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117 Cytogenetic study of MDS
Posters / Leukemia Research 35 (2011) S27–S142
Results: All samples were reclassified according FAB, 2001 WHO and 2008 WHO classifications. Regarding 2008 WHO categories, 6 cases had refractory cytopenia with unilineage dysplasia (RCUD), 8 refractory anaemia with ring sideroblasts (RARS), 51 RC multilineage dysplasia, (RCMD), 14 RA with excess blasts-1 (RAEB-1), 9 RAEB-2, 3 MDS del(5q), 3 MDS-unclassified (MDS-U), and 4 MDS/ myeloproliferative neoplasms (MDS/MPN). Seventeen cases were considered as MDS-U by the 2001 WHO classification. Only two remained as MDS-U by the 2008 WHO proposal. Ninety five samples were considered suitable by the second observers. Discordance was observed in 27/95 (28%) (range 21–35%). The interobserver agreement regarding specimens with and without excess of blasts was 94%. The inter-observer concordace was 84% with RAEB-1, 89% RAEB-2, 74% RCMD, 100% MDS/MPN, and 100% MDS-U. The observers only found concordance in 1/7 cases with RARS (14%), in 2/5 with CRDU (40%) and in 2/3 MDS del(5q) (66%). Diagnosis of MDS with unilineage dysplasia (CRDU, or RARS, or MDS-U) was assessed in 21 patients, by at least one observer. There was consensus only in 4 cases. (19%) Conclusions: In our series, the 2008 WHO criteria were able to diagnose most of patients considered unclassified by the 2001 WHO proposal. We found high inter-observer agreement regarding cases with and without excess of blasts. The 2008 WHO categories with unilineage dysplasia were not reproducible by a second observer in most of cases. Cases with inter-observer discrepancy First review (n cases)
Second review
RCUD (3) RARS (6) RCMD (13) RAEB-1 (2) RAEB-2 (1) MDS del(5q) (1)
RCMD (2) RA (1) RCMD (6) RARS (3) RCUD (5) RAEB-1 (5) RAEB-2 (2) RAEB-1(1) RAEB-1 (1)
117 Cytogenetic study of MDS B. Ganguly1 , M. Ghosh2 , D. Banerjee3 , S. Chandra3 . 1 Genetics, Genetics Center, Navi Mumbai, 2 Hemetology, NRS Medical College, 3 Hematology, Clinical Hematology Service, Kolkata, India Myelodysplastic syndrome (MDS) is a heterogeneous hematopoietic clonal disorder of the elderly characterized mostly by anemia, thrombocytopenia and bone marrow dysplasia. Primary and secondary MDS are classified on the basis of patient-history, previous treatments and chemo- and/or radiotherapy or occupational exposure to toxic substances. MDS is generally resulted from disturbances in proliferation, differentiation and apoptotic processes of hematopoietic precursor cells and frequently evolves into acute myeloid leukemia (AML). Cytogenetically, MDS is prominently associated with del(5q), monosomy 7, del(7q), trisomy 8 and del(20q). In primary MDS, approximately 50% of karyotypes are abnormal with a single abnormal chromosome; however, actual frequency would be much higher. Higher incidences of normal karyotype are primarily due to limitation of resolution of conventional G-banding technique and culture condition followed by the diagnostic laboratories. In secondary MDS, multiple and complex chromosomal rearrangements are frequently seen. In the present study, conventional karyotyping was carried out on a total of 108 cases with provisional diagnosis of primary MDS. The mean age of the population was 56 years ranging from 22 to 80. Four cultures were processed for each individual following unstimulated bone marrow culture technique for different durations, viz. 2h, 24h, 48h and 72h. Initially five metaphases were analysed from each culture to select the culture duration with some expressed abnormality, which was considered for further analysis of 20 metaphases. It was experienced that culture duration is related to expression of aberrations.
S45
Karyotyopes were normal in 2h cultures, whereas abnormalities were expressed in 24 h or 48 h cultures in most of the cases. In fact, 48 h or 72 h cultures would have been ideal culture duration; however, in vitro cell death appears as limiting factor. In the present group, chromosomal picture appeared as 26% and 74% with normal and abnormal karyotypes respectively. The classified aberrations, including del(5q)/del(7q) (8% each), −7 (11%), +8 (3%), del(20q) (3%), −Y (3%), +21 (13%), chromatid breaks (8%), t(13;19) (3%), t(12;17) (3%) and hyperdiplidy (tetra- and octaploidy) (10%) were recorded. In addition, a constitutive pericentric inversion in chromosome 9 was detected in 8% of the cases, which may affect prognosis and influence disease evolution as found in CML. This result highlights the importance of culture duration in cytogenetic characterization of MDS and role of conventional cytogenetic technique in understanding the clonal and constitutive aberrations at whole genome level for prognostication and evolution of the disease. 118 Comprehensive genetic characterization of MDS patients by CD34+ bone marrow and peripheral blood combining FISH-, SNP- and chromosome banding analysis C. Ganster1 , K. Shirneshan1 , G. Salinas-Riester2 , F. Braulke1 , 3 K. Gotze ¨ , U. Platzbecker4 , D. Haase1 . 1 Department of Haematology and Oncology, 2 DNA Microarray Facility, University Medicine Goettingen, Goettingen, 3 Department of Haematology and Oncology, Tech. University of Munich, Munich, 4 Medical Clinic I, University Hospital, Dresden, Germany Introduction: Usually, chromosomal banding analysis (CBA) of bone marrow metaphases is used to identify chromosomal abnormalities in myelodysplastic syndromes (MDS). In ongoing studies we perform serial fluorescence in situ hybridisation (FISH) analysis on CD34+ peripheral blood cells to detect and follow chromosomal abnormalities without the need of repeated bone marrow biopsies. To identify additional abnormalities not detectable by FISH, we asked whether SNP array analysis (SNP-A) can be applied complementary to CD34+ peripheral blood cells. Material and Methods: We included eleven MDS and two AML patients with known karyotypes in our pilot study. Immunomagnetically enriched CD34+ peripheral blood cells were available in nine patients. CD34+ bone marrow cells from four additional patients were investigated. The CD34+ cells were analyzed by FISH and SNP-A. Either the Affymetrix Genome-Wide Human SNP Array 6.0 or the Affymetrix Cytogenetics Whole-Genome 2.7 M Array were used. Three patients were analysed using both arrays. Results: By both, FISH and SNP-A, we could confirm all gains and losses in CD34+ blood cells detected by CBA of bone marrow cells in three patients and verify CBA in CD34+ bone marrow cells in one patient. In two patients clones smaller than 15% could be confirmed by FISH only but not by SNP-A. Though 5q− was identified by CBA and by FISH in one patient, poor quality array results hampered the confirmation of this aberration by SNP-A. SNP-A of three patients identified additional copy number variations and uniparental disomies not detected by CBA or FISH: upd(7q), del(9)(q22.33q31.1), del(15)(q15.1), upd(17pterp11.2). In two patients with complex abnormalities SNP-A allowed the identification of breakpoints and of additional aberrations, including del(15)(q11.2q21.1) and upd(20p). In the first of serial samples of one patient, a small del(13q14) not detectable by CBA was identified in a subclone by FISH and SNP-A. Four months later without clinical signs of progression, SNP-A revealed that this subclone had disappeared, but two further abnormalities were identified indicating clonal evolution (del(2)(q31q32), del(4)(q24q26)). Conclusion: Parallel FISH and SNP-A of CD34+ peripheral blood cells identify most chromosomal aberrations detectable by CBA of bone marrow metaphases and even increase the detection rate. As reliable detection of chromosomal aberrations is possible in peripheral blood applying FISH and SNP-A to the same specimen, comprehensive
Results: All samples were reclassified according FAB, 2001 WHO and 2008 WHO classifications. Regarding 2008 WHO categories, 6 cases had refractory cytopenia with unilineage dysplasia (RCUD), 8 refractory anaemia with ring sideroblasts (RARS), 51 RC multilineage dysplasia, (RCMD), 14 RA with excess blasts-1 (RAEB-1), 9 RAEB-2, 3 MDS del(5q), 3 MDS-unclassified (MDS-U), and 4 MDS/ myeloproliferative neoplasms (MDS/MPN). Seventeen cases were considered as MDS-U by the 2001 WHO classification. Only two remained as MDS-U by the 2008 WHO proposal. Ninety five samples were considered suitable by the second observers. Discordance was observed in 27/95 (28%) (range 21–35%). The interobserver agreement regarding specimens with and without excess of blasts was 94%. The inter-observer concordace was 84% with RAEB-1, 89% RAEB-2, 74% RCMD, 100% MDS/MPN, and 100% MDS-U. The observers only found concordance in 1/7 cases with RARS (14%), in 2/5 with CRDU (40%) and in 2/3 MDS del(5q) (66%). Diagnosis of MDS with unilineage dysplasia (CRDU, or RARS, or MDS-U) was assessed in 21 patients, by at least one observer. There was consensus only in 4 cases. (19%) Conclusions: In our series, the 2008 WHO criteria were able to diagnose most of patients considered unclassified by the 2001 WHO proposal. We found high inter-observer agreement regarding cases with and without excess of blasts. The 2008 WHO categories with unilineage dysplasia were not reproducible by a second observer in most of cases. Cases with inter-observer discrepancy First review (n cases)
Second review
RCUD (3) RARS (6) RCMD (13) RAEB-1 (2) RAEB-2 (1) MDS del(5q) (1)
RCMD (2) RA (1) RCMD (6) RARS (3) RCUD (5) RAEB-1 (5) RAEB-2 (2) RAEB-1(1) RAEB-1 (1)
117 Cytogenetic study of MDS B. Ganguly1 , M. Ghosh2 , D. Banerjee3 , S. Chandra3 . 1 Genetics, Genetics Center, Navi Mumbai, 2 Hemetology, NRS Medical College, 3 Hematology, Clinical Hematology Service, Kolkata, India Myelodysplastic syndrome (MDS) is a heterogeneous hematopoietic clonal disorder of the elderly characterized mostly by anemia, thrombocytopenia and bone marrow dysplasia. Primary and secondary MDS are classified on the basis of patient-history, previous treatments and chemo- and/or radiotherapy or occupational exposure to toxic substances. MDS is generally resulted from disturbances in proliferation, differentiation and apoptotic processes of hematopoietic precursor cells and frequently evolves into acute myeloid leukemia (AML). Cytogenetically, MDS is prominently associated with del(5q), monosomy 7, del(7q), trisomy 8 and del(20q). In primary MDS, approximately 50% of karyotypes are abnormal with a single abnormal chromosome; however, actual frequency would be much higher. Higher incidences of normal karyotype are primarily due to limitation of resolution of conventional G-banding technique and culture condition followed by the diagnostic laboratories. In secondary MDS, multiple and complex chromosomal rearrangements are frequently seen. In the present study, conventional karyotyping was carried out on a total of 108 cases with provisional diagnosis of primary MDS. The mean age of the population was 56 years ranging from 22 to 80. Four cultures were processed for each individual following unstimulated bone marrow culture technique for different durations, viz. 2h, 24h, 48h and 72h. Initially five metaphases were analysed from each culture to select the culture duration with some expressed abnormality, which was considered for further analysis of 20 metaphases. It was experienced that culture duration is related to expression of aberrations.
S45
Karyotyopes were normal in 2h cultures, whereas abnormalities were expressed in 24 h or 48 h cultures in most of the cases. In fact, 48 h or 72 h cultures would have been ideal culture duration; however, in vitro cell death appears as limiting factor. In the present group, chromosomal picture appeared as 26% and 74% with normal and abnormal karyotypes respectively. The classified aberrations, including del(5q)/del(7q) (8% each), −7 (11%), +8 (3%), del(20q) (3%), −Y (3%), +21 (13%), chromatid breaks (8%), t(13;19) (3%), t(12;17) (3%) and hyperdiplidy (tetra- and octaploidy) (10%) were recorded. In addition, a constitutive pericentric inversion in chromosome 9 was detected in 8% of the cases, which may affect prognosis and influence disease evolution as found in CML. This result highlights the importance of culture duration in cytogenetic characterization of MDS and role of conventional cytogenetic technique in understanding the clonal and constitutive aberrations at whole genome level for prognostication and evolution of the disease. 118 Comprehensive genetic characterization of MDS patients by CD34+ bone marrow and peripheral blood combining FISH-, SNP- and chromosome banding analysis C. Ganster1 , K. Shirneshan1 , G. Salinas-Riester2 , F. Braulke1 , 3 K. Gotze ¨ , U. Platzbecker4 , D. Haase1 . 1 Department of Haematology and Oncology, 2 DNA Microarray Facility, University Medicine Goettingen, Goettingen, 3 Department of Haematology and Oncology, Tech. University of Munich, Munich, 4 Medical Clinic I, University Hospital, Dresden, Germany Introduction: Usually, chromosomal banding analysis (CBA) of bone marrow metaphases is used to identify chromosomal abnormalities in myelodysplastic syndromes (MDS). In ongoing studies we perform serial fluorescence in situ hybridisation (FISH) analysis on CD34+ peripheral blood cells to detect and follow chromosomal abnormalities without the need of repeated bone marrow biopsies. To identify additional abnormalities not detectable by FISH, we asked whether SNP array analysis (SNP-A) can be applied complementary to CD34+ peripheral blood cells. Material and Methods: We included eleven MDS and two AML patients with known karyotypes in our pilot study. Immunomagnetically enriched CD34+ peripheral blood cells were available in nine patients. CD34+ bone marrow cells from four additional patients were investigated. The CD34+ cells were analyzed by FISH and SNP-A. Either the Affymetrix Genome-Wide Human SNP Array 6.0 or the Affymetrix Cytogenetics Whole-Genome 2.7 M Array were used. Three patients were analysed using both arrays. Results: By both, FISH and SNP-A, we could confirm all gains and losses in CD34+ blood cells detected by CBA of bone marrow cells in three patients and verify CBA in CD34+ bone marrow cells in one patient. In two patients clones smaller than 15% could be confirmed by FISH only but not by SNP-A. Though 5q− was identified by CBA and by FISH in one patient, poor quality array results hampered the confirmation of this aberration by SNP-A. SNP-A of three patients identified additional copy number variations and uniparental disomies not detected by CBA or FISH: upd(7q), del(9)(q22.33q31.1), del(15)(q15.1), upd(17pterp11.2). In two patients with complex abnormalities SNP-A allowed the identification of breakpoints and of additional aberrations, including del(15)(q11.2q21.1) and upd(20p). In the first of serial samples of one patient, a small del(13q14) not detectable by CBA was identified in a subclone by FISH and SNP-A. Four months later without clinical signs of progression, SNP-A revealed that this subclone had disappeared, but two further abnormalities were identified indicating clonal evolution (del(2)(q31q32), del(4)(q24q26)). Conclusion: Parallel FISH and SNP-A of CD34+ peripheral blood cells identify most chromosomal aberrations detectable by CBA of bone marrow metaphases and even increase the detection rate. As reliable detection of chromosomal aberrations is possible in peripheral blood applying FISH and SNP-A to the same specimen, comprehensive