Building a Healthcare Alliance for Resourceful Medicine Offensive Against Neoplasms in Hematology Added Value Framework for Hematologic Malignancies: A Comparative Analysis of Existing Tools.

OBJECTIVES: The Innovative Medicines Initiative-funded, multistakeholders project Healthcare Alliance for Resourceful Medicine Offensive Against Neoplasms in Hematology (HARMONY) created a task force involving patient organizations, medical associations, pharmaceutical companies, and health technology assessment/regulator agencies' representatives to evaluate the suitability of previously established value frameworks (VFs) for assessing the clinical and societal impact of new interventions for hematologic malignancies (HMs). METHODS: Since the HARMONY stakeholders identified the inclusion of patients' points of view on evaluating VFs as a priority, surveys were conducted with the patient organizations active in HMs and part of the HARMONY network, together with key opinion leaders, pharmaceutical companies, and regulators, to establish which outcomes were important for each HM. Next, to evaluate VFs against the sources of information taken into account (randomized clinical trials, registries, real-world data), structured questionnaires were created and filled by HARMONY health professionals to specify preferred data sources per malignancy. Finally, a framework evaluation module was built to analyze existing clinical VFs (American Society of Clinical Oncology, European Society of Medical Oncology, Magnitude of Clinical Benefit Scale, Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen, Institute for Clinical and Economic Review, National Comprehensive Cancer Network Evidence Blocks, and patient-perspective VF). RESULTS: The comparative analysis describes challenges and opportunities for the use of each framework in the context of HMs and drafts possible lines of action for creating or integrating a more specific, patient-focused clinical VF for HMs. CONCLUSIONS: None of the frameworks meets the HARMONY goals for a tool that applies to HMs and assesses in a transparent, reproducible, and systematic way the therapeutic value of innovative health technologies versus available alternatives, taking a patient-centered approach and using real-world evidence.

Core outcome set measurement for future clinical trials in acute myeloid leukemia: the HARMONY study protocol using a multi-stakeholder consensus-based Delphi process and a final consensus meeting.

BACKGROUND: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has an unacceptably low cure rate. In recent years, a number of new treatment strategies and compounds were developed for the treatment of AML. There were several randomized controlled clinical trials with the objective to improve patients' management and patients' outcome in AML. Unfortunately, these trials are not always directly comparable since they do not measure the same outcomes, and currently there are no core outcome sets that can be used to guide outcome selection and harmonization in this disease area. The HARMONY (Healthcare Alliance for Resourceful Medicine Offensive against Neoplasms in Hematology) Alliance is a public-private European network established in 2017 and currently includes 53 partners and 32 associated members from 22 countries. Amongst many other goals of the HARMONY Alliance, Work Package 2 focuses on defining outcomes that are relevant to each hematological malignancy. Accordingly, this pilot study will be performed to define a core outcome set in AML. METHODS: The pilot study will use a three-round Delphi survey and a final consensus meeting to define a core outcome set. Participants will be recruited from different stakeholder groups, including patients, clinicians, regulators and members of the European Federation of Pharmaceutical Industries and Associations. At the pre-Delphi stage, a literature research was conducted followed by several semi-structured interviews of clinical public and private key opinion leaders. Subsequently, the preliminary outcome list was discussed in several multi-stakeholder face-to-face meetings. The Delphi survey will reduce the preliminary outcome list to essential core outcomes. After completion of the last Delphi round, a final face-to-face meeting is planned to achieve consensus about the core outcome set in AML. DISCUSSION: As part of the HARMONY Alliance, the pilot Delphi aims to define a core outcome set in AML on the basis of a multi-stakeholder consensus. Such a core outcome set will help to allow consistent comparison of future clinical trials and real-world evidence research and ensures that appropriate outcomes valued by a range of stakeholders are measured within future trials.

Multi-centre national audit of juvenile localised scleroderma: describing current UK practice in disease assessment and management.

OBJECTIVE: To describe current United Kingdom practice in assessment and management of patients with juvenile localised scleroderma (JLS) compared to Paediatric Rheumatology European Society (PRES) scleroderma working party recommendations. METHODS: Patients were included if they were diagnosed with JLS and were under the care of paediatric rheumatology between 04/2015-04/2016. Retrospective data was collected in eleven UK centres using a standardised proforma and collated centrally. RESULTS: 149 patients were included with a median age of 12.5 years. The outcome measures recommended by the PRES scleroderma working party were not utilised widely. The localised scleroderma cutaneous assessment tool was only used in 37.6% of patients. Screening for extracutaneous manifestations did not meet recommendations that patients with head involvement have regular screening for uveitis and baseline magnetic resonance imaging (MRI) brain: only 38.5% of these patients were ever screened for uveitis; 71.2% had a MRI brain. Systemic treatment with disease-modifying anti-rheumatic drugs (DMARDs) or biologics was widely used (96.0%). In keeping with the recommendations, 95.5% of patients were treated with methotrexate as first-line therapy. 82.6% received systemic corticosteroids and 34.2% of patients required two or more DMARDs/biologics, highlighting the significant treatment burden. Second-line treatment was mycophenolate mofetil in 89.5%. CONCLUSION: There is wide variation in assessment and screening of patients with JLS but a consistent approach to systemic treatment within UK paediatric rheumatology. Improved awareness of PRES recommendations is required to ensure standardised care. As recommendations are based on low level evidence and consensus opinion, further studies are needed to better define outcome measures and treatment regimens for JLS.

Topographical study of O(6)-alkylguanine DNA alkyltransferase repair activity and N7-methylguanine levels in resected lung tissue.

BACKGROUND: Tobacco specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are genotoxic alkylating agents found within cigarette smoke that induce lung adenocarcinomas in animal models. In humans, adenocarcinomas originate most frequently in the lung periphery. The aim of this study was to determine whether peripheral lung has increased susceptibility to the genotoxic effects of alkylating agents by comparing DNA alkylation damage (N7-methylguanine: N7-meG) and repair (O(6)-alkylguanine DNA alkyltransferase: MGMT) in peripheral relative to central lung tissue. METHODS: Macroscopically normal lung tissue, resected from patients undergoing surgery for lung cancer, was sampled at equidistant points from central to peripheral lung along a bronchus. N7-meG levels were determined using an immunoslotblot technique and MGMT activity with a [32P]-labelled oligodeoxynucleotide cleavage assay. RESULTS: A total of 20 subjects were recruited, 12 males and 8 females with a mean age of 68.7±5.8years. There were 14 former and 6 current smokers with a mean smoking exposure of 34.0±18.3packyears. N7-meG (mean 0.75±0.57/10(6)dG, n=65 samples from 14 patients) and MGMT repair (geometric mean 9.57±1.62fmol/µg DNA, n=79 samples from 16 patients) were detected in all samples assayed. MGMT activity increased towards the lung periphery (r=0.28, p=0.023; n=16) with a highly significant association in current (r=0.53, p=0.008; n=6) but not former smokers (r=0.13; p=0.41; n=10). No correlation was seen with N7-meG levels and lung position (r=-0.18; p=0.21; n=14). N7-meG levels were higher in current compared to former smokers reaching significance in two lung positions including peripheral lung (p=0.047). CONCLUSIONS: The findings in this study do not support the hypothesis that peripheral tissue is more susceptible to the genotoxic effects of alkylating agents than central lung tissue. In addition exposure to cigarette smoke reduced the level of MGMT in central bronchial tissue possibly through increased alkylating agent exposure.

GSTM1 copy number and lung cancer risk.

The GSTM1 null genotype is associated with a small increased lung cancer risk when compared to controls with at least one copy of the GSTM1 gene. As two copies of the GSTM1 gene might provide more protection than a single copy, we have determined GSTM1 copy number in a lung cancer case-control study. Cases with incident lung cancer were identified through a Bronchoscopy Unit and two separate hospital based control groups with non-malignant disease were selected with one from the same Bronchoscopy Unit and the other from a chest clinic at the same hospital. Subjects with at least one GSTM1 copy had a decreased lung cancer risk whatever the control group: the odds ratio (95% CI), after adjustment for age, gender and smoking duration, was 0.64 (0.41-0.98) and 0.54 (0.32-0.91) with bronchoscopy and chest clinic controls, respectively. Lung cancer risk varied with GSTM1 copy number with chest clinic controls only: the OR was 0.56 (0.32-0.97) for one copy of the GSTM1 gene and with two copies 0.43 (0.15-1.22), a trend that was significant (p=0.02): with bronchoscopy controls the trend was not significant (p=0.07). Results then confirm that the presence of GSTM1 provides protection against the risk of lung cancer. In addition there is equivocal evidence that this protection varies with the number of gene copies.

Association between lung cancer risk and single nucleotide polymorphisms in the first intron and codon 178 of the DNA repair gene, O6-alkylguanine-DNA alkyltransferase.

The association between lung cancer risk and 2 polymorphisms, rs12268840 and rs2308327 (codon K178R), in the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase, which are associated with interindividual differences in activity, have been investigated in 3 hospital-based case-control studies. Genotyping was carried out on 617 subjects of whom 255 had lung cancer. In 2 of the 3 series, there was a significant inverse association between the 178R allele and case status (p < 0.05). In a meta-analysis, the odds ratio (95% CI) associated with the 178R allele relative to the 178K allele was 0.64 (0.45-0.92, p = 0.01) and 0.51 (0.24-1.11, p = 0.09) in fixed effects and random effects models, respectively. In a pooled analysis, after adjustment for sex, age, pack years and series, the OR (95% CI) for a heterozygote was 0.67 (0.45-1.01) and for a 178R homozygote was 0.10 (0.01-0.94); the trend for a decreased risk with the number of R alleles was significant (p = 0.008). This trend was particularly pronounced in heavy smokers (trend test p = 0.003), but not significant in light smokers (p = 0.73). There was no evidence of an association between rs12268840 and lung cancer risk. These results suggest that the R allele may protect against lung cancer, specifically in heavy smokers, an effect that may result from this polymorphism affecting the function of the MGMT protein and/or levels in MGMT activity.

No association between N7-methyldeoxyguanosine and 8-oxodeoxyguanosine levels in human lymphocyte DNA.

To examine associations between two different classes of DNA damage that can occur through endogenous processes or exogenous exposures such as smoking, N7-methyldeoxyguanosine (N7-MedG) and 8-oxodeoxyguanosine (8-oxodG) levels were measured in lymphocyte DNA from 22 bronchoscopy patients. 8-OxodG and N7-MedG was detected in 100% and 91% of samples, respectively with 8-oxodG levels being approx 20 times higher (mean 8.39+/-3.578-oxodG/10(6)dG versus 0.41+/-0.33 N7-MedG/10(6) dG) which provides an indication of the relative importance of the agents that induce oxidative DNA damage or alkylation damage. The sources of these genotoxic lesions remain to be established but N7-MedG and 8-oxodG levels were not correlated (r(2)<0.01) suggesting that there is no association between alkylating agent and reactive oxygen species exposure, their metabolism and/or the DNA repair processes that can remove this DNA damage.

N7-methyldeoxyguanosine levels in DNA isolated from cervical cytology samples are associated with smoking.

Smoking has been associated, in epidemiological studies, with an increased risk of cervical neoplasia. This may be in part due to the presence of tobacco-specific nitrosamines in cervical mucous of smokers, which may result in carcinogenic DNA damage. We have thus examined whether cervical DNA contains alkylation damage arising from exposure to methylating agents (N7-methyldeoxyguanosine, N7-MedG). DNA was extracted from cervical cytology samples and N7-MedG levels were measured using an immunoslotblot assay. Ninety percentage of the DNA samples were alkylated and N7-MedG levels (mean, 95% CI) in ever-smokers (1.27, 0.90-1.81 micromol/mol dG) were significantly higher than those in nonsmokers (0.42, 0.20-0.91 micromol/mol dG: p = 0.005). N7-MedG adduct levels were significantly correlated with number of cigarettes smoked per day and pack years of cigarette smoking in current smokers. There was no association with N7-MedG levels and cervical intraepithelial neoplasia status, age, parity or contraception use. Our study suggests that cervical DNA contains alkylation damage that can arise from exposure to cigarette smoke.

Quantitative trait locus analysis reveals two intragenic sites that influence O6-alkylguanine-DNA alkyltransferase activity in peripheral blood mononuclear cells.

The repair of specific types of DNA alkylation damage by O6-alkylguanine-DNA alkyltransferase (MGMT) is a major mechanism of resistance to the carcinogenic and chemotherapeutic effects of certain alkylating agents. MGMT expression levels vary widely between individuals but the underlying causes of this variability are not known. To address this, we used an expressed single nucleotide polymorphism (SNP) and demonstrated that the MGMT alleles are frequently expressed at different levels in peripheral blood mononuclear cells (PBMC). This suggests that there is a genetic component of inter-allelic variation of MGMT levels that maps close to or within the MGMT locus. We then used quantitative trait locus (QTL) analysis using intragenic SNPs and found that there are at least two sites influencing inter-individual variation in PBMC MGMT activity. One is characterized by an SNP at the 3' end of the first intron and the second by two SNPs in the last exon. The latter are in perfect disequilibrium and both result in amino acid substitutions-one of them, Ile143Val, affecting an amino acid close to the Cys145 residue at the active site of MGMT. Using in vitro assays, we further showed that while the Val143 variant did not affect the activity of the protein on methylated DNA substrate, it was more resistant to inactivation by the MGMT pseudosubstrate, O6-(4-bromothenyl)guanine. These findings suggest that further investigations of the potential epidemiological and clinical significance of inherited differences in MGMT expression and activity are warranted.

Reduced MGMT activity in human colorectal adenomas is associated with K-ras GC->AT transition mutations in a population exposed to methylating agents.

There is increasing evidence to suggest that O(6)-alkyl guanine DNA-alkyltransferase (MGMT) activity provides protection against alkylating agent induced formation of GC-->AT transition mutations in the K-ras oncogene of colorectal tumours. As this mutagenic event occurs during the growth of adenomas, both biomarkers of exposure (N7-methylguanine levels in DNA) and susceptibility (MGMT activity) were measured in biopsy samples obtained from normal and adenomatous tissue from 34 patients with large adenomas (>10 mm in size). There was no correlation between MGMT activity in the adenoma and in matched normal tissue. However, MGMT activity was significantly lower in adenoma tissue than in adjacent normal mucosa (5.18 versus 7.05 fmol/microg DNA, P = 0.01), particularly in men and those whose age was greater than the median. Upon stratification by K-ras mutational status, MGMT activity was lower in adenomas bearing a K-ras GC-->AT transition mutation (mean 4.21 fmol/microg DNA) than in adjacent normal tissue (mean 7.7 fmol/microg DNA; P < 0.004). In contrast, there was no significant difference in MGMT activity in adenomas lacking a K-ras GC-->AT transition mutation and adjacent normal mucosa. N7-methylguanine levels however did not vary with age, gender, K-ras mutational status or MGMT activity. These results are consistent with the acquisition of K-ras GC-->AT transition mutations in adenomas with low MGMT activity as a result of unavoidable exposure to methylating agents.

Inhibition of gamma interferon decreases bacterial load in peritonitis by accelerating peritoneal fibrin deposition and tissue repair.

Bowel perforation can lead to significant bacterial spillage, which may then cause septic peritonitis, characterized by a systemic inflammatory response and organ dysfunction. There are several reports that have shown that the development of peritoneal adhesions is dependent on inflammatory cytokine levels and that these adhesions can reduce bacterial spread, possibly by sealing off the cecum in the cecal ligation and puncture (CLP) model of septic peritonitis. There have not, however, been any studies that have utilized a strategy to accelerate tissue repair in order to seal off the injured cecum and reduce bacterial spread as well as ameliorate systemic inflammation. In the present study, we demonstrate that the administration of anti-gamma interferon (IFN-gamma) antibody (1.2 mg/kg of body weight, intravenously) accelerated tissue repair via increased fibrin deposition 12 and 24 h after CLP in rats. This increase in fibrin deposition was associated with peritoneal adhesion 24 h after CLP and a reduction in bacterial load compared to the bacterial load of rats given irrelevant antibody. Plasma fibrin levels, however, were not altered after IFN-gamma antibody administration, suggesting that the inhibition of IFN-gamma activity specifically increased fibrin deposition to the site of injury. Furthermore, plasma interleukin-6, used as a marker of systemic inflammatory response, was reduced in CLP rats given IFN-gamma antibody compared to that found in those given irrelevant antibody. These results suggest that the early inhibition of IFN-gamma activity in the CLP model is beneficial by accelerating fibrin deposition in cecal tissue to prevent bacterial spread and reduce the systemic inflammatory response. Importantly, increased fibrin deposition in the ceca was not associated with increased plasma fibrin whereas the latter may have detrimental effects associated with coagulation disorders.

DNA alkylation and repair in the large bowel: animal and human studies.

O6-methylguanine (O6-MeG), a procarcinogenic DNA adduct that arises from exposure to methylating agents, has been detected in human colorectal DNA at levels comparable to those that cause adverse effects in model systems. O6-MeG levels vary within the colon, being higher in the cancer-prone regions of the large bowel. In rats and mice, O6-MeG persistence in colon DNA is associated with the induction of colon tumors after treatment with methylating agents. These tumors frequently contain K-ras GC-->AT transition mutations, which is consistent with the mutagenic properties of O6-MeG: such mutations are also commonly found in human colorectal cancers. O6-Alkylguanine adducts are removed by the DNA repair protein, O6-alkylguanine DNA-alkyltransferase (MGMT). MGMT overexpression in transgenic mice reduces the formation of K-ras GC-->AT mutations and tumors induced by methylating agents. Interindividual variations in human colon MGMT activity are large and large bowel tumors can occur in regions of low activity. Low MGMT activity in normal mucosa has been associated with the occurrence of K-ras GC-->AT mutations, whereas reduced MGMT expression and an increased frequency of K-ras GC-->AT mutations in colorectal cancers have been linked to MGMT promoter methylation. MGMT activity is also lower in adenomas than in adjacent normal tissue but only in those adenomas with this specific mutation. These results are entirely consistent with the hypothesis that GC-->AT mutations in the K-ras oncogene result from the formation and persistence of O6-alkylguanine lesions in colorectal DNA. Human exposure to endogenous or exogenous alkylating agents may thus be an environmental determinant of colorectal cancer risk.

Total N-nitroso compounds and their precursors in hot dogs and in the gastrointestinal tract and feces of rats and mice: possible etiologic agents for colon cancer.

We review evidence that red and processed meat are causes of colon cancer and that processed meat is a risk factor for childhood cancer and type 2 diabetes. Associations could be due to N-nitroso compounds (NOCs) derived from nitrosation of NOC precursors (NOCPs). We review our survey of total NOC and NOCP content of foods. Only rapidly nitrosated amines, including a glycosyl amino acid, were efficiently determined as NOCPs. NOCPs in hot dogs and rat feces were partly purified by adsorption-desorption and HPLC. After nitrosation, purified hot dog fractions were directly mutagenic in Ames test. The main NOCPs in these materials may be N-glycosyl amino acids and peptides. NOC levels in rat gastrointestinal tract rose steadily from stomach to feces. NOCP levels showed similar trend but with sharp increases from stomach to duodenum. One day after Min and C57BL/6J mice were fed 4% dextran sulfate sodium to induce acute colitis, fecal NOC levels increased 1.9-fold compared with untreated mice (P < 0.05). For 7 d Swiss mice received semipurified diet, 180 g beef-pork hot dogs mixed with 820 g diet or 180 g sautéed beef mixed with 820 g diet. Fecal NOC outputs on day 7 were 3.7-5.0 (hot dog) and 2.0-2.9 (beef) times those for control groups (P < 0.002 for combined groups), perhaps reflecting higher dietary NOC intakes. Feeding a similar hot dog mixture to mice did not affect normal 7-methyldeoxyguanosine level in colonic mucosal DNA. Overall, results support the hypothesis that colonic NOCs are a cause of colon cancer.

Heterogeneity of O(6)-alkylguanine-DNA alkyltransferase activity in colorectal cancer: implications for treatment.

OBJECTIVES: MGMT (O(6)-alkylguanine-DNA alkyltransferase) reverses the carcinogenic, mutagenic and cytotoxic effects of alkylating agents. Measurement of MGMT activity in tumours might thus be of use in selecting those patients with colorectal cancer who may be sensitive to adjuvant alkylating agent therapy. The aim of this study was to assess whether measurement of MGMT activity in a single tumour biopsy is representative of the whole tumour. METHODS: Multiple symmetrically spaced biopsies were taken from colorectal cancers obtained from 9 patients. MGMT activity was then measured in cell-free extracts by quantifying the transfer of [(3)H]methyl group from calf thymus DNA methylated in vitro with N-nitroso-N-[(3)H]-methylurea to the MGMT protein. RESULTS: MGMT activity was detected in all tumour samples with the activity ranging between 3.6 and 36.2 fmol/microg DNA and 202-1,986 fmol/mg protein. Heterogeneity in MGMT activity (ratio of maximum/minimum MGMT levels per tumour) varied between 1.3 and 5.4. CONCLUSIONS: Measurement of MGMT activity in a single biopsy is not necessarily indicative of the level throughout the tumour. The response of colorectal cancers to alkylating agent treatment is likely to be non-uniform both within the tumour and between patients.