Analysis of lymphatic and blood vessel invasion biomarkers in T1 esophagogastric adenocarcinomas for improved patient prognostication.

Lymphovascular invasion (LVI) in T1 esophagogastric adenocarcinoma may predict risk of recurrence despite definitive treatment with surgery or endoscopic resection. Podoplanin and CD34 are emerging biomarkers of lymphatic and blood vessel invasion, respectively, and could be adopted to refine LVI assessment. A consecutive series of 65 patients with T1 adenocarcinomas diagnosed at Nottingham University Hospitals were investigated. T1 tumors from 43/65 patients who received primary surgery only were suitable for LVI evaluation by hematoxylin and eosin (H&E) staining as well as by CD34 and Podoplanin immunohistochemistry. LVI was correlated to clinicopathological features and recurrence free survival. H&E staining detected LVI in 11.6% (5/43) of T1 tumors. CD34 and Podoplanin immunohistochemistry significantly improved LVI detection to 25.6% (11/43). Compared with LVI by H&E, immunohistochemical evaluation of blood vessel invasion (CD34) or lymphatic vessel invasion (Podoplanin) was significantly associated with higher grade (P = 0.005), submucosal invasion (T1b) (P = 0.018), lymph node positivity (N1) (P = 0.029) and poor recurrence free survival (P = 0.0003). Our study provides evidence that CD34 and Podoplanin immunohistochemistry could improve LVI detection and allow better prognostication of patients and optimum selection of definitive treatment. Larger multicenter studies are required for further validation that could have significant clinical implications.

A spectrum of susceptibility to rheumatoid arthritis within HLA-DRB1: stratification by autoantibody status in a large UK population.

Previously-proposed rheumatoid arthritis (RA) HLA-DRB1 susceptibility and protective models were compared, based on amino acids at positions 67-74 and autoantibody combinations. 3 657 RA patients and 1 357 controls were studied using logistic regression, with secondary stratification by anti-citrullinated peptide antibodies(ACPA) and rheumatoid factor(RF). Susceptibility models were based on previously defined HLA-DRB1 shared epitope(SE) subgroups. (70)DERAA(74), D(70) and I(67) protective models were compared, adjusting for HLA-DRB1 SE. A hierarchy of risk was observed within the HLA-DRB1 SE, particularly for ACPA-positive and RF-positive RA: HLA-DRB1(*)0401∼(*)0404>(*)0101∼(*)1001 ((*)0404>(*)0101: P=0.0003). HLA-DRB1(*)0401/(*)0404 compound heterozygosity conferred a risk similar to (*)0401 homozygosity (P=0.70). Protective effects of D(70) and I(67) were similar. Predictions of the D(70) model fitted the data better than those of the I(67) model. The protective effect of D(70) showed a gene-dose effect (OR 0.82, 95% CI 0.73-0.92, P=5.8 × 10(-4)), but was only seen in RA patients positive for RF or ACPA. HLA-DRB1 SE alleles were also associated with ACPA-negative, RF-positive RA (OR 1.42 (1.15-1.76), P=0.0012). In conclusion, HLA-DRB1 SE alleles show heterogeneity in RA susceptibility; their major effect appears to be mediated by ACPA positivity, but a significant association of HLA-DRB1 SE with RF-positive, ACPA-negative RA was also observed. D(70) specifically protected against antibody-positive RA.

Calpain system protein expression in basal-like and triple-negative invasive breast cancer.

BACKGROUND: Basal-like and triple-negative breast tumours encompass an important clinical subgroup and biomarkers that can prognostically stratify these patients are required. MATERIALS AND METHODS: We investigated two breast cancer tissue microarrays for the expression of calpain-1, calpain-2 and calpastatin using immunohistochemistry. The first microarray was comprised of invasive tumours from 1371 unselected patients, and the verification microarray was comprised of invasive tumours from 387 oestrogen receptor (ER)-negative patients. RESULTS: The calpain system contains a number of proteases and an endogenous inhibitor, calpastatin. Calpain activity is implicated in important cellular processes including cytoskeletal remodelling, apoptosis and survival. Our results show that the expression of calpastatin and calpain-1 are significantly associated with various clinicopathological criteria including tumour grade and ER expression. High expression of calpain-2 in basal-like or triple-negative disease was associated with adverse breast cancer-specific survival (P = 0.003 and <0.001, respectively) and was verified in an independent cohort of patients. Interestingly, those patients with basal-like or triple-negative disease with a low level of calpain-2 expression had similar breast cancer-specific survival to non-basal- or receptor- (oestrogen, progesterone or human epidermal growth factor receptor 2 (HER2)) positive disease. CONCLUSIONS: Expression of the large catalytic subunit of m-calpain (calpain-2) is significantly associated with clinical outcome of patients with triple-negative and basal-like disease.

Vascular invasion in breast cancer; an overview of recent prognostic developments and molecular pathophysiological mechanisms.

Vascular invasion (VI) is an essential step in breast cancer metastasis and the main cause of morbidity and mortality from the disease. Detection of VI in the primary tumour is a marker of metastatic potential. The prognostic value of VI in breast cancer has been known for more than four decades, but its application in clinical practice is still fraught with difficulties due to the limited number of studies conducted on large numbers of well-characterized patients with long-term follow-up. Detection of VI in the primary tumour is currently assessed using sections stained with haematoxylin and eosin, which has some disadvantages. A number of vascular markers have been used to improve detection of VI; however, their sensitivity and specificity, as endothelial markers, vary considerably. In this review we describe the evolution of the prognostic importance of VI and the recent pathomolecular mechanisms that contribute to the ability of breast cancers to invade through vessels, in addition to the types, locations and methods of detection of vascular invasion.

Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres.

The mammalian Ku70 and Ku86 proteins form a heterodimer that binds to the ends of double-stranded DNA in vitro and is required for repair of radiation-induced strand breaks and V(D)J recombination [1,2]. Deletion of the Saccharomyces cerevisiae genes HDF1 and HDF2--encoding yKu70p and yKu80p, respectively--enhances radiation sensitivity in a rad52 background [3,4]. In addition to repair defects, the length of the TG-rich repeat on yeast telomere ends shortens dramatically [5,6]. We have shown previously that in yeast interphase nuclei, telomeres are clustered in a limited number of foci near the nuclear periphery [7], but the elements that mediate this localization remained unknown. We report here that deletion of the genes encoding yKu70p or its partner yKu80p altered the positioning of telomeric DNA in the yeast nucleus. These are the first mutants shown to affect the subnuclear localization of telomeres. Strains deficient for either yKu70p or yKu80p lost telomeric silencing, although they maintained repression at the silent mating-type loci. In addition, the telomere-associated silencing factors Sir3p and Sir4p and the TG-repeat-binding protein Rap1p lost their punctate pattern of staining and became dispersed throughout the nucleoplasm. Our results implicate the yeast Ku proteins directly in aspects of telomere organization, which in turn affects the repression of telomere-proximal genes.

Vascular endothelial growth factor expression predicts outcome after primary radiotherapy for head and neck squamous cell cancer.

AIMS: To establish whether the expression of vascular endothelial growth factors (VEGFs) predicts prognosis in patients treated with primary radiotherapy for cancers of the upper aerodigestive tract. MATERIALS AND METHODS: A retrospective analysis was undertaken of VEGF and VEGF-D expression in tumour tissue in pre-treatment biopsies from 27 patients who had been treated with primary radiotherapy for stage II-IV squamous head and neck carcinomas. Serial sections (4 microm) were cut from formalin-fixed, paraffin-embedded specimens and stained with monoclonal antibodies using standard immunoperoxidase methods. Two independent investigators assessed the staining intensity in a randomised, blind manner. Both negative and positive controls (placenta and/or tonsil) were included in the staining procedure. All patients were followed for a minimum of 5 years, or until death. Local control and overall survival were taken as end points for the comparative analysis between patients whose tumours expressed low levels and those that expressed high levels of the two growth factors. Comparisons were made using the Log-rank test with Kaplan-Meier actuarial survival analysis. RESULTS: In patients with tumours expressing low levels of VEGF, 5-year local control was seen in 75% compared with 18% for those with high levels; overall survival was 75 and 23%, respectively. For those with low levels of VEGF-D, 5-year local control was 64% compared with 17% for those with high levels; overall survival was 58 and 20%, respectively. CONCLUSION: Our results suggest that the expression of endothelial growth factors in squamous head and neck cancers may predict outcome after radiotherapy.

Nuclear displacement and fluorescence recovery after photobleaching (FRAP) assays to study division site placement and cytokinesis in fission yeast.

Cytokinesis is an essential cellular event that completes the cell division cycle. It begins with the assembly of an actomyosin contractile ring that undergoes constriction concomitant with the septum formation to divide the cell in two. Placement of the septum at the right position is important to ensure fidelity of the division process. In fission yeast, the medially placed nucleus is a major spatial cue to position the site of division. In this chapter, we describe a simple synthetic biology-based approach to displace the nucleus and study the consequence on division site positioning. We also describe how to perform fluorescence recovery after photobleaching to follow the dynamics of cytokinetic proteins at defined time points by live-cell microscopy.

Gene-transfer systems for human endothelial cells. stewart.martin@nottingham.ac.uk.

By virtue of its location and importance in a number of pathophysiological processes the endothelium represents an attractive target tissue for gene-transfer and gene-therapy strategies. Although it is important to maximise gene-transfer to endothelial cells in such strategies primary human endothelial cells have proven to be rather intransigent to a variety of transfection techniques both in vitro and in vivo. We report on the variety of techniques in current use, revealing their strengths and weaknesses, indicate the steps that should ideally be taken to optimise expression and discuss the usefulness and future directions for viral mediated transduction.

Photoneutrons from medical linear accelerators--radiobiological measurements and risk estimates.

PURPOSE: To assess the oncogenic potential of the photoneutrons produced by high energy medical linear accelerators. METHODS AND MATERIALS: An established line of cells of rodent origin (C3H 10T1/2) was used to assess the oncogenic potential of the radiation dose received in the breast of an anthropomorphic "randoman" phanton, while the cervix received a dose of 70 Gy. Experiments were performed at 6 MV, below the threshold for the production of photoneutrons, and at 20 MV where the dose includes about 0.01 Gy of photoneutrons as well as scattered x-rays. RESULTS: A significantly higher transformation incidence was observed for the 20-MV machine, consistent with the measured neutron dose of about 0.01 Gy and a quality factor of 20. CONCLUSION: An estimate can be made of the additional deaths from second malignancies that might result from the photoneutrons generated by higher energy linear accelerators (Linacs), which must be offset against the possible improvements in survival that might result from the higher tumor doses made possible by the increased percentage depth doses.

The biological effectiveness of radon-progeny alpha particles. IV. Morphological transformation of Syrian hamster embryo cells at low doses.

Primary explants of Syrian hamster embryo (SHE) cells were exposed to either low-LET 250 kVp X rays or graded single doses of defined high-LET alpha particles (90, 100, 120, 150, 180 and 200 keV/microns), simulating those produced by radon progeny, and monitored for cell inactivation and oncogenic transformation. For the alpha particles the doses delivered ranged from 1 cGy to 1 Gy with an emphasis on doses less than 20 cGy, while for the X rays the doses ranged from 20 cGy to 4 Gy. The dose-response curves for cell killing by alpha particles approximated an exponential function of dose, whereas the X rays produced a curve with a shoulder characteristic of linear-quadratic relationships seen for low-LET radiations. The RBE at 10% survival varied between 3.6-7.0 depending on the LET of the alpha particles, with the RBEm ranging between 7-12. The most effective alpha particles were those with an LET of 120 keV/microns. All radiations produced initial increases in the frequency of morphological transformants, as a function of dose, with a rise to a maximum followed by a plateau in the response which was relatively constant at approximately 2-6 x 10(-3) transformants frequency, expressed per initial cell at risk, had a tendency to decline to parallel the cell survival response. Both the dose at which the maximum frequency of transformants was expressed and the initial slope of the dose-response relationship differed substantially between the different radiation qualities. Maximal transformation per initial cell at risk occurred at doses as low as 1-4 cGy for the 90 and 100 keV/microns particles with the maximum occurring at higher doses (to 16 cGy) as the LET increased toward 200 keV/microns. In contrast, the maximal transformation for 250 kVp X rays was at 50 cGy. The 90 and 100 keV/microns particles, with an RBEm of 60 and 37, respectively, based on the ratios of the initial slopes of the dose-response curves, were the most effective LETs in terms of the ability to induce morphological transformation of SHE cells. When expressed in terms of particle fluence, it appears that in the LET range of radon progeny approximately two to four particle traversals per nucleus are required per killing event, whereas it is at doses corresponding to less than one particle per nucleus that maximal oncogenic transformation is expressed.(ABSTRACT TRUNCATED AT 400 WORDS)

Neutron-induced cell cycle-dependent oncogenic transformation of C3H 10T1/2 cells.

Exposure of synchronized populations of mouse C3H 10T1/2 cells to a single dose (0.6 Gy) of 5.9 MeV neutrons at intervals after mitotic shake-off results in a distinctive variation in the oncogenic transformation frequency through the cell cycle. Previous findings show a sensitive window for X-ray-induced oncogenic transformants at late times after mitotic shake-off (14-16 h). Optimal sensitivity to neutrons was observed for cell populations irradiated soon after mitotic shake-off (4-6 h), where the majority of cells would be in the G1 phase of the cell cycle. Additionally, enhanced sensitivity was also found for that period after shake-off (14-16 h) which was maximally sensitive to X rays corresponding to cell populations with a high proportion of G2-phase cells. That is, low-LET radiation (250 kVp X rays) largely appears to produce oncogenic transformants in G2-phase cells, while intermediate-LET radiation (5.9 MeV neutrons) is effective principally on G1- and, to a somewhat lesser extent, G2-phase cells. Cells irradiated with neutrons showed less variation for lethality through the cell cycle than those irradiated with X rays, in agreement with previous findings. The mechanistic basis for the difference in the response of cells in the different phases of the cell cycle to radiations of different quality is unknown but is suggestive of distinct ("signature") molecular changes leading to the observed oncogenic transformation response.

Collagen metabolism in the murine colon following X irradiation.

Female CBA mice, aged 16 weeks, were irradiated to the total pelvic region with either single doses (5-20 Gy) or two equal fractions (10- to 30-Gy total dose, 24-h interval) of 240 kV X rays. Total protein and collagen synthesis rates, collagen breakdown, and net collagen content of the colon were measured at various times postirradiation using a radioisotope incorporation method and HPLC analysis. Immunohistochemical staining and computerized image analysis were used to assess the relative amounts of collagen types I and III at various times postirradiation, in various regions of the colon. Total protein and collagen synthesis rates were elevated above control levels at 4 and 8 weeks postirradiation, as was collagen degradation. Values had returned to control levels by 16 weeks postirradiation, and there were no further changes up to 71 weeks postirradiation. The net amount of collagen in the colon did not change relative to controls at any time during the investigation. There was, however, increased immunohistochemical staining for collagen type I 52 weeks postirradiation in all regions of the colon and decreased staining of type III in the circular muscle layer and villi. Altered ratios of these two collagen isotypes are consistent with changes in mechanical properties of the tissue.

The biological effectiveness of radon-progeny alpha particles. II. Oncogenic transformation as a function of linear energy transfer.

Epidemiological studies have established an association between exposure to radon and carcinoma of the lung. However, based on data for either lung cancer in uranium miners exposed to radon or bronchial epithelial carcinomas in Japanese A-bomb survivors, it has not been possible to assign estimates of risk of lung cancer for the general population exposed to radon in their homes. Based on past success with the excellent quantitative properties of the C3H 10T1/2 in vitro oncogenic transformation assay system, the relative biological effectiveness (RBE) for radiation-induced transformation for charged particles of defined LET has been determined. As the LET of the radiation was increased, the rate of induction of oncogenic transformation increased and the RBEm approached 20. At higher LETs, RBE dropped precipitously. The rapid drop in effectiveness for alpha particles with LETs between 120 and 265 keV/microns implies a lower quality factor than the 20-25 currently considered appropriate when estimating lung cancer mortality.

Determination of X-ray-induced damage to the murine colon using tissue compliance measurements.

A proctometroscope has been developed to measure the mechanical functioning of the colon in a murine model. A balloon-tipped probe is inserted into the colorectal region of anaesthetized mice and inflated hydraulically, at a constant rate, by a motor-driven syringe. Balloon pressure and volume are monitored, processed and plotted on a X-Y/t recorder. The balloon is inserted within the irradiated area, such that the centre of the balloon is 1 cm proximal to the anus, and inflated up to either a maximum volume of 100 microliters or a maximum pressure of 100 cmH2O, depending on which occurs first. Compliance (delta V/delta P) of the colon was measured at various intervals following X-irradiation, a dose-dependent decrease being observed at 24 weeks. This decrease was progressive with time out to 72 weeks postirradiation. The compliance measured, in both control and irradiated groups, was not dependent on the inflation rate used to make the determination, and no iatrogenic effects have been detected after repeated probing during a long-term study. The use of different anaesthetic agents was also investigated, no significant difference between the compliance of mice anaesthetized with the different agents being detected. Use of this technique provides fully quantitative data on the function of the colon following radiation injury, and provides an alternative to other physiological assays. The technique is non-destructive, rapid, easy to use and non-invasive to the tissue, thus the onset and progression of damage can be followed in each mouse over long postirradiation periods.

Transfection and transduction of primary human endothelial cells.

The endothelium is involved in a number of normal physiological processes (regulating circulating levels of vasoactive agents, blood/gas exchange, regulating cellular traffic between intavascular and extravascular compartments of tissues, maintenance of the blood brain barrier, and so forth) and pathophysiological conditions characterized either by increased angiogenesis (arthritis, diabetic retinopathy, atherosclerosis, tumor growth, and metastasis) or inadequate angiogenesis [failure of ulcers to heal (inadequate wound healing in general), myocardial infarction, limb ischaemia secondary to arterial occlusive diseases and others]. Its location immediately adjacent to the blood stream and the fact that it has a large surface area makes it an attractive target for genetransfer/gene-therapy strategies.

Neutron-energy-dependent cell survival and oncogenic transformation.

Both cell lethality and neoplastic transformation were assessed for C3H10T1/2 cells exposed to neutrons with energies from 0.040 to 13.7 MeV. Monoenergetic neutrons with energies from 0.23 to 13.7 MeV and two neutron energy spectra with average energies of 0.040 and 0.070 MeV were produced with a Van de Graaff accelerator at the Radiological Research Accelerator Facility (RARAF) in the Center for Radiological Research of Columbia University. For determination of relative biological effectiveness (RBE), cells were exposed to 250 kVp X rays. With exposures to 250 kVp X rays, both cell survival and radiation-induced oncogenic transformation were curvilinear. Irradiation of cells with neutrons at all energies resulted in linear responses as a function of dose for both biological endpoints. Results indicate a complex relationship between RBEm and neutron energy. For both survival and transformation, RBEm was greatest for cells exposed to 0.35 MeV neutrons. RBEm was significantly less at energies above or below 0.35 MeV. These results are consistent with microdosimetric expectation. These results are also compatible with current assessments of neutron radiation weighting factors for radiation protection purposes. Based on calculations of dose-averaged LET, 0.35 MeV neutrons have the greatest LET and therefore would be expected to be more biologically effective than neutrons of greater or lesser energies.

An analysis of hospital case mix, cost, and payment differences for Medicare, Medicaid, and Blue Cross Plan patients using DRGs.

To increase our understanding of case mix as a major contributor to variability in hospital costs, we examined the relationship among case mix, resource consumption, and payments for all Medicare, Medicaid, and Blue Cross Plan patients discharged from 28 hospitals in New York State. Case mix differences among the three payers were found to contribute to differences in overall average cost per case, although residual differences in costs existed at the DRG level. Medicare and Medicaid payments more often covered the actual costs of their patients than did Blue Cross Plan payments. Our results indicate the importance of payer-specific data in the design of effective and equitable reimbursement and cost containment strategies.

Effect of track structure and radioprotectors on the induction of oncogenic transformation in murine fibroblasts by heavy ions.

The oncogenic potential of high-energy 56Fe particles (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at the Brookhaven National Laboratory was examined utilizing the mouse C3H 10T1/2 cell model. The dose-averaged LET for high-energy 56Fe is estimated to be 143 keV/micrometer with the exposure conditions used in this study. For 56Fe ions, the maximum relative biological effectiveness (RBEmax) values for cell survival and oncogenic transformation were 7.71 and 16.5 respectively. Compared to 150 keV/micrometer 4He nuclei, high-energy 56Fe nuclei were significantly less effective in cell killing and oncogenic induction. The prostaglandin E1 analog misoprostol, an effective oncoprotector of C3H 10T1/2 cells exposed to X rays, was evaluated for its potential as a radioprotector of oncogenic transformation with high-energy 56Fe. Exposure of cells to misoprostol did not alter 56Fe cytotoxicity or the rate of 56Fe-induced oncogenic transformation.

Redox proteins and radiotherapy.

Although conventional radiotherapy can directly damage DNA and other organic molecules within cells, most of the damage and the cytotoxicity of such ionising radiation, comes from the production of ions and free radicals produced via interactions with water. This 'indirect effect', a form of oxidative stress, can be modulated by a variety of systems within cells that are in place to, in normal situations, maintain homeostasis and redox balance. If cancer cells express high levels of antioxidant redox proteins, they may be more resistant to radiation and so targeting such systems may be a profitable strategy to increase therapeutic efficacy of conventional radiotherapy. An overview, with exemplars, of the main systems regulating redox homeostasis is supplied and discussed in relation to their use as prognostic and predictive biomarkers, and how targeting such proteins and systems may increase radiosensitivity and, potentially, improve the radiotherapeutic response.