Preliminary Clinical Evaluation of Intrafraction Prostate Displacements for Two Immobilization Systems.

Immobilization systems and their corresponding set-up errors influence the clinical target volume to the planning target volume (CTV-PTV) margins, which is critical for hypofractionated prostate stereotactic body radiotherapy (SBRT). This preliminary study evaluates intrafraction prostate displacement for two immobilization systems (A and B). Six consecutive patients having localized prostate cancer and implanted prostate marker seeds were studied. Planar X-ray images were acquired pre- and post-treatment to find the intrafraction prostate displacement. The average absolute displacements (lateral, longitudinal, vertical) were 0.9 ± 0.4 mm, 1.7 ± 0.1 mm, 1.3 ± 0.3 mm (system A), and 0.5 ± 0.2 mm, 0.6 ± 0.1 mm, 0.8 ± 0.3 mm (system B), with average three-dimensional displacements of 2.6 ± 0.2 mm (system A) and 1.3 ± 0.2 mm (system B). The computed CTV-PTV margins (lateral, longitudinal, vertical) were 2.5 mm, 2.5 mm, 3.6 mm and 1.4 mm, 1.6 mm, 2.4 mm for systems A and B, respectively. This suggests that the immobilization system influences intrafraction prostate displacement and, therefore, the margins applied. However, the margins found for both systems are comparable to the margins used for hypofractionated prostate SBRT.

Prospective Study of Use of Edmonton Symptom Assessment Scale Versus Routine Symptom Management During Weekly Radiation Treatment Visits.

PURPOSE: During radiotherapy (RT), patient symptoms are evaluated and managed weekly during physician on-treatment visits (OTVs). The Edmonton Symptom Assessment Scale (ESAS) is a 9-symptom validated self-assessment tool for reporting common symptoms in patients with cancer. We hypothesized that implementation and physician review of ESAS during weekly OTVs may result in betterment of symptom severity during RT for certain modifiable domains. METHODS: As an institutional quality improvement project, patients were partitioned into 2 groups: (1) 85 patients completing weekly ESAS (preintervention) but blinded to their providers who gave routine symptom management and (2) 170 completing weekly ESAS (postintervention group) reviewed by providers during weekly OTVs with possible intervention. To determine the independent association with symptom severity of the intervention, multivariate logistic regression was performed. At study conclusion, provider assessments of ESAS utility were also collected. RESULTS: Compared with the preintervention group, stable or improved symptom severity was seen in the postintervention group for pain (70.7% v 85.6%; P = .005) and anxiety (79.3% v 92.9%; P = .002). The postintervention group had decreased association (on multivariate analysis) with worsening severity of pain (OR, 0.13; P < .001), nausea (OR, 0.25; P = .023), loss of appetite (OR, 0.30; P = .024), and anxiety (OR, 0.19; P = .005). Most physicians (87.5%) and nurses (75%) found ESAS review useful in symptom management. CONCLUSION: Incorporation of ESAS for OTVs was associated with stable or improved symptom severity where therapeutic intervention is more readily available, such as counseling, pain medication, anti-emetics, appetite stimulants, and anti-anxiolytics. The incorporation of validated patient-reported symptom-scoring tools may improve provider management.

Vascular-targeted cancer gene therapy.

As a consequence of the dramatic progress that has been made in recent years towards elucidating the diverse molecular events involved in the development and pathogenesis of malignant disease, there is now no shortage of genes that can be exploited or targeted in the context of cancer gene therapy. Many of these have been shown to be effective both in vitro and in various animal models, and a number have progressed to the clinic. The results of these later studies, although generally encouraging, are perhaps less dramatic than one might have hoped. Although a number of factors undoubtedly contribute to this finding, it is evident that a major reason relates to the difficulties implicit in achieving efficient in vivo gene transfer, particularly in a clinical context. Targeting gene therapy, not to the malignant population, but instead to the vasculature upon which the survival and growth of a tumour depends constitutes an alternative approach that overcomes some of the delivery problems associated with established tumour cell-directed strategies.

Molecular mechanisms regulating the tumor-targeting potential of splice-activated gene expression.

Previous studies have suggested that differences in the ability of normal and malignant cells to process certain alternatively spliced pre-mRNA transcripts can be exploited as a potentially powerful means of targeting the expression of therapeutic genes to tumor cells in vivo and in vitro. Specifically, it was shown that efficient processing of minigene constructs containing the alternatively spliced CD44 exons v9 and v10 only occurs in tumor cells that express CD44 isoforms that incorporate these exons (e.g. CD44R1). In the present study, efforts were made to define the molecular mechanisms that underlie the apparent specificity of this process. RT-PCR analysis and DNA sequencing were used to characterize the various splicing events that occur between CD44 exons v8, v9 and v10 following transfection of minigene constructs containing these various exons into CD44R1-positive (PC3) and CD44R1-negative (T24) cell lines. The results obtained confirm that although the v8-v9 intron is efficiently removed in both CD44R1-positive and CD44R1-negative cells, the corresponding v9-v10 intron is accurately spliced and the exons appropriately joined only in lines that express v10-containing CD44 isoforms (e.g. PC3). In CD44R1-negative cell lines (e.g. T24) alternative 5' and 3' splice sites located within the v9-v10 intron are preferentially used, resulting in various portions of the intron being retained within the final processed mRNA product. It is proposed that identification of these functionally important intronic sequence elements will facilitate the development of second generation "splice activated gene expression" vectors that may prove useful in various cancer gene therapy applications.

Exploiting the differential production of angiogenic factors within the tumor microenvironment in the design of a novel vascular-targeted gene therapy-based approach to the treatment of cancer.

PURPOSE: The aim of this study is to explore a novel strategy through which the differential production of pro-angiogenic cytokines within the tumor microenvironment can be exploited as a means of selectively killing the vascular endothelial cells upon which the survival and growth of a tumor depend. METHODS AND MATERIALS: Adenoviral vectors encoding a chimeric cell surface receptor composed of the extracellular domain of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR fused in frame to the membrane spanning and cytoplasmic domain of Fas were constructed and used to transduce primary human endothelial cells in vitro. The apoptotic response of these cells induced upon ligation of the chimeric receptor with VEGF was determined by measuring caspase-3 activation, AnnexinV-FITC binding, and the release of glucose-6-phosphate dehydrogenase. RESULTS: The chimeric Flk-1/Fas protein is stable and expressed at high levels on the surface of adenovirally transduced cells. Upon the addition of exogenous VEGF, these cells undergo rapid apoptosis. CONCLUSIONS: Receptor/Fas chimeras that recognize and bind pro-angiogenic cytokines represent a novel means by which the signal transduction events normally triggered in vascular endothelial cells upon the binding of angiogenic cytokines may be redirected toward the induction of apoptotic cell death. It is proposed that these constructs will prove of value in the further development of safe and effective vascular-targeted gene therapy-based approaches to the treatment of cancer.

Identification of sequence motifs responsible for the adhesive interaction between exon v10-containing CD44 isoforms.

Previous studies have demonstrated that CD44 isoforms containing the alternatively spliced exon v10 promote cell-cell adhesion via a mechanism that involves the recognition of chondroitin sulfate side chains presented on the surface of interacting cells in association with other CD44 molecules. Sequence analysis revealed the presence within exon v10 of two motifs that may be relevant to this interaction, a B[X(7)]B motif that may contribute to the recognition and binding of chondroitin sulfate and a serine-glycine motif that may serve as a site of chondroitin sulfate attachment. To determine whether either of these two motifs explain the unique adhesive activity of exon v10-containing CD44 isoforms, each was targeted by site-directed mutagenesis, and the adhesive activity of the resultant mutants was determined using a quantitative cell-cell binding assay. The data obtained demonstrate conclusively that it is the exon v10-encoded B[X(7)]B motif that is solely responsible for the enhanced adhesive activity of exon v10-containing CD44 isoforms.

Alternative splicing as a novel of means of regulating the expression of therapeutic genes.

In order to determine the potential of alternative splicing as a means of targeting the expression of therapeutic genes to tumor cells in vivo, a series of episomal plasmid-based "splice-activated gene expression" (pSAGE) vectors was generated, which contain minigene cassettes composed of various combinations of the three alternatively spliced exons present in the differentially expressed adhesion protein CD44R1 (v8, v9, and v10) with or without their corresponding intronic sequences, positioned in-frame between the CD44 leader sequence and a "leaderless" human liver/bone/kidney alkaline phosphatase (ALP) cDNA. Because both the v8-v9 and v9-v10 introns contain multiple in-frame stop codons, the expression and enzymatic activity of ALP are dependent upon the accurate removal of intronic sequences from the pre-mRNA transcripts encoded by these constructs. The various pSAGE constructs were introduced into CD44H-positive (T24) and CD44R1-positive (PC3) target cells by electroporation and transfectants selected in hygromycin B. ALP expression was determined by staining with the ALP substrate, BCIP/INT, and the transfected cells tested for their sensitivity to the inactive prodrug, etoposide phosphate. ALP-mediated dephosphorylation of etoposide phosphate generates the potent topoisomerase II inhibitor etoposide. The data obtained indicate that whereas the v8-v9 intron is spliced in both CD44H- and CD44R1-positive cells, the v9-v10 intron is efficiently and accurately removed only in CD44R1-positive cells. Furthermore, only CD44R1-positive cells were sensitized to etoposide phosphate when transfected with the v9-v10.ALP construct. These data emphasize the potential usefulness of alternative splicing as a novel means of targeting gene expression to tumor cells in vivo.