Zelanna Goldberg

Human in vivo dose-response to controlled, low-dose low linear energy transfer ionizing radiation exposure.

Purpose: The effect of low doses of low–linear energy transfer (photon) ionizing radiation (LDIR, <10 cGy) on human tissue when exposure is under normal physiologic conditions is of significant interest to the medical and scientific community in therapeutic and other contexts. Although, to date, there has been no direct assessment of the response of human tissue to LDIR when exposure is under normal physiologic conditions of intact three-dimensional architecture, vasculature, and cell-cell contacts (between epithelial cells and between epithelial and stromal cells). Experimental Design: In this article, we present the first data on the response of human tissue exposed in vivo to LDIR with precisely controlled and calibrated doses. We evaluated transcriptomic responses to a single exposure of LDIR in the normal skin of men undergoing therapeutic radiation for prostate cancer (research protocol, Health Insurance Portability and Accountability Act–compliant, Institutional Review Board–approved). Using newly developed biostatistical tools that account for individual splice variants and the expected variability of temporal response between humans even when the outcome is measured at a single time, we show a dose-response pattern in gene expression in a number of pathways and gene groups that are biologically plausible responses to LDIR. Results: Examining genes and pathways identified as radiation-responsive in cell culture models, we found seven gene groups and five pathways that were altered in men in this experiment. These included the Akt/phosphoinositide-3-kinase pathway, the growth factor pathway, the stress/apoptosis pathway, and the pathway initiated by transforming growth factor-β signaling, whereas gene groups with altered expression included the keratins, the zinc finger proteins and signaling molecules in the mitogen-activated protein kinase gene group. We show that there is considerable individual variability in radiation response that makes the detection of effects difficult, but still feasible when analyzed according to gene group and pathway. Conclusions: These results show for the first time that low doses of radiation have an identifiable biosignature in human tissue, irradiated in vivo with normal intact three-dimensional architecture, vascular supply, and innervation. The genes and pathways show that the tissue (a) does detect the injury, (b) initiates a stress/inflammatory response, (c) undergoes DNA remodeling, as suggested by the significant increase in zinc finger protein gene expression, and (d) initiates a “pro-survival” response. The ability to detect a distinct radiation response pattern following LDIR exposure has important implications for risk assessment in both therapeutic and national defense contexts.

Integration of Novel Therapeutics into Combined Modality Therapy of Locally Advanced Non–Small Cell Lung Cancer

Novel therapeutic agents (NTA) directed against a wide array of newly described molecular targets are now entering clinical investigation, many in the treatment of non–small cell lung cancer (NSCLC). The great majority of these clinical trials have been directed toward patients with advanced stage (metastatic) disease. More recently, study of NTAs has turned toward earlier-stage disease. Locally advanced, or stage III, NSCLC represents a large and heterogeneous group of patients and several clinically distinct substages. During the last 15 years, randomized clinical trials have shown improved survival with sequential chemoradiation compared with radiation alone and, more recently, the superiority of concurrent versus sequential chemoradiation. As NTAs have increasingly shown clinical activity against NSCLC, questions of how to incorporate them into clinical trials in stage III disease, whether they should be given together with radiotherapy, substituting for chemotherapy, or whether they should be added to current chemoradiation strategies, all remain as issues. Here, we describe conceptual issues, preclinical rationale, and ongoing or planned clinical trials incorporating NTAs into current treatment paradigms for unresectable stage III NSCLC.

Phase II Study of Tirapazamine, Cisplatin, and Etoposide and Concurrent Thoracic Radiotherapy for Limited-Stage Small-Cell Lung Cancer: SWOG 0222

PURPOSE A SWOG pilot study (S0004) showed that tirapazamine (TPZ) when combined with concurrent chemoradiotherapy yielded a promising median survival of 22 months in limited-stage small-cell lung cancer (LSCLC). We report results of the phase II study designed to confirm this result. PATIENTS AND METHODS The concurrent phase consisted of two cycles of cisplatin, etoposide, and once-daily radiation to 61 Gy. TPZ was given at 260 mg/m(2) on days 1, 29, and at 160 mg/m(2) on days 8, 10, 12, 36, 38, and 40. Consolidation consisted of two cycles of cisplatin and etoposide. Complete responders received prophylactic cranial irradiation. Results were considered promising if the median survival time was at least 21 months and of no further interest if < or = 14 months. RESULTS S0222 was closed early due to a report of excess toxicity for TPZ in a head and neck cancer trial elsewhere. Of planned 85 patients, 69 were accrued. In 68 assessable patients, 17 (25%) had grade 3 to 4 esophagitis and eight (12%) had grade 3 febrile neutropenia during the concurrent phase. There were three possible treatment-related deaths, two in concurrent phase (one progressive disease not otherwise specified within 30 days, one pericardial effusion) and one in consolidation phase (esophageal hemorrhage). At a median follow-up of 35 months, median progression-free survival was 11 months (95% CI, 10 to 13 months) and median overall survival was 21 months (95% CI, 17 to 33 months). CONCLUSION S0222 showed acceptable levels of toxicity and similar promising median survival as S0004. Further study of hypoxia-targeted therapy is warranted in LSCLC.

A Phase II Study of Paclitaxel, Carboplatin, and Radiation with or without Surgery for Esophageal Cancer

Background: Cisplatin-based chemoradiotherapy (CRT) has been a standard treatment for patients with locally advanced esophageal cancer. However, cisplatin is associated with significant toxicity. We conducted a phase II clinical trial of concurrent paclitaxel, carboplatin, and radiation with or without surgery as an alternative to the standard cisplatin-based CRT for localized and metastatic esophageal cancer. Methods: Fifty patients with esophageal cancer were enrolled: 16 patients with stage II, eight patients with stage III, and 26 patients with stage IV disease. Two thirds (67%) of patients had adenocarcinoma and one third (33%) with squamous histology. Patients with resectable disease were treated with paclitaxel 30 mg/m2, twice weekly for 10 doses, carboplatin AUC (area under the curve) 1.5 weekly for five doses; and concurrent radiation, 1.8 Gy/day, for a total of 45 Gy, followed by esophagectomy. Without surgery, patients received an additional dose each of paclitaxel and carboplatin with concurrent radiation for a total of 50.4 Gy, followed by two consolidation cycles of paclitaxel (200 mg/m2) and carboplatin (AUC 6). Results: During CRT, common stage III/IV toxicities included nausea/emesis (19%), esophagitis (9%), and neutropenia (4%). For consolidation chemotherapy, neutropenia (23%), neuropathy (8%) and nausea/emesis (4%) were the most common stage III/IV side effects. After CRT, 26% had a complete response, 17% had a partial response, and 41% had stable disease. Ninety-one percent of patients had clinical improvement of dysphagia. With a median follow-up of 32 months, the median survival was 12 months for patients with metastatic disease, 44 months for localized disease treated with esophagectomy, and >44 months for localized disease treated with definitive CRT. Conclusions: The regimen of paclitaxel, carboplatin, and radiation with or without surgery is well tolerated with promising efficacy for patients with esophageal cancer.

A method for detection of differential gene expression in the presence of inter-individual variability in response

MOTIVATION Many stimuli to biological systems result in transcriptional responses that vary across the individual organism either in type or in timing. This creates substantial difficulties in detecting these responses. This is especially the case when the data for any one individual are limited and when the number of genes, probes or probe sets is large. RESULTS We have developed a procedure that allows for sensitive detection of transcriptional responses that differ between individuals in type or in timing. This consists of four steps: one is to identify a group of genes, probes or probe sets that detect genes that belong to a molecular class or to a common pathway. The second is to conduct a statistical test of the hypothesis that the gene is differentially expressed for each individual and for each gene in the set. The third is to examine the collection of these statistics to see if there is a detectable signal in the aggregate of them. The final step is to assess the significance of this by resampling to avoid correlational bias. AVAILABILITY Software in the form of R code to perform the required test is available from the first author or from his website http://www.idav.ucdavis.edu/~dmrocke/software; however the procedures are also easily performed using any standard statistical software.

Dosimetry for quantitative analysis of the effects of low-dose ionizing radiation in radiation therapy patients

Abstract Lehmann, J., Stern, R. L., Daly, T. P., Rocke, D. M., Schwietert, C. W., Jones, G. E., Arnold, M. L., Hartmann Siantar, C. L. and Goldberg, Z. Dosimetry for Quantitative Analysis of the Effects of Low-Dose Ionizing Radiation in Radiation Therapy Patients. Radiat. Res. 165, 240–247 (2006). We have developed and validated a practical approach to identifying the location on the skin surface that will receive a prespecified biopsy dose (ranging down to 1 cGy) in support of in vivo biological dosimetry in humans. This represents a significant technical challenge since the sites lie on the patients surface outside the radiation fields. The PEREGRINE Monte Carlo simulation system was used to model radiation dose delivery, and TLDs were used for validation on phantoms and for confirmation during patient treatment. In the developmental studies, the Monte Carlo simulations consistently underestimated the dose at the biopsy site by approximately 15% (of the local dose) for a realistic treatment configuration, most likely due to lack of detail in the simulation of the linear accelerator outside the main beam line. Using a single, thickness-independent correction factor for the clinical calculations, the average of 36 measurements for the predicted 1-cGy point was 0.985 cGy (standard deviation: 0.110 cGy) despite patient breathing motion and other real-world challenges. Since the 10-cGy point is situated in the region of high-dose gradient at the edge of the field, patient motion had a greater effect, and the six measured points averaged 5.90 cGy (standard deviation: 1.01 cGy), a difference that is equivalent to approximately a 6-mm shift on the patients surface.

Proteomic analysis of low dose arsenic and ionizing radiation exposure on keratinocytes.

Human exposure to arsenic and ionizing radiation (IR) occur environmentally at low levels. While the human health effects of arsenic and IR have been examined separately, there is little information regarding their combined effects at doses approaching environmental levels. Arsenic toxicity may be affected by concurrent IR especially given their known individual carcinogenic actions at higher doses. We found that keratinocytes responded to either low dose arsenic and/or low dose IR exposure, resulting in differential proteomic expression based on 2‐DE, immunoblotting and statistical analysis. Seven proteins were identified that passed a rigorous statistical screen for differential expression in 2‐DE and also passed a strict statistical screen for follow‐up immunoblotting. These included: α‐enolase, epidermal‐fatty acid binding protein, heat shock protein 27, histidine triad nucleotide‐binding protein 1, lactate dehydrogenase A, protein disulfide isomerase precursor, and S100A9. Four proteins had combined effects that were different than would be expected based on the response to either individual toxicant. These data demonstrate a possible reaction to the combined insult that is substantially different from that of either separate treatment. Several proteins had different responses than what has been seen from high dose exposures, adding to the growing literature suggesting that the cellular responses to low dose exposures are distinct.

Enhancement of Radiation Cytotoxicity by UCN-01 in Non-small Cell Lung Carcinoma Cells

Abstract Mack, P. C., Jones, A. A., Gustafsson, M. H., Gandara, D. R., Gumerlock, P. H. and Goldberg, Z. Enhancement of Radiation Cytotoxicity by UCN-01 in Non-small Cell Lung Carcinoma Cells. Radiat. Res. 162, 623–634 (2004). Thoracic ionizing radiation is a standard component of combined-modality therapy for locally advanced non-small cell lung cancer. To improve low 5-year survival rates (5– 15%), new strategies for enhancing the effectiveness of ionizing radiation are needed. The kinase inhibitor UCN-01 has multiple cell cycle effects, including abrogation of DNA damage-induced S- and G2-phase arrest, which may limit DNA repair prior to mitosis. To test the hypothesis that therapy-induced cell cycle effects would have an impact on the efficacy of a combination of UCN-01 plus ionizing radiation, the cell cycle responses of the non-small cell lung cancer cell lines Calu1 (TP53-null) and A549 (wild-type TP53) to 2 Gy ionizing radiation were correlated with clonogenic survival after irradiation plus UCN-01. Irradiated cells were exposed to UCN-01 simultaneously and at 3-h increments after irradiation. In Calu1 cells but not A549 cells, sequence-dependent potentiation of radiation by UCN-01 was observed, with maximal interaction occurring when UCN-01 was administered 6 h after irradiation. This coincided with the postirradiation time with the greatest depletion of cells from G1. Abrogation of G2 arrest was observed regardless of TP53 status. The role of TP53 was investigated using siRNA to achieve gene silencing. These studies demonstrated that radiation plus UCN-01 was more effective in cells with diminished TP53 activity, associated with a reduced G1 checkpoint arrest. These studies indicate that simultaneous elimination of multiple DNA damage-induced checkpoints in G1, S and G2 may enhance the effects of radiation and that drug scheduling may have an impact on clinical efficacy.

Assessing probe-specific dye and slide biases in two-color microarray data.

BackgroundA primary reason for using two-color microarrays is that the use of two samples labeled with different dyes on the same slide, that bind to probes on the same spot, is supposed to adjust for many factors that introduce noise and errors into the analysis. Most users assume that any differences between the dyes can be adjusted out by standard methods of normalization, so that measures such as log ratios on the same slide are reliable measures of comparative expression. However, even after the normalization, there are still probe specific dye and slide variation among the data. We define a method to quantify the amount of the dye-by-probe and slide-by-probe interaction. This serves as a diagnostic, both visual and numeric, of the existence of probe-specific dye bias. We show how this improved the performance of two-color array analysis for arrays for genomic analysis of biological samples ranging from rice to human tissue.ResultsWe develop a procedure for quantifying the extent of probe-specific dye and slide bias in two-color microarrays. The primary output is a graphical diagnostic of the extent of the bias which called ECDF (Empirical Cumulative Distribution Function), though numerical results are also obtained.ConclusionWe show that the dye and slide biases were high for human and rice genomic arrays in two gene expression facilities, even after the standard intensity-based normalization, and describe how this diagnostic allowed the problems causing the probe-specific bias to be addressed, and resulted in important improvements in performance. The R package LMGene which contains the method described in this paper has been available to download from Bioconductor.

Film dosimetry in the peripheral region using multiple sensitometric curves

We present a method for applying film dosimetry to the peripheral region utilizing multiple sensitometric curves. There are many instances when the dose to the peripheral region outside the field edges is of clinical and/or research interest. Published peripheral dose data may be insufficient if detailed dose modeling is required, and in those cases measurements must be performed. Film dosimetry is an attractive approach for dose measurement in the peripheral region because it integrates dose, overcoming the low-dose-rate problem, and is time efficient, as it acquires an entire plan of data in a single exposure. However, film response increases at energies below approximately 300 keV. As the scattered photon spectrum changes with distance from the field edge, this increased film sensitivity causes changes in the film response along profiles perpendicular to the field edge. A single sensitometric curve is therefore no longer sufficient for accurate conversion of the optical density to dose. Our new method uses multiple sensitometric curves defined at increasing distances from the field edge. To convert an optical density profile, the dose at each point in the profile is defined as a linear combination of the doses calculated using the two sensitometric curves that bracket the point of interest. A single set of sensitometric curves derived at one field size and source-to-surface distance (SSD) can be applied to density profiles for other field sizes and SSDs. We verified our new method by comparison to ion chamber measurements using three different types of film. Agreement with chamber measurements was within 7%, or less than 2 mm in regions of high gradient, over a wide range of field sizes and SSDs.