CAR virus receptor mediates erythroid differentiation and migration and is downregulated in MDS.

Myelodysplastic syndromes (MDS) are myeloid neoplasms presenting with dysplasia in the bone marrow (BM) and peripheral cytopenia. In most patients anemia develops. We screened for genes that are expressed abnormally in erythroid progenitor cells (EP) and contribute to the pathogenesis of MDS. We found that the Coxsackie-Adenovirus receptor (CAR = CXADR) is markedly downregulated in CD45low/CD105+ EP in MDS patients compared to control EP. Correspondingly, the erythroblast cell lines HEL, K562, and KU812 stained negative for CAR. Lentiviral transduction of the full-length CXADR gene into these cells resulted in an increased expression of early erythroid antigens, including CD36, CD71, and glycophorin A. In addition, CXADR-transduction resulted in an increased migration against a serum protein gradient, whereas truncated CXADR variants did not induce expression of erythroid antigens or migration. Furthermore, conditional knock-out of Cxadr in C57BL/6 mice resulted in anemia and erythroid dysplasia. Finally, decreased CAR expression on EP was found to correlate with high-risk MDS and decreased survival. Together, CAR is a functionally relevant marker that is down-regulated on EP in MDS and is of prognostic significance. Decreased CAR expression may contribute to the maturation defect and altered migration of EP and thus their pathologic accumulation in the BM in MDS.

Impact of age on the cumulative risk of transformation in patients with chronic myelomonocytic leukaemia.

In older patients with chronic myelomonocytic leukaemia (CMML) and limited life expectancy due to age and or comorbidities, it is particularly important to consider the risk of transformation for individualised treatment decisions. There is limited information on potential differences between younger and older CMML patients regarding the cumulative risk of transformation as well as haematological, molecular and biologic characteristics. We analysed data from the Austrian Biodatabase for CMML (ABCMML) to compare these parameters in 518 CMML patients. Categorisation of patients into 3 age-related groups: <60 years, 60-79 years and ≥80 years, showed a significantly lower risk of transformation at higher age by competing risk analysis, with a 4-year risk of 39%, 23% and 13%, respectively (P < .0001). The lower probability of transformation was associated with a lower percentage of blast cells in the peripheral blood (PB) of older patients. Furthermore, we provide a simple score based on age, PB blasts and platelet counts that allowed us to define subgroups of CMML patients with a different cumulative transformation risk, including a low-risk group with a transformation risk of only 5%. Our findings may facilitate reasonable treatment decisions in elderly patients with CMML.

Correlation of RAS-Pathway Mutations and Spontaneous Myeloid Colony Growth with Progression and Transformation in Chronic Myelomonocytic Leukemia-A Retrospective Analysis in 337 Patients.

Although the RAS-pathway has been implicated as an important driver in the pathogenesis of chronic myelomonocytic leukemia (CMML) a comprehensive study including molecular and functional analyses in patients with progression and transformation has not been performed. A close correlation between RASopathy gene mutations and spontaneous in vitro myeloid colony (CFU-GM) growth in CMML has been described. Molecular and/or functional analyses were performed in three cohorts of 337 CMML patients: in patients without (A, n = 236) and with (B, n = 61) progression/transformation during follow-up, and in patients already transformed at the time of sampling (C, n = 40 + 26 who were before in B). The frequencies of RAS-pathway mutations (variant allele frequency ≥ 20%) in cohorts A, B, and C were 30%, 47%, and 71% (p < 0.0001), and of high colony growth (≥20/105 peripheral blood mononuclear cells) 31%, 44%, and 80% (p < 0.0001), respectively. Increases in allele burden of RAS-pathway mutations and in numbers of spontaneously formed CFU-GM before and after transformation could be shown in individual patients. Finally, the presence of mutations in RASopathy genes as well as the presence of high colony growth prior to transformation was significantly associated with an increased risk of acute myeloid leukemia (AML) development. Together, RAS-pathway mutations in CMML correlate with an augmented autonomous expansion of neoplastic precursor cells and indicate an increased risk of AML development which may be relevant for targeted treatment strategies.

The Austrian biodatabase for chronic myelomonocytic leukemia (ABCMML) : A representative and useful real-life data source for further biomedical research.

In the Austrian biodatabase for chronic myelomonocytic leukemia (ABCMML) clinicolaboratory real-life data have been captured from 606 CMML patients from 14 different hospitals over the last 30 years. It is the only large biodatabase worldwide in which functional methods such as semisolid in vitro cultures complement modern molecular methods such as next generation sequencing. This provides the possibility to comprehensively study the biology of CMML. The aim of this study was to compare patient characteristics with published CMML cohorts and to validate established prognostic parameters in order to examine if this real-life database can serve as a representative and useful data source for further research. After exclusion of patients in transformation characteristics of 531 patients were compared with published CMML cohorts. Median values for age, leukocytes, hemoglobin, platelets, lactate dehydrogenase (LDH) and circulating blasts were within the ranges of reported CMML series. Established prognostic parameters including leukocytes, hemoglobin, blasts and adverse cytogenetics were able to discriminate patients with different outcome. Myeloproliferative (MP) as compared to myelodysplastic (MD)-CMML patients had higher values for circulating blasts, LDH, RAS-pathway mutations and for spontaneous myelomonocytic colony growth in vitro as well as more often splenomegaly. This study demonstrates that the patient cohort of the ABCMML shares clinicolaboratory characteristics with reported CMML cohorts from other countries and confirms phenotypic and genotypic differences between MP-CMML and MD-CMML. Therefore, results obtained from molecular and biological analyses using material from the national cohort will also be applicable to other CMML series and thus may have a more general significance.

Karyotype plus NPM1 mutation status defines a group of elderly patients with AML (≥60 years) who benefit from intensive post-induction consolidation therapy.

Although it is generally appreciated that a subset of elderly patients with acute myeloid leukemia (AML) may benefit from intensive consolidation, little is known about variables predicting such benefit. We analyzed 192 consecutive patients with de novo AML aged ≥60 years who were treated with intensive chemotherapy. About 115 patients (60%) achieved complete hematologic remission (CR). Among several parameters, the karyotype was the only independent variable predicting CR (P < 0.05). About 92% (105/115) of the CR-patients received up to four consolidation cycles of intermediate dose ARA-C. Median continuous CR (CCR) and disease-free survival (DFS) were 1.3 and 1.1 years, respectively. CCR, DFS, and survival at 5 years were 23%, 18%, and 15%, respectively. Only karyotype and mutated NPM1 (NPM1mut) were independent predictors of survival. NPM1mut showed a particular prognostic impact in patients with normal (CN) or non-monosomal (Mkneg) karyotype by Haemato-Oncology Foundation for Adults in the Netherlands (HOVON)-criteria, or intermediate karyotype by Southwest Oncology Group (SWOG)-criteria. The median CCR was 0.94, 1.6, 0.9, and 0.5 years for core-binding-factor, CN/Mkneg-NPM1mut, CN/Mkneg-NPM1-wild-type AML, and AML with monosomal karyotype, respectively, and the 5-year survival was 25%, 39%, 2%, and 0%, respectively (P < 0.05). Similar results (0.9, 1.5, 0.9, and 0.5 years) were obtained using modified SWOG criteria and NPM1 mutation status (P < 0.05). In summary, elderly patients with CN/Mkneg-NPM1mut or CBF AML can achieve long term CCR when treated with intensive induction and consolidation therapy whereas most elderly patients with CN/Mkneg-NPM1wt or Mkpos AML may not benefit from intensive chemotherapy. For these patients either hematopoietic-stem-cell-transplantation or alternative treatments have to be considered. Am. J. Hematol. 91:1239-1245, 2016. © 2016 Wiley Periodicals, Inc.

BRCA-1 methylation and TP53 mutation in triple-negative breast cancer patients without pathological complete response to taxane-based neoadjuvant chemotherapy.

INTRODUCTION: Triple-negative breast cancer (TNBC) patients without pathological complete response (pCR) to neoadjuvant chemotherapy have an unfavourable prognosis. TNBC harbouring BRCA-1 germline mutations may be less responsive to taxanes, while sensitivity to DNA-damaging agents is retained. A similar effect was seen in tumours with epigenetic BRCA-1 silencing. Patients without pCR to neoadjuvant chemotherapy consisting of epirubicin plus docetaxel routinely received post-operative CMF at our centre. Here, we investigated the effect of adjuvant CMF in patients with or without BRCA-1 methylation or TP53 mutation. METHODS: DNA was extracted from formalin-fixed paraffin-embedded tissue. For determining BRCA-1 methylation status, quantitative methylation-specific PCR was performed. For the investigation of TP53 mutation status, DNA was PCR amplified and sequenced by Sanger sequencing. RESULTS: Twenty-four patients were included; BRCA-1 methylation was present in 41.7 %, while TP53 mutations were observed in 66.7 %. At a median follow-up of 27.5 months, 20 % of patients with BRCA-1 methylation had a disease-free survival (DFS) event, as compared to 64.3 % in the non-methylated group (p = 0.0472). Median DFS in the non-methylated group was 16 months and was not reached in the methylated group (n.s.). No association TP53 mutation status with clinical outcome was observed. CONCLUSIONS: Adjuvant CMF is of limited activity in TNBC refractory to taxane-based neoadjuvant chemotherapy. In this population, BRCA-1 methylation was associated with a significant decrease in DFS events suggesting a better prognosis and potentially retained activity of DNA-damaging agents.

Pathways involved in Drosophila and human cancer development: the Notch, Hedgehog, Wingless, Runt, and Trithorax pathway.

Animal models are established tools to study basic questions of biology in a systematic way. They have greatly facilitated our understanding of the mechanisms by which nature forms and maintains organisms. Much of the knowledge on molecular changes underlying the development of organisms originates from research in the fruit fly model Drosophila melanogaster. Vertebrate models including the mouse and zebrafish model, but also other animal models coming from different corners of the animal kingdom have shown that much of the basic machinery of development is essentially identical, not just in all vertebrates but in all major phyla of invertebrates too. Moreover, key elements of this machinery have been demonstrated to be involved in recurrent molecular abnormalities detected in tumor-tissue from patients, indicating their implication in the genesis of human cancer. Thus, research in this field has become a common topic for both biologists and hemato-oncologists. In this review, we summarize current knowledge on some of these key elements and molecular pathways such as Notch, Hedgehog, Wingless, Runt, and Trithorax that have been originally described and studied in animal models and which seem to play a major role in the pathophysiology and targeted management of human cancer.

The clinical impact of DNA methylation frequencies of JAK2 negative regulators in patients with essential thrombocythemia.

Suppressors of cytokine signalling (SOCS) and protein tyrosine phosphatase (PTPN) proteins are negative regulators of Janus Kinase 2 (JAK2). They are thought to be involved in the molecular pathogenesis of essential thrombocythaemia (ET) particularly in patients with unmutated JAK2. In this study we compared DNA methylation of SOCS1, SOCS3 and PTPN6 in peripheral blood cells between 39 ET patients (24 JAK2 V617F mutated) and 22 healthy controls by methylation specific PCR (MSP) and analysed the clinical outcome of patients with respect to DNA methylation. In SOCS1, ET patients showed significantly less methylation (P<0.05) than healthy controls, and in SOCS3 and PTPN6 such a tendency was shown. However, there were no significant differences in the methylation frequencies between JAK2 wildtype and mutated ET patients. In addition, no correlation was detected between methylation of SOCS and PTPN and any clinical outcome parameters. Taken together, regarding the genomic regions investigated our data indicate a minor role of methylation of JAK2 negative regulators for the clinical course of ET.

A novel 3' splice-site mutation and a novel gross deletion in leukocyte adhesion deficiency (LAD)-1.

A patient was diagnosed with leukocyte adhesion deficiency-1. She was born in 1996 and her parents are not known to be related. Her leukocytes expressed less than 2% of the CD18 antigens relative to normal individuals. Molecular analysis revealed that she is a compound heterozygote. She inherited a 27,703bp deletion from her father (g.43201_PTTG1IP:10890del27703), spanning from intron 11 of the gene for the ß2 integrin (ITGB2, CD18, NG_007270.2) to intron 2 of the gene for the Pituitary Tumor-Transforming Gene 1 Interacting Protein (PTTG1IP, NC_000021.8). The maternal allele has a g.23457C>A mutation at position -10 in intron 2 of the ITGB2 gene, resulting in the activation of a cryptic 3' splice site in intron 2 to include 43 intronic nucleotides (r.[59-43_59-1ins;59-10C>A]).

Immunodetection array.

A novel procedure for DNA methylation analysis is described that characterizes the extent of DNA methylation in CpG islands. The basic concept relies on direct immunodetection of 5' methylcytosines (5' mCs) without the need for bisulfite treatment utilizing a microarray format. This system is designed for the application of immunofluorescence using a monoclonal antibody that specifically recognizes 5' mC in single-stranded DNA hybridized to oligonucleotide microarrays. An ultrasensitive fluorescence scanner and 170-mum thin aldehyde-functionalized glass slides are used to optimize the signal-to-noise ratio and to minimize autofluorescence. These methodological improvements allow for the direct detection of 5' mC in genomic DNA hybridized to microarrays without prior PCR amplification with high analytical sensitivity.

In vitro detection of methylated DNA via recombinant protein MBD2b.

Members of the methyl binding domain (MBD) protein family are known for binding to methylated DNA by recognizing methylated cytosines. Their original function is to regulate protein biosynthesis by recruitment of transcriptional repression complexes to silence gene expression. The aim of the presented work was to detect methylated DNA spotted onto nitrocellulose membranes with recombinant proteins MBD2b, MBD2b-GFP and directly labeled protein MBD2b. Proteins were affinity purified and tested for functionality before application. We were able to show that these functional recombinant proteins bind to unilaterally and symmetrically methylated oligonucleotides and genomic DNA in vitro and thus can be used in various detection assays.

A single nucleotide polymorphism at chromosome 2q21.3 (LCT -13910C>T) associates with clinical outcome after allogeneic hematopoietic stem cell transplantation.

A single nucleotide polymorphism (SNP) responsible for lactase persistence (LCT -13910C>T) changes intestinal microflora. Considering the influence of bacterial microflora on various immune effects, we tested DNA from 111 recipients/donors and analyzed whether this SNP interferes with survival and the incidence of acute graft-versus-host disease (aGVHD) after allogeneic hematopoetic stem cell transplantations (HSCT). Median overall survival (OS) was significantly longer when donors had a CC genotype (not reached after 133 vs 11.1 months, P = .004). Multivariate analysis identified a donor T allele (hazard ratio 2.63, 95% confidence interval 1.29-5.33, P = .008) as independent risk factor for death. Surprisingly, recipient genotypes did not influence outcome and there were no differences regarding aGVHD. Transplantation-related mortality (TRM), relapse and pneumonia were significantly less frequent in patients with CC donors. These findings add to the growing list of non-HLA polymorphisms with impact on outcome after allogeneic HSCT.

Ultra-sensitive immunodetection of 5'methyl cytosine for DNA methylation analysis on oligonucleotide microarrays.

For the determination of methylation levels in genomic regulatory DNA sequences a high-sensitive assay for detecting 5'methyl-cytosines (5'mC) in non-bisulfite-treated DNA has been established. The system is designed for the application of immunofluorescence using a monoclonal antibody that specifically recognizes 5'mC in single-stranded DNA hybridized to oligonucleotide microarrays. For assay readout an ultra-sensitive fluorescence scanner with submicrometer resolution was used. To minimize autofluorescence 150-microm thin glass slides with an aldehyde-functionalized surface were developed. These methodological improvements allowed the detection of 5'mC in synthetic oligonucleotides hybridized to microarrays with atto molar analytical sensitivity. Using enzymatic fragmented genomic DNA from myeloid leukemia tumor cell lines differences in the methylation status of gene regulatory sequences for E-cadherin, p15/CDKN2b and p16/CDKN2a were demonstrated. Thus, this novel technique can potentially be used for DNA methylation analysis in various scientific fields.

Skin transplantation to monitor clinical donor-related tolerance in mixed hematopoietic chimerism.

Mixed hematopoietic chimerism usually carries with it the tolerance to any other tissue from the same donor. Consequently, the establishment of a sustained chimerism may allow long-term acceptance of transplanted organs without immunosuppression. We report a girl with refractory severe aplastic anemia who developed low recipient level hematopoietic chimerism following transplantation of maternal highly purified CD34+ cells without prophylactic immunosuppression. Renal thrombotic microangiopathy led to chronic renal failure and she received skin allografts from her mother in view of a future kidney donation. The maternal skin grafts were accepted without immunosuppression and the hematopoietic chimerism remained stable. Skin transplantation may be a helpful and easily applicable tool to monitor donor-related tolerance in hematopoietic chimerism clinically. It should contribute to minimize the risks of subsequent solid organ transplantation from the same donor without immunosuppression.

Tumor cell detection in peripheral blood and bone marrow.

PURPOSE OF REVIEW: Whether the occurrence of tumor cells in peripheral blood or bone marrow from patients with solid tumors is predictive for disease recurrence or of any other prognostic relevance remains unknown. This article reviews recently published results focusing on the various methods used, their correlations with clinical or biological parameters and their potential prognostic value. RECENT FINDINGS: An increasing number of marker genes and different techniques, alone or in combinations, have been used for the detection of tumor cells in peripheral blood and bone marrow. Various results obtained are hardly comparable, most often due to the different methods in use. The frequency of circulating tumor cells in peripheral blood varied within a broad range and their clinical relevance appeared to be contradictory, at least in part. Disseminated tumor cells in bone marrow reached an independent prognostic value in breast cancer patients, but several investigations led to inconsistent correlations with clinical or prognostic criteria. SUMMARY: Still many questions remain unanswered; hence, the detection of tumor cells in peripheral blood or bone marrow cannot yet be taken into account for therapeutic decisions.