A new biodosimetric method: branched DNA-based quantitative detection of B1 DNA in mouse plasma.

A simple and accurate method for measuring the biological effects of radiation is of increasing importance, especially in mass casualty scenarios. We have therefore developed a new biodosimetric technique targeting circulating B1 DNA in mouse plasma by branched DNA signal amplification for rapid quantification of plasma DNA. This technology targets repetitive elements of the B1 retrotransposon in the mouse genome, followed by signal amplification using Panomics Quantigene 2.0 reagents. Evaluation was conducted concerning precision, accuracy and linearity. Plasma samples were collected from mice 0-24 h after 0-10 Gy total body irradiation (TBI). The average inter- and intra-assay coefficients of variance were 8.7% and 12.3%, respectively. The average recovery rate of spiked DNA into plasma was 89.5%. This assay revealed that when BALB/c and NIH Swiss mice were exposed to 6 Gy TBI, plasma B1 DNA levels increased significantly at 3 h post-TBI, peaked at 9 h and gradually returned toward baseline levels in 24 h. A dose-dependent change in plasma DNA was observed at 9 h post-TBI; the dose-response relation was monotonic, exhibiting linearity for BALB/c mice from 3 to 6 Gy (r = 0.993) and NIH Swiss mice from 3 to 7 Gy (r = 0.98). This branched DNA-based assay is reliable, accurate and sensitive in detecting plasma B1 DNA quantitatively. A radiation dose-correlated increase in plasma B1 DNA was demonstrated in BALB/c and NIH Swiss mice in the dose range from 3 to 6 Gy, suggesting that plasma B1 DNA has potential as a biomarker for radiation biological effect.

Tumor Detection in vivo with Optical Spectroscopy.

Ultrasound induced blood stasis has been observed for a long time, but to date most experimental observations have been in vitro. In this paper we discuss a possible diagnostic use for this previously undesirable effect of ultrasound - tumor detection in vivo. We demonstrate that, using optical spectroscopy, effects of ultrasound can be used to differentiate tumor from non-tumor in murine tissue. Finally, we propose a novel diagnostic algorithm that quantitatively differentiates tumor from non-tumor with maximum specificity 0.83, maximum sensitivity 0.79, and area under ROC curve 0.90.

In vivo Optical Spectroscopy of Acoustically Induced Blood Stasis.

Ultrasound-induced blood stasis has been observed for more than thirty years. Most of the literature has been focused on the health risks associated with this phenomenon and methods employed to prevent stasis from occurring during ultrasound imaging. To date, experimental observations have been either in vitro or invasive. The current work demonstrates ultrasound- induced blood stasis in murine tumor and nontumor tissue, observed through noninvasive measurements of optical spectroscopy, and discusses possible diagnostic uses for this previously undesirable effect of ultrasound.

Mutant, wild type, or overall p53 expression: freedom from clinical progression in tumours of astrocytic lineage.

Abnormalities of the p53 tumor-suppressor gene are found in a significant proportion of astrocytic brain tumours. We studied tumour specimens from 74 patients evaluated over 20 years at the Massachusetts General Hospital, where clinical outcome could be determined and sufficient pathologic material was available for immunostaining. p53 expression studies employed an affinity-purified p53 monoclonal antibody, whose specificity was verified in absorption studies and, in a minority of cases, a second antibody recognising a different epitope of p53. Significant overexpression of p53 protein was found in 48% of the 74 tumours included in this series and high levels of expression were associated with higher mortality from astrocytic tumours (P<0.001, log rank). Multivariate analyses revealed that immunohistochemically detected p53 was an independent marker of shortened progression-free and overall actuarial survival in patients with astrocytic tumours, suggesting that increased expression of p53 plays an important role in the pathobiology of these tumours. In a subset of 36 cases, coding regions of the p53 gene were completely sequenced via SSCP and direct DNA sequencing, revealing that overexpression of p53 protein is not always associated with point mutations in conserved exons of the p53 gene. Finally, we confirmed p53 protein expression in early-passage human glioma cell lines of known p53 mutational status and immunostaining scores. Although grade continues to be the strongest prognostic variable, the use of p53 staining as a prognostic indicator, in contrast to mutational DNA analyses, may be a useful adjunct in identifying patients at higher risk of treatment failure.

Loss of p16 expression is of prognostic significance in locally advanced prostate cancer: an analysis from the Radiation Therapy Oncology Group protocol 86-10.

PURPOSE: The retinoblastoma (RB) cell cycle regulatory pathway is known to be deregulated in virtually all known human tumors. The protein product of the RB gene, pRB, and its upstream regulator, p16, are among the most commonly affected members of this pathway. We investigated the prognostic significance of both pRB and p16 expression in locally advanced prostate cancers, from patients treated on the Radiation Therapy Oncology Group (RTOG) protocol 86-10. MATERIALS AND METHODS: Sixty-seven cases from RTOG 86-10 had immunohistochemically stained slides, judged interpretable for both p16 and pRB, available for analysis. Median follow-up was 8.9 years (range, 6.0 to 11.8 years) for surviving patients. Staining for each marker was then correlated with overall survival, local progression, distant metastasis, and disease-specific survival. RESULTS: Loss of p16 expression, as defined by expression was significantly associated with reduced overall survival (P =.039), disease-specific survival (P =.006), and higher risk of local progression (P =.0007) and distant metastasis (P =.026) in the univariate analysis. In the multivariate analysis, loss of p16 was significantly associated with reduced disease-specific survival (P =.0078) and increased risk of local failure (P =.0035) and distant metastasis (P =.026). A borderline association with reduced overall survival (P =.07) was also evident. Loss of pRB was associated with improved disease-specific survival on univariate (P =.028) and multivariate analysis (P =.043), but carried no other significant outcome associations. CONCLUSION: Loss of p16 is significantly associated with adverse clinical outcome in cases of locally advanced prostate cancer.

Predictive value of histologic tumor necrosis after radiation.

Postsurgical evaluation of histologic changes of tumors after preoperative chemotherapy and/or radiotherapy has been a routine clinical practice of pathologists and oncologists. There appears to be secure evidence that the extent of tumor necrosis vs. viable tumor cells postchemotherapy is a clinically useful predictor of outcome. The significance of histologic tumor necrosis after radiotherapy, however, has not been clearly established and deserves further investigation. We investigated the correlation between histological extent of tumor necrosis, survival of tumor transplants, and radiation doses in an experimental model using three human tumor xenografts. Three human tumor cell lines were investigated: STS-26, SCC-21, and HGL-21. Tumors were grown subcutaneously in athymic nude mice and received external beam radiation of different doses. Tumors were excised 2 weeks postirradiation. One-half of the tumor was divided into 1-mm(3) fragments and transplanted to naive mice. The other half was examined for histologic tumor necrosis. Transplant survival was strongly correlated with radiation dose, TCD(p) (radiation dose that results in local tumor control in proportion, p, to irradiated tumors). In contrast, there was no clear association between transplant survival rate and the extent of tumor necrosis. The experimental model demonstrated a strong inverse correlation between radiation doses and tumor transplant survival. Histologic tumor necrosis did not correlate well with radiation doses or transplant survival rates. Despite common practices in histologic examination of tumors posttherapy, clinical interpretations and implications of histologic tumor necrosis after radiotherapy should be considered with caution.

Schedule-dependent pulsed paclitaxel radiosensitization for thoracic malignancy.

The objective of this study was to apply preclinical research of paclitaxel radiosensitization to the treatment of thoracic malignancy. Human lung cancer cell line NCI520 and epidermoid cell line A431 were investigated in vitro for radiosensitizing effects of paclitaxel. Optimal schedule of paclitaxel treatment was applied to a clinical protocol as well as off-protocol treatment of thoracic malignancy. Pulsed paclitaxel with concurrent once-daily radiation was delivered every 48 hours during the week using doses of 15 mg/m2, 20 mg/m2, or 25 mg/m2 in a phase I clinical trial of dose escalation. Preclinical data support the finding that low-dose paclitaxel is sufficient for radiosensitization. Data also support that delaying radiation is better than immediate radiation after drug treatment. Twenty-three patients have enrolled in the phase I clinical trial. Seventeen patients completed treatment (6 at 15 mg/m2; 5 at 20 mg/m2; and 6 at 25 mg/m2). Mean tumor shrinkage at 4 to 6 weeks after therapy was 82%, 84%, and 84% for dose levels I, II, and III, respectively [average primary tumor shrinkage was 83% +/- 8% (95% C.I.)]. Locoregional tumor response rate was 100% [12% (2/17) complete response and 88% (15/17) partial response] with low rates of toxicity. It is concluded that pulsed low-dose paclitaxel and radiation is a very effective and well-tolerated regimen for thoracic malignancy.

Characterization of the effects of antiangiogenic agents on tumor pathophysiology.

A variety of strategies have been proposed to control tumor growth and metastasis by inhibiting tumor angiogenesis. To optimally combine such antiangiogenic approaches with conventional therapy, improved methods are needed to characterize the underlying pathophysiologic changes. The objective of the current work was to demonstrate the utility of a combination of recently developed immunohistochemical and image analysis techniques in quantitating changes in tumor vasculature and hypoxia. Murine MCa-35 mammary carcinomas were frozen after administration of two COX-2 inhibitors: meloxicam and celecoxib (Celebrex). Total blood vessels were visualized using anti-CD31 staining, perfused vessels by intravenous injection of DiOC7, and tumor hypoxia by EF5 uptake. Although both agents produced similar reductions in tumor volume compared with untreated tumors, varied effects on tumor vasculature and hypoxia were noted. Meloxicam reduced total vessel numbers significantly, whereas celecoxib had no effect. Both drugs substantially increased perfused vessel densities. Although mean hypoxic marker uptake was unchanged from matched controls, intratumor EF5 heterogeneities were significantly different between drugs. The results suggest that COX-2 inhibitors can have varying effects on tumor pathophysiology. Successful use of these drugs to enhance radiation response will likely require optimization of drug choice, dose schedule, and direct physiologic monitoring.

Keratinocyte growth factors radioprotect bowel and bone marrow but not KHT sarcoma.

Various members of the fibroblast growth factor (FGF) family of proteins have been shown to protect against acute and late radiation damage of normal tissues. Protection of the small bowel, for example, occurs via both increased proliferation and reduced apoptosis. Other beneficial effects of FGFs include promotion of bone growth, pneumonitis prevention, and apoptosis suppression of endothelium in vivo and in vitro after irradiation. This protection against radiation requires only low and infrequent doses of FGFs. Two newly identified members of the FGF family, FGF7 and FGF10, have effects similar to many of the other FGF family proteins, but with more specificity for normal epithelial structures. For this reason, they have also been named keratinocyte growth factors one and two (KGF1 and KGF2, respectively). We therefore examined the potential utility of KGFs for radioprotection of the bone marrow and small bowel and examined safety issues concerning their adverse effects on KHT sarcoma. The results suggest that KGFs could be safely used to prevent radiation toxicity of the abdomen or pelvis and may in fact improve tumor response to radiation.

External beam irradiation for inhibition of intimal hyperplasia following prosthetic bypass: preliminary results.

To determine whether external beam irradiation delivered immediately after graft implantation can inhibit anastomotic intimal hyperplasia (IH) 1 month following polytetrafluoroethylene (PTFE) bypass in a sheep carotid artery model, 23 sheep underwent bilateral bypass of the ligated common carotid artery with 8-mm PTFE immediately followed by a single dose of irradiation (15, 21, or 30 Gy) to one side. The 15 animals with bilaterally patent grafts were euthanized at 1 month and graft-arterial anastomoses harvested. Using computer-aided image analysis, IH areas and thicknesses were measured. Graft patency in this model was 83% at 1 month and did not differ according to treatment administered. In the control animals, IH was greatest at mid-anastomosis, but minimal within the native vessel. All three radiation doses markedly inhibited mid-anastomotic IH area and thickness. At the proximal anastomosis, 30 Gy reduced the IH area 20-fold, from 2.06 to 0.14 mm2 (p < 0.0001 by ANOVA), and IH thickness 70-fold, from 29.0 to 0.4 micron (p < 0.0002); similar effects were seen at the distal anastomosis. No adverse effects of radiation treatment were observed. External beam irradiation in doses of 15 to 30 Gy delivered in a single fraction immediately after operation markedly inhibits development of intimal hyperplasia 1 month following end-to-side anastomosis with PTFE in sheep.

The role of autocrine growth factors in radiation damage to the epiphyseal growth plate.

Radiation therapy plays an important role as part of the multimodality treatment for a number of childhood malignancies. Dose-limiting complications of radiotherapy include skeletal abnormalities and disturbances in skeletal development within the irradiated field. The current study was undertaken to investigate the molecular mechanisms involved in radiation-induced arrest of bone growth. Our hypotheses were: (1) Expression of autocrine growth factors that regulate chondrocyte proliferation is inhibited by radiation in a specific pattern; (2) the disparity in radiosensitivity of growth plate chondrocytes and epiphyseal chondrocytes is due to differential modulation of autocrine growth factor expression by radiation. Given the important role these cells play in skeletal growth and development, we examined the comparative effects of radiation on expression of specific mitogenic growth factors in growth plate chondrocytes. The effect of radiation on the expression of autocrine/paracrine growth factors was examined in an established avian model of epiphyseal growth plate maturation. Exposure of growth plate chondrocytes to radiation resulted in a specific pattern of biochemical and morphological alterations that were dependent on dose and were progressive over time. While radiation did not affect the mRNA expression of some of the autocrine and paracrine factors important in endochondral ossification (such as FGF2 and TGFB isoforms), it did lead to a decrease in the mRNA expression of PTHrP, a critically important mitogen in growth plate chondrocytes, and a dose-dependent decrease in the PTH/PTHrP receptor mRNA. Interestingly, PTHrP mRNA levels were not affected in irradiated epiphyseal chondrocytes, the main source of PTHrP. Given evidence indicating a role for intracellular calcium levels in regulating PTHrP expression, basal calcium levels in irradiated growth plate chondrocytes and epiphyseal chondrocytes were examined 24 h after treatment. While cytosolic calcium levels were significantly higher in irradiated growth plate chondrocytes, they were not significantly affected in irradiated epiphyseal chondrocytes. The importance of calcium in mediating radiation damage to growth plate chondrocytes was further demonstrated by the finding that the addition of 4.0 mM EGTA (a calcium chelator) to the cell cultures before irradiation prevented the decrease in PTHrP mRNA levels. Since PTHrP up-regulates BCL2 levels and prevents growth plate chondrocyte maturation and apoptosis, BCL2 mRNA levels were examined in irradiated growth plate chondrocytes, and a dose-dependent decrease was found. An increase in apoptosis was further confirmed by a fivefold increase in caspase 3 levels in irradiated growth plate chondrocytes. The results of the current study suggest that radiation may interfere with proliferation of growth plate chondrocytes in part by causing an increase in cytosolic calcium levels which in turn leads to a decrease in PTHrP mRNA. Growth plate chondrocyte PTHrP receptor mRNA expression is also inhibited by radiation, further decreasing PTHrP signaling. Despite subtle differences between the chick and mammalian growth plates, further studies should provide an enhanced understanding of the mechanism(s) of radiation injury to the growth plate, as well as possibilities for new therapeutic strategies to protect the growing skeleton from the detrimental effects of radiotherapy.

Erythrocyte membrane ATP binding cassette (ABC) proteins: MRP1 and CFTR as well as CD39 (ecto-apyrase) involved in RBC ATP transport and elevated blood plasma ATP of cystic fibrosis.

In addition to the better-known roles of the erythrocyte in the transport of oxygen and carbon dioxide, the concept that the red blood cell is involved in the transport and release of ATP has been evolving (J. Luthje, Blut 59, 367, 1989; G. R. Bergfeld and T. Forrester, Cardiovasc. Res. 26, 40, 1992; M. L. Ellsworth et al., Am. J. Physiol. 269, H2155, 1995; R. S. Sprague et al., Am. J. Physiol. 275, H1726, 1998). Membrane proteins involved in the release of ATP from erythrocytes now appear to include members of the ATP binding cassette (ABC) family (C. F. Higgins, Annu. Rev. Cell Biol. 8, 67, 1992; C. F. Higgins, Cell 82, 693, 1995). In addition to defining physiologically the presence of ABC proteins in RBCs, accumulating gel electrophoretic evidence suggests that the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance-associated protein (MRP1), respectively, constitute significant proteins in the red blood cell membrane. As such, this finding makes the mature erythrocyte compartment a major mammalian repository of these important ABC proteins. Because of its relative structural simplicity and ready accessibility, the erythrocyte offers an ideal system to explore details of the physiological functions of ABC proteins. Moreover, the presence of different ABC proteins in a single membrane implies that interaction among these proteins and with other membrane proteins may be the norm and not the exception in terms of modulation of their functions.

Cellular and biophysical evidence for interactions between adenosine triphosphate and P-glycoprotein substrates: functional implications for adenosine triphosphate/drug cotransport in P-glycoprotein overexpressing tumor cells and in P-glycoprotein low-level expressing erythrocytes.

P-glycoprotein is involved with the removal of drugs, most of them cations, from the plasma membrane and cytoplasm. Pgp is also associated with movement of ATP, an anion, from the cytoplasm to the extracellular space. The central question of this study is whether drug and ATP transport associated with the expression of Pgp are in any way coupled. We have measured the stoichiometry of transport coupling between drug and ATP release. The drug and ATP transport that is inhibitable by the sulfonylurea compound, glyburide (P. E. Golstein, A. Boom, J. van Geffel, P. Jacobs, B. Masereel, and R. Beauwens, Pfluger's Arch. 437, 652, 1999), permits determination of the transport coupling ratio, which is close to 1:1. In view of this result, we asked whether ATP interacts directly with Pgp substrates. We show by measuring the movement of Pgp substrates in electric fields that ATP and drug movement are coupled. The results are compatible with the view that substrates for Pgp efflux are driven by the movement of ATP through electrostatic interaction and effective ATP-drug complex formation with net anionic character. This mechanism not only pertains to drug efflux from tumor cells overexpressing Pgp, but also provides a framework for understanding the role of erythrocytes in drug resistance. The erythrocyte consists of a membrane surrounding a millimolar pool of ATP. Mammalian RBCs have no nucleus or DNA drug/toxin targets. From the perspective of drug/ATP complex formation, the RBC serves as an important electrochemical sink for toxins. The presence in the erythrocyte membrane of approximately 100 Pgp copies per RBC provides a mechanism for eventual toxin clearance. The RBC transport of toxins permits their removal from sensitive structures and ultimate clearance from the organism via the liver and/or kidneys.

Circulating IL-6 as a predictor of radiation pneumonitis.

PURPOSE: We report results from a clinical research protocol investigating circulating pro-inflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNFalpha]) in relation to radiation pulmonary injury. METHODS AND MATERIALS: In a protocol for cytokine measurement, 25 patients had clinical follow-up longer than 12 months, and 24 had serial cytokine data. Serial plasma specimens before, during, and after thoracic radiotherapy were analyzed for IL-6 and TNFalpha using enzyme-linked immunosorbent assay (ELISA). Radiation pulmonary injury was defined using National Cancer Institute Common Toxicity Criteria. RESULTS: Of the 24 patients, 6 had Grade 1 pneumonitis, and 13 had Grade 2 pneumonitis. There was no Grade 3/4 pneumonitis. Median time of radiation pneumonitis was between 8 and 12 weeks post-therapy. IL-6 levels before, during, and after thoracic radiation therapy were significantly higher in those who developed pneumonitis. In contrast, we did not detect a significant correlation between plasma TNFalpha and radiation pneumonitis. CONCLUSIONS: High pretreatment plasma levels of IL-6 predisposed patients to the risk of radiation pneumonitis. Pretreatment IL-6 level may serve as a predictor for radiation pneumonitis. Serial plasma IL-6 was consistently higher for the pneumonitis group. The role of IL-6 in the cytokine cascades that promote radiation pulmonary injury deserves further investigation.

Intratumoral administration of endostatin plasmid inhibits vascular growth and perfusion in MCa-4 murine mammary carcinomas.

Endostatin, a fragment of the COOH-terminal domain of mouse collagen XVIII is a recently demonstrated endogenous inhibitor of tumor angiogenesis and endothelial cell growth. Antiangiogenic therapy with endostatin in animals requires multiple and prolonged administration of the protein. Gene therapy could provide an alternative approach to continuous local delivery of this antiangiogenic factor in vivo. Established MCa-4 murine mammary carcinomas, grown in immunodeficient mice, were treated with intratumoral injection of endostatin plasmid at 7-day intervals. At the time of sacrifice, 14 days after the first injection, endostatin-treated tumor weights were 51% of controls (P < 0.01). Tumor growth inhibition was accompanied by a marked reduction in total vascular density. Specifically, computerized image analysis showed a 18-21% increase in the median distances between tumor cells and both the nearest anatomical (CD31-stained) vessel [48.1 +/- 3.8 versus 38.3 +/- 1.6 microm (P < 0.05)] and the nearest tumor-specific (CD105-stained) vessel [48.5 +/- 1.5 versus 39.8 +/- 1.5 microm (P < 0.01)]. An increased apoptotic index of tumor cells in endostatin-treated tumors [3.2 +/- 0.5% versus 1.9 +/- 0.3% (P < 0.05)] was observed in conjunction with a significant decrease in tumor perfused vessels (DiOC7 staining), and an increase in tumor cell hypoxia (EF5 staining). Hypoxia resulting from endostatin therapy most likely caused a compensatory increase of in situ vascular endothelial growth factor (VEGF) and VEGF receptor mRNA expression. Increased immunoreactivity of endostatin staining in endostatin-treated tumors was also associated with an increased thrombospondin-1 staining [1.12 +/- 0.16 versus 2.44 +/- 0.35]. Our data suggest that intratumoral delivery of the endostatin gene efficiently suppresses murine mammary carcinoma growth and support the potential utility of the endostatin gene for cancer therapy.

Multi-objective optimization in radiotherapy: applications to stereotactic radiosurgery and prostate brachytherapy.

Treatment planning for radiation therapy is a multi-objective optimization process. Here we present a machine intelligent scheme for treatment planning based on multi-objective decision analysis (MODA) and genetic algorithm (GA) optimization. Multi-objective ranking strategies are represented in the L(p) metric under the displaced ideal model. Goal setting, protocol satisficing and fuzzy ranking of objective importance can be incorporated into the decision scheme to assimilate clinical decision making. For distance measures in the L(p) metric, a dynamic gauge function is defined based on the state energy of the decision system, which is assumed to undergo thermodynamic cooling with iteration time. The MODA scheme interacts with a robust GA engine, which adaptively evolves in the multi-modal landscape that defines the treatment plan quality. A conventionally challenging case of stereotactic radiosurgery of a brain lesion was selected for GA optimization. The resulting dose distributions are compared to human-developed plans, which are commonly regarded as clinically relevant and empirically optimal. The GA-optimized plans achieve substantially better sparing of critical normal neuroanatomy surrounding the brain lesion while respecting the preset constraints on tumor dose uniformity. In addition, machine optimization tends to produce novel treatment strategies which complements expert knowledge. The run time for producing an optimal plan is considerably shorter than the typical planning time for human experts, thus GA can also be used to aid the human treatment planning process. In prostate brachytherapy, MODA-GA was specifically applied to non-ideal conditions in which typical surgical uncertainties in seed implant positioning occur, where noisy objectives were introduced into the optimization scheme. The noisy system is found to be manageable by MODA-GA at uncertainty levels corresponding to reasonably proficient surgery teams. In contrast, noisy objectives would be very difficult to explore by human expert planners. Potential use of noisy optimization with time series analysis is being explored for error-corrective computer guidance in the operating room for prostate seed implantation. In conclusion, the combination of MODA and GA optimization offers both a solution to practical treatment planning tasks and the potential for real time applications in radiotherapy.

Irradiation for intimal hyperplasia: implications for peripheral arterial bypass.

Irradiation has been shown to inhibit postangioplasty intimal hyperplasia ("restenosis") in unbranched tubes. It seems likely that irradiation will similarly be able to inhibit intimal hyperplasia after a surgical anastomosis at a biochemical and cellular level, but whether it will produce a clinically relevant or even clinically detectable difference is unproved. One possibility is that no clinical effect may occur; the search for a "cure" for intimal hyperplasia has been long and, as yet, unsuccessful. On the other hand, if a strong effect without insurmountable logistical problems could be produced, one major cause of bypass graft failure would be preventable. Not only would the incidence of late graft occlusion, need for reoperation, and limb loss be reduced, but, if patency of prosthetics could be sufficiently improved, the initial operation could be made much easier, faster, and perhaps safer.