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Intensity modulated radiation therapy (IMRT) has recently been proposed for the treatment of malignant pleural mesothelioma (MPM). Here, we describe our experience with a multimodality approach for the treatment of mesothelioma, incorporating extrapleural pneumonectomy, intraoperative photodynamic therapy and postoperative hemithoracic IMRT. From 2004-2007, we treated 11 MPM patients with hemithoracic IMRT, 7 of whom had undergone porfimer sodium-mediated PDT as an intraoperative adjuvant to surgical debulking. The median radiation dose to the planning treatment volume (PTV) ranged from 45.4-54.5 Gy. For the contralateral lung, V20 ranged from 1.4-28.5%, V5 from 42-100% and MLD from 6.8-16.5 Gy. In our series, 1 patient experienced respiratory failure secondary to radiation pneumonitis that did not require mechanical ventilation. Multimodality therapy combining surgery with increased doses of radiation using IMRT, and newer treatment modalities such as PDT , appears safe. Future prospective analysis will be needed to demonstrate efficacy of this approach in the treatment of malignant mesothelioma. Efforts to reduce lung toxicity and improve dose delivery are needed and provide the promise of improved local control and quality of life in a carefully chosen multidisciplinary approach.

To compare clinical and treatment characteristics and outcomes in locally advanced anal cancer, a potentially curable disease, in patients referred from a public or private hospital.We retrospectively reviewed 112 anal cancer patients from a public and a private hospital who received definitive chemoradiotherapy at the same cancer center between 2004 and 2013. Tumor stage, radiotherapy delay, radiotherapy duration, and unplanned treatment breaks ≥10 days were compared using t-test and χ(2) test. Overall survival (OS), disease free survival (DFS), and colostomy free survival (CFS) were examined using the Kaplan-Meier method and compared with the log-rank test. Cox proportional hazard models for OS and DFS were developed.The follow-up was 14.9 months (range, 0.7-94.8 months). Public hospital patients presented with significantly higher clinical T stage (P0.05) and clinical stage group (P0.05), had significantly longer radiotherapy delays (P0.05) and radiotherapy duration (P0.05), and had more frequent radiation therapy (RT) breaks ≥10 days (P0.05). Three-year OS showed a marked trend in favor of private hospital patients for 3-year OS (72.8% vs. 48.9%; P=0.171), 3-year DFS (66.3% vs. 42.7%, P=0.352), and 3-year CFS (86.4% vs. 68.9%, P=0.299). Referral hospital was not predictive of OS or DFS on multivariate analysis.Public hospital patients presented at later stage and experienced more delays in initiating and completing radiotherapy, which may contribute to the trend in poorer DFS and OS. These findings emphasize the need for identifying clinical and treatment factors that contribute to decreased survival in low socioeconomic status (SES) populations.

Background and Objectives: Interstitial photodynamic therapy (PDT) is an emerging modality for the treatment of solid organ disease. Our group at the University of Pennsylvania has performed extensive studies that demonstrate the feasibility of interstitial PDT for prostate cancer. Our preclinical and clinical experience is herein detailed. StudyDesign/MaterialsandMethods: We have treated 16 canines in preclinical studies, and 16 human subjects in a Phase I study, using motexafin lutetium-mediated PDT for recurrent prostate adenocarcinoma. Dosimetry of light fluence, drug level and oxygen distribution for these patients were performed. Results: We demonstrate the safe and comprehensive treatment of the prostate using PDT. However, there is significant variability in the dose distribution and the subsequent tissue necrosis throughout the prostate. Conclusions: PDT is an attractive option for the treatment of prostate adenocarcinoma. However, the observed variation in PDT dose distribution translates into uncertain therapeutic reproducibility. Our future focus will be on the development of an integrated system that is able to both detect and compensate for dose variations in real-time, in order to deliver a consistent overall PDT dose distribution. Lasers Surg. Med. 38:427–434, 2006. 2006 Wiley-Liss, Inc.

Cytoskeletal proteins play important roles in regulating cellular morphology, cytokinesis and intracellular signaling. In this report, we describe a developmentally regulated gene encoding a novel cell lineage-restricted cytoskeletal protein, designated SM22beta. SM22beta shares high-grade sequence identity with the smooth muscle cell (SMC)-specific protein, SM22alpha, the neuron-specific protein, NP25, and the Drosophila melanogaster flight muscle-specific protein, mp20. The mouse SM22beta cDNA encodes a 199-amino acid polypeptide that contains a single conserved calponin-like repeat domain. During mouse embryonic development, the SM22beta gene is expressed in a temporally and spatially regulated pattern in the tunica media of arteries and veins, endocardium and compact layer of the myocardium, bronchial epithelium and mesenchyme of the lung, gastrointestinal epithelium and cartilaginous primordia. During postnatal development, SM22beta is co-expressed with SM22alpha in arterial and venous SMCs. In addition, SM22beta is expressed at high levels in the bronchial epithelium and lung mesenchyme, gastrointestinal epithelial cells and in the cartilagenous and periosteal layer of bones. Three-dimensional deconvolution microscopic analyses of A7r5 SMCs revealed that SM22beta co-localizes with SM22alpha to cytoskeletal actin filaments. Taken together, these data demonstrate that SM22beta is a novel actin-associated protein with a unique cell lineage-restricted pattern of expression.

Serum response factor (SRF) plays an important role in regulating smooth muscle cell (SMC) development and differentiation. To understand the molecular mechanisms underlying the activity of SRF in SMCs, the two CArG box-containing elements in the arterial SMC-specific SM22alpha promoter, SME-1 and SME-4, were functionally and biochemically characterized. Mutations that abolish binding of SRF to the SM22alpha promoter totally abolish promoter activity in transgenic mice. Moreover, a multimerized copy of either SME-1 or SME-4 subcloned 5' of the minimal SM22alpha promoter (base pairs -90 to +41) is necessary and sufficient to restrict transgene expression to arterial SMCs in transgenic mice. In contrast, a multimerized copy of the c-fos SRE is totally inactive in arterial SMCs and substitution of the c-fos SRE for the CArG motifs within the SM22alpha promoter inactivates the 441-base pair SM22alpha promoter in transgenic mice. Deletion analysis revealed that the SME-4 CArG box alone is insufficient to activate transcription in SMCs and additional 5'-flanking nucleotides are required. Nuclear protein binding assays revealed that SME-4 binds SRF, YY1, and four additional SMC nuclear proteins. Taken together, these data demonstrate that binding of SRF to specific CArG boxes is necessary, but not sufficient, to restrict transgene expression to SMCs in vivo.

SM22alpha is a 22-kDa smooth muscle cell (SMC) lineage-restricted protein that physically associates with cytoskeletal actin filament bundles in contractile SMCs. To examine the function of SM22alpha, gene targeting was used to generate SM22alpha-deficient (SM22(-/-LacZ)) mice. The gene targeting strategy employed resulted in insertion of the bacterial lacZ reporter gene at the SM22alpha initiation codon, permitting precise analysis of the temporal and spatial pattern of SM22alpha transcriptional activation in the developing mouse. Northern and Western blot analyses confirmed that the gene targeting strategy resulted in a null mutation. Histological analysis of SM22(+/-LacZ) embryos revealed detectable beta-galactosidase activity in the unturned embryonic day 8.0 embryo in the layer of cells surrounding the paired dorsal aortae concomitant with its expression in the primitive heart tube, cephalic mesenchyme, and yolk sac vasculature. Subsequently, during postnatal development, beta-galactosidase activity was observed exclusively in arterial, venous, and visceral SMCs. SM22alpha-deficient mice are viable and fertile. Their blood pressure and heart rate do not differ significantly from their control SM22alpha(+/-) and SM22alpha(+/+) littermates. The vasculature and SMC-containing tissues of SM22alpha-deficient mice develop normally and appear to be histologically and ultrastructurally similar to those of their control littermates. Taken together, these data demonstrate that SM22alpha is not required for basal homeostatic functions mediated by vascular and visceral SMCs in the developing mouse. These data also suggest that signaling pathways that regulate SMC specification and differentiation from local mesenchyme are activated earlier in the angiogenic program than previously recognized.

Photodynamic therapy (PDT) takes advantage of the generation of reactive oxygen species and subsequent oxidative effects to generate focal tissue damage. It involves the local activation of a chemical photosensitizer by a specific light wavelength corresponding to the absorption characteristics of the photosensitizer. The excited photosensitizer then transfers its energy to oxygen within the tissue, thereby creating the cytotoxic oxygen species. PDT is an established treatment modality for cancer, and its feasibility has been demonstrated for a variety of cancer types. Here, we briefly detail the known mechanisms of the photodynamic reaction, with particular attention to the role of reactive oxygen species in PDT-mediated cell death. We then describe the application of PDT in recent clinical studies for the treatment of prostate cancer.

Previous studies by our group and others have demonstrated the importance of sociodemographic factors in cancer-related outcomes. The identification of these factors has led to novel approaches to the care of the high-risk cancer patient, specifically in the adoption of clinical interventions that convey similar benefits as favorable sociodemographic characteristics. This study examined the importance of marital status and race as prognostic indicators in men with prostate cancer. This report is a meta-analysis of 3,570 patients with prostate cancer treated in three prospective RTOG clinical trials. The Kaplan–Meier method was used to estimate the survival rate and the cumulative incidence method was used to analyze biochemical failure rate. Hazard ratios were calculated for all covariates using either the Cox or Fine and Gray’s proportional hazards model or logistic regression model with associated 95% confidence intervals and p values. Hazard ratio (HR) for overall survival (OS) for single status compared to married status was 1.36 (95% CI, 1.2 to 1.53). OS HR for non-White compared to White patients was 1.05 (CI 0.92 to 1.21). In contrast, the disease-free survival (DFS) HR and biochemical failure (BF) HR were both not significantly different neither between single and married patients nor between White patients and non-White patients. Median time to death for married men was 5.68 years and for single men was 4.73 years. Median time for DFS for married men was 7.25 years and for single men was 6.56 years. Median time for BF for married men was 7.81 years and for single men was 7.05 years. Race was not associated with statistically significant differences in this analysis. Congruent with our previous work in other cancer sites, marital status predicted improved prostate cancer outcomes including overall survival. Prostate cancer is the most common visceral cancer in men in the USA. The stratification of prostate cancer risk is currently modeled solely on pathologic prognostic factors including PSA and Gleason Score. Independent of these pathologic prognostic factors, our paper describes the central sociodemographic factor of being single as a negative prognostic indicator. Single men are at high risk of poorer outcomes after prostate cancer treatment. Intriguingly, in our group of patients, race was not a significant prognostic factor. The findings in this paper add to the body of work that describes important sociodemographic prognostic factors that are currently underappreciated in patients with cancer. Future steps will include the validation of these findings in prospective studies, and the incorporation of clinical strategies that identify and compensate for sociodemographic factors that predict for poorer cancer outcomes.

Purpose EF5, a 2-nitroimidazole hypoxia marker, was used to study the presence, levels, and prognostic significance of hypoxia in primary head and neck squamous cell tumors. Methods and Materials Twenty-two patients with newly diagnosed squamous cell carcinoma of the oral cavity, oropharynx, or larynx with at least 2 years of clinical follow-up were included in this study. Quantitative analyses of EF5 immunofluorescence was carried out, and these data were compared with patient outcome. Results EF5 immunostaining showed substantial intra- and intertumoral hypoxic heterogeneity. The majority of cells in all tumors were well oxygenated. Three patterns of EF5 binding in cells were identified using criteria based on the cellular region that was stained (peripheral or central) and the relationship of binding to necrosis. We tested the association between EF5-binding levels with event-free and overall survival irrespective of the pattern of cellular binding or treatment regimen. Patients with tumors containing EF5-binding regions corresponding to severe hypoxia (≤0.1% oxygen) had a shorter event-free survival time than patients with pO 2 values greater than 0.1% ( p = 0.032). Nodal status was also predictive for outcome. Conclusions These data illustrate the potential utility of EF5 binding based on quantitative immunohistochemistry of tissue pO 2 and provide support for the development of noninvasive hypoxia positron emission tomographic studies with fluorine 18–labeled EF5.

Members of the myocardin-related family of transcription factors play critical roles in regulating vascular smooth muscle and cardiac differentiation. To examine the function of myocardin-related transcription factor (MRTF)-B, mice were generated from ES cells harboring a conditional insertional mutation, or gene trap, of the MRTF-B gene. Expression of the MRTF-B mutant allele results in a fusion protein consisting of the N terminus of MRTF-B fused to β-galactosidase, which is functionally null. Homozygous MRTF-B gene trap mice ( MRTF-B –/– ) die between embryonic day (E)17.5 and postnatal day 1 from cardiac outflow tract defects. MRTF-B is expressed in the premigratory neural crest, in rhombomeres 3 and 5, and in the neural crest-derived mesenchyme surrounding the aortic arch arteries. Consistent with the pattern of expression, E10.5 and E11.5 MRTF-B –/– mutants exhibit deformation of aortic arch arteries 3, 4, and 6 and severe attenuation of smooth muscle cell differentiation in the arch arteries and the aorticopulmonary septum, despite normal migration and initial patterning of cardiac neural crest cells. Remarkably, the observed pathology was rescued and viable mice generated by intercrossing MRTF-B mutants with mice expressing Cre recombinase under the transcriptional control of the neural crest-restricted Wnt-1 promoter, which results in restoration of normal MRTF-B expression in the neural crest. Taken together, these studies reveal that MRTF-B plays a critical role in regulating differentiation of cardiac neural crest cells into smooth muscle and demonstrate that neural crest-derived smooth muscle differentiation is specifically required for normal cardiovascular morphogenesis.

The diverse functions mediated by smooth muscle cells (SMCs) in organ systems throughout the body are ultimately dependent upon the expression of a unique set of SMC-restricted contractile and cytoskeletal proteins that distinguish this cell lineage from cardiac and skeletal myocytes. A distinguishing feature of the SMC lineage is the capacity of SMCs to reversibly modulate their phenotype and proliferate in response to a variety of stimuli during postnatal development (for a review, see reference 35). In the vasculature, SMCs in the tunica medium of arteries and veins are cell cycle arrested and express a set of lineage-restricted genes, including those for smooth muscle (SM) myosin heavy chain, SM α-actin, SM22α, and calponin, which together define the unique contractile properties of this muscle cell lineage. However, in response to arterial injury, SMCs downregulate expression of contractile genes and concomitantly upregulate a set of genes required for synthetic, migratory, and proliferative functions. This phenotypic modulation has been implicated in the pathogenesis of diseases, including atherosclerosis, restenosis following coronary angioplasty and/or stent implantation, pulmonary hypertension, and asthma (14, 33, 39, 41). Because it is expressed exclusively and abundantly in SMCs during postnatal development (23, 42), our group and others have utilized the SMC-restricted SM22α promoter as a model system to elucidate the molecular mechanisms that regulate SMC differentiation and modulation of the SMC phenotype (4, 19, 24, 28, 34, 42, 44). The 441-bp mouse SM22α promoter functions in an SMC-specific fashion and restricts gene expression to arterial SMCs in transgenic mice (19, 24, 28). The SM22α promoter contains six nuclear protein binding sites, designated SME 1 to 6, two of which contain consensus CArG motifs (SME-1 and SME-4) that bind the MADS box transcription factor serum response factor (SRF) (19). Mutations that abolish binding of SRF to the SM22α promoter totally abolish promoter activity in transgenic mice (19). Moreover, a multimerized copy of the proximal SM22α CArG motif and 21 nucleotides of 5′-flanking sequence (bp −171 to −136) is necessary and sufficient to restrict gene expression to arterial SMCs in F0 transgenic mouse embryos (44). Consistent with these data, functionally important CArG boxes have been identified in multiple other SMC-restricted genes, including the SM myosin heavy chain (MyHC) promoter and intragenic enhancer, the SM α-actin promoter and intragenic enhancer, the telokin promoter, and the γ-enteric actin promoter (12, 25, 26, 38). Moreover, SRF plays an important role in regulating SMC differentiation and specification from undifferentiated mesenchyme (22). However, it remains unclear how SRF, a transcription factor that is expressed ubiquitously and which activates expression of growth-responsive genes, including c-fos and egr-1, can differentially activate genes expressed in an SMC lineage-restricted pattern. In this regard, it is noteworthy that SRF may be activated by posttranslational modifications and that alternative SRF isoforms have been identified, some of which function in a dominant-negative fashion (3, 17). SRF binds to the CArG box as a homodimer, via its MADS domain, and recruits transcriptional cofactors in order to integrate combinatorial signaling and modulate target gene expression (for a review, see reference 45). In skeletal muscle, SRF physically associates with MyoD and synergistically activates skeletal muscle-specific transcriptional regulatory elements (40). Similarly, in cardiac myocytes, SRF physically associates with the cardiac lineage-restricted transcription factors Nkx-2.5 and GATA4 and synergistically activates cardiac-specific transcriptional regulatory elements (5). Consistent with this paradigm, UV-crosslinking analyses of nuclear proteins that bind to the SME-4 CArG box in the SM22α promoter with SRF revealed an 80- to 90-kDa band that was expressed in an SMC lineage-restricted fashion (44). These data suggested that one, or more, SMC lineage-restricted SRF cofactors may function in concert with SRF and activate transcription of SMC lineage-restricted genes. Therefore, it was noteworthy when Olson and colleagues performed a search in silico and identified a novel cardiac-restricted transcription factor, designated myocardin, which binds to and functionally synergizes with SRF in the heart (48). Myocardin shows high-level sequence identity with members of the SAP (SAF-A/B, Acinus, PIAS) family of nuclear proteins, including the recently identified proteins MRTF-A/MAL/MKL1 and MRTF-B (1, 49). Each member of this family contains a conserved SAP domain that binds to A/T-rich genomic sequences or scaffold attachment regions (SARs). Related SAP family members have been shown to play a role in high-order transcriptional regulation, chromatin remodeling, and apoptosis-mediated condensation and fragmentation of chromosomal DNA (1, 10, 20). Olson and colleagues reported that myocardin is an early marker of the cardiac muscle cell lineage, where it continues to be expressed throughout development (48). In addition, they reported that myocardin is expressed transiently in some SMCs during embryonic development. Interestingly, myocardin transactivates multiple cardiac-restricted promoters but notably only those containing consensus CArG boxes or SRF binding sites (48). Consistent with this finding, the N-terminal domain of myocardin binds directly to the MADS box of SRF (48). In Xenopus laevis, forced expression of a dominant negative myocardin mutant protein resulted in a dramatic reduction in genes encoding cardiac myocyte-restricted contractile protein isoforms and the cardiac-restricted homeobox transcription factor Nkx-2.5 (48). Given these data and the previously defined role of SRF in the SMC lineage, it was of interest to determine what role, if any, myocardin plays in regulating differentiation of the SMC lineage. In the studies described in this report, we demonstrated that the myocardin gene is expressed in visceral and vascular SMCs at levels equivalent to or exceeding those observed in the heart, the myocardin gene is developmentally regulated in visceral and vascular SMCs during embryonic development, myocardin transactivates multiple SMC-specific transcriptional regulatory elements in an SRF-dependent fashion, SM22α promoter activity in SMCs is myocardin dependent, and, remarkably, forced expression of myocardin in undifferentiated embryonic stem (ES) cells activates transcription and expression of the endogenous SM22α gene as well as expression of the endogenous SM-α-actin and calponin-h1 genes. Taken together, these data demonstrate that myocardin plays a previously unrecognized and important role in the transcriptional program regulating SMC development and differentiation.

Unlike human L1 retrotransposons, the 5′ UTR of mouse L1 elements contains tandem repeats of ∼200 bp in length called monomers. Multiple L1 subfamilies exist in the mouse which are distinguished by their monomer sequences. We previously described a young subfamily, called the TF subfamily, which contains ∼1800 active elements among its 3000 full-length members. Here we characterize a novel subfamily of mouse L1 elements, GF, which has unique monomer sequence and unusual patterns of monomer organization. A majority of these GF elements also have a unique length polymorphism in ORF1. Polymorphism analysis of GF elements in various mouse subspecies and laboratory strains revealed that, like TF, the GF subfamily is young and expanding. About 1500 full-length GF elements exist in the diploid mouse genome and, based on the results of a cell culture assay, ∼400 GF elements are potentially capable of retrotransposition. We also tested 14 A-type subfamily elements in the assay and estimate that about 900 active A elements may be present in the mouse genome. Thus, it is now known that there are three large active subfamilies of mouse L1s; TF, A, and GF, and that in total ∼3000 full-length elements are potentially capable of active retrotransposition. This number is in great excess to the number of L1 elements thought to be active in the human genome.

chemoradiation therapy regimens, cisplatin is one of the most commonly utilized agents. Although cisplatin is highly effective as a radiosensitizer, its use has been limited by toxicity. Therefore, one of the goals of radiation oncology research has been identifying methods to improve cisplatin’s efficacy while lowering its toxicity. While traditional drug delivery strategies such as intra-arterial delivery have failed, the development of nanoparticle (NP) drug delivery vehicles offers an unprecedented opportunity. NPs are known to preferentially accumulate in tumors while depositing lower dose to normal tissues such as kidneys. Such unique biodistribution is ideally suited for chemoradiation therapy. We hypothesized that the development of a NP formulation of cisplatin can improve therapeutic efficacy and lower the toxicity of cisplatin-based chemoradiation therapy. Materials/Methods: We have developed a novel formulation of cisplatin using the Polysilsesquoxane (PSQ) NP platform. The cisplatin-PSQ NPs were characterized physically using transmission electron microscopy. Its drug release profile was determined. First, we evaluated cisplatin-PSQ NP as a radiosensitizer in vitro using non-small cell lung cancer (NSCLC) cells lines H460 and A549. We then compared the toxicity profiles of cisplatin-PSQ NP to that of cisplatin in vivo. The efficacy of cisplatin-PSQ NP in chemoradiation therapy was compared to that of cisplatin using mouse models of NSCLC. Results: Cisplatin-PSQ NPs were w65 nm in diameter with cisplatin loading of up to 42% (weight) of NP. The cisplatin-PSQ NPs were highly stable with minimal drug release in neutral pH. Cisplatin release was rapid from NPs in reducing environments similar to that of tumor. In vitro evaluation showed cisplatin-PSQ NP is an excellent radiosensitizer with radiation survival curves similar to that of cisplatin. Toxicity evaluations showed cisplatin-PSQ NP has a significantly lower toxicity overall toxicity profile than that of cisplatin. Furthermore, cisplatin-PSQ NP has lower renal toxicity than cisplatin. Lastly, we demonstrated that cisplatin-PSQ NP is more effective than cisplatin in chemoradiation therapy using two mouse xenograft models of NSCLC. Conclusions: We have engineered a novel NP formulation of cisplatin. Cisplatin-PSQ NP has been shown to have lower toxicity and higher efficacy than cisplatin in chemoradiation therapy using NSCLC as a model disease. We believe cisplatin-PSQ is a promising radiosensitizer with high potential for clinical translation. Author Disclosure: A.Z. Wang: P. Ownership Other; Co-Founder, Coordination Therapeutics, Inc. J. Della Rocca: None. N.D. Cummings: None. R. Sukumar: None. M.E. Werner: None. W. Lin: P. Ownership Other; CoFounder, Coordination Therapeutics, Inc.