Proton therapy reduces the likelihood of high-grade radiation-induced lymphopenia in glioblastoma patients: phase II randomized study of protons vs photons.

BACKGROUND: We investigated differences in radiation-induced grade 3+ lymphopenia (G3+L), defined as an absolute lymphocyte count (ALC) nadir of <500 cells/µL, after proton therapy (PT) or X-ray (photon) therapy (XRT) for patients with glioblastoma (GBM). METHODS: Patients enrolled in a randomized phase II trial received PT (n = 28) or XRT (n = 56) concomitantly with temozolomide. ALC was measured before, weekly during, and within 1 month after radiotherapy. Whole-brain mean dose (WBMD) and brain dose-volume indices were extracted from planned dose distributions. Univariate and multivariate logistic regression analyses were used to identify independent predictive variables. The resulting model was evaluated using receiver operating characteristic (ROC) curve analysis. RESULTS: Rates of G3+L were lower in men (7/47 [15%]) versus women (19/37 [51%]) (P < 0.001), and for PT (4/28 [14%]) versus XRT (22/56 [39%]) (P = 0.024). G3+L was significantly associated with baseline ALC, WBMD, and brain volumes receiving 5‒40 Gy(relative biological effectiveness [RBE]) or higher (ie, V5 through V40). Stepwise multivariate logistic regression analysis identified being female (odds ratio [OR] 6.2, 95% confidence interval [CI]: 1.95‒22.4, P = 0.003), baseline ALC (OR 0.18, 95% CI: 0.05‒0.51, P = 0.003), and whole-brain V20 (OR 1.07, 95% CI: 1.03‒1.13, P = 0.002) as the strongest predictors. ROC analysis yielded an area under the curve of 0.86 (95% CI: 0.79-0.94) for the final G3+L prediction model. CONCLUSIONS: Sex, baseline ALC, and whole-brain V20 were the strongest predictors of G3+L for patients with GBM treated with radiation and temozolomide. PT reduced brain volumes receiving low and intermediate doses and, consequently, reduced G3+L.

Memantine prevents acute radiation-induced toxicities at hippocampal excitatory synapses.

Background: Memantine has shown clinical utility in preventing radiation-induced cognitive impairment, but the mechanisms underlying its protective effects remain unknown. We hypothesized that abnormal glutamate signaling causes radiation-induced abnormalities in neuronal structure and that memantine prevents synaptic toxicity. Methods: Hippocampal cultures expressing enhanced green fluorescent protein were irradiated or sham-treated and their dendritic spine morphology assessed at acute (minutes) and later (days) times using high-resolution confocal microscopy. Excitatory synapses, defined by co-localization of the pre- and postsynaptic markers vesicular glutamate transporter 1 and postsynaptic density protein 95, were also analyzed. Neurons were pretreated with vehicle, the N-methyl-d-aspartate-type glutamate receptor antagonist memantine, or the glutamate scavenger glutamate pyruvate transaminase to assess glutamate signaling. For animal studies, Thy-1-YFP mice were treated with whole-brain radiotherapy or sham with or without memantine. Results: Unlike previously reported long-term losses of dendritic spines, we found that the acute response to radiation is an initial increase in spines and excitatory synapses followed by a decrease in spine/synapse density with altered spine dynamics. Memantine pre-administration prevented this radiation-induced synaptic remodeling. Conclusion: These results demonstrate that radiation causes rapid, dynamic changes in synaptic structural plasticity, implicate abnormal glutamate signaling in cognitive dysfunction following brain irradiation, and describe a protective mechanism of memantine.

Spatial mapping of the biologic effectiveness of scanned particle beams: towards biologically optimized particle therapy.

The physical properties of particles used in radiation therapy, such as protons, have been well characterized, and their dose distributions are superior to photon-based treatments. However, proton therapy may also have inherent biologic advantages that have not been capitalized on. Unlike photon beams, the linear energy transfer (LET) and hence biologic effectiveness of particle beams varies along the beam path. Selective placement of areas of high effectiveness could enhance tumor cell kill and simultaneously spare normal tissues. However, previous methods for mapping spatial variations in biologic effectiveness are time-consuming and often yield inconsistent results with large uncertainties. Thus the data needed to accurately model relative biological effectiveness to guide novel treatment planning approaches are limited. We used Monte Carlo modeling and high-content automated clonogenic survival assays to spatially map the biologic effectiveness of scanned proton beams with high accuracy and throughput while minimizing biological uncertainties. We found that the relationship between cell kill, dose, and LET, is complex and non-unique. Measured biologic effects were substantially greater than in most previous reports, and non-linear surviving fraction response was observed even for the highest LET values. Extension of this approach could generate data needed to optimize proton therapy plans incorporating variable RBE.

Is there an impact of heart exposure on the incidence of radiation pneumonitis? Analysis of data from a large clinical cohort.

BACKGROUND: The goal of the present study was to determine, in a large clinical cohort, whether incidental radiation exposure to the heart during definitive radiotherapy of inoperable non-small cell lung cancer (NSCLC) detectably increased the risk of radiation pneumonitis (RP) beyond that resulting from radiation exposure to lung. MATERIAL AND METHODS: Data were analyzed from all patients who received definitive three-dimensional (3D) concurrent radiotherapy or intensity-modulated radiotherapy for the treatment of NSCLC over a 10-year period at our institution, except those who had previous lung cancer or for whom radiation treatment plans were unavailable for calculation of heart and lung dose-volume histograms (DVHs). Parameters computed from heart and lung DVHs included mean lung dose (MLD), effective lung dose computed using volume parameter n = 0.5 (Deff), mean heart dose (MHD), percentage of heart receiving > 65 Gy (V65), and minimum dose to the hottest 10% of heart (D10). Univariate and multivariate normal-tissue complication probability (NTCP) models were used to analyze incidence of Grade ≥ 2 or Grade ≥ 3 RP as a function of these and other parameters. RESULTS: The study cohort included 629 patients, with crude rates of Grade ≥ 2 RP and Grade ≥ 3 RP of N = 263 (42%) and N = 124 (20%), respectively. Univariate NTCP models based on dosimetric lung parameters (MLD and Deff) fit the data better than models based on univariate heart parameters (heart D10, heart V65 or MHD). In multivariate modeling, incorporation of heart parameters did not significantly improve the fit of RP risk models based on lung parameters alone (p > 0.38 in each case). CONCLUSIONS: In this large clinical cohort, there was no evidence that incidental heart exposure during radiotherapy of NSCLC had a detectable impact on the occurrence of moderate or severe RP.

Comparison of proton therapy techniques for treatment of the whole brain as a component of craniospinal radiation.

BACKGROUND: For treatment of the entire cranium using passive scattering proton therapy (PSPT) compensators are often employed in order to reduce lens and cochlear exposure. We sought to assess the advantages and consequences of utilizing compensators for the treatment of the whole brain as a component of craniospinal radiation (CSI) with PSPT. Moreover, we evaluated the potential benefits of spot scanning beam delivery in comparison to PSPT. METHODS: Planning computed tomography scans for 50 consecutive CSI patients were utilized to generate passive scattering proton therapy treatment plans with and without Lucite compensators (PSW and PSWO respectively). A subset of 10 patients was randomly chosen to generate scanning beam treatment plans for comparison. All plans were generated using an Eclipse treatment planning system and were prescribed to a dose of 36 Gy(RBE), delivered in 20 fractions, to the whole brain PTV. Plans were normalized to ensure equal whole brain target coverage. Dosimetric data was compiled and statistical analyses performed using a two-tailed Student's t-test with Bonferroni corrections to account for multiple comparisons. RESULTS: Whole brain target coverage was comparable between all methods. However, cribriform plate coverage was superior in PSWO plans in comparison to PSW (V95%; 92.9 ± 14 vs. 97.4 ± 5, p < 0.05). As predicted, PSWO plans had significantly higher lens exposure in comparison to PSW plans (max lens dose Gy(RBE): left; 24.8 ± 0.8 vs. 22.2 ± 0.7, p < 0.05, right; 25.2 ± 0.8 vs. 22.8 ± 0.7, p < 0.05). However, PSW plans demonstrated no significant cochlear sparing vs. PSWO (mean cochlea dose Gy(RBE): 36.4 ± 0.2 vs. 36.7 ± 0.1, p = NS). Moreover, dose homogeneity was inferior in PSW plans in comparison to PSWO plans as reflected by significant alterations in both whole brain and brainstem homogeneity index (HI) and inhomogeneity coefficient (IC). In comparison to both PSPT techniques, multi-field optimized intensity modulated (MFO-IMPT) spot scanning treatment plans displayed superior sparing of both lens and cochlea (max lens: 12.5 ± 0.6 and 12.9 ± 0.7 right and left respectively; mean cochlea 28.6 ± 0.5 and 27.4 ± 0.2), although heterogeneity within target volumes was comparable to PSW plans. CONCLUSIONS: For PSPT treatments, the addition of a compensator imparts little clinical advantage. In contrast, the incorporation of spot scanning technology as a component of CSI treatments, offers additional normal tissue sparing which is likely of clinical significance.

The threshold level of the sensor histidine kinase KinA governs entry into sporulation in Bacillus subtilis.

Sporulation in Bacillus subtilis is controlled by a complex gene regulatory circuit that is activated upon nutrient deprivation. The initial process is directed by the phosphorelay, involving the major sporulation histidine kinase (KinA) and two additional phosphotransferases (Spo0F and Spo0B), that activates the master transcription factor Spo0A. Little is known about the initial event and mechanisms that trigger sporulation. Using a strain in which the synthesis of KinA is under the control of an IPTG (isopropyl-beta-d-thiogalactopyranoside)-inducible promoter, here we demonstrate that inducing the synthesis of the KinA beyond a certain level leads to the entry of the irreversible process of sporulation irrespective of nutrient availability. Moreover, the engineered cells expressing KinA under a sigma(H)-dependent promoter that is similar to but stronger than the endogenous kinA promoter induce sporulation during growth. These cells, which we designated COS (constitutive sporulation) cells, exhibit the morphology and properties of sporulating cells and express sporulation marker genes under nutrient-rich conditions. Thus, we created an engineered strain displaying two cell cycles (growth and sporulation) integrated into one cycle irrespective of culture conditions, while in the wild type, the appropriate cell fate decision is made depending on nutrient availability. These results suggest that the threshold level of the major sporulation kinase acts as a molecular switch to determine cell fate and may rule out the possibility that the activity of KinA is regulated in response to the unknown signal(s).

Single-cell measurement of the levels and distributions of the phosphorelay components in a population of sporulating Bacillus subtilis cells.

Upon nutrient starvation, the Gram-positive bacterium Bacillus subtilis switches from growth to sporulation by activating a multicomponent phosphorelay consisting of a major sensor histidine kinase (KinA), two phosphotransferases (Spo0F and Spo0B) and a response regulator (Spo0A). Although the primary sporulation signal(s) produced under starvation conditions is not known, it is believed that the reception of a signal(s) on the sensor kinase results in the activation of autophosphorylation of the enzyme. The phosphorylated kinase transfers the phosphate group to Spo0A via the phosphorelay and thus triggers sporulation. With a combination of quantitative immunoblot analysis, microscopy imaging and computational analysis, here we found that each of the phosphorelay components tested increased gradually over the period of sporulation, and that Spo0F was expressed in a more heterogeneous pattern than KinA and Spo0B in a sporulating cell population. We determined molecule numbers and concentrations of each phosphorelay component under physiological sporulation conditions at the single-cell level. Based on these results, we suggest that successful entry into the sporulation state is manifested by a certain critical level of each phosphorelay component, and thus that only a subpopulation achieves a sufficient intracellular quorum of the phosphorelay components to activate Spo0A and proceed successfully to the entry into sporulation.

Joint effect of MCP-1 genotype GG and MMP-1 genotype 2G/2G increases the likelihood of developing pulmonary tuberculosis in BCG-vaccinated individuals.

We previously reported that the -2518 MCP-1 genotype GG increases the likelihood of developing tuberculosis (TB) in non-BCG-vaccinated Mexicans and Koreans. Here, we tested the hypothesis that this genotype, alone or together with the -1607 MMP-1 functional polymorphism, increases the likelihood of developing TB in BCG-vaccinated individuals. We conducted population-based case-control studies of BCG-vaccinated individuals in Mexico and Peru that included 193 TB cases and 243 healthy tuberculin-positive controls from Mexico and 701 TB cases and 796 controls from Peru. We also performed immunohistochemistry (IHC) analysis of lymph nodes from carriers of relevant two-locus genotypes and in vitro studies to determine how these variants may operate to increase the risk of developing active disease. We report that a joint effect between the -2518 MCP-1 genotype GG and the -1607 MMP-1 genotype 2G/2G consistently increases the odds of developing TB 3.59-fold in Mexicans and 3.9-fold in Peruvians. IHC analysis of lymph nodes indicated that carriers of the two-locus genotype MCP-1 GG MMP-1 2G/2G express the highest levels of both MCP-1 and MMP-1. Carriers of these susceptibility genotypes might be at increased risk of developing TB because they produce high levels of MCP-1, which enhances the induction of MMP-1 production by M. tuberculosis-sonicate antigens to higher levels than in carriers of the other two-locus MCP-1 MMP-1 genotypes studied. This notion was supported by in vitro experiments and luciferase based promoter activity assay. MMP-1 may destabilize granuloma formation and promote tissue damage and disease progression early in the infection. Our findings may foster the development of new and personalized therapeutic approaches targeting MCP-1 and/or MMP-1.