On-line cone beam CT image guidance for vocal cord tumor targeting

Background and purpose: We are developing a technique for highly focused vocal cord irradiation in early glottic carcinoma to optimally treat a target volume confined to a single cord. This technique, in contrast with the conventional methods, aims at sparing the healthy vocal cord. As such a technique requires sub-mm daily targeting accuracy to be effective, we investigate the accuracy achievable with on-line kV-cone beam CT (CBCT) corrections. Materials and methods: CBCT scans were obtained in 10 early glottic cancer patients in each treatment fraction. The grey value registration available in X-ray volume imaging (XVI) software (Elekta, Synergy) was applied to a volume of interest encompassing the thyroid cartilage. After application of the thus derived corrections, residue displacements with respect to the planning CT scan were measured at clearly identifiable relevant landmarks. The intra- and inter-observer variations were also measured. Results: While before correction the systematic displacements of the vocal cords were as large as 2.4 ± 3.3 mm (cranial-caudal population mean ± SD Σ), daily CBCT registration and correction reduced these values to less than 0.2 ± 0.5 mm in all directions. Random positioning errors (SD σ) were reduced to less than 1 mm. Correcting only for translations and not for rotations did not appreciably affect this accuracy. The residue random displacements partly stem from intra-observer variations (SD = 0.2-0.6 mm). Conclusion: The use of CBCT for daily image guidance in combination with standard mask fixation reduced systematic and random set-up errors of the vocal cords to <1 mm prior to the delivery of each fraction dose. Thus, this facilitates the high targeting precision required for a single vocal cord irradiation. © 2009 Elsevier Ireland Ltd. All rights reserved.

Tumor-derived CXCL8 signaling augments stroma-derived CCL2-promoted proliferation and CXCL12-mediated invasion of PTEN-deficient prostate cancer cells

Impaired PTEN function is a genetic hallmark of aggressive prostate cancers (CaP) and is associated with increased CXCL8 expression and signaling. The current aim was to further characterize biological responses and mechanisms underpinning CXCL8-promoted progression of PTEN-depleted prostate cancer, focusing on characterizing the potential interplay between CXCL8 and other disease-promoting chemokines resident within the prostate tumor microenvironment. Autocrine CXCL8-stimulation (i) increased expression of CXCR1 and CXCR2 in PTEN-deficient CaP cells suggesting a self-potentiating signaling axis and (ii) induced expression of CXCR4 and CCR2 in PTEN-wild-type and PTEN-depleted CaP cells. In contrast, paracrine CXCL8 signaling induced expression and secretion of the chemokines CCL2 and CXCL12 from prostate stromal WPMY-1 fibroblasts and monocytic macrophage-like THP-1 cells. In vitro studies demonstrated functional co-operation of tumor-derived CXCL8 with stromal-derived chemokines. CXCL12-induced migration of PC3 cells and CCL2-induced proliferation of prostate cancer cells were dependent upon intrinsic CXCL8 signaling within the prostate cancer cells. For example, in co-culture experiments, CXCL12/CXCR4 signaling but not CCL2/CCR2 signaling supported fibroblast-mediated migration of PC3 cells while CXCL12/CXCR4 and CCL2/CCR2 signaling underpinned monocyte-enhanced migration of PC3 cells. Combined inhibition of both CXCL8 and CXCL12 signaling was more effective in inhibiting fibroblast-promoted cell motility while repression of CXCL8 attenuated CCL2-promoted proliferation of prostate cancer cells. We conclude that tumor-derived CXCL8 signaling from PTEN-deficient tumor cells increases the sensitivity and responsiveness of CaP cells to stromal chemokines by concurrently upregulating receptor expression in cancer cells and inducing stromal chemokine synthesis. Combined chemokine targeting may be required to inhibit their multi-faceted actions in promoting the invasion and proliferation of aggressive CaP.

Mechanistic Rationale to Target PTEN-Deficient Tumor Cells with Inhibitors of the DNA Damage Response Kinase ATM

Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response (DDR). ATM loss of function can produce a synthetic lethal phenotype in combination with tumor-associated mutations in FA/BRCA pathway components. In this study, we took an siRNA screening strategy to identify other tumor suppressors that, when inhibited, similarly sensitized cells to ATM inhibition. In this manner, we determined that PTEN and ATM were synthetically lethal when jointly inhibited. PTEN-deficient cells exhibited elevated levels of reactive oxygen species, increased endogenous DNA damage, and constitutive ATM activation. ATM inhibition caused catastrophic DNA damage, mitotic cell cycle arrest, and apoptosis specifically in PTEN-deficient cells in comparison with wild-type cells. Antioxidants abrogated the increase in DNA damage and ATM activation in PTEN-deficient cells, suggesting a requirement for oxidative DNA damage in the mechanism of cell death. Lastly, the ATM inhibitor KU-60019 was specifically toxic to PTEN mutant cancer cells in tumor xenografts and reversible by reintroduction of wild-type PTEN. Together, our results offer a mechanistic rationale for clinical evaluation of ATM inhibitors in PTEN-deficient tumors.

Time and Cell Type Dependency of Survival Responses in Co-cultured Tumor and Fibroblast Cells after Exposure to Modulated Radiation Fields

Advanced radiotherapy techniques such as intensity-modulated radiation therapy (IMRT) achieve high levels of conformity to the target volume through the sequential delivery of highly spatially and temporally modulated radiation fields, which have been shown to impact radiobiological response. This study aimed to characterize the time and cell type dependency of survival responses to modulated fields using single cell type (SCT) and mixed cell type (MCT) co-culture models of transformed fibroblast (AG0-1522b) cells, and prostate (DU-145) and lung (H460) cancer cells. In SCT cultures, in-field responses showed no significant time dependency while out-of-field responses occurred early, and plateaued 6 h after irradiation in both DU-145 and H460 cells. Under modulated beam configurations MCT co-cultures showed cell-specific, differential out-of-field responses depending on the irradiated in-field and responding out-of-field cell type. The observed differential out-of-field responses may be due to the genetic background of the cells, in particular p53 status, which has been shown to mediate radiation-induced bystander effects (RIBEs). These data provide further insight into the radiobiological parameters that influence out-of-field responses, which have potential implications for advanced radiotherapy modalities and may provide opportunities for biophysical optimization in radiotherapy treatment planning.

Independent genomewide screens identify the tumor suppressor VTRNA2-1 as a human epiallele responsive to periconceptional environment

Background: Interindividual epigenetic variation that occurs systemically must be established prior to gastrulation in the very early embryo and, because it is systemic, can be assessed in easily biopsiable tissues. We employ two independent genome-wide approaches to search for such variants.

Results: First, we screen for metastable epialleles by performing genomewide bisulfite sequencing in peripheral blood lymphocyte (PBL) and hair follicle DNA from two Caucasian adults. Second, we conduct a genomewide screen for genomic regions at which PBL DNA methylation is affected by season of conception in rural Gambia. Remarkably, both approaches identify the genomically imprinted VTRNA2-1 as a top environmentally responsive epiallele. We demonstrate systemic and stochastic interindividual variation in DNA methylation at the VTRNA2-1 differentially methylated region in healthy Caucasian and Asian adults and show, in rural Gambians, that periconceptional environment affects offspring VTRNA2-1 epigenotype, which is stable over at least 10 years. This unbiased screen also identifies over 100 additional candidate metastable epialleles, and shows that these are associated with cis genomic features including transposable elements.

Conclusions: The non-coding VTRNA2-1 transcript (also called nc886) is a putative tumor suppressor and modulator of innate immunity. Thus, these data indicating environmentally induced loss of imprinting at VTRNA2-1 constitute a plausible causal pathway linking early embryonic environment, epigenetic alteration, and human disease. More broadly, the list of candidate metastable epialleles provides a resource for future studies of epigenetic variation and human disease.

A selective antagonist of histamine H₄ receptors prevents antigen-induced airway inflammation and bronchoconstriction in guinea pigs:involvement of lipocortin-1

BACKGROUND AND PURPOSE: Among the pathogenic mechanisms of asthma, a role for oxidative/nitrosative stress has been well documented. Recent evidence suggests that histamine H₄ receptors play a modulatory role in allergic inflammation. Here we report the effects of compound JNJ 7777120 (JNJ), a selective H4 receptor antagonist, on antigen-induced airway inflammation, paying special attention to its effects on lipocortin-1 (LC-1/annexin-A1), a 37 kDA anti-inflammatory protein that plays a key role in the production of inflammatory mediators.

EXPERIMENTAL APPROACH: Ovalbumin (OA)-sensitized guinea pigs placed in a respiratory chamber were challenged with antigen. JNJ (5, 7.5 and 10 mg.kg⁻¹) was given i.p. for 4 days before antigen challenge. Respiratory parameters were recorded. Bronchoalveolar lavage (BAL) fluid was collected and lung specimens taken for further analyses 1 h after antigen challenge. In BAL fluid, levels of LC-1, PGD2 , LTB4 and TNF-α were measured. In lung tissue samples, myeloperoxidase, caspase-3 and Mn-superoxide dismutase activities and 8-hydroxy-2-deoxyguanosine levels were measured.

KEY RESULTS: OA challenge decreased LC-1 levels in BAL fluid, induced cough, dyspnoea and bronchoconstriction and increased PGD2 , LTB4 and TNF-α levels in lung tissue. Treatment with JNJ dose-dependently increased levels of LC-1, reduced respiratory abnormalities and lowered levels of PGD2 , LTB4 and TNF-α in BAL fluid.

CONCLUSIONS AND IMPLICATIONS: Antigen-induced asthma-like reactions in guinea pigs decreased levels of LC-1 and increased TNF-α and eicosanoid production. JNJ pretreatment reduced allergic asthmatic responses and airway inflammation, an effect associated with LC-1 up-regulation.

Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70

The Wilms tumor suppressor WT1 encodes a zinc finger transcription factor that is expressed in glomerular podocytes during a narrow window in kidney development. By immunoprecipitation and protein microsequencing analysis, we have identified a major cellular protein associated with endogenous WT1 to be the inducible chaperone Hsp70. WT1 and Hsp70 are physically associated in embryonic rat kidney cells, in primary Wilms tumor specimens and in cultured cells with inducible expression of WT1. Colocalization of WT1 and Hsp70 is evident within podocytes of the developing kidney, and Hsp70 is recruited to the characteristic subnuclear clusters that contain WT1. The amino-terminal transactivation domain of WT1 is required for binding to Hsp70, and expression of that domain itself is sufficient to induce expression of Hsp70 through the heat shock element (HSE). Substitution of a heterologous Hsp70-binding domain derived from human DNAJ is sufficient to restore the functional properties of a WT1 protein with an amino-terminal deletion, an effect that is abrogated by a point mutation in DNAJ that reduces binding to Hsp70. These observations indicate that Hsp70 is an important cofactor for the function of WT1, and suggest a potential role for this chaperone during kidney differentiation.

CD38 expression level and pattern of expression remains a reliable and robust marker of progressive disease in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) follows a variable clinical course which is difficult to predict at diagnosis. We assessed somatic mutation (SHM) status, CD38 and ZAP-70 expression in 87 patients (49 male, 38 female) with stage A CLL and known cytogenetic profile to compare their role in predicting disease progression, which was assessed by the treatment free interval (TFI) from diagnosis. Sixty (69%) patients were SHM+, 24 (28%) were CD38+ and ten (12%) were ZAP-70+. The median TFI for: (i) SHM + versus SHM- patients was 124 versus 26 months; hazard ratio (HR) = 3.6 [95% confidence interval (CI) = 1.8 - 7.3; P = 0.001]: (ii) CD38- versus CD38+ patients was 120 versus 34 months; HR = 2.4 (95% CI = 1.4 - 5.3; P = 0.02); and (iii) ZAP70- versus ZAP70+ was 120 versus 16 months; HR = 3.4 (95% CI = 1.4 - 8.7; P = 0.01). SHM status and CD38 retained prognostic significance on multivariate analysis whereas ZAP-70 did not. We conclude that ZAP-70 analysis does not provide additional prognostic information in this group of patients.

Wilms' tumor gene 1 (WT1) expression in childhood acute lymphoblastic leukemia:a wide range of WT1 expression levels, its impact on prognosis and minimal residual disease monitoring

Wilms' tumor gene 1 (WT1) is overexpressed in the majority (70-90%) of acute leukemias and has been identified as an independent adverse prognostic factor, a convenient minimal residual disease (MRD) marker and potential therapeutic target in acute leukemia. We examined WT1 expression patterns in childhood acute lymphoblastic leukemia (ALL), where its clinical implication remains unclear. Using a real-time quantitative PCR designed according to Europe Against Cancer Program recommendations, we evaluated WT1 expression in 125 consecutively enrolled patients with childhood ALL (106 BCP-ALL, 19 T-ALL) and compared it with physiologic WT1 expression in normal and regenerating bone marrow (BM). In childhood B-cell precursor (BCP)-ALL, we detected a wide range of WT1 levels (5 logs) with a median WT1 expression close to that of normal BM. WT1 expression in childhood T-ALL was significantly higher than in BCP-ALL (P<0.001). Patients with MLL-AF4 translocation showed high WT1 overexpression (P<0.01) compared to patients with other or no chromosomal aberrations. Older children (> or =10 years) expressed higher WT1 levels than children under 10 years of age (P<0.001), while there was no difference in WT1 expression in patients with peripheral blood leukocyte count (WBC) > or =50 x 10(9)/l and lower. Analysis of relapsed cases (14/125) indicated that an abnormal increase or decrease in WT1 expression was associated with a significantly increased risk of relapse (P=0.0006), and this prognostic impact of WT1 was independent of other main risk factors (P=0.0012). In summary, our study suggests that WT1 expression in childhood ALL is very variable and much lower than in AML or adult ALL. WT1, thus, will not be a useful marker for MRD detection in childhood ALL, however, it does represent a potential independent risk factor in childhood ALL. Interestingly, a proportion of childhood ALL patients express WT1 at levels below the normal physiological BM WT1 expression, and this reduced WT1 expression appears to be associated with a higher risk of relapse.

Activated protein C inhibits tumor necrosis factor and macrophage migration inhibitory factor production in monocytes

The precise regulatory mechanisms of amplification and downregulation of the pro- and anti-inflammatory cytokines in the inflammatory response have not been fully delineated. Although activated protein C (APC) and its precursor protein C (PC) have recently been reported to be promising therapeutic agents in the management of meningococcal sepsis, direct evidence for the anti-inflammatory effect remains scarce. We report that APC inhibits in vitro the release of tumor necrosis factor (TNF) and macrophage migration inhibitory factor (MIF), two known cytokine mediators of bacterial septic shock, from lipopolysaccharide (LPS)-stimulated human monocytes. The THP-1 monocytic cell line, when stimulated with LPS and concomitant APC, exhibited a marked reduction in the release of TNF and MIF protein in a concentration-dependent manner compared to cells stimulated with LPS alone. This effect was observed only when incubations were performed in serum-free media, but not in the presence of 1-10% serum. Serum-mediated inhibition could only be overcome by increasing APC concentrations to far beyond physiological levels, suggesting the presence of endogenous serum-derived APC inhibitors. Inhibition of MIF release by APC was found to be independent of TNF, as stimulation of MIF release by LPS was unaltered in the presence of anti-TNF antibodies. Our data confirm that the suggested anti-inflammatory properties of APC are due to direct inhibition of the release of the pro-inflammatory monokine TNF, and imply that the anti-inflammatory action of APC is also mediated via inhibition of MIF release.

Automated tumor analysis for molecular profiling in lung cancer

The discovery and clinical application of molecular biomarkers in solid tumors, increasingly relies on nucleic acid extraction from FFPE tissue sections and subsequent molecular profiling. This in turn requires the pathological review of haematoxylin & eosin (H&E) stained slides, to ensure sample quality, tumor DNA sufficiency by visually estimating the percentage tumor nuclei and tumor annotation for manual macrodissection. In this study on NSCLC, we demonstrate considerable variation in tumor nuclei percentage between pathologists, potentially undermining the precision of NSCLC molecular evaluation and emphasising the need for quantitative tumor evaluation. We subsequently describe the development and validation of a system called TissueMark for automated tumor annotation and percentage tumor nuclei measurement in NSCLC using computerized image analysis. Evaluation of 245 NSCLC slides showed precise automated tumor annotation of cases using Tissuemark, strong concordance with manually drawn boundaries and identical EGFR mutational status, following manual macrodissection from the image analysis generated tumor boundaries. Automated analysis of cell counts for % tumor measurements by Tissuemark showed reduced variability and significant correlation (p < 0.001) with benchmark tumor cell counts. This study demonstrates a robust image analysis technology that can facilitate the automated quantitative analysis of tissue samples for molecular profiling in discovery and diagnostics.

Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

A commercial Bacillus anthracis (Anthrax) whole genome protein microarray has been used to identify immunogenic Anthrax proteins (IAP) using sera from groups of donors with (a) confirmed B. anthracis naturally acquired cutaneous infection, (b) confirmed B. anthracis intravenous drug use-acquired infection, (c) occupational exposure in a wool-sorters factory, (d) humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups. Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However, a number of other chromosomally-located and plasmid encoded open reading frame proteins were also recognized by infected or exposed groups in comparison to controls. Some of these antigens e.g., BA4182 are not recognized by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo that are not currently found in the UK licensed Anthrax Vaccine (AVP). These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis "infectome." These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesized, tested in mouse immunogenicity studies and validated in parallel using human sera from the same study.

Infrequent Expression of the Cancer-Testis Antigen, PASD1, in Ovarian Cancer

Ovarian cancer is very treatable in the early stages of disease; however, it is usually detected in the later stages, at which time, treatment is no longer as effective. If discovered early (Stage I), there is a 90% chance of five-year survival. Therefore, it is imperative that early-stage biomarkers are identified to enhance the early detection of ovarian cancer. Cancer-testis antigens (CTAs), such as Per ARNT SIM (PAS) domain containing 1 (PASD1), are unique in that their expression is restricted to immunologically restricted sites, such as the testis and placenta, which do not express MHC class I, and cancer, making them ideally positioned to act as targets for immunotherapy as well as potential biomarkers for cancer detection where expressed. We examined the expression of PASD1a and b in a number of cell lines, as well as eight healthy ovary samples, eight normal adjacent ovarian tissues, and 191 ovarian cancer tissues, which were predominantly stage I (n = 164) and stage II (n = 14) disease. We found that despite the positive staining of skin cancer, only one stage Ic ovarian cancer patient tissue expressed PASD1a and b at detectable levels. This may reflect the predominantly stage I ovarian cancer samples examined. To examine the restriction of PASD1 expression, we examined endometrial tissue arrays and found no expression in 30 malignant tumor tissues, 23 cases of hyperplasia, or 16 normal endometrial tissues. Our study suggests that the search for a single cancer-testes antigen/biomarker that can detect early ovarian cancer must continue.