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133 Specificity of features of the bone marrow CD34+ cell fraction for the diagnosis of MDS
Posters / Leukemia Research 35 (2011) S27–S142
131 Frequency of karyotype and FISH results in myelodysplastic syndromes: A single institutional experience S. Capetillo-Contreras1,2 , P.N. Rao1 , F. Quintero-Rivera1 . 1 Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, USA; 2 Departments of Human Genetics and Pediatrics, Universidad de Nuevo Leon, Monterrey, Mexico Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral cytopenias, and substantial risk for progression to acute myeloid leukemia. The chromosomal aberrations for MDS are well-known and are associated with good (−5/5q−, −Y, +8), poor (>3aberrations, chromosome 7), and intermediate prognosis (other). However, the frequency of aberrations detected by interphase FISH, karyotype, or both, and the frequency in each of the MDS subtypes as listed in the WHO 2008 classification, is not well documented. We identified 189 patients with a de novo MDS (by bone marrow morphology) between 2008–2010; 101 males and 88 females (9–93 years: median age 69). We analyzed the frequency of: (a) normal karyotype and normal FISH (59%), (b) normal karyotype and abnormal FISH (9%), (c) abnormal karyotype and abnormal FISH (16.4%), (d) abnormal karyotype and normal FISH (6.4%), and (e) abnormal karyotype only (10%) performed at a single institution. The following MDS subtypes were identified: Refractory cytopenias with unilineage dysplasia (RCUD) – 2.6%, refractory anemia with ring sideroblasts (RARS) – 0.5%, refractory anemia with multilineage dysplasia (RCMD) – 5.3%, Refractory anemia with excess blast-1 (RAEB-1) – 2.6%, RA with excess blast-2 (RAEB-2) – 6.6%, MDSunclassified (MDS-U) – 80.4%, MDS with isolated deletion 5q – 1%. Overall the most common aberration was 5q31 deletion detected by FISH in 11%, MDS-U (47%), RAEB2 −2 (37%), MDS-5q (10%), RCMD (5%), and trisomy 8 in 11% [MDS-U (65%), RCMD (15%)], followed by deletion 20q12 in 9.8% [(MDS-U (41%), RCMD (18%), RAEB-1/2 (12% each)], deletion 7q31 in 8.1% [MDS-U (67%)], and monosomy 7 in 5.8% [MDS-U (70%), RCUD (10%), RAEB-1 (10%)]. The frequency of +8, −7 or del(7q), −5 or del(5q) in MDS is similar to that in the WHO 2008 (~10%). However, del 20q is slightly higher in our cohort. In addition, the frequency of abnormal aberrations in the different subtypes of MDS, excluding MDS-U, is 15–20% higher than that of the WHO. Our findings expand the cytogenetic spectrum of MDS, reaffirm the importance of karyotype and highlight the utility of combining FISH and standard chromosome analyses. Karyotype alone has a higher yield compared to FISH alone, while the detection of aberrations increased when combined together. The frequency of aberration type cannot help to sub classify patients, as each type is more frequent in MDS-U. More than half of our patients have normal karyotype. This suggests that other submicroscopic chromosomal aberrations that go undetected by current clinical testing and that can stratify patients into different risk categories are likely frequent. 132 Chromosomal abnormalities in primary myelodysplastic syndrome performed by conventional cytogenetics – tertiary care center experience A. Rashid. Department of Pathology and Microbiology, Aga Khan University and Hospital, Karachi, Pakistan Introduction: Myelodysplastic syndromes (MDS) consist of a heterogeneous group of haematological disorders characterized by peripheral blood cytopenias in the presence of hypercellular bone marrow with features of ineffective haematopoiesis. Chromosomal anomalies are detected in approximately 50% of patients with de novo MDS and in up to 80% of patients with MDS secondary to chemotherapy or other toxic agents. Cytogenetic abnormalities are major determinants in the pathogenesis, diagnosis, prognosis, and the basis for selection of drugs in individual patients with MDS. Objective: Aim of this study was to determine the frequency of cytogenetic abnormalities in patients diagnosed as primary
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myelodysplastic syndrome using conventional karyotyping in Pakistani population. Method: Patients who fulfilled the WHO criteria for MDS were included. Cytogenetic analysis was conducted at the time of diagnosis. Patients who had previously received chemo/radiotherapy, and those with MDS secondary to a previous malignancy were excluded from analysis. Chromosome identification and karyotype description was done according to the International System for Chromosome Nomenclature (ISCN, 1995). Results: Total of n = 112 patients were diagnosed as primary myelodysplastic syndrome. N = 50 patients had their karyotype done at the time of diagnosis. Of the n = 50 patients, n = 33 were males (66%) and n = 17 were females (34%). The median age was 58 years (range 1–75 years). Only one patient was under 15 years of age. Out of n = 50, n = 5 (10%) were classified as RA, n = 2 (4%) as RARS, n = 23 (46%) as RCMD, n = 1 (2%) as RCMD-RS, n = 9 (18%) as RAEB-I and n = 10 (20%) as RAEB-II. Among the 50 patients, n = 29 (58%) showed normal karyotype and n = 21 (42%) showed clonal karyotypic abnormalities at diagnosis, of which, n = 7 (14%) had single, n = 6 (12%) had double and n = 9 (18%) has complex cytogenetics. The common abnormalities found were, triosomy 8 in n = 3 cases (6%), complex triosomy 8 in n = 1 case (2%), −7/del(7q) (1/1cases) 4%, −Y in n = 2 cases (4%), complex 5q in n = 2 cases(4%), del 11q in n = 1 case (2%), inversion 9 in n = 1case (2%), trisomy 19 in n = 1 case (2%) and other abnormalities in n = 8 cases (16%). Conclusion: In contrast to del 5q which is the most reported cytogenetic abnormality in India, USA and European countries, triosomy 8 is found to be most common in our study population followed by the complex cytogenetics. RA has the highest number of abnormalities followed by RAEBII, ie 60% and 55% respectively, in contrast to RAEBII. This is an ongoing study. Large prospective studies are needed to report most common abnormality prevalent according to classification of Myelodysplastic syndrome. 133 Specificity of features of the bone marrow CD34+ cell fraction for the diagnosis of MDS S.C. Reis-Alves1 , P.M. Campos2 , F.G. Pereira-Cunha2 , F. Traina2 , I. Lorand-Metze2 . 1 State Healthcare Institution, Tula Regional Clinical Hospital, University of Campinas, 2 Internal Medicine, State University of Campinas, Campinas, Brazil Introduction: immunophenotyping has been used as a complimentary technique for the confirmation of the diagnosis of MDS, especially in cases with a normal karyotype. Methods: using a previously described four-color-combination panel designed to detect abnormalities of myelomonocytic precursors and CD34+ cells, we standardized the normal values for CD34+ cell fractions according to the guidelines of ELN, examining their co-expression of CD19/CD10, CD13, CD117, CD15, CD11b, CD7 and CD56 (30 normal BM donors of allogeneic BMT). These values were compared with the findings obtained for confirmed cases of MDS (28) and with cases of PB cytopenias due to non-clonal disorders (16) that performed the analysis in their diagnostic work-up. Results: Donors of normal BM: 33 years (15–63), patients with nonclonal disorder: 63 years (22–86); cases of MDS: 70 years (28–84). WHO types: 3 RA; 12 RCDM; 3 RCDM-SA; 5 RAEB-I; 5 RAEB-II. CD34+ cells/total non-erythroid cells were significantly increased in MDS as compared to reactive and normal cases (2.1%, 0.44%, and 0.80% respectively). Myeloblasts (CD34+/CD13+CD117+) were increased in MDS with BM blasts < 5% compared to reactive cases, although there was some overlap. The number of B-cell precursors was correlated with age (MDS = −0.38, p = 0.02; reactive = −0.55, p = 0.01), but their number was higher in reactive cases than in MDS (p = 0.02). The analysis of the co-expression of CD11b and CD15 in CD34+ cells was not able to discriminate the 3 groups. But, expression of CD56 and/or that of CD7 in CD34+ cells was significantly increased in MDS compared to reactive and normal BM.
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Posters / Leukemia Research 35 (2011) S27–S142
Conclusions: the analysis of BM subsets of CD34+ cells is useful in the differential diagnosis of MDS with BM blasts < 5% and non-clonal disorders. The most useful features are the number of B-cell precursors and aberrant co-expressions, but not asynchronous maturation. Support: MDS Foundation, FAPESP and CNPq 134 Hidden genomic abnormalities detected in MDS patients with a normal karyotype have prognostic relevance B. Royer-Pokora1 , A. Thiel1 , M. Beier1 , D. Ingenhag1 , K. Servan1 , M. Hein1 , U. Germing2 , B. Hildebrandt1 . 1 Institute of Human Genetics, 2 Department of Haematology, Oncology and Clinical Immunology, Heinrich Heine University Duesseldorf, Duesseldorf, Germany Approximately 50% of MDS patients have a normal karyotype. Our aim was to study whether recurrent or individual abnormalities hidden can be detected in MDS with a normal Karyotype and to determine their prognostic significance. To address this question we have analyzed 107 MDS patients of various FAB/WHO subgroups and a normal karyotype using aCGH. For this study we have used Agilent microarrays consisting of 44K, 105K or 244K 60-mer oligonucleotide probes spanning the human genome with an average spatial resolution of approximately 43, 22 and 9 kb, respectively. Array CGH identified hidden recurrent chromosomal aberrations in ≥2 cases: del5q, del7q, del21q and del4q. In 34 cases we have identified individual imbalances. Of the 107 cases 42 (39%) had hidden alterations. Larger alterations were verified by FISH and smaller alterations with Q-PCR. So far 40 of the identified aberrations were verified with other methods. Zoom in custom arrays were designed to characterize the breakpoints. In the imbalances several interesting genes were localized, involved in epigenetic and chromatin modification, such as DNMT3a, ASXL2, TET2, HDAC3, BRD8 or several genes with a role in DNA repair, e.g. DNAJC 16, 18 and 27, MLH3, RAD50, as well as members of the RAS signalling pathway, for example RAB10, 38, 39, RABL5, RAPGEF6 and RIN3. Gene functional classification using DAVID revealed an enrichment of metallopeptidases, Apoptosis genes and genes regulating haematopoiesis. Comparing the survival of patients with and without additional hidden alterations showed that those with additional genomic imbalances had an inferior survival (p = 0.002). In conclusion, hidden recurrent and individual aberrations in cytogenetically normal MDS can be identified with aCGH and these have prognostic relevance. 135 Lower bone marrow myeloid and plasmacytoid dendritic cell counts in high-risk than in low-risk MDS patients and controls 1 2 L. Saft1 , E. Bjorklund ¨ , E. Hellstrom-Lindberg ¨ , A. Porwit1 . 1 Department of Pathology, 2 Department of Medicine, Center for Experimental Hematology, Karolinska University Hospital and Institute, Stockholm, Sweden
Background: Dendritic cells (DC) are professional antigenpresenting cells and play a pivotal role in coordinating functions of the immune system by the initiation and regulation of T-cell mediated responses. Laboratory and clinical studies suggest that bone marrow (BM) failure in MDS may be, at least in part, immune-mediated. The observation that MDS patients can respond to immunosuppressive therapy sparked a renewed interest in T-cell mediated mechanisms. DCs originate from BM hematopoietic stem cells and clonal involvement of precursor DC has been demonstrated in both MDS and AML. Aims: To determine whether levels of myeloid (mDC) and plasmacytoid (pDC) dendritic cells in BM samples from MDS patients differ from those in reactive BM. Patients and Methods: BM samples from eighty MDS patients (IPSS: low-risk 25, INT-1 39, INT-2 11, high-risk 5, all WHO subtypes included) diagnosed at the Karolinska University Hospital between
January 2006 and November 2010 were included. In 67 patients (84%) the BM samples were taken before treatment and in 20/67 patients at least one additional follow-up biopsy was available prior to treatment start. BM samples from 13/80 (16%) patients were analyzed after therapy start. DC levels (expressed as percentage of BM cells) were measured by 4-color flow cytometry (FCM) using Lineage cocktail 1(lin)− FITC/CD123-PE/HLA-DR-PerCP/CD11cAPC antibody combination, FACS-Calibur flow cytometer and Paint-a Gate Pro software (Becton&Dickinson). pDC immunophenotype was characterized as lin−/HLA−DR+/CD123+/CD11c− and mDC as lin−/HLA−DR+/CD11c+/CD123−. In addition, BM samples from 36 hospital controls were investigated. Results: Average DC levels were significantly lower in MDS (mDC 0.039%; pDC 0.118%) as compared to the control group (mDC 0.089%; pDC 0.178%; p < 0.001). Lower DC levels were found in treated MDS patients than in untreated patients (p < 0.01). High-risk MDS and patients with higher BM blast percentages (>5%) had significantly lower DC levels than low-risk MDS patients. Progression to AML was seen in 15/80 (18.7%) patients (one del(5q), 6 RCMD, 5 RAEB-1, 3 RAEB-2); no significant difference in DC levels at diagnosis were seen in patients who developed AML in the follow-up period but in sequentially analyzed patients we could see lower DC levels at progression. Conclusions: DC levels are significantly lower in high-risk MDS and inversely correlated with the percentage of BM blasts. Our findings may indicate involvement of the DC compartment in the MDS disease process. FCM-determined DC levels may assist in the diagnostic work-up and risk-assessment of MDS patients. 136 Using flow cytometry for differential diagnosis of thrombocytopenia D. Subira, D. de Miguel, I. San Roman, ´ M. D´ıaz Morfa, N. Golbano, D. Morales, J. Arbeteta, S. Herrero, F. Fuertes, B. Pinedo. Hematology, Hospital Universitario de Guadalajara, Guadalajara, Spain Differential diagnosis between immune thrombocytopenia (ITP) and myelodysplastic syndrome (MDS) may be difficult, especially in old people. Altered immunophenotypes in maturing cells have been described in bone marrow samples from patients with MDS. However, flow cytometry immunophenotyping (FCI) detractors criticize lack of standard procedures of analyses and subjective identification of deviation from normal patterns of differentiation. Aim: To determine the utility of semi-quantitative FCI for identifying MDS within a group of patients with thrombocytopenia. Patients: From May 2010 to January 2011, 20 patients (9M/11F, median age 72, range 16–89) were studied for differential diagnosis of thrombocytopenia. Median platelet count was 45×109 (range 3.5–118); median hemoglobin was 12 g/dl, and leukocyte 6.4×109 /l. The following diagnoses were established: ITP (n = 6), refractory ITP (n = 1); MDS (n = 6); non-MDS thrombocytopenia (n = 7). Methods: FCI using 4-colour combinations. Data studied in bone marrow samples were: myeloid granularity, abnormalities in the phenotypic pattern of maturation of the myeloid (CD16/ CD13/HLADR/CD11b) and monocytic (CD36/CD14/HLADR/CD64) compartment, distribution of CD10 on mature granulocytes, quantification of CD10+ B-cell precursors and myeloid CD34+ cells. Deviations from normal patterns were calculated using reference images with the Infinicyt software program for FCI analysis. The following deviations were considered as abnormalities: >30% in myeloid granularity, >19% in myeloid maturation, >11% in monocytic maturation, < 70% of CD10 mean fluorescence intensity on granulocytes, <1% of CD10+ lymphocytes within the B-cell compartment, and >2% CD34+ myeloid cells. Results: Median number of FCI abnormalities was significantly higher in patients with MDS (n = 3.5) as compared to patients with ITP (n = 0.4) and non-MDS thrombocytopenia (n = 0.6). These
131 Frequency of karyotype and FISH results in myelodysplastic syndromes: A single institutional experience S. Capetillo-Contreras1,2 , P.N. Rao1 , F. Quintero-Rivera1 . 1 Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, USA; 2 Departments of Human Genetics and Pediatrics, Universidad de Nuevo Leon, Monterrey, Mexico Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral cytopenias, and substantial risk for progression to acute myeloid leukemia. The chromosomal aberrations for MDS are well-known and are associated with good (−5/5q−, −Y, +8), poor (>3aberrations, chromosome 7), and intermediate prognosis (other). However, the frequency of aberrations detected by interphase FISH, karyotype, or both, and the frequency in each of the MDS subtypes as listed in the WHO 2008 classification, is not well documented. We identified 189 patients with a de novo MDS (by bone marrow morphology) between 2008–2010; 101 males and 88 females (9–93 years: median age 69). We analyzed the frequency of: (a) normal karyotype and normal FISH (59%), (b) normal karyotype and abnormal FISH (9%), (c) abnormal karyotype and abnormal FISH (16.4%), (d) abnormal karyotype and normal FISH (6.4%), and (e) abnormal karyotype only (10%) performed at a single institution. The following MDS subtypes were identified: Refractory cytopenias with unilineage dysplasia (RCUD) – 2.6%, refractory anemia with ring sideroblasts (RARS) – 0.5%, refractory anemia with multilineage dysplasia (RCMD) – 5.3%, Refractory anemia with excess blast-1 (RAEB-1) – 2.6%, RA with excess blast-2 (RAEB-2) – 6.6%, MDSunclassified (MDS-U) – 80.4%, MDS with isolated deletion 5q – 1%. Overall the most common aberration was 5q31 deletion detected by FISH in 11%, MDS-U (47%), RAEB2 −2 (37%), MDS-5q (10%), RCMD (5%), and trisomy 8 in 11% [MDS-U (65%), RCMD (15%)], followed by deletion 20q12 in 9.8% [(MDS-U (41%), RCMD (18%), RAEB-1/2 (12% each)], deletion 7q31 in 8.1% [MDS-U (67%)], and monosomy 7 in 5.8% [MDS-U (70%), RCUD (10%), RAEB-1 (10%)]. The frequency of +8, −7 or del(7q), −5 or del(5q) in MDS is similar to that in the WHO 2008 (~10%). However, del 20q is slightly higher in our cohort. In addition, the frequency of abnormal aberrations in the different subtypes of MDS, excluding MDS-U, is 15–20% higher than that of the WHO. Our findings expand the cytogenetic spectrum of MDS, reaffirm the importance of karyotype and highlight the utility of combining FISH and standard chromosome analyses. Karyotype alone has a higher yield compared to FISH alone, while the detection of aberrations increased when combined together. The frequency of aberration type cannot help to sub classify patients, as each type is more frequent in MDS-U. More than half of our patients have normal karyotype. This suggests that other submicroscopic chromosomal aberrations that go undetected by current clinical testing and that can stratify patients into different risk categories are likely frequent. 132 Chromosomal abnormalities in primary myelodysplastic syndrome performed by conventional cytogenetics – tertiary care center experience A. Rashid. Department of Pathology and Microbiology, Aga Khan University and Hospital, Karachi, Pakistan Introduction: Myelodysplastic syndromes (MDS) consist of a heterogeneous group of haematological disorders characterized by peripheral blood cytopenias in the presence of hypercellular bone marrow with features of ineffective haematopoiesis. Chromosomal anomalies are detected in approximately 50% of patients with de novo MDS and in up to 80% of patients with MDS secondary to chemotherapy or other toxic agents. Cytogenetic abnormalities are major determinants in the pathogenesis, diagnosis, prognosis, and the basis for selection of drugs in individual patients with MDS. Objective: Aim of this study was to determine the frequency of cytogenetic abnormalities in patients diagnosed as primary
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myelodysplastic syndrome using conventional karyotyping in Pakistani population. Method: Patients who fulfilled the WHO criteria for MDS were included. Cytogenetic analysis was conducted at the time of diagnosis. Patients who had previously received chemo/radiotherapy, and those with MDS secondary to a previous malignancy were excluded from analysis. Chromosome identification and karyotype description was done according to the International System for Chromosome Nomenclature (ISCN, 1995). Results: Total of n = 112 patients were diagnosed as primary myelodysplastic syndrome. N = 50 patients had their karyotype done at the time of diagnosis. Of the n = 50 patients, n = 33 were males (66%) and n = 17 were females (34%). The median age was 58 years (range 1–75 years). Only one patient was under 15 years of age. Out of n = 50, n = 5 (10%) were classified as RA, n = 2 (4%) as RARS, n = 23 (46%) as RCMD, n = 1 (2%) as RCMD-RS, n = 9 (18%) as RAEB-I and n = 10 (20%) as RAEB-II. Among the 50 patients, n = 29 (58%) showed normal karyotype and n = 21 (42%) showed clonal karyotypic abnormalities at diagnosis, of which, n = 7 (14%) had single, n = 6 (12%) had double and n = 9 (18%) has complex cytogenetics. The common abnormalities found were, triosomy 8 in n = 3 cases (6%), complex triosomy 8 in n = 1 case (2%), −7/del(7q) (1/1cases) 4%, −Y in n = 2 cases (4%), complex 5q in n = 2 cases(4%), del 11q in n = 1 case (2%), inversion 9 in n = 1case (2%), trisomy 19 in n = 1 case (2%) and other abnormalities in n = 8 cases (16%). Conclusion: In contrast to del 5q which is the most reported cytogenetic abnormality in India, USA and European countries, triosomy 8 is found to be most common in our study population followed by the complex cytogenetics. RA has the highest number of abnormalities followed by RAEBII, ie 60% and 55% respectively, in contrast to RAEBII. This is an ongoing study. Large prospective studies are needed to report most common abnormality prevalent according to classification of Myelodysplastic syndrome. 133 Specificity of features of the bone marrow CD34+ cell fraction for the diagnosis of MDS S.C. Reis-Alves1 , P.M. Campos2 , F.G. Pereira-Cunha2 , F. Traina2 , I. Lorand-Metze2 . 1 State Healthcare Institution, Tula Regional Clinical Hospital, University of Campinas, 2 Internal Medicine, State University of Campinas, Campinas, Brazil Introduction: immunophenotyping has been used as a complimentary technique for the confirmation of the diagnosis of MDS, especially in cases with a normal karyotype. Methods: using a previously described four-color-combination panel designed to detect abnormalities of myelomonocytic precursors and CD34+ cells, we standardized the normal values for CD34+ cell fractions according to the guidelines of ELN, examining their co-expression of CD19/CD10, CD13, CD117, CD15, CD11b, CD7 and CD56 (30 normal BM donors of allogeneic BMT). These values were compared with the findings obtained for confirmed cases of MDS (28) and with cases of PB cytopenias due to non-clonal disorders (16) that performed the analysis in their diagnostic work-up. Results: Donors of normal BM: 33 years (15–63), patients with nonclonal disorder: 63 years (22–86); cases of MDS: 70 years (28–84). WHO types: 3 RA; 12 RCDM; 3 RCDM-SA; 5 RAEB-I; 5 RAEB-II. CD34+ cells/total non-erythroid cells were significantly increased in MDS as compared to reactive and normal cases (2.1%, 0.44%, and 0.80% respectively). Myeloblasts (CD34+/CD13+CD117+) were increased in MDS with BM blasts < 5% compared to reactive cases, although there was some overlap. The number of B-cell precursors was correlated with age (MDS = −0.38, p = 0.02; reactive = −0.55, p = 0.01), but their number was higher in reactive cases than in MDS (p = 0.02). The analysis of the co-expression of CD11b and CD15 in CD34+ cells was not able to discriminate the 3 groups. But, expression of CD56 and/or that of CD7 in CD34+ cells was significantly increased in MDS compared to reactive and normal BM.
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Posters / Leukemia Research 35 (2011) S27–S142
Conclusions: the analysis of BM subsets of CD34+ cells is useful in the differential diagnosis of MDS with BM blasts < 5% and non-clonal disorders. The most useful features are the number of B-cell precursors and aberrant co-expressions, but not asynchronous maturation. Support: MDS Foundation, FAPESP and CNPq 134 Hidden genomic abnormalities detected in MDS patients with a normal karyotype have prognostic relevance B. Royer-Pokora1 , A. Thiel1 , M. Beier1 , D. Ingenhag1 , K. Servan1 , M. Hein1 , U. Germing2 , B. Hildebrandt1 . 1 Institute of Human Genetics, 2 Department of Haematology, Oncology and Clinical Immunology, Heinrich Heine University Duesseldorf, Duesseldorf, Germany Approximately 50% of MDS patients have a normal karyotype. Our aim was to study whether recurrent or individual abnormalities hidden can be detected in MDS with a normal Karyotype and to determine their prognostic significance. To address this question we have analyzed 107 MDS patients of various FAB/WHO subgroups and a normal karyotype using aCGH. For this study we have used Agilent microarrays consisting of 44K, 105K or 244K 60-mer oligonucleotide probes spanning the human genome with an average spatial resolution of approximately 43, 22 and 9 kb, respectively. Array CGH identified hidden recurrent chromosomal aberrations in ≥2 cases: del5q, del7q, del21q and del4q. In 34 cases we have identified individual imbalances. Of the 107 cases 42 (39%) had hidden alterations. Larger alterations were verified by FISH and smaller alterations with Q-PCR. So far 40 of the identified aberrations were verified with other methods. Zoom in custom arrays were designed to characterize the breakpoints. In the imbalances several interesting genes were localized, involved in epigenetic and chromatin modification, such as DNMT3a, ASXL2, TET2, HDAC3, BRD8 or several genes with a role in DNA repair, e.g. DNAJC 16, 18 and 27, MLH3, RAD50, as well as members of the RAS signalling pathway, for example RAB10, 38, 39, RABL5, RAPGEF6 and RIN3. Gene functional classification using DAVID revealed an enrichment of metallopeptidases, Apoptosis genes and genes regulating haematopoiesis. Comparing the survival of patients with and without additional hidden alterations showed that those with additional genomic imbalances had an inferior survival (p = 0.002). In conclusion, hidden recurrent and individual aberrations in cytogenetically normal MDS can be identified with aCGH and these have prognostic relevance. 135 Lower bone marrow myeloid and plasmacytoid dendritic cell counts in high-risk than in low-risk MDS patients and controls 1 2 L. Saft1 , E. Bjorklund ¨ , E. Hellstrom-Lindberg ¨ , A. Porwit1 . 1 Department of Pathology, 2 Department of Medicine, Center for Experimental Hematology, Karolinska University Hospital and Institute, Stockholm, Sweden
Background: Dendritic cells (DC) are professional antigenpresenting cells and play a pivotal role in coordinating functions of the immune system by the initiation and regulation of T-cell mediated responses. Laboratory and clinical studies suggest that bone marrow (BM) failure in MDS may be, at least in part, immune-mediated. The observation that MDS patients can respond to immunosuppressive therapy sparked a renewed interest in T-cell mediated mechanisms. DCs originate from BM hematopoietic stem cells and clonal involvement of precursor DC has been demonstrated in both MDS and AML. Aims: To determine whether levels of myeloid (mDC) and plasmacytoid (pDC) dendritic cells in BM samples from MDS patients differ from those in reactive BM. Patients and Methods: BM samples from eighty MDS patients (IPSS: low-risk 25, INT-1 39, INT-2 11, high-risk 5, all WHO subtypes included) diagnosed at the Karolinska University Hospital between
January 2006 and November 2010 were included. In 67 patients (84%) the BM samples were taken before treatment and in 20/67 patients at least one additional follow-up biopsy was available prior to treatment start. BM samples from 13/80 (16%) patients were analyzed after therapy start. DC levels (expressed as percentage of BM cells) were measured by 4-color flow cytometry (FCM) using Lineage cocktail 1(lin)− FITC/CD123-PE/HLA-DR-PerCP/CD11cAPC antibody combination, FACS-Calibur flow cytometer and Paint-a Gate Pro software (Becton&Dickinson). pDC immunophenotype was characterized as lin−/HLA−DR+/CD123+/CD11c− and mDC as lin−/HLA−DR+/CD11c+/CD123−. In addition, BM samples from 36 hospital controls were investigated. Results: Average DC levels were significantly lower in MDS (mDC 0.039%; pDC 0.118%) as compared to the control group (mDC 0.089%; pDC 0.178%; p < 0.001). Lower DC levels were found in treated MDS patients than in untreated patients (p < 0.01). High-risk MDS and patients with higher BM blast percentages (>5%) had significantly lower DC levels than low-risk MDS patients. Progression to AML was seen in 15/80 (18.7%) patients (one del(5q), 6 RCMD, 5 RAEB-1, 3 RAEB-2); no significant difference in DC levels at diagnosis were seen in patients who developed AML in the follow-up period but in sequentially analyzed patients we could see lower DC levels at progression. Conclusions: DC levels are significantly lower in high-risk MDS and inversely correlated with the percentage of BM blasts. Our findings may indicate involvement of the DC compartment in the MDS disease process. FCM-determined DC levels may assist in the diagnostic work-up and risk-assessment of MDS patients. 136 Using flow cytometry for differential diagnosis of thrombocytopenia D. Subira, D. de Miguel, I. San Roman, ´ M. D´ıaz Morfa, N. Golbano, D. Morales, J. Arbeteta, S. Herrero, F. Fuertes, B. Pinedo. Hematology, Hospital Universitario de Guadalajara, Guadalajara, Spain Differential diagnosis between immune thrombocytopenia (ITP) and myelodysplastic syndrome (MDS) may be difficult, especially in old people. Altered immunophenotypes in maturing cells have been described in bone marrow samples from patients with MDS. However, flow cytometry immunophenotyping (FCI) detractors criticize lack of standard procedures of analyses and subjective identification of deviation from normal patterns of differentiation. Aim: To determine the utility of semi-quantitative FCI for identifying MDS within a group of patients with thrombocytopenia. Patients: From May 2010 to January 2011, 20 patients (9M/11F, median age 72, range 16–89) were studied for differential diagnosis of thrombocytopenia. Median platelet count was 45×109 (range 3.5–118); median hemoglobin was 12 g/dl, and leukocyte 6.4×109 /l. The following diagnoses were established: ITP (n = 6), refractory ITP (n = 1); MDS (n = 6); non-MDS thrombocytopenia (n = 7). Methods: FCI using 4-colour combinations. Data studied in bone marrow samples were: myeloid granularity, abnormalities in the phenotypic pattern of maturation of the myeloid (CD16/ CD13/HLADR/CD11b) and monocytic (CD36/CD14/HLADR/CD64) compartment, distribution of CD10 on mature granulocytes, quantification of CD10+ B-cell precursors and myeloid CD34+ cells. Deviations from normal patterns were calculated using reference images with the Infinicyt software program for FCI analysis. The following deviations were considered as abnormalities: >30% in myeloid granularity, >19% in myeloid maturation, >11% in monocytic maturation, < 70% of CD10 mean fluorescence intensity on granulocytes, <1% of CD10+ lymphocytes within the B-cell compartment, and >2% CD34+ myeloid cells. Results: Median number of FCI abnormalities was significantly higher in patients with MDS (n = 3.5) as compared to patients with ITP (n = 0.4) and non-MDS thrombocytopenia (n = 0.6). These