Doublesex and mab-3 related transcription factor 1 (DMRT1) is a sex-specific genetic determinant of childhood-onset asthma and is expressed in testis and macrophages.

BACKGROUND: Asthma is a disease affecting more boys than girls in childhood and more women than men in adulthood. The mechanisms behind these sex-specific differences are not yet understood. OBJECTIVE: We analyzed whether and how genetic factors contribute to sex-specific predisposition to childhood-onset asthma. METHODS: Interactions between sex and polymorphisms on childhood asthma risk were evaluated in the Multicentre Asthma Genetics in Childhood Study (MAGICS)/Phase II International Study of Asthma and Allergies in Childhood (ISAAC II) population on a genome-wide level, and findings were validated in independent populations. Genetic fine mapping of sex-specific asthma association signals was performed, and putatively causal polymorphisms were characterized in vitro by using electrophoretic mobility shift and luciferase activity assays. Gene and protein expression of the identified gene doublesex and mab-3 related transcription factor 1 (DMRT1) were measured in different human tissues by using quantitative real-time PCR and immunohistochemistry. RESULTS: Polymorphisms in the testis-associated gene DMRT1 displayed interactions with sex on asthma status in a population of primarily clinically defined asthmatic children and nonasthmatic control subjects (lowest P = 5.21 × 10(-6)). Replication of this interaction was successful in 2 childhood populations clinically assessed for asthma but showed heterogeneous results in other population-based samples. Polymorphism rs3812523 located in the putative DMRT1 promoter was associated with allele-specific changes in transcription factor binding and promoter activity in vitro. DMRT1 expression was observed not only in the testis but also in lung macrophages. CONCLUSION: DMRT1 might influence sex-specific patterns of childhood asthma, and its expression in testis tissue and lung macrophages suggests a potential involvement in hormone or immune cell regulation.

BMI and lifetime changes in BMI and cancer mortality risk.

Body Mass Index (BMI) is known to be associated with cancer mortality, but little is known about the link between lifetime changes in BMI and cancer mortality in both males and females. We studied the association of BMI measurements (at baseline, highest and lowest BMI during the study-period) and lifetime changes in BMI (calculated over different time periods (i.e. short time period: annual change in BMI between successive surveys, long time period: annual change in BMI over the entire study period) with mortality from any cancer, and lung, colorectal, prostate and breast cancer in a large cohort study (n=8,645. Vlagtwedde-Vlaardingen, 1965-1990) with a follow-up on mortality status on December 31st 2008. We used multivariate Cox regression models with adjustments for age, smoking, sex, and place of residence. Being overweight at baseline was associated with a higher risk of prostate cancer mortality (hazard ratio (HR) =2.22; 95% CI 1.19-4.17). Obesity at baseline was associated with a higher risk of any cancer mortality [all subjects (1.23 (1.01-1.50)), and females (1.40 (1.07-1.84))]. Chronically obese females (females who were obese during the entire study-period) had a higher risk of mortality from any cancer (2.16 (1.47-3.18), lung (3.22 (1.06-9.76)), colorectal (4.32 (1.53-12.20)), and breast cancer (2.52 (1.15-5.54)). We found no significant association between long-term annual change in BMI and cancer mortality risk. Both short-term annual increase and decrease in BMI were associated with a lower mortality risk from any cancer [all subjects: (0.67 (0.47-0.94)) and (0.73 (0.55-0.97)), respectively]. In conclusion, a higher BMI is associated with a higher cancer mortality risk. This study is the first to show that short-term annual changes in BMI were associated with lower mortality from any type of cancer.

Objective allergy markers and risk of cancer mortality and hospitalization in a large population-based cohort.

PURPOSE: There are indications that a history of allergy may offer some protection against cancer. We studied the relation of three objectively determined allergy markers with cancer mortality and hospitalization risk. METHODS: Associations between three allergy markers (number of peripheral blood eosinophil counts, skin test positivity, and serum total IgE) with mortality and hospitalization from any type and four common types of cancer (lung, colorectal, prostate, and breast cancer) were assessed in the Vlagtwedde-Vlaardingen cohort (1965-1990), with follow-up of mortality until 31 December 2008. Hospitalization data were available since 1 January 1995. RESULTS: There were no significant associations between objective allergy markers and cancer mortality or hospitalization. We found several associations in specific subgroups. A higher number of eosinophils was associated with a decreased risk of colorectal cancer mortality in ever smokers HR (95 % CI) = 0.61 (0.45-0.83) and in males 0.59 (0.42-0.83); however, no overall association was observed 0.84 (0.64-1.09). Skin test positivity was associated with a decreased risk of any cancer mortality only among females 0.59 (0.38-0.91) and showed no overall association 0.83 (0.67-1.04). Serum total IgE levels were associated with an increased risk of lung cancer mortality among females 4.64 (1.04-20.70), but with a decreased risk of cancer hospitalization in ever smokers 0.77 (0.61-0.97) and males 0.72 (0.55-0.93); however, no overall associations were observed [mortality 0.99 (0.79-1.25), and hospitalization 0.86 (0.71-1.04)]. CONCLUSIONS: We found no associations between objective allergy markers and cancer in the total population. However, skin test positivity and a high number of eosinophils were associated with a reduced risk to die of cancer in specific subgroups. Hence, it seems important to study specific subgroups defined by gender and smoking habits in order to identify allergy markers of predictive value for cancer mortality.

Routine application of a novel MLPA-based first-line screening test uncovers clinically relevant copy number aberrations in haematological malignancies undetectable by conventional cytogenetics.

OBJECTIVE: The presence of numerical and/or structural chromosomal abnormalities is a frequent finding in clonal hematopoietic malignant disease, typically diagnosed through routine karyotyping and/or fluorescent in situ hybridization (FISH) analysis. Recently, the application of array comparative genomic hybridization (aCGH) has uncovered many new cryptic genomic copy number imbalances, most of which are now recognized as clinically useful markers of haematological malignancies. In view of the limitations of both FISH and aCGH techniques, in terms of their routine application as a first line screening test, we designed a new multiple ligation-dependent probe amplification (MLPA) probemix for use in addition to classic karyotype analysis. METHODS: A novel MLPA probemix was developed to interrogate copy number changes involving chromosomal regions: 2p23-24 (MYCN, ALK), 5q32-34 (MIR145A, EBF1, MIR146A), 6q21-27, 7p12.2 (IKZF1), 7q21-36, 8q24.21 (MYC), 9p24 (JAK2 V617F point mutation), 9p21.3 (CDKN2A/2B), 9p13.2 (PAX5), 10q23 (PTEN), 11q22.3 (ATM), 12p13.2 (ETV6), 13q14 (RB1, MIR15A, DLEU2, DLEU1), 17p13.1 (TP53), and 21q22.1 (RUNX1/AML1) and was applied to DNA extracted from 313 consecutive bone marrow patient samples, referred for routine karyotype analysis. RESULTS: More than half of the samples originated from newly investigated patients. We discovered clinically relevant genomic aberrations, involving a total of 24 patients (8%) all with a normal karyotype, which would have remained undiagnosed. DISCUSSION: Our data clearly indicate that routine application of this MLPA screening panel, as an adjunct to karyotype analysis, provides a sensitive, robust, rapid and low-cost approach for uncovering clinically important genomic abnormalities, which would have otherwise remained undetected.

A methylation assay for the detection of non-muscle-invasive bladder cancer (NMIBC) recurrences in voided urine.

OBJECTIVE: To develop a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay for the detection of non-muscle invasive bladder cancer (NMIBC) recurrences in voided urine. PATIENTS AND METHODS: Genes frequently methylated in NMIBC tumours (n= 37) were selected to develop a BC-specific MS-MLPA assay. Genes methylated in blood from patients with BC (n= 29) and genes methylated in urine from patients with no history of BC (n= 46) were excluded. A four-gene panel with the highest predictive value was selected from the initial assay. This four-gene panel was tested and validated on urine from patients with a histologically confirmed recurrence (n= 68 test set; n= 49 validation set) and urine samples from patients without BC (n= 91, test set) and urine from recurrence-free BC (rec-free BC) patients (n= 60, validation set). A model was developed to predict the probability of having a recurrence based on methylation of the four-gene panel and a threshold probability with the highest sensitivity and specificity was determined. The outcome of the model was validated on BC urine samples (n= 65) and on urine samples from rec-free BC patients (n= 29). RESULTS: The BC MS-MLPA assay consisted of 23 methylation probes. The selected four-gene panel included: APC_a, TERT_a, TERT_b, and EDNRB. This panel reached an area under the receiver operating characteristic curve (AUC) of 0.82 (test set) and AUC 0.69 (validation set). Sensitivity and specificity for the detection of a concomitant tumour were 63.3% and 58.3% respectively (test set) and 72.3% and 55.2%, respectively (validation set). CONCLUSIONS: We have developed a methylation detection assay specifically for the detection of recurrences in patients with NMIBC in voided urine. The findings are promising and improvement of this test could eventually contribute to a more individualized patient friendly surveillance.

Paradoxical responses after start of antimicrobial treatment in Mycobacterium ulcerans infection.

BACKGROUND: Antimicrobial killing in mycobacterial infections may be accompanied by (transient) clinical deterioration, known as paradoxical reaction. To search for patterns reflecting such reactions in the treatment of Buruli ulcer (Mycobacterium ulcerans infection), the evolution of lesions of patients treated with antimicrobials was prospectively assessed. METHODS: The lesion size of participants of the BURULICO antimicrobial trial (with lesions ≤10 cm cross-sectional diameter) was assessed by careful palpation and recorded by serial acetate sheet tracings. Patients were treated with antimicrobials for 8 weeks. For the size analysis, participants whose treatment had failed, had skin grafting, or were coinfected with human immunodeficiency virus were excluded. For every time point, surface area was compared with the previous assessment. A generalized additive mixed model was used to study lesion evolution. Nonulcerative lesions were studied using digital images recording possible subsequent ulceration. RESULTS: Of 151 participants, 134 were included in the lesion size analysis. Peak paradoxical response occurred at week 8; >30% of participants showed an increase in lesion size as compared with the previous (week 6) assessment. Seventy-five of 90 (83%) of nonulcerative lesions ulcerated after start of treatment. Nine participants developed new lesions during or after treatment. All lesions subsequently healed. CONCLUSIONS: After start of antimicrobial treatment for Buruli ulcer, new or progressive ulceration is common before healing sets in. This paradoxical response, most prominent at the end of the 8-week antimicrobial treatment, should not be misinterpreted as failure to respond to treatment. Clinical Trials Registration. ClinicalTrials.gov, NCT00321178.

Tumor Vascular Morphology Undergoes Dramatic Changes during Outgrowth of B16 Melanoma While Proangiogenic Gene Expression Remains Unchanged.

In established tumors, angiogenic endothelial cells (ECs) coexist next to "quiescent" EC in matured vessels. We hypothesized that angio-gene expression of B16.F10 melanoma would differ depending on the growth stage. Unraveling the spatiotemporal nature thereof is essential for drug regimen design aimed to affect multiple neovascularization stages. We determined the angiogenic phenotype-represented by 52 angio-genes-and vascular morphology of small, intermediate, and large s.c. growing mouse B16.F10 tumors and demonstrated that expression of these genes did not differ between the different growth stages. Yet vascular morphology changed dramatically from small vessels without lumen in small to larger vessels with increased lumen size in intermediate/large tumors. Separate analysis of these vascular morphologies revealed a significant difference in αSMA expression in relation to vessel morphology, while no relation with VEGF, HIF-1α, nor Dll4 expression levels was observed. We conclude that the tumor vasculature remains actively engaged in angiogenesis during B16.F10 melanoma outgrowth and that the major change in tumor vascular morphology does not follow molecular concepts generated in other angiogenesis models.

Antimicrobial treatment for early, limited Mycobacterium ulcerans infection: a randomised controlled trial.

BACKGROUND: Surgical debridement was the standard treatment for Mycobacterium ulcerans infection (Buruli ulcer disease) until WHO issued provisional guidelines in 2004 recommending treatment with antimicrobial drugs (streptomycin and rifampicin) in addition to surgery. These recommendations were based on observational studies and a small pilot study with microbiological endpoints. We investigated the efficacy of two regimens of antimicrobial treatment in early-stage M ulcerans infection. METHODS: In this parallel, open-label, randomised trial undertaken in two sites in Ghana, patients were eligible for enrolment if they were aged 5 years or older and had early (duration <6 months), limited (cross-sectional diameter <10 cm), M ulcerans infection confirmed by dry-reagent-based PCR. Eligible patients were randomly assigned to receive intramuscular streptomycin (15 mg/kg once daily) and oral rifampicin (10 mg/kg once daily) for 8 weeks (8-week streptomycin group; n=76) or streptomycin and rifampicin for 4 weeks followed by rifampicin and clarithromycin (7.5 mg/kg once daily), both orally, for 4 weeks (4-week streptomycin plus 4-week clarithromycin group; n=75). Randomisation was done by computer-generated minimisation for study site and type of lesion (ulceration or no ulceration). The randomly assigned allocation was sent from a central site by cell-phone text message to the study coordinator. The primary endpoint was lesion healing at 1 year after the start of treatment without lesion recurrence or extensive surgical debridement. Analysis was by intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT00321178. FINDINGS: Four patients were lost to follow-up (8-week streptomycin, one; 4-week streptomycin plus 4-week clarithromycin, three). Since these four participants had healed lesions at their last assessment, they were included in the analysis for the primary endpoint. 73 (96%) participants in the 8-week streptomycin group and 68 (91%) in the 4-week streptomycin plus 4-week clarithromycin group had healed lesions at 1 year (odds ratio 2.49, 95% CI 0.66 to infinity; p=0.16, one-sided Fisher's exact test). No participants had lesion recurrence at 1 year. Three participants had vestibulotoxic events (8-week streptomycin, one; 4-week streptomycin plus 4-week clarithromycin, two). One participant developed an injection abscess and two participants developed an abscess close to the initial lesion, which was incised and drained (all three participants were in the 4-week streptomycin plus 4-week clarithromycin group). INTERPRETATION: Antimycobacterial treatment for M ulcerans infection is effective in early, limited disease. 4 weeks of streptomycin and rifampicin followed by 4 weeks of rifampicin and clarithromycin has similar efficacy to 8 weeks of streptomycin and rifampicin; however, the number of injections of streptomycin can be reduced by switching to oral clarithromycin after 4 weeks. FUNDING: European Union (EU FP6 2003-INCO-Dev2-015476) and Buruli Ulcer Groningen Foundation.

Detecting 22q11.2 deletions by use of multiplex ligation-dependent probe amplification on DNA from neonatal dried blood spot samples.

The 22q11 deletion syndrome, which is caused by a 1.5- to 3.0-megabase hemizygous deletion in chromosome 22q11.2, has a prevalence of 1/2000 to 1/4000. However, the syndrome presents with highly variable phenotypes and thus may be underestimated among Danish newborns. To establish a true incidence of 22q11.2 deletions among certain manifestations, eg, congenital heart disease, on selected Danes, a multiplex ligation-dependant probe amplification (MLPA) analysis was designed. The analysis was planned to be performed on DNA extracted from dried blood spot samples (DBSS) obtained from Guthrie cards collected during neonatal screening programs. However, the DNA concentration necessary for a standard MLPA analysis (20 ng) could not be attained from DBSS, and a novel MLPA design was developed to permit for analysis on limited amounts of DNA (2 ng). A pilot study is reported here that validates the new MLPA design using nine patients diagnosed with the 22q11.2 deletion and 101 controls. All deletions were identified using DNA extracted from DBSS, and no copy number variations were detected in the controls, resulting in a specificity and sensitivity of 100%. It is thereby concluded that the novel MLPA probe design is successful and reliable using minimal amounts of DNA. This allows for use of DBSS samples in a retrospective study of 22q11.2 deletion among certain manifestations associated with DiGeorge Syndrome.

Level and course of FEV1 in relation to polymorphisms in NFE2L2 and KEAP1 in the general population.

BACKGROUND: The metabolism of xenobiotics plays an essential role in smoking related lung function loss and development of Chronic Obstructive Pulmonary Disease. Nuclear Factor Erythroid 2-Like 2 (NFE2L2 or NRF2) and its cytosolic repressor Kelch-like ECH-associated protein-1 (KEAP1) regulate transcription of enzymes involved in cellular detoxification processes and Nfe2l2-deficient mice develop tobacco-induced emphysema. We assessed the impact of Single Nucleotide Polymorphisms (SNPs) in both genes on the level and longitudinal course of Forced Expiratory Volume in 1 second (FEV1) in the general population. METHODS: Five NFE2L2 and three KEAP1 tagging SNPs were genotyped in the population-based Doetinchem cohort (n = 1,152) and the independent Vlagtwedde-Vlaardingen cohort (n = 1,390). On average 3 FEV1 measurements during 3 surveys, respectively 7 FEV1 measurements during 8 surveys were present. Linear Mixed Effect models were used to test cross-sectional and longitudinal genetic effects on repeated FEV1 measurements. RESULTS: In the Vlagtwedde-Vlaardingen cohort SNP rs11085735 in KEAP1 was associated with a higher FEV1 level (p = 0.02 for an additive effect), and SNP rs2364723 in NFE2L2 was associated with a lower FEV1 level (p = 0.06). The associations were even more significant in the pooled cohort analysis. No significant association of KEAP1 or NFE2L2 SNPs with FEV1 decline was observed. CONCLUSION: This is the first genetic study on variations in key antioxidant transcriptional regulators KEAP1 and NFE2L2 and lung function in a general population. It identified 2 SNPs in NFE2L2 and KEAP1 which affect the level of FEV1 in the general population. It additionally shows that NFE2L2 and KEAP1 variations are unlikely to play a role in the longitudinal course of FEV1 in the general population.

Urinary creatinine excretion, an indirect measure of muscle mass, is an independent predictor of cardiovascular disease and mortality in the general population.

OBJECTIVE: Low muscle mass often indicates poor health, but the relation with cardiovascular disease (CVD) is unknown. Skeletal muscles are responsible for approximately 75% of insulin stimulated whole body glucose disposal and therefore insulin resistance could underlie the relation between muscle mass and CVD. We aimed to determine whether muscle mass, as reflected by 24h urinary creatinine excretion, is associated with CVD and whether this depends on insulin resistance. METHODS: The study was performed in the prospective, community-based, observational cohort of the PREVEND study in Groningen, the Netherlands. 24h creatinine excretion was assessed in 4044 women and 4048 men. Outcome events were incidence of major adverse cardiovascular events (MACE) and all-cause mortality, with a follow-up of 7.5 [7.3-7.9] years. Insulin resistance was estimated using fasting insulin and HOMA. RESULTS: In women every doubling of creatinine excretion was associated with an approximate 60% decrease in risk for MACE (hazard ratio (HR) 0.41 [95%CI 0.26-0.64], P<0.001) and 50% decrease in risk for all-cause mortality (HR: 0.52 [0.31-0.90], P=0.02) independent of age, smoking, CVD history, race, fasting insulin concentrations and components of the metabolic syndrome. In men every doubling of creatinine excretion was borderline associated with an approximately 25% decrease in risk for MACE (HR: 0.74 [0.53-1.03], P=0.07) and independently associated with a 55% decreased risk for all-cause mortality (HR: 0.45 [0.32-0.62], P<0.001). CONCLUSIONS: Low creatinine excretion, as indirect measure of low muscle mass, is associated with MACE and all-cause mortality in the general population, independent of insulin resistance. Perhaps protein-calorie malnutrition or physical activity could underlie the association between muscle mass and CVD.

Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer.

We have studied in vivo responses of "spontaneous" Brca1- and p53-deficient mammary tumors arising in conditional mouse mutants to treatment with doxorubicin, docetaxel, or cisplatin. Like human tumors, the response of individual mouse tumors varies, but eventually they all become resistant to the maximum tolerable dose of doxorubicin or docetaxel. The tumors also respond well to cisplatin but do not become resistant, even after multiple treatments in which tumors appear to regrow from a small fraction of surviving cells. Classical biochemical resistance mechanisms, such as up-regulated drug transporters, appear to be responsible for doxorubicin resistance, rather than alterations in drug-damage effector pathways. Our results underline the promise of these mouse tumors for the study of tumor-initiating cells and of drug therapy of human cancer.

Activated intrinsic apoptosis pathway is a key related prognostic parameter in acute myeloid leukemia.

PURPOSE: By parallel assessment of multiple apoptosis-related transcripts, we aimed to refine the current concept of apoptosis resistance in acute myeloid leukemia (AML) and identify the combination of genes best predicting overall survival (OS). PATIENTS AND METHODS: The reverse transcriptase multiplex ligation-dependent probe amplification technique was used for simultaneous quantification of 31 apoptosis-related transcripts in viable (7AAD-/AnnexinV-) blasts (CD45dim) from bone marrow aspirates of 120 newly diagnosed AML patients. By forward selection, a prognosis-predicting gene expression profile was constructed. The predictive validity of this profile was assessed by cross validation. RESULTS: High transcript levels were associated with poor OS for seven of 31 genes, three of which were proapoptotic. The average expression of all 12 antiapoptotic genes was associated with poor OS (P = .029). A similar association with poor OS was found for the average expression of all 19 proapoptotic genes (P = .009). Forward selection and cross validation revealed the antiapoptotic gene BIRC3 and the proapoptotic genes BAX-(l) and BMF to optimally predict OS. Three equally sized patient groups, constructed by ranking the cross-validated prognoses of the patients, were clearly distinct (median OS times were 8.2, 16.7, and 85.6 months). CONCLUSION: High expression of both pro- and antiapoptotic genes predicted poor OS, which postulates a mechanism of activation of the apoptosis pathway as a whole. This mechanism, which culminates in a three-gene expression signature, allows accurate clinical outcome prediction in AML and puts efforts to target single antiapoptosis genes in a new perspective.

Incidence of chronic obstructive pulmonary disease in a cohort of young adults according to the presence of chronic cough and phlegm.

RATIONALE: The few prospective studies aimed at assessing the incidence of chronic obstructive pulmonary disease (COPD) in relation to the presence of chronic cough/phlegm have produced contrasting results. OBJECTIVES: To assess the incidence of COPD in a cohort of young adults and to test whether chronic cough/phlegm and dyspnea are independent predictors of COPD. METHODS: An international cohort of 5,002 subjects without asthma (ages 20-44 yr) with normal lung function (FEV(1)/FVC ratio >/= 70%) from 12 countries was followed from 1991-2002 in the frame of the European Community Respiratory Health Survey II. Incident cases of COPD were those who had an FEV(1)/FVC ratio less than 70% at the end of the follow-up, but did not report having had a doctor diagnose asthma during the follow-up. MAIN RESULTS: The incidence rate of COPD was 2.8 cases/1,000/yr (95% confidence interval [CI], 2.3-3.3). Chronic cough/phlegm was an independent and statistically significant predictor of COPD (incidence rate ratio [IRR], 1.85; 95% CI, 1.17-2.93) after adjusting for smoking habits and other potential confounders, whereas dyspnea was not associated with the disease (IRR = 0.98; 95% CI, 0.64-1.50). Subjects who reported chronic cough/phlegm both at baseline and at the follow-up had a nearly threefold-increased risk of developing COPD with respect to asymptomatic subjects (IRR = 2.88; 95% CI, 1.44-5.79). CONCLUSIONS: The incidence of COPD is substantial even in young adults. The presence of chronic cough/phlegm identifies a subgroup of subjects with a high risk of developing COPD, independently of smoking habits.

Epigenetic events of disease progression in head and neck squamous cell carcinoma.

OBJECTIVE: To examine the promoter methylation status of the 22 cancer genes and their contribution to disease progression in 6 head and neck squamous cell carcinoma (HNSCC) cell lines. DESIGN: A panel of 41 gene probes, designed to interrogate 35 unique genes with known associations to cancer including HNSCC, was interrogated for alterations in gene copy number and aberrant methylation status (22 genes) using the methylation-specific multiplex ligation-dependent probe amplification assay. SUBJECTS: Primary (A) and recurrent or metastatic (B) HNSCC cell lines UMSCC-11A/11B, UMSCC-17A/17B, and UMSCC-81A/81B are described. RESULTS: Nine genes, TIMP3, APC, KLK10, TP73, CDH13, IGSF4, FHIT, ESR1, and DAPK1, were aberrantly methylated. The most frequently hypermethylated genes were APC and IGSF4, observed in 3 of 6 cell lines, and TP73 and DAPK1, observed in 2 of 6. For KLK10 and IGSF4, TIMP3 and FHIT, and TP73, in UMSCC-11B, UMSCC-17B, and UMSCC-81B, respectively, promoter hypermethylation was a disease progression event, indicating complete abrogation of tumor suppressor function for KLK10, IGSF4, and TIMP3 and gene silencing of 1 of 2 copies of TP73. Hypermethylation of IGSF4, TP73, CDH13, ESR1, DAPK1, and APC were primary events in UMSCC-17A. CONCLUSIONS: Gene silencing through promoter hypermethylation was observed in 5 of 6 cell lines and contributed to primary and progressive events in HNSCC. In addition to genetic alterations of gains and losses, epigenetic events appear to further undermine a destabilized genomic repertoire in HNSCC.

Fine-mapping loss of gene architecture at the CDKN2B (p15INK4b), CDKN2A (p14ARF, p16INK4a), and MTAP genes in head and neck squamous cell carcinoma.

OBJECTIVE: To identify the extent and the smallest region of loss for CDKN2B(INK4b), CDKN2A(ARF,INK4a), and MTAP. Homozygous deletions of human chromosome 9p21 occur frequently in malignant cell lines and are common in squamous cell carcinoma of the head and neck (HNSCC). This complex region encodes the tumor suppressor genes cyclin-dependent kinase 2B (CDKN2B) (p15(INK4b)) and CDKN2A (p14(ARF), p16(INK4a)) and the housekeeping gene methylthioadenosine phosphorylase (MTAP). DESIGN: A targeted probe panel designed to finely map the region of 9p21 loss comprised 3 probes for CDKN2B(INK4b), 7 for CDKN2A(ARF, INK4a), and 3 for MTAP and was interrogated using the multiplex ligation-dependent probe amplification assay (MLPA). The MLPA genomic copy number alterations for CDKN2A were validated using real-time polymerase chain reaction. SUBJECTS: Six HNSCC primary (A) and recurrent or metastatic (B) cell lines were examined: UMSCC-11A/11B, UMSCC-17A/17B, and UMSCC-81A/81B. RESULTS: Cell line UMSCC-11B retained all 9p loci tested in the region. Cell lines UMSCC-17A/B indicated homozygous deletion of CDKN2A(ARF, INK4a) starting at p16(INK4) exon 1alpha to include exons 2 and 3. Homozygous loss was indicated for CDKN2B(INK4b) and CDKN2A(ARF,INK4a) in UMSCC-11A, and UMSCC-81A. Cell line UMSCC-81B indicated retention of all 9p loci except for exon 1alpha (p16(INK4a)). Selective loss of the 3' end of MTAP was observed in UMSCC-11A. Genomic alterations by fine-mapping MLPA were validated at the DNA level for CDKN2A. CONCLUSIONS: We identified exon 1alpha (p16(INK4a)) as the smallest region of loss in the CDKN2A(ARF, INK4a) gene. The frequency and precise loss of CDKN2B(INK4b), CDKN2A(ARF, INK4a), and MTAP in the prognosis of 9p21-deleted HNSCC may provide impetus for use of these targets as therapeutic biomarkers in head and neck cancer.

Iodine-123 metaiodobenzylguanidine scintigraphy in localising phaeochromocytomas--experience and meta-analysis.

BACKGROUND: Recommendations on the use of I-123 metaiodobenzylguanidine (MIBG) scintigraphy in localising phaeochromocytomas vary. The accuracy of I-123 MIBG scintigraphy was determined by evaluating our own I-123 MIBG scans and performing a meta-analysis. MATERIALS AND METHODS: Between January 1992 and May 2002, the I-123 MIBG scans of consecutive patients suspected of a phaeochromocytoma were re-evaluated. For the meta-analysis, studies with more than 5 I-123 MIBG scans were selected. RESULTS: Thirty patients were evaluated. The sensitivity in our own population was 92% and the specificity was 100%. Twelve articles were selected for our meta-analysis. The overall sensitivity and specificity were 96% and 100%, respectively. The sensitivity and specificity for tumours in the adrenal gland was 98% for both. For tumours located outside the adrenal gland, the sensitivity was 98% and the sensitivity for malignancies was 79%. CONCLUSION: 1-123 MIBG scintigraphy has an excellent sensitivity and specificity in localising phaeochromocytomas, except for malignant tumours. 1-123 MIBG scintigraphy is superior in localising tumours outside the adrenal gland.

High-resolution mapping of molecular events associated with immortalization, transformation, and progression to breast cancer in the MCF10 model.

BACKGROUND: A comprehensive and consistent picture of the genetic changes that underlie breast cancer initiation, development, and progression remains unresolved. The MCF10 series of cell lines represents many steps in that progression. We performed high resolution mapping of the MCF10 series of cell lines to identify specific gene targets to elucidate the molecular correlates of immortalization, development, and progression of breast cancer at the level of individual genes. DESIGN: We evaluated the initial untransformed outgrowths (MCF-10MS and MCF-10A) with six transformed cell lines with benign proliferations (MCF-10AT1, MCF-10AT1kcl2), carcinoma in situ (MCF-10CA1h cl13), and invasive carcinoma (MCF-10CA1h cl2, MCF-10CA1a cl1, MCF-10CA1d cl1). Losses and gains of loci at 112 unique human genome sites were interrogated using the multiplex ligation-dependent probe amplification assay (MLPA). RESULTS: Cytogenetic alterations in the four benign progenitors that persisted in the CIS and invasive cell lines corresponded to gains and losses of genes by MLPA. MCF-10MS had only normal gene copies. The untransformed MCF-10A had cytogenetic gain of 5q13-qter with corresponding gains of the IL3, IL4 and IL12B genes at 5q31-q33; gain of distal 19q12-qter was reflected in gains in KLK3 and BAX gene loci at 19q13-q13.4. The observed genic gain of cMYC at 8q24.12 was not indicated by cytogenetics. The apparently balanced t(3;9) component of the t(3;9)(p13;p22)t(3;5)(p26;q31) resulted in complete loss of the CDKN2A and CDKN2B genes at 9p21. Additional clonal cytogenetic changes in the DCIS cell line (MCF-10A1h cl13) involving chromosomes 1, 3 and 10 persisted in the invasive progeny, with gain of corresponding genes at 1p13 (BCAR2, BCAR3, NRAS, TGFB2), at 3p12-13 (IL12A), and 3q21-27 (MME, PIK3CA, BCL6). CONCLUSIONS: Our study adopted a comprehensive exploration of genetic changes using high resolution molecular probes applied to the MCF10 family of cell lines to identify individual genes in a continuum starting from normal breast epithelial cells and progressing through immortalization, transformation and invasive malignancy. Homozygous loss of CDKN2A and CDKN2B genes and gain of MYC were initiating immortalization events. Transformation and progression to malignancy event were marked by gains of IL13, VEGF, HRAS, TRAF2, and BCAS2, IL12A, and MME, respectively.

Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.

Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA-probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA-probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader-Willy syndrome, Angelman syndrome or acute myeloid leukemia.

An increase in bronchial responsiveness is associated with continuing or restarting smoking.

RATIONALE: Bronchial responsiveness (BHR) has been found to be associated with smoking, atopy, and lower lung function in cross-sectional studies, but there is little information on determinants of change in adults. OBJECTIVES: To analyze change in bronchial responsiveness in an international longitudinal community study. METHODS: The study was performed in 3,993 participants in the European Community Respiratory Health Survey who had bronchial responsiveness measured in 1991-1993, when aged 20 to 44 yr, and in 1998-2002. MEASUREMENTS: Bronchial responsiveness was assessed by methacholine challenge. Serum samples were tested for total IgE, and for specific IgE to four common allergens. Smoking information was obtained from detailed administered questionnaires. Change in bronchial responsiveness was analyzed by change in IgE sensitization, smoking, and lung function, with tests of interaction terms with age and sex. MAIN RESULTS: Continuing and restarting smokers had increasing bronchial responsiveness, approximately equivalent to a mean reduction in PD20 of 0.68 and 0.75 doubling doses, respectively, over 10 yr, in addition to a small increase explained by decline in FEV1. No other risk factor for change in bronchial responsiveness was identified. CONCLUSIONS: Smoking is a risk factor for increasing bronchial responsiveness over and above the effect of decreasing lung function. Neither baseline IgE sensitization nor change in sensitization was shown to be a risk factor for increasing BHR, the latter possibly due to little overall increase or decrease in sensitization.