In vitro characterization of human AML-reactive CTL clones generated from the naive subset of healthy donors and adoptive transfer into leukemia-engrafted NSG mice

Acute myeloid leukemia (AML) is a very aggressive cancer of the hematopoietic system. Chemotherapy and immunotherapeutical approaches including hematopoietic stem cell transplantation (HSCT) and donor lymphocyte infusion (DLI) are the only curative options available. The beneficial graft-versus-leukemia (GVL) effect of cellular immunotherapy is mostly mediated by donor-derived CD8+ T lymphocytes that recognize minor histocompatibility antigens (mHags) and leukemia-associated antigens (LAAs) presented on the surface of AML blasts (Falkenburg et al. 2008; Kolb 2008). A main complication is graft-versus-host disease (GVHD) that can be induced when cytotoxic T lymphocytes (CTLs) recognize broadly expressed antigens. To reduce the risk of GVHD, specific allogeneic T-cell therapy inducing selective GVL responses could be an option (Barrett & Le Blanc 2010; Parmar et al. 2011; Smits et al. 2011). This requires efficient in vitro strategies to generate AML-reactive T cells with an early differentiation phenotype as well as vigorous effector functions and humanized mouse models to analyze the anti-leukemic potential of adoptively transferred T cells in vivo. In this study, AML-reactive CTL clones and oligoclonal T-cell lines could be reliably generated from the naive subset of healthy HLA-class I-identical donors by stimulation with primary AML blasts in mini-mixed-lymphocyte / leukemia cultures (MLLCs) in eight different patient / donor pairs. These CTLs were promising candidates for cellular immunotherapy because of their relatively early differentiation phenotype and strong proliferative and lytic capabilities. The addition of the common γ-chain cytokine IL-21 to the stimulation protocol enabled more precursors to develop into potent leukemia-reactive CTLs, presumably by its beneficial effects on cell survival and antigen-specific proliferation during the first weeks of cultures. It also strengthened the early-stage phenotype. Three long-term cultured CTLs exemplarily transferred into leukemia-engrafted immunodeficient NSG mice mediated a significant reduction of the leukemic burden after a single transfusion. These results demonstrate that CTL clones with reactivity to patient-derived AML blasts can be isolated from the naive compartment of healthy donors and show potent anti-leukemic effects in vivo. The herein described allo-MLLC approach with in vitro “programmed” naive CTL precursors independent of a HSCT setting is a valuable alternative to the conventional method of isolating in vivo primed donor CTLs out of patients after transplantation (Kloosterboer et al. 2004; Warren et al. 2010). This would make leukemia-reactive CTLs already available at the time point of HSCT, when residual leukemia disease is minimal and the chances for complete leukemia eradication are high. Furthermore, leukemia-reactive CTLs effectively expanded by this in vitro protocol can be used as screening populations to identify novel candidate LAAs and mHags for antigen-specific immunotherapy.

Retinal thickness in Parkinson's disease

BACKGROUND: Visual symptoms are common in Parkinson's disease with studies consistently demonstrating reductions in visual acuity, contrast sensitivity, colour and motion perception as well as alterations in electroretinogram latencies and amplitudes. Optical coherence tomography can examine retinal structure non-invasively and retinal thinning has been suggested as a potential biomarker for neurodegeneration in Parkinson's disease. Our aim was to examine the retinal thickness of a cohort of Parkinson's disease subjects (and age-matched controls) to establish the practical utility of optical coherence tomography in a representative older Parkinson's disease group. METHODS: Fifty-one established Parkinson's disease subjects and 25 healthy controls were subjected to ophthalmological assessment and optical coherence tomography (Zeiss Stratus 3000™) of macular thickness and volume and retinal nerve fibre thickness around the optic nerve head. Twenty four percent of control and 20% of Parkinson's disease subjects were excluded from final analysis due to co-morbid ocular pathology. Further data was excluded either due to poor tolerability of optical coherence tomography or poor quality scans. RESULTS: Despite a reduction in both visual acuity and contrast sensitivity in the residual evaluable Parkinson's disease cohort, we did not detect any differences between the two study groups for any measures of retinal thickness, in contrast to previously published work. CONCLUSIONS: In addition to technical problems inherent in the evaluation, the lack of difference between Parkinson's disease and healthy control subjects suggests longitudinal studies, employing newer techniques, will be required to define the role of optical coherence tomography as a potential diagnostic biomarker.

Incidence of spinal compressive lesions in chondrodystrophic dogs with abnormal recovery after hemilaminectomy for treatment of thoracolumbar disc disease: a prospective magnetic resonance imaging study

OBJECTIVE: To investigate causes of the lack of clinical improvement after thoracolumbar disc surgery. STUDY DESIGN: Case-control magnetic resonance imaging (MRI) study. ANIMALS: Chondrodystrophic dogs with acute thoracolumbar disc disease treated by hemilaminectomy: 10 that had no short-term clinical improvement and 12 with "normal" clinical improvement. METHODS: Dogs that had surgery for treatment of intervertebral disc extrusion (2003-2008) where thoracolumbar disc disease was confirmed by MRI were evaluated to identify dogs that had lack of clinical improvement after surgery. Ten dogs with delayed recovery or clinical deterioration were reexamined with MRI and compared with 12 dogs with normal recovery and MRI reexamination after 6 weeks (control group). RESULTS: Of 173 dogs, 10 (5.8%) had clinical deterioration within 1-10 days after surgery. In 8 dogs, residual spinal cord compression was identified on MRI. Bleeding was present in 1 dog. In 3 dogs, the cause was an incorrect approach and insufficient disc material removal. In 3 dogs, recurrence occurred at the surgical site. In 1 dog, the centrally located extruded material was shifted to the contralateral side during surgery. These 8 dogs had repeat surgery and recovery was uneventful. In 2 dogs, deterioration could not be associated with a compressive disc lesion. Hemorrhagic myelomalacia was confirmed by pathologic examination in 1 dog. The other dog recovered after 6 months of conservative management. CONCLUSION: Delayed postsurgical recovery or deterioration is commonly associated with newly developed and/or remaining compressive disc lesion. CLINICAL RELEVANCE: We recommend early MRI reexamination to assess the postsurgical spinal canal and cord, and to plan further therapeutic measures in chondrodystrophic dogs with delayed recovery after decompressive hemilaminectomy for thoracolumbar disc disease.

Rates of disease progression according to initial highly active antiretroviral therapy regimen: a collaborative analysis of 12 prospective cohort studies

BACKGROUND: No large clinical end-point trials have been conducted comparing regimens among human immunodeficiency virus type 1-positive persons starting antiretroviral therapy. We examined clinical progression according to initial regimen in the Antiretroviral Therapy Cohort Collaboration, which is based on 12 European and North American cohort studies. METHODS: We analyzed progression to death from any cause and to AIDS or death (AIDS/death), comparing efavirenz (EFV), nevirapine (NVP), nelfinavir, idinavir, ritonavir (RTV), RTV-boosted protease inhibitors (PIs), saquinavir, and abacavir. We also compared nucleoside reverse-transcriptase inhibitor pairs: zidovudine/lamivudine (AZT/3TC), stavudine (D4T)/3TC, D4T/didanosine (DDI), and others. RESULTS: A total of 17,666 treatment-naive patients, 55,622 person-years at risk, 1,617 new AIDS events, and 895 deaths were analyzed. Compared with EFV, the adjusted hazard ratio (HR) for AIDS/death was 1.28 (95% confidence interval [CI], 1.03-1.60) for NVP, 1.31 (95% CI, 1.01-1.71) for RTV, and 1.45 (95% CI, 1.15-1.81) for RTV-boosted PIs. For death, the adjusted HR for NVP was 1.65 (95% CI, 1.16-2.36). The adjusted HR for death for D4T/3TC was 1.35 (95% CI, 1.14-1.59), compared with AZT/3TC. CONCLUSIONS: Outcomes may vary across initial regimens. Results are observational and may have been affected by bias due to unmeasured or residual confounding. There is a need for large, randomized, clinical end-point trials.

Proposed minimal standards for the description of genera, species and subspecies of the Pasteurellaceae

Principles and guidelines are presented to ensure a solid scientific standard of papers dealing with the taxonomy of taxa of Pasteurellaceae Pohl 1981. The classification of the Pasteurellaceae is in principle based on a polyphasic approach. DNA sequencing of certain genes is very important for defining the borders of a taxon. However, the characteristics that are common to all members of the taxon and which might be helpful for separating it from related taxa must also be identified. Descriptions have to be based on as many strains as possible (inclusion of at least five strains is highly desirable), representing different sources with respect to geography and ecology, to allow proper characterization both phenotypically and genotypically, to establish the extent of diversity of the cluster to be named. A genus must be monophyletic based on 16S rRNA gene sequence-based phylogenetic analysis. Only in very rare cases is it acceptable that monophyly can not be achieved by 16S rRNA gene sequence comparison. Recently, the monophyly of genera has been confirmed by sequence comparison of housekeeping genes. In principle, a new genus should be recognized by a distinct phenotype, and characters that separate the new genus from its neighbours should be given clearly. Due to the overall importance of accurate classification of species, at least two genotypic methods are needed to show coherence and for separation at the species level. The main criterion for the classification of a novel species is that it forms a monophyletic group based on 16S rRNA gene sequence-based phylogenetic analysis. However, some groups might also include closely related species. In these cases, more sensitive tools for genetic recognition of species should be applied, such as DNA-DNA hybridizations. The comparison of housekeeping gene sequences has recently been used for genotypic definition of species. In order to separate species, phenotypic characters must also be identified to recognize them, and at least two phenotypic differences from existing species should be identified if possible. We recommend the use of the subspecies category only for subgroups associated with disease or similar biological characteristics. At the subspecies level, the genotypic groups must always be nested within the boundaries of an existing species. Phenotypic cohesion must be documented at the subspecies level and separation between subspecies and related species must be fully documented, as well as association with particular disease and host. An overview of methods previously used to characterize isolates of the Pasteurellaceae has been given. Genotypic and phenotypic methods are separated in relation to tests for investigating diversity and cohesion and to separate taxa at the level of genus as well as species and subspecies.

Minimal extracorporeal circulation is a promising technique for coronary artery bypass grafting

BACKGROUND: Minimal extracorporeal circulation (MECC) is a promising perfusion technology, taking the advantage of an ECC while having a significantly reduced priming volume. We analyzed the actual possible benefits of using MECC in patients undergoing CABG procedures and compared the results with conventional extracorporeal circulation (CECC). METHODS: One thousand fifty-three consecutive patients underwent CABG surgery using the MECC perfusion technique. Subgroup analyses focused on perioperative myocardial markers (cardiac troponin I [cTnI]), incidence of atrial fibrillation (AF), and perioperative evaluation of inflammatory markers and data were compared with those of patients who underwent CABG using CECC. A propensity score analysis was performed. RESULTS: Patient characteristics and distribution of EuroSCORE risk were similar in both groups. Severity of coronary artery disease and extent of revascularization were also comparable in both groups (number of distal anastomoses: 3.2 +/- 1.1 in CECC vs 3.2 +/- 0.9 in MECC; p = not significant [ns]). The cTnI was significantly lower in the MECC group (11.0 +/- 10.8 microg/L in MECC vs 24.7 +/- 25.3 microg/L in CECC; p < 0.05). Incidence of AF was 11.1% in MECC and 39.0% in CECC (p < 0.05). Inflammatory markers (interleukin-6, SC5b-9) were lower in MECC patients (p < 0.05). Propensity score analysis confirmed faster recovery in MECC patients and lower incidence of AF. CONCLUSIONS: Minimal extracorporeal circulation is a safe perfusion technique for CABG and may therefore concurrence OPCAB and traditional CABG under CECC.

Influence of residual pockets on progression of periodontitis and tooth loss: results after 11 years of maintenance

BACKGROUND: Limited evidence exists on the significance of residual probing pocket depth (PPD) as a predictive parameter for periodontal disease progression and tooth loss. AIM: The aim of this study was to investigate the influence of residual PPD >or=5 mm and bleeding on probing (BOP) after active periodontal therapy (APT) on the progression of periodontitis and tooth loss. MATERIAL AND METHODS: In this retrospective cohort, 172 patients were examined after APT and supportive periodontal therapy (SPT) for 3-27 years (mean 11.3 years). Analyses were conducted using information at site, tooth and patient levels. The association of risk factors with tooth loss and progression of periodontitis was investigated using multilevel logistic regression analysis. RESULTS: The number of residual PPD increased during SPT. Compared with PPDor=7 mm 37.9 and 64.2, respectively. At patient level, heavy smoking, initial diagnosis, duration of SPT and PPD>or=6 mm were risk factors for disease progression, while PPD>or=6 mm and BOP>or=30% represented a risk for tooth loss. CONCLUSION: Residual PPD>or=6 mm represent an incomplete periodontal treatment outcome and require further therapy.

Percutaneous coronary intervention for stable coronary artery disease

Patients with significant coronary artery stenoses are at increased risk of future cardiac events. However, in the absence of acute coronary syndrome or recent myocardial infarction and residual ischemia, elective percutaneous coronary intervention has not been shown to improve prognosis. Possible explanations for this might be the limited follow-up time adopted by most randomized trials comparing percutaneous coronary intervention with medical therapy, limited number of patients with proven ischemia enrolled in these trials, and adoption of complex, elaborate techniques that have not proved their usefulness. Published evidence identifies certain indications for percutaneous coronary intervention in patients with stable coronary lesions: demonstration of significant inducible ischemia, particularly in the context of a recent myocardial infarction; detection of unequivocally reduced fractional flow reserve; and specific angiographic features of coronary stenoses. Operators should take into account long-term consequences of adopted techniques rather than immediate angiographic results. We review existing evidence and provide our recommendations in this setting.

Interferon-gamma regulates idiopathic pneumonia syndrome, a Th17+CD4+ T-cell-mediated graft-versus-host disease

RATIONALE: Pulmonary complications of hematopoietic stem cell transplantation include infections and graft-versus-host diseases, such as idiopathic pneumonia syndrome (IPS). Conflicting data exist regarding the role of the interferon (IFN)-gamma-producing Th1 CD4(+) T-cell subset and IL-17A in IPS. OBJECTIVES: To determine the role of IFN-gamma and IL-17A in the establishment of pulmonary graft-versus-host disease. METHODS: A semiallogeneic murine model based on C57BL/6 x BALB/c as recipients with transplantation of BALB/c RAG2(-/-) bone marrow and transfer of different genetic knockout T cells (T-bet(-/-), IFN-gamma(-/-), IFN-gammaR(-/-)) on a BALB/c background. Lung tissue was examined for parenchymal changes and infiltrating cells by histology and fluorescence-activated cell sorter analysis. MEASUREMENTS AND MAIN RESULTS: After transfer of semiallogeneic bone marrow together with donor CD4(+) T cells lacking IFN-gamma or T-bet-a T-box transcription factor controlling Th1 commitment-we found severe inflammation in the lungs, but no enhancement in other organs. In contrast, wild-type donor CD4(+) T cells mediated minimal inflammation only, and donor CD8(+) T cells were not required for IPS development. Mechanistically, the absence of IFN-gamma or IFN-gamma signaling in pulmonary parenchymal cells promoted expansion of IL-17A-producing CD4(+) T cells and local IL-17A release. In vivo depletion of IL-17A reduced disease severity. CONCLUSIONS: One mechanism of IFN-gamma protection against IPS is negative regulation of the expansion of pathogenic IL-17A-producing CD4(+) T cells through interaction with the IFN-gamma receptor on the pulmonary parenchymal cell population.

Percutaneous PFO closure with Amplatzer PFO occluder: predictors of residual shunts at 6 months follow-up

OBJECTIVE: The objective of this study was to assess predictors of residual shunts after percutaneous patent foramen ovale (PFO) closure with Amplatzer PFO occluder (AGA Medical Corporation, Golden Valley, MN, USA). METHODS: All percutaneous PFO closures, using Amplatzer PFO occluder performed at a tertiary center between May 2002 and August 2006, were reviewed. Follow-up, including saline contrast transesophageal echocardiography, was performed in all patients 6 months after the intervention. PATIENTS: A total of 135 procedures were performed. Mean age of the patients was 51 years. The indication for PFO closure was an ischemic cerebrovascular event in 92%, paradoxical systemic embolism in 4%, and a diving accident in 4%. Recurrent events prior to PFO closure were noted in 34%. A concomitant atrial septal aneurysm was present in 61%. RESULTS: At 6 months follow-up, a residual shunt was detected in 26 patients (19%). Residual shunts were more common in patients with an atrial septal aneurysm (27 vs. 8%, P= .01) and in patients treated with a 35-mm compared with a 25-mm device (39 vs. 15%, P= .01). A concomitant atrial septal aneurysm remained independently associated with residual shunts when controlled for body mass index, gender, age, atrial dimensions, and presence of a Chiari network (odds ratio 4.1, 95% confidence intervals 1.1-15.0). CONCLUSION: The presence of atrial septal aneurysms in patients undergoing percutaneous PFO closure with an Amplatzer PFO occluder significantly increases the rate of residual shunts at 6 months follow-up, even if 35-mm devices are used.

Benign prostatic obstruction and parkinson's disease--should transurethral resection of the prostate be avoided?

PURPOSE: According to the literature transurethral resection of the prostate in patients with Parkinson's disease has an increased risk of postoperative urinary incontinence. However, this conclusion might have been reached because some patients with multiple system atrophy incorrectly diagnosed as Parkinson's disease were included in these reports. Therefore, we investigated the outcome of transurethral prostate resection in patients with a secure neurological diagnosis of Parkinson's disease. MATERIALS AND METHODS: A total of 23 patients with Parkinson's disease who underwent transurethral prostate resection for benign prostatic obstruction were evaluated retrospectively. Subsequent neurological developments in patients were followed, ensuring that those with multiple system atrophy had not been included in analysis. RESULTS: At transurethral prostate resection median patient age was 73 years, median duration of Parkinson's disease before the resection was 3 years, and median Hoehn and Yahr scale was 2. Of the 14 patients with a preoperative indwelling urinary catheter transurethral prostate resection restored voiding in 9 (64%) and only 5 (36%) required catheterization postoperatively. Of the 10 patients with preoperative urge urinary incontinence, continence was restored in 5 and improved in 3 following transurethral prostate resection. There were no cases of de novo urinary incontinence after transurethral prostate resection. At a median postoperative followup of 3 years transurethral prostate resection was successful in 16 of the 23 patients (70%). CONCLUSIONS: Transurethral prostate resection for benign prostatic obstruction in patients with Parkinson's disease may be successful in up to 70% and the risk of de novo urinary incontinence seems minimal. Thus, Parkinson's disease should no longer be considered a contraindication for transurethral prostate resection provided that preoperative investigations including urodynamic assessment indicate prostatic bladder outlet obstruction.

Testing visual perception in dementia with Lewy bodies and Alzheimer disease

OBJECTIVE Visuoperceptual deficits are common in dementia with Lewy bodies (DLB) and Alzheimer disease (AD). Testing visuoperception in dementia is complicated by decline in other cognitive domains and extrapyramidal features. To overcome these issues, we developed a computerized test, the Newcastle visuoperception battery (NEVIP), which is independent of motor function and has minimal cognitive load.We aimed to test its utility to identify visuoperceptual deficits in people with dementia. PARTICIPANTS AND MEASUREMENTS We recruited 28 AD and 26 DLB participants with 35 comparison participants of similar age and education. The NEVIP was used to test angle, color, and form discrimination along with motion perception to obtain a composite visuoperception score. RESULTS Those with DLB performed significantly worse than AD participants on the composite visuoperception score (Mann-Whitney U = 142, p = 0.01). Visuoperceptual deficits (defined as 2 SD below the performance of comparisons) were present in 71% of the DLB group and 40% of the AD group. Performance was not significantly correlated with motor impairment, but was significantly related to global cognitive impairment in DLB (rs = -0.689, p <0.001), but not in AD. CONCLUSION Visuoperceptual deficits can be detected in both DLB and AD participants using the NEVIP, with the DLB group performing significantly worse than AD. Visuoperception scores obtained by the NEVIP are independent of participant motor deficits and participants are able to comprehend and perform the tasks.

Saccadic eye movement changes in Parkinson's disease dementia and dementia with Lewy bodies.

Neurodegeneration in Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) affect cortical and subcortical networks involved in saccade generation. We therefore expected impairments in saccade performance in both disorders. In order to improve the pathophysiological understanding and to investigate the usefulness of saccades for differential diagnosis, saccades were tested in age- and education-matched patients with PDD (n = 20) and DLB (n = 20), Alzheimer's disease (n = 22) and Parkinson's disease (n = 24), and controls (n = 24). Reflexive (gap, overlap) and complex saccades (prediction, decision and antisaccade) were tested with electro-oculography. PDD and DLB patients had similar impairment in all tasks (P > 0.05, not significant). Compared with controls, they were impaired in both reflexive saccade execution (gap and overlap latencies, P < 0.0001; gains, P < 0.004) and complex saccade performance (target prediction, P < 0.0001; error decisions, P < 0.003; error antisaccades: P < 0.0001). Patients with Alzheimer's disease were only impaired in complex saccade performance (Alzheimer's disease versus controls, target prediction P < 0.001, error decisions P < 0.0001, error antisaccades P < 0.0001), but not reflexive saccade execution (for all, P > 0.05). Patients with Parkinson's disease had, compared with controls, similar complex saccade performance (for all, P > 0.05) and only minimal impairment in reflexive tasks, i.e. hypometric gain in the gap task (P = 0.04). Impaired saccade execution in reflexive tasks allowed discrimination between DLB versus Alzheimer's disease (sensitivity > or =60%, specificity > or =77%) and between PDD versus Parkinson's disease (sensitivity > or =60%, specificity > or =88%) when +/-1.5 standard deviations was used for group discrimination. We conclude that impairments in reflexive saccades may be helpful for differential diagnosis and are minimal when either cortical (Alzheimer's disease) or nigrostriatal neurodegeneration (Parkinson's disease) exists solely; however, they become prominent with combined cortical and subcortical neurodegeneration in PDD and DLB. The similarities in saccade performance in PDD and DLB underline the overlap between these conditions and underscore differences from Alzheimer's disease and Parkinson's disease.

The cam-type deformity - what is it: SCFE, osteophyte, or a new disease?

Cam-type deformity of the proximal femur is a risk factor for the development of cam-type femoroacetabular impingement and a prearthrotic condition of the hip. The etiology of cam-type deformity remains unclear. There are a number of causes of cam-type deformity including sequellae of slipped capital femoral epiphysis, Legg-Calvé-Perthes disease or Perthes-like deformities, postinfectious, and traumatic. However, the majority of cam-type deformities arise without any apparent preexisting hip disease. These "idiopathic" cam-type deformities likely represent a majority of cases, and show clear racial and sex differences, as well as developmental and genetic influences. Idiopathic cam-type deformity also seems to be a distinct entity from residual or silent slipped capital femoral epiphysis, as well as osteoarthritis-induced osteophytes. In this paper we examine the different pathogenetic aspects of the proximal femur that contribute to cam-type deformity and/or symptomatic cam-type femoroacetabular impingement.

Osteopontin (OPN) and neoplastic disease: Circulating levels and tissue distribution

OPN is a secreted phosphate containing protein which is expressed by osteoblasts and a variety of other cells in vivo. Data from in vitro studies has accumulated which relates OPN to cellular transformation. We hypothesize that OPN expression is associated with neoplastic disease in humans as suggested by cell culture models. The overall objective of the current study was to determine the tissue distribution of OPN in human malignancy and to determine whether or not a correlation exists between OPN serum levels and malignancy. At the inception of this project, no study had been made demonstrating the relevance of OPN expression with naturally occurring neoplastic disease in humans. To date, few studies have reported OPN distribution in human neoplasia and are limited by either the number of specimens analyzed or the technique used in analysis. In this dissertation study, OPN was purified from human milk and $\alpha$-OPN antiserum developed and characterized. Following antibody development, the distribution and prevalence of OPN in human oral squamous cell carcinoma and human prostate carcinoma was evaluated using immunohistochemical localization. OPN immunolocalization was found in a high percentage of oral epithelial dysplasia and oral squamous cell carcinoma in humans. One oral squamous cell carcinoma cells line, UMSCC-1, was found to express OPN mRNA using Northern blotting. OPN localized to a high percentage of primary prostate adenocarcinomas. OPN localized to 52% of androgen dependent cases and 100% of androgen independent cases. Androgen dependent cell lines such as LNCap and NbE showed minimal OPN mRNA expression while the androgen independent lines C4-2 and PC3 produced ample OPN mRNA. An OPN sandwich assay was developed and used to determine the serum level of OPN in normal males, patients with BPH (benign prostate hypertrophy), and patients with prostate carcinoma. No statistically significant difference was found in OPN serum levels among the three groups. However, a trend of increasing OPN in the serum was noted in patients with BPH and prostate cancer. ^