Kathryn A. Mason

Animal Models for Medical Countermeasures to Radiation Exposure

Abstract Since September 11, 2001, there has been the recognition of a plausible threat from acts of terrorism, including radiological or nuclear attacks. A network of Centers for Medical Countermeasures against Radiation (CMCRs) has been established across the U.S.; one of the missions of this network is to identify and develop mitigating agents that can be used to treat the civilian population after a radiological event. The development of such agents requires comparison of data from many sources and accumulation of information consistent with the “Animal Rule” from the Food and Drug Administration (FDA). Given the necessity for a consensus on appropriate animal model use across the network to allow for comparative studies to be performed across institutions, and to identify pivotal studies and facilitate FDA approval, in early 2008, investigators from each of the CMCRs organized and met for an Animal Models Workshop. Working groups deliberated and discussed the wide range of animal models available for assessing agent efficacy in a number of relevant tissues and organs, including the immune and hematopoietic systems, gastrointestinal tract, lung, kidney and skin. Discussions covered the most appropriate species and strains available as well as other factors that may affect differential findings between groups and institutions. This report provides the workshop findings.

Is the therapeutic index better with gemcitabine-based chemoradiation than with 5-fluorouracil-based chemoradiation in locally advanced pancreatic cancer?

PURPOSE To retrospectively compare the toxicity and efficacy of concurrent gemcitabine-based chemoradiation with that of concurrent 5-fluorouracil (5-FU)-based chemoradiation in patients with unresectable pancreatic cancer. PATIENTS AND METHODS Between September 1996 and May 2000, 114 patients with localized unresectable adenocarcinoma of the pancreas were treated with concurrent chemoradiation. Locally advanced unresectable disease was defined as low-density tumor in contact with the superior mesenteric artery (SMA) or celiac artery, or occlusion of the superior mesenteric-portal venous confluence. Fifty-three patients were selected to receive gemcitabine in 7 weekly cycles (250-500 mg/m(2)) with concurrent radiotherapy (median dose 30 Gy, range 30-33 Gy in 10-11 fractions). The remaining 61 patients received continuous-infusion 5-FU (200-300 mg/m(2)) with concurrent radiotherapy (30 Gy in 10 fractions). Radiotherapy was delivered to the primary tumor and regional lymphatics. Patients receiving gemcitabine and those receiving 5-FU had a similar mean Karnofsky performance status (KPS, 89% vs. 86%), distribution of tumor grade (43% vs. 33% poorly differentiated), and percent weight loss (all p = NS). However, patients treated with gemcitabine had a significantly larger median maximum cross-sectional tumor area (TA, 8.8 cm(2) vs. 5.7 cm(2), p = 0.046) and were significantly younger (median age 60 vs. 68 years, p <0.001). Severe acute toxicity (ST) was defined as toxicity requiring a hospital stay of more than 5 days, mucosal ulceration with bleeding, more than 3 dose deletions of gemcitabine or discontinuation of 5-FU, or toxicity resulting in surgical intervention or death. Kaplan-Meier analysis was used to calculate the actuarial rate of local progression on imaging (LP), the rate of distant metastasis (DM), and the overall survival (OS) rate. The imaging was reviewed in resected patients. RESULTS Patients receiving gemcitabine developed significantly more ST during treatment (23% vs. 2%, p < 0.0001) than did those receiving 5-FU. Patients treated with gemcitabine had a similar 10-month LP rate (62% vs. 61%), 10-month DM rate (55% vs. 47%), 1-year OS rate (42% vs. 28%), and median OS duration (11 months vs. 9 months) to patients treated with 5 FU (all p = NS). Five patients who received gemcitabine and 1 patient who received 5-FU underwent margin-negative pancreaticoduodenectomy after chemoradiation. Three patients had a short segment (<or= 1 cm in length) of low-density tumor abutting the SMA, 1 had involvement of the common hepatic artery, and 1 had a short-segment occlusion of the superior mesenteric vein, amenable to venous resection and reconstruction. The other patient was thought to have inflammatory changes discontiguous with the tumor surrounding the SMA, which resolved after therapy. TA >10 cm(2) (p = 0.03) and poor differentiation (p = 0.07) were associated with a worse survival duration; however, other factors, such as KPS and weight loss >10% and age did not influence OS. CONCLUSION Despite the selection of healthier patients to receive gemcitabine, there was a significantly higher severe toxicity rate than with 5-FU. The median and 1-year survivals were not significantly different with the use of concurrent gemcitabine; however, the tumors treated were significantly larger. Additionally, a small number of patients with minimal arterial involvement whose disease met our radiographic definition of unresectable disease had margin-negative resections after treatment with gemcitabine-based chemoradiation. These possible benefits and the high rate of severe toxicity define a very narrow therapeutic index for concurrent gemcitabine-based chemoradiation given by this schedule of administration.

Kinetics of mitotic arrest and apoptosis in murine mammary and ovarian tumors treated with taxol

The kinetics of taxol-induced mitotic arrest and apoptosis in murine mammary carcinoma MCA-4 and ovarian carcinoma OCA-I tumors were determined to establish a possible causative relationship between mitotic arrest and apoptosis and to see whether these cellular effects of taxol would correlate with the extent of its antitumor efficacy. Mice bearing 8-mm tumors in a hind leg were given taxol i.v. at a dose of 10–80 mg/kg. Both tumors responded to taxol by significant growth delay or transient regression; in general, the response was greater as the dose of taxol was increased. For kinetics studies the mice were treated with 60 mg/kg taxol given once when tumors were 8 mm in size or twice, with the second dose being given 3 days after the first. At various times ranging from 1 to 96 h after treatment with taxol, tumors were histologically analyzed to quantify mitotic and apoptotic activity. After a single dose of taxol, mitotic arrest was visible at 1 h, and the mitotic index increased with time to reach peak values of 36% in MCA-4 tumors and 22% in OCA-I tumors at 9 h. The index then declined to a baseline of 1%–3% at 3 days for MCA-4 tumors and 1 day for OCA-I tumors. Apoptosis followed mitotic arrest, beginning at the time of peak mitotic arrest, increasing to the highest level of about 20% at 18–24 h after treatment and gradually declining to the normal level of 3%–6% after 3–4 days. Nuclear material progressively condensed in mitotically arrested cells, culminating in the frank appearance of multiple apoptotic bodies. The change in cell morphology plus the dynamics of apoptosis development imply that a large percentage of tumor cells arrested in mitosis by taxol die by apoptosis. Kinetic analysis undertaken after the second dose of taxol showed a considerably lower percentage of cells arrested in mitosis as compared with that seen after a single dose, and the induction of apoptosis by the second dose was minimal. However, the antitumor efficacy of the second dose of taxol was similar to or better than that of the first dose, implying that in addition to mitotic arrest and apoptosis, there exist other mechanisms by which taxol exerts its antitumor action.

MK-1775, a novel wee1 kinase inhibitor, radiosensitizes p53-defective human tumor cells

Purpose: Radiotherapy is commonly used to treat a variety of solid tumors. However, improvements in the therapeutic ratio for several disease sites are sorely needed, leading us to assess molecularly targeted therapeutics as radiosensitizers. The aim of this study was to assess the wee1 kinase inhibitor, MK-1775, for its ability to radiosensitize human tumor cells. Experimental Design: Human tumor cells derived from lung, breast, and prostate cancers were tested for radiosensitization by MK-1775 using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of MK-1775 to abrogate the radiation-induced G2 block, thereby allowing cells harboring DNA lesions to prematurely progress into mitosis, was determined using flow cytometry and detection of γ-H2AX foci. The in vivo efficacy of the combination of MK-1775 and radiation was assessed by tumor growth delay experiments using a human lung cancer cell line growing as a xenograft tumor in nude mice. Results: Clonogenic survival analyses indicated that nanomolar concentrations of MK-1775 radiosensitized p53-defective human lung, breast, and prostate cancer cells but not similar lines with wild-type p53. Consistent with its ability to radiosensitize, MK-1775 abrogated the radiation-induced G2 block in p53-defective cells but not in p53 wild-type lines. MK-1775 also significantly enhanced the antitumor efficacy of radiation in vivo as shown in tumor growth delay studies, again for p53-defective tumors. Conclusions: These results indicate that p53-defective human tumor cells are significantly radiosensitized by the potent and selective wee1 kinase inhibitor, MK-1775, in both the in vitro and in vivo settings. Taken together, our findings strongly support the clinical evaluation of MK-1775 in combination with radiation. Clin Cancer Res; 17(17); 5638–48. ©2011 AACR.

Chemoradiotherapy: Emerging treatment improvement strategies

The use of chemotherapeutic drugs in combination with radiotherapy has become a common strategy for the treatment of advanced cancer. Solid evidence exists showing that chemotherapy administered during the course of radiotherapy (concurrent chemoradiotherapy) increases both local tumor control and patient survival in a number of cancer sites, including head and neck cancer. These therapy improvements, however, have been achieved at the expense of considerable toxicity, which underscores the need for further improvements.

Toxicity and Efficacy of Concurrent Gemcitabine and Radiotherapy for Locally Advanced Pancreatic Cancer.

SummaryBackground. Gemcitabine and radiotherapy are a potent combination. A clinical assessment of the therapeutic ratio for locally advanced pancreatic cancer patients has not yet been reported.Aim of Study. To assess the toxicity, survival, and pattern of failure of locally advanced pancreatic cancer patients treated with concurrent gemcitabine-based chemoradiation.Patients and Methods. Between the dates of December 1996 and August 2000 51 patients with locally advanced unresectable adenocarcinoma of the pancreas were treated with concurrent gemcitabine and radiotherapy at MDACC. Patients received 250–500 mg/m2 of gemcitabine weekly ×7 over 30 min and 30–33 Gy in 10–11 fractions over two weeks to the primary tumor and regional lymphatics. Severe toxicity was defined as admission >5 d, mucosal ulceration, >3 dose deletions of gemcitabine or toxicity resulting in surgical intervention or that resulted in death.Results. The median survival was 11 mo. Overall, 37 of 51 patients had objective evidence of local progression. The actuarial rate of local progression rate at 9 mo was 70%. The 9-mo distant metastasis rate was 52%. Tumors ≥ 10 cm2 had worse local control, distant control, and overall survival. Six patients underwent pancreaticoduodenectomy after therapy. After review of the imaging, only four of these patients had minimal arterial involvement, one was incorrectly staged, and one had initial inflammatory change on CT that resolved. Twelve of 51 (24%) patients suffered severe acute toxicity, and 17 of 51 (33%) patients were admitted for supportive care.Conclusion. Concurrent gemcitabine and radiotherapy can be a very difficult combination to administer safely. Our results do not suggest a prolongation of median survival for patients with localized pancreatic cancer treated with this therapy. It is possible that gemcitabine-based chemoradiation contributes to the margin-negative resectability of a small number of patients with minimal arterial involvement, but this benefit is obscured by the frequent toxicity encountered in most patients. Locally advanced pancreatic cancer patients should continue to be enrolled on prospective studies investigating novel combinations of cytotoxic and/or biologic agents with concurrent radiotherapy.

Lack of correlation between mitotic arrest or apoptosis and antitumor effect of docetaxel

Purpose: To determine, as we did for paclitaxel, whether mitotic arrest and apoptosis induced in murine tumors in vivo by docetaxel correlate with the drugs antitumor effect and whether the antitumor efficacy of docetaxel depends on p53 mutational status of tumors. Methods: C3Hf/Kam mice were implanted with one of the following 15 syngeneic tumors: seven adenocarcinomas (MCa-4, MCa-29, MCa-35, MCa-K, OCa-I, ACa-SG, and HCa-I), two squamous cell carcinomas (SCC-IV and SCC-VII), five sarcomas (FSa, FSa-II, Sa-NH, NFSa, and Sa-4020) and one lymphoma (Ly-TH). When the tumors had grown to 8 mm in diameter, the mice were treated with 31.3 mg/kg docetaxel i.v. Tumor growth delay was the endpoint of docetaxels antitumor effect. In separate groups of mice, mitotic arrest and apoptosis were determined micromorphometrically 1 to 72 h after docetaxel treatment. Tumors were assayed for their p53 status by sequence analysis of RNA prepared from freshly excised tumors. Results: Docetaxel caused statistically significant growth delay in six of seven adenocarcinomas, three of five sarcomas, and the lymphoma, but not in either of the squamous cell carcinomas. The drug induced mitotic arrest in all tumor types, but to various degrees ranging from 6.4 +/− 0.4% to 25.1 +/− 0.1%. In contrast, docetaxel induced appreciable apoptosis in only 5 of 15 tumors, with 10.3 +/− 1.6% being the highest apoptotic value. Neither mitotic arrest nor apoptosis were significantly correlated with tumor growth delay. However, tumors that responded to docetaxel by significant tumor growth delay histologically displayed massive cell destruction by cell lysis, and four of these tumors also showed marked infiltration with mononuclear lymphoid cells. Of the 15 tumors only 3 had mutant p53. Conclusions: Docetaxel exhibited a strong antitumor effect in two-thirds of murine tumors, and on a milligram per kilogram basis was more effective than paclitaxel against the same tumors. The drug was a potent inducer of mitotic arrest but a weak inducer of apoptosis, neither of which correlated with its antitumor effect. Tumor cell lysis appeared to be a major mode of tumor cell destruction and can be regarded as the main mechanism underlying antitumor efficacy of docetaxel. In contrast, paclitaxels antitumor efficacy is related to its ability to induce apoptosis. At the molecular level, there was no dependency of antitumor efficacy of docetaxel on p53 mutational status of tumors.

Comparison of the gastrointestinal syndrome after total-body or total-abdominal irradiation

In pathogen-free mice, but not standard conventionally housed laboratory rodents, two distinctly different modes of early radiation lethality can be identified by modifying the irradiation technique (total-body versus abdominal irradiation) or by therapeutic intervention such as rescue of total-body-irradiated mice with syngeneic bone marrow or spleen. While damage to the gastrointestinal tract is usually designated as the predominant cause of death occurring within 10 days of radiation exposure, it was demonstrated that damage to the hematopoietic/lymphopoietic system can result in animal lethality over the same period as the gastrointestinal syndrome and that this target cell population is more radiation-sensitive than the gastrointestinal epithelium.

Cyclo-oxygenase-2 and its inhibition in cancer: is there a role?

Despite recent improvements in chemotherapy and radiation therapy in cancer management with the addition of biological agents, novel treatment approaches are needed to further benefit patients. Cyclo-oxygenase (COX)-2 inhibition represents one such possibility. COX-2 is an enzyme induced in pathological states such as inflammatory disorders and cancer, where it mediates production of prostanoids. The enzyme is commonly expressed in both premalignant lesions and malignant tumours of different types. A growing body of evidence suggests an association of COX-2 with tumour development, aggressive biological tumour behaviour, resistance to standard cancer treatment, and adverse patient outcome. COX-2 may be related to cancer development and propagation through multiple mechanisms, including stimulation of growth, migration, invasiveness, resistance to apoptosis, suppression of the immunosurveillance system, and enhancement of angiogenesis. Epidemiological data suggest that NSAIDs and selective COX-2 inhibitors might prevent the development of cancers, including colorectal, oesophageal and lung cancer. Preclinical investigations have demonstrated that inhibition of this enzyme with selective COX-2 inhibitors enhances tumour response to radiation and chemotherapeutic agents. These preclinical findings have been rapidly advanced to clinical oncology. Clinical trials of the combination of selective COX-2 inhibitors with radiotherapy, chemotherapy or both in patients with a number of cancers have been initiated, and preliminary results are encouraging. This review discusses the role of COX-2, its products (prostaglandins) and its inhibitors in tumour growth and treatment.

Effect of paclitaxel (taxol) alone and in combination with radiation on the gastrointestinal mucosa.

PURPOSE Paclitaxel is a potentially useful drug for augmenting the cytotoxic action of radiotherapy because it has independent cytotoxic activity against certain cancers and blocks cells in the radiosensitive mitotic phase of the cell cycle. However, all rapidly proliferating tissues, both normal and neoplastic, may be affected by this therapeutic strategy. The aim of this study was to define the in vivo response of rapidly dividing cells of the small bowel mucosa to paclitaxel given alone and in combination with radiation. METHODS AND MATERIALS Mice were given single IV doses of 10 or 40 mg/kg paclitaxel or four doses of 10 mg/kg paclitaxel at 6, 12, or 24 h intervals. The kinetics of mitotic arrest and apoptosis in jejunal crypts of mice at 1-24 h after treatment were defined histologically. An in vivo stem cell microcolony assay was used to assess the radiosensitizing potential of paclitaxel when radiation was delivered at the peak of mitosis and at 24 h after drug treatment. RESULTS Paclitaxel blocked jejunal crypt cells in mitosis and induced apoptosis in a dose-dependent manner. Fractionating the paclitaxel dose over 1-4 days did not result in any greater accumulation of mitotically blocked cells than did a single dose. Mitosis peaked 2-4 h after paclitaxel and returned to near normal by 24 h. Apoptosis lagged several hours behind mitosis and peaked about 6 h later than mitosis. Despite these kinetic perturbations, there was little or no enhancement of radiation effect when single doses were delivered 2-4 h after paclitaxel administration. The maximum sensitizer enhancement ratio of 1.07 observed after a single paclitaxel dose of 40 mg/kg is consistent with independent crypt cell killing. Conversely, when radiation was given 24 h after paclitaxel, a significant protective effect of the drug (SER 0.89-0.92), most probably due to a regenerative overshoot induced by paclitaxel, was observed. CONCLUSION Stem cells of the jejunal mucosa determining radiation response were not radiosensitized by paclitaxel with the drug concentrations and dose delivery schedules used, although additive cytotoxicity was observed with the highest drug dose. A radioprotective effect was observed when radiation was given 24 h after paclitaxel administration.