Merging and imputation of flow cytometry data: A critical assessment.

Although most modern techniques and analysis methods in multiparameter flow cytometry (MFC) allow for increased dimensionality for the characterization and quantification of cell populations, most MFC applications depend on flow cytometers measuring relatively small (<16) numbers of parameters. When more markers than the available parameters need to be acquired, these are commonly distributed over multiple independent measurements that include a backbone of common markers. Several methods have been proposed to impute values for combinations of markers that were not measured simultaneously. These imputation methods are frequently used without proper validation and knowledge of their effects on data analysis. We evaluated the performance of existing imputation software (Infinicyt, CyTOFmerge, CytoBackBone, and cyCombine) in approximating known measured expression data in terms of similarity in visual appearance, cell expression, and gating in different datasets by splitting MFC samples into separate measurements with partially overlapping markers and re-calculating missing marker expression. Out of the assessed packages, CyTOFmerge showed the most accurate approximation of the known expression in terms of similar expression values and concordance with manual gating, with a mean F-score between 0.53 and 0.87 when retrieving cell populations in different datasets. Performance remained inadequate for all methods, with only limited similarity at the cell level. In conclusion, the use of imputed MFC data should take such limitations into account and include independent validation of results to justify conclusions.

Ex vivo cultures and drug testing of primary acute myeloid leukemia samples: Current techniques and implications for experimental design and outcome.

New treatment options of acute myeloid leukemia (AML) are rapidly emerging. Pre-clinical models such as ex vivo cultures are extensively used towards the development of novel drugs and to study synergistic drug combinations, as well as to discover biomarkers for both drug response and anti-cancer drug resistance. Although these approaches empower efficient investigation of multiple drugs in a multitude of primary AML samples, their translational value and reproducibility are hampered by the lack of standardized methodologies and by culture system-specific behavior of AML cells and chemotherapeutic drugs. Moreover, distinct research questions require specific methods which rely on specific technical knowledge and skills. To address these aspects, we herein review commonly used culture techniques in light of diverse research questions. In addition, culture-dependent effects on drug resistance towards commonly used drugs in the treatment of AML are summarized including several pitfalls that may arise because of culture technique artifacts. The primary aim of the current review is to provide practical guidelines for ex vivo primary AML culture experimental design.

[In process]. / Insomnie, anxiété: faut-il réguler la prescription des benzodiazépines?

Benzodiazepine hypnotics bear a higher risk of high dose dependence than benzodiazepine anxiolytics, according to a recent study in Luxemburg. This article summarizes the main indications of these molecules and the current treatment recommendations. It provides an overview of public health actions of the past and the future to reduce their excessive consumption.

Implementation of a multi-institutional diffuse intrinsic pontine glioma autopsy protocol and characterization of a primary cell culture.

AIMS: Diffuse intrinsic pontine glioma (DIPG) is a fatal paediatric malignancy. Tumour resection is not possible without serious morbidity and biopsies are rarely performed. The resulting lack of primary DIPG material has made preclinical research practically impossible and has hindered the development of new therapies for this disease. The aim of the current study was to address the lack of primary DIPG material and preclinical models by developing a multi-institutional autopsy protocol. METHODS: An autopsy protocol was implemented in the Netherlands to obtain tumour material within a brief post mortem interval. A team of neuropathologists and researchers was available at any time to perform the autopsy and process the material harvested. Whole brain autopsy was performed and primary DIPG material and healthy tissue were collected from all affected brain areas. Finally, the study included systematic evaluation by parents. RESULTS: Five autopsies were performed. The mean time interval between death and time of autopsy was 3 h (range 2-4). All tumours were graded as glioblastoma. None of the parents regretted their choice to participate, and they all derived comfort in donating tissue of their child in the hope to help future DIPG patients. In addition, we developed and characterized one of the first DIPG cell cultures from post mortem material. CONCLUSION: Here we show that obtaining post mortem DIPG tumour tissue for research purposes is feasible with short delay, and that the autopsy procedure is satisfying for participating parents and can be suitable for the development of preclinical DIPG models.

[Benzodiazepines : known risks and recent data]. / Dangers des benzodiazépines risques connus et données récentes.

Benzodiazepines have been considered the treatment of choice for a variety of neuropsychiatric disorders. They are currently much more controversial and drugs considered less dangerous are generally preferred. This article summarizes the characteristics of the different benzodiazepines present on the Belgian market. It describes abuse and dependence, as well as the risks of these substances in specific populations or situations. New data suggest that there is a much higher risk of decease in case of a chronic use. Finally, recommendations on rational use and withdrawal are given.

Lenalidomide added to standard intensive treatment for older patients with AML and high-risk MDS.

More effective treatment modalities are urgently needed in patients with acute myeloid leukemia (AML) of older age. We hypothesized that adding lenalidomide to intensive standard chemotherapy might improve their outcome. After establishing a safe lenalidomide, dose elderly patients with AML were randomly assigned in this randomized Phase 2 study (n = 222) to receive standard chemotherapy ("3 + 7") with or without lenalidomide at a dose of 20 mg/day 1-21. In the second cycle, patients received cytarabine 1000 mg/m2 twice daily on days 1-6 with or without lenalidomide (20 mg/day 1-21). The CR/CRi rates in the two arms were not different (69 vs. 66%). Event-free survival (EFS) at 36 months was 19% for the standard arm versus 21% for the lenalidomide arm and overall survival (OS) 35% vs. 30%, respectively. The frequencies and grade of adverse events were not significantly different between the treatment arms. Cardiovascular toxicities were rare and equally distributed between the arms. The results of the present study show that the addition of lenalidomide to standard remission induction chemotherapy does not improve the therapeutic outcome of older AML patients. This trial is registered as number NTR2294 in The NederlandsTrial Register (www.trialregister.nl).

Correction: Lenalidomide added to standard intensive treatment for older patients with AML and high-risk MDS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hypermethylation of the FANCC and FANCL promoter regions in sporadic acute leukaemia.

OBJECTIVE: Inactivation of the FA-BRCA pathway results in chromosomal instability. Fanconi anaemia (FA) patients have an inherited defect in this pathway and are strongly predisposed to the development of acute myeloid leukaemia (AML). Studies in sporadic cancers have shown promoter methylation of the FANCF gene in a significant proportion of various solid tumours. However, only a single leukaemic case with methylation of one of the FA-BRCA genes has been described to date, i.e. methylation of FANCF in cell line CHRF-288. We investigated the presence of aberrant methylation in 11 FA-BRCA genes in sporadic cases of leukaemia. METHODS: We analyzed promoter methylation in 143 AML bone marrow samples and 97 acute lymphoblastic leukaemia (ALL) samples using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Samples with aberrant methylation were further analyzed by bisulphite sequencing and tested for mitomycin C sensitivity using Colony Forming Units assays. RESULTS: MS-MLPA showed promoter methylation of FANCC in one AML and three ALL samples, while FANCL was found methylated in one ALL sample. Bisulphite sequencing of promoter regions confirmed hypermethylation in all cases. In addition, samples with hypermethylation of either FANCC or FANCL appeared more sensitive towards mitomycin C in Colony Forming Units assays, compared to controls. CONCLUSION: Hypermethylation of promoter regions from FA-BRCA genes does occur in sporadic leukaemia, albeit infrequently. Hypermethylation was found to result in hypersensitivity towards DNA cross-linking agents, a hallmark of the FA cellular phenotype, suggesting that these samples displayed chromosomal instability. This instability may have contributed to the occurrence of the leukaemia. In addition, this is the first report to describe hypermethylation of FANCC and FANCL. This warrants the investigation of multiple FA-BRCA genes in other malignancies.

Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples.

In acute myeloid leukemia (AML), activating mutations in the fms-like tyrosine kinase 3 (FLT3) gene predict poor prognosis. We determined FLT3 internal tandem duplications (FLT3/ITD) and D835 point mutations in paired initial and relapse samples from 80 pediatric and adult AML patients. One D835 point mutation was found in an initial pediatric AML sample. Fms-like tyrosine kinase 3/ITDs were present in 21 initial and 22 relapse samples (26.3 and 27.5%, respectively). Interestingly, FLT3/ITD positivity was related to a significantly shorter time to relapse, most pronounced when the ITD-positive status was found at relapse (P<0.001). However, FLT3/ITD status changed between diagnosis and relapse in 14 cases. In four patients, the FLT3/ITD became undetectable at relapse in five patients FLT3/ITDs were only detected at relapse, and in five patients the length or number of FLT3/ITDs changed. Gain of FLT3/ITDs may suggest oligoclonality with selective outgrowth of the FLT3/ITD-positive clone, whereas losses may reflect ITDs in the more mature leukemic cells rather than in the leukemic stem cell, or, alternatively, that other genetic aberrations provided a greater selective advantage. Studying FLT3/ITD kinetics in minimal residual disease setting may provide some answers for the changes we observed. Fms-like tyrosine kinase 3/ITD is a relevant marker for prognosis, and remains an important target for therapeutic inhibition.

Expression profiling and prediction of distant metastases in head and neck squamous cell carcinoma.

BACKGROUND: For breast and prostate cancer, a gene expression signature of the tumour is associated with the development of distant metastases. Regarding head and neck squamous cell carcinoma (HNSCC), the only known risk factor is the presence of > or =3 tumour-positive lymph nodes. AIM: To evaluate whether a HNSCC gene expression signature can discriminate between the patients with and without distant metastases. METHODS: Patients with HNSCC with and without distant metastases had >3 tumour-positive lymph nodes, and did not differ with respect to other risk factors. Statistical analysis was carried out using Student's t test, as well as statistical analysis of microarrays (SAM), to assess the false discovery rate for each gene. These analyses were supplemented with a newly developed method that computed deviations from gaussian-order statistics (DEGOS). To validate the platform, normal mucosa of the head and neck was included as control. RESULTS: 2963 genes were differently expressed between HNSCC and normal mucosa (t test; p<0.01). More rigorous statistical analysis with SAM confirmed the differential expression of most genes. The comparison of genes in HNSCC with and without metastases showed 150 differently expressed genes (t test; p<0.01), none of which, however, could be confirmed using SAM or DEGOS. CONCLUSIONS: No evidence for a metastasis signature is found, and gene expression profiling of HNSCC has seemingly no value in determining the risk of developing distant metastases. The absence of such a signature can be understood when it is realised that, for HNSCC in contrast with breast cancer, the lymph nodes are a necessary in-between station for haematogenous spread.

Inherited susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes.

BACKGROUND: Susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes may reflect the way a person deals with carcinogenic challenges. This susceptibility (also referred to as mutagen sensitivity) has been found to be increased in patients with environmentally related cancers, including cancers of the head and neck, lung, and colon, and, in combination with carcinogenic exposure, this susceptibility can greatly influence cancer risk. The purpose of this study was to assess the heritability of mutagen sensitivity. METHODS: Heritability was determined by use of a maximum likelihood method that employed the FISHER package of pedigree analysis. Bleomycin-induced breaks per cell values for 135 healthy volunteers without cancer were determined. These individuals were from 53 different pedigrees and included 25 monozygotic twin pairs (n = 50), 14 pairs of dizygotes (twin pairs and siblings, n = 28), and 14 families selected on the basis of a first-degree relative who was successfully treated for head and neck cancer and who had no sign of recurrence for at least 1 year. All data were analyzed simultaneously, and different models of familial resemblance were fitted to the data. All P values are two-sided. RESULTS: Our results showed no evidence for the influence of a shared family environment on bleomycin-induced chromatid breaks. Genetic influences, however, were statistically significant (P =. 036) and accounted for 75% of the total variance. CONCLUSIONS: The high heritability estimate of the susceptibility to bleomycin-induced chromatid breaks indicates a clear genetic basis. The findings of this study support the notion that a common genetic susceptibility to DNA damage--and thereby a susceptibility to cancer--may exist in the general population.

Genetic susceptibility to head and neck squamous cell carcinoma.

BACKGROUND: In addition to influences of exposure to carcinogenic compounds, the development of cancer may depend on an individual intrinsic cancer susceptibility. Biomarkers for cancer susceptibility can be powerful additions to epidemiologic analyses. PURPOSE: This multicenter, case-control analysis combines previously published data and new data to substantiate the value of mutagen sensitivity as a biomarker of susceptibility to head and neck squamous cell carcinoma and, more importantly, to gain insight into the interaction between susceptibility and exposure to carcinogens. METHODS: Mutagen sensitivity (mean number of chromatid breaks per cell of cultured lymphocytes treated with bleomycin in the late S-G2 phase of the cell cycle) was determined in 313 patients with head and neck cancer and in 334 control subjects at two major U.S. medical institutions and one European institution, yielding a unique study population. The ages of the case and control subjects, as well as their history of use of tobacco and alcohol, were also recorded. The relationships between variables were analyzed by use of Student's t tests, Spearman's rank correlations, and multiple linear regression. For estimation of cancer risk, crude odds rations (ORs) were measured and multiple logistic regression was performed. All P values were based on two-sided tests. RESULTS: There were no differences across institutions in the distribution of mutagen sensitivity (Kruskal-Wallis test) for both case subjects and control subjects. Values for case subjects were consistently and significantly (P<.0001) higher than values for control subjects in the overall analyses. Age and tobacco or alcohol use did not influence the outcome in terms of mutagen-sensitivity values for either the case or the control subjects. A mean number of breaks per cell dichotomized at 1.0 was found to be the best predictor of a hypersensitive phenotype. For nonsensitive, heavy smokers, the OR was 11.5 (95% confidence interval [CI] = 5.0-26.6). This risk increased dramatically in mutagen-hypersensitive, heavy smokers to 44.5 (95% CI = 17.4-114.0). Multiple logistic regression analysis confirmed these results, and a significant trend was found (P<.01) for the dose-dependent increase in cancer risk by smoking. The consumption of alcohol potentiated the effects of smoking, resulting in an OR of 57.5 (95% CI = 17.5-188.0) in hypersensitive persons. CONCLUSIONS: Mutagen sensitivity was found to be a biomarker of cancer susceptibility. This study underscores the importance of utilizing both susceptibility markers and the exposure data for the identification of persons at high risk of developing cancer. IMPLICATIONS: More accurate risk estimation can define susceptible subgroups who might be targeted for intensive behavioral interventions, surveillance through screening, and enrollment in chemoprevention programs.

Cost of disorders of the brain in Luxembourg.

Brain disorders (psychiatric, neurological and neurosurgical diseases) are leading causes of disease and disability. According to WHO data they cause 35% of the burden of all diseases in Europe. The present study aims to estimate the cost of defined brain disorders and adds all selected disorders to arrive at the total cost for Luxembourg. A model combining published economic and epidemiological data retrieved from the OECD (Organization for Economic Co-operation and Development) and Eurostat databases on brain disorders in Europe (EU member countries, Iceland, Norway and Switzerland) was used. We transformed and converted data for a defined period into the same currency (Euro 2004) and adjusted country specific economic data for purchasing power and relative size of economy and imputed data where no local data were available. There are an estimated 123000 people in Luxembourg currently living with a brain disorder. The total annual cost of brain disorders is estimated at Euro 500 million in 2004 or an average of Euro 1100 per inhabitant. Mental disorders constitute 62% of the total cost (excluding dementia), followed by neurological diseases (excluding dementia) 22%, neurosurgical diseases excluding herniated discs 2.2%. Direct medical expenditures (outpatient care, hospitalization, drugs) have a share of 32%, direct non-medical costs (social services, informal care, adaptation, transportation) 18% and indirect costs (sick leave, early retirement and premature death) 51%.

A clinical update on the role of carfilzomib in the treatment of relapsed or refractory multiple myeloma.

Even though the prognosis of patients with multiple myeloma is continuing to improve, all patients eventually develop relapsed refractory disease. Several novel therapeutics have been developed in the last few years including the second-generation proteasome inhibitor carfilzomib which has been approved for patients with relapsed and refractory multiple myeloma in the United States since 2012. Recently data from several phase III studies have become available showing the promising efficacy of carfilzomib in combination with lenalidomide, which led to the renewed approval of carfilzomib in combination with lenalidomide and dexamethasone for relapsed myeloma in 2015. Furthermore carfilzomib showed superiority over bortezomib on both efficacy and toxicity profiles, especially a profoundly lower incidence in polyneuropathy. Carfilzomib has been shown to partially overcome the negative effects of high-risk cytogenetics. Promising combinations of carfilzomib with histone deacetylase (HDAC) inhibitors, pomalidomide and several other novel therapeutics have been presented in early studies. The optimal dosing regimen and sequence of treatment regimens remain important questions for the future.

A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia.

Relapses after initial successful treatment in acute myeloid leukemia are thought to originate from the outgrowth of leukemic stem cells. Their flow cytometrically assessed frequency is of importance for relapse prediction and is therefore assumed to be implemented in future risk group profiling. Since current detection methods are complex, time- and bone marrow consuming (multiple-tubes approach), it would be advantageous to have a broadly applicable approach that enables to quantify leukemia stem cells both at diagnosis and follow-up. We compared 15 markers in 131 patients concerning their prevalence, usefulness and stability in CD34(+)CD38(-) leukemic stem cell detection in healthy controls, acute myeloid leukemia diagnosis and follow-up samples. Ultimately, we designed a single 8-color detection tube including common markers CD45, CD34 and CD38, and specific markers CD45RA, CD123, CD33, CD44 and a marker cocktail (CLL-1/TIM-3/CD7/CD11b/CD22/CD56) in one fluorescence channel. Validation analyses in 31 patients showed that the single tube approach was as good as the multiple-tube approach. Our approach requires the least possible amounts of bone marrow, and is suitable for multi-institutional studies. Moreover, it enables detection of leukemic stem cells both at time of diagnosis and follow-up, thereby including initially low-frequency populations emerging under therapy pressure.

Peripheral blood minimal residual disease may replace bone marrow minimal residual disease as an immunophenotypic biomarker for impending relapse in acute myeloid leukemia.

As relapses are common in acute myeloid leukemia (AML), early relapse prediction is of high importance. Although conventional minimal residual disease (MRD) measurement is carried out in bone marrow (BM), peripheral blood (PB) would be an advantageous alternative source. This study aims to investigate the specificity of leukemia-associated immunophenotypes used for MRD detection in blood samples. Consistency of PB MRD as compared with BM MRD was determined in flow cytometric data of 205 paired BM and PB samples of 114 AML patients. A significant correlation was found between PB and BM MRD (r=0.67, P<0.001), while median PB MRD percentage was factor 4-5 lower compared with BM MRD. Primitive blast (CD34+/CD117+/CD133+) frequency was significantly lower in PB (median factor 23.7), indicating that PB MRD detection is more specific than BM. Cumulative incidence of relapse 1 year after induction therapy was 29% for PB MRD-negative and 89% for PB MRD-positive patients (P<0.001). Three-year OS was 52% for MRD-negative and 15% for MRD-positive patients (P=0.034). Similar differences were found after consolidation therapy. As PB MRD appeared to be an independent predictor for response duration, the highly specific PB MRD assay may have a prominent role in future MRD assessment in AML.

EphB1 Suppression in Acute Myelogenous Leukemia: Regulating the DNA Damage Control System.

UNLABELLED: Loss of ephrin receptor (EphB1) expression may associate with aggressive cancer phenotypes; however, the mechanism of action remains unclear. To gain detailed insight into EphB1 function in acute myelogenous leukemia (AML), comprehensive analysis of EphB1 transcriptional regulation was conducted. In AML cells, EphB1 transcript was inversely correlated with EphB1 promoter methylation. The presence of EphB1 allowed EfnB1 ligand-mediated p53 DNA binding, leading to restoration of the DNA damage response (DDR) cascade by the activation of ATR, Chk1, p53, p21, p38, CDK1(tyr15), and Bax, and downregulation of HSP27 and Bcl2. Comparatively, reintroduction of EphB1 expression in EphB1-methylated AML cells enhanced the same cascade of ATR, Chk1, p21, and CDK1(tyr15), which consequently enforced programmed cell death. Interestingly, in pediatric AML samples, EphB1 peptide phosphorylation and mRNA expression were actively suppressed as compared with normal bone marrow, and a significant percentage of the primary AML specimens had EphB1 promoter hypermethylation. Finally, EphB1 repression associated with a poor overall survival in pediatric AML. Combined, the contribution of EphB1 to the DDR system reveals a tumor-suppressor function for EphB1 in pediatric AML. IMPLICATIONS: The tumor-suppressor function of EphB1 is clinically relevant across many malignancies, suggesting that EphB1 is an important regulator of common cancer cell transforming pathways.

Lack of effect of daily N-acetylcysteine supplementation on mutagen sensitivity.

The European Organization for Research and Treatment of Cancer multicenter Euroscan trial was set up to prevent the occurrence of second primary tumors in the upper aerodigestive and respiratory tract in patients cured for early stage head and neck squamous cell carcinoma. One randomized group of patients receive daily N-acetylcysteine, an antioxidant that may be protective especially in the early steps of carcinogenesis. Mutagen sensitivity, measured as sensitivity to bleomycin in peripheral blood lymphocytes, has been found to be increased in head and neck squamous cell carcinoma and is hypothesized to reflect cancer susceptibility. The aim of this study was to investigate whether mutagen sensitivity is influenced by oral N-acetylcysteine supplementation and can therefore be used as intermediate end point in chemoprevention. Patients (n = 19) who had various periods of N-acetylcysteine supplementation (600 mg daily for 3-9 months) were analyzed. In addition, a patient group (n = 14) that did not receive N-acetylcysteine supplementation was analyzed for comparison. Our results show no evidence that administration of N-acetylcysteine did influence the mutagen sensitivity level. The only explanatory variable in the analysis of the difference between two samples of one person was the b/c value of the first measurement. Moreover, the variability in these repeated measurements (coefficient of variation of 14%) indicates that additional studies should be performed to minimize this variability and to optimize the testing of mutagen sensitivity to accurately identify individual patients at high risk for the development of multiple primary tumors.

Mutagen sensitivity as a biomarker for second primary tumors after head and neck squamous cell carcinoma.

The occurrence of second primary tumors after curative treatment of early stage head and neck squamous cell carcinoma negatively influences the overall survival. Our aim was to prospectively evaluate whether mutagen sensitivity (mean number of chromatid breaks per cell in cultured lymphocytes exposed to bleomycin) could be used as a biomarker to predict which patients will develop second malignancies in the respiratory or upper digestive tract. Patients treated for head and neck squamous cell carcinoma (n = 218) were followed for approximately 6 years. Nineteen patients developed a second primary tumor, and each of these patients was matched on age, gender, cumulative smoking, tumor site, and tumor stage to two patients who did not develop any second malignancy. No difference between the groups was found with respect to mutagen sensitivity. Smoking at the time of the index tumor had a significant influence on the occurrence of second primary tumors (log-rank, P = 0.019). There was a significantly (P = 0.005) higher mean breaks-per-cell value in those patients who had developed their second primary tumor > or = 3 years after the first tumor (0.97 +/- 0.24; n = 10) compared with early second primary tumor patients (0.69 +/- 0.09; n = 9). Conditional on a more than 3-year second primary tumor-free survival (n = 38), there is a significantly (log-rank, P = 0.036) higher probability of a second primary tumor for mutagen-sensitive patients [relative risk, 7.8 (95% confidence interval, 0.99-61.74; P = 0.05)]. Mutagen sensitivity is a potential biomarker for the occurrence of 'late' second malignancies (> 3 years between tumors), and additional studies on the inclusion of this biomarker in chemoprevention trials is commendable because it would greatly improve their efficiency.