(5) FSA tumor cells were inoculated into the center of the irradiated field. Radiation dose-response curves were obtained by calculating the time required for tumors to reach 12 mm diameter. No recovery occurred within 6 h of the radiation delivery as measured by this assay. Some recovery, 3.2-4.6 Gy above a single radiation dose, occurred when the interval between two fractions was 24 h. With increasing interfraction intervals of 6 and 12 weeks further dose sparing occurred in the amount of 5.0-6.9 and 7.5-8.3 Gy, respectively. The data suggest that repopulation is the major contributor to the radiation dose-sparing recovery of stromal tissue and that some proliferative response may occur as early as 1 day after the first irradiation.