Clinical Utility of Multiparameter Flow Cytometry in the Diagnosis of 1013 Patients with Suspected Myelodysplastic Syndrome: Correlation to Cytomorphology, Cytogenetic, and Clinical Data.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 595-595
Author(s):  
Wolfgang Kern ◽  
Claudia Haferlach ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Flow Cytometry ◽  
Myelodysplastic Syndrome ◽  
Expression Pattern ◽  
Antigen Expression ◽  
Refractory Anemia ◽  
Multiparameter Flow Cytometry ◽  
Multilineage Dysplasia ◽  
Cytogenetic Aberrations ◽  
Ring Sideroblasts ◽  
Equity Ownership

Abstract Abstract 595 Diagnosis and classification of myelodysplastic syndromes (MDS) is based on cytomorpholgy (CM) and cytogenetics (CG). By the identification of MDS-related aberrant antigen expression multiparameter flow cytometry (MFC) may add important diagnostic information. Examples include an aberrant expression pattern of CD13 and CD16 in granulocytes, an aberrante expression pattern of HLA-DR and CD11b in monocytes and the expression of lymphoid markers on myeloid blasts (Haematologica 2009:94:1124). To evaluate the potential role of MFC in the diagnostic setting of MDS we analyzed 1013 cases with suspected MDS by CM, CG, and MFC in parallel. Cases were classified by CM as refractory anemia (RA, n=31, 3.1%), refractory anemia with ring sideroblasts (RARS, n=27, 2.7%), refractory cytopenia with multilineage dysplasia (RCMD, n=64, 6.3%), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS, n=49, 4.8%), refractory anemia with excess of blasts 1 (RAEB-1, n=133, 13.1%), RAEB-2 (n=81, 8.0%), 5q- syndrome (n=24, 2.4%), chronic myelomonocytic leukemia (CMML, n=65, 6.4%), myelodysplastic syndrome unspecified (MDS-u, n=15, 1.5%), MDS borderline to acute myeloid leukemia (MDS/AML, n=6, 0.6%), MDS/myeloproliferative neplasia overlap (MDS/MPN, n=16, 1.6%), suspected MDS (n=225, 22.2%), reactive condition (n=266, 26.3%), and normal findings (n=11, 1.1%). Cytogenetic findings were normal karyotype (n=768, 75.8%), isolated deletion of long arm of chromosome 5 (del(5q), n=43, 4.2%), isolated aberrations of chromosome 7 (n=14, 1.4%), isolated trisomy 8 (n=30, 3.0%), isolated deletion of long arm of chromosome 20 (del(20q), n=21, 2.1%), complex karyotype (n=23, 2.3%), loss of Y-chromosome (n=43, 4.2%), other aberrations (n=71, 7.0%). Concordance between CM and MFC was 82.0% for diagnostic results in 788 cases with unequivocal CM. 277 of these 788 cases were classified by CM as not having MDS, 13 (4.7%) of which showed MDS-typical features by MFC. Additional 225 cases showed only minor dysplastic features by CM, 51 (22.7%) of which showed clear evidence of MDS by MFC. To further analyze the significance of MDS-related findings by MFC we then focused on cytogenetically aberrant cases without unequivocal MDS by CM. In 6/12 (50.0%) cases with no indication of MDS by CM and MDS-typical cytogenetic aberrations MFC revealed MDS characteristics. In another 11/23 (47.8%) cases with minor dysplastic features by CM and MDS-typical cytogenetic aberrations MFC revealed MDS characteristics. Furthermore, we compared blast counts as determined by CM and MFC and found a strong correlation (p<0.001) although the mean±SD percentage was higher as determined by CM as compared to MFC (4.67±4.18 vs. 3.78±2.97). Frequencies of aberrantly expressed antigens significantly differed between cases rated by CM as MDS (median number of aberrantly expressed antigens: 3), suspected MDS (1), and no MDS (0, p<0.001). In various cases MFC identified MDS-typical aberrant antigen expression in cell compartments not rated dysplastic by CM. Spearman rank correlation confirmed a highly significant relation between the number of aberrantly expressed antigens and IPSS (r=0.409, p<0.001). In 257 cases with data on overall survival (OS) the presence of MDS-related findings (≥3 aberrantly expressed antigens or a blast count >5% in MFC or a reduced side-scatter signal) resulted in significantly inferior 6-year-OS (68% vs. 100% p=0.008). The present analysis clearly demonstrates a diagnostic yield of MFC in addition to cytomorphology and cytogenetics in cases with suspected MDS. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Prognostic Impact of Multiparameter Flow Cytometry in Patients Analyzed for Suspected MDS.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2806-2806
Author(s):  
Wolfgang Kern ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Expression Pattern ◽  
Multivariable Analysis ◽  
Multiparameter Flow Cytometry ◽  
Prognostic Impact ◽  
Cd16 Expression ◽  
Equity Ownership ◽  
Cox Analysis ◽  
Bone Marrow Blasts

Abstract Abstract 2806 Background: Multiparameter flow cytometry (MFC) is increasingly used to evaluate patients with suspected myelodysplastic syndromes (MDS). The prognostic impact of distinct MFC findings has been controversial yet. Aims: Assess the respective impact of different antigen expression aberrancies on overall survival (OS) in suspected MDS in relation to established prognostic parameters. Methods: We studied 804 patients (pts) who were analyzed for suspected MDS by cytomorphology (CM), cytogenetics and MFC in parallel (f/m 463/341; median age 70 yrs, range 2–89). Pts had been included in a previous study evaluating the diagnostic role of MFC (Kern et al., Cancer 2010). Data on OS was available in all pts (median OS 6.2 yrs, median follow-up 3.2 yrs). CM revealed MDS in 493 (61.3%) pts; 170 (21.1%) pts had evidence of dysplasia which was not sufficient to diagnose MDS by CM; in 141 (17.5%) pts MDS was excluded by CM. Karyotypes were favorable / intermediate / unfavorable according to IPSS in 684 (85.1%) / 89 (11.1%) / 31 (3.9%) pts. MFC was performed following recent ELN Working Group recommendations (Westers et al., Leukemia 2012) in myeloid progenitor cells (MPC), granulocytes, monocytes and erythroid cells. MFC parameters included, each compared to normal bone marrow, increased or decreased expression of antigens, expression of normally not expressed antigens, aberrant expression pattern of pairs of antigens as well as cross-lineage expression of lymphatic antigens. Results: Considering all 804 pts, i.e. regardless of confirmation of MDS by CM, the following MFC parameters were associated with OS: MPC >5% (p<0.001, hazard ratio (HR) 2.5), expression of CD5 (p<0.001, HR 4.1), CD56 (p=0.043, HR 2.0), CD7 (p=0.015, HR 2.2) in MPC; reduced side-scatter signal (p<0.001, HR 1.9), aberrant CD13/CD16 expression pattern (p=0.007, HR 1.4), aberrant CD11b/CD16 expression pattern (p=0.003, HR 1.6), expression of CD56 (p<0.001, HR 2.1), reduced expression of CD33 (p=0.018, HR 1.6) in granulocytes; expression of CD56 (p<0.001, HR 1.6) in monocytes; reduced expression of CD71 (p<0.001, HR 2.1) in erythroid cells. A score was devised calculating for each pt the sum of all HRs for the respective parameters found positive. Pts were separated into 4 groups: group 1 (n=263 pts) had a score of 0; group 2 (n=259) had a score >0 and below the median (2.135); group 3 (n=149) had a score above the median and below the 75th percentile (4.961); group 4 had a score above the 75thpercentile. Significant differences in OS were observed: OS at 4 years in groups 1, 2, 3, and 4 amounted to 82.4%, 67.1%, 54.7%, and 36.2%, respectively (p=0.001 for 1 vs 2, p=0.022 for 2 vs 3, p=0.003 for 3 vs 4, p<0.001 for all other comparisons). Cox analysis of the MFC score revealed a significant association with OS (p<0.001, HR 1.4 per group). Other parameters univariably related to OS were: diagnosis of MDS by CM (p<0.001, HR 2.2), percentage of bone marrow blasts by CM (p<0.001, HR 1.9 per 10% increment), cytogenetic group according to IPSS (p<0.001, HR 5.1 per group), WBC count (p<0.001, HR 1.2 per 10 G/L increment), hemoglobin level (p<0.001, HR 0.8 per g/L), platelet count (p<0.001, HR 1.4 per 100 G/L increment), and age (p<0.001, HR 1.5 per decade). Multivariable Cox analysis including diagnostic markers as covariates revealed an independent impact on OS for all of them: MFC score (p<0.001, HR 1.3), diagnosis of MDS by CM (p=0.003, HR 1.4), bone marrow blasts by CM (p=0.015, HR 1.4), and cytogenetics according to IPSS (p<0.001, HR 2.9). The addition of age or of peripheral blood counts as covariates into the multivariable analysis still indicated an independent impact of the MFC score on OS. Limiting the multivariable analysis to cases with MDS proven by CM still resulted in an independent impact of the MFC score on OS (p=0.001, HR 1.2). Interestingly, if only cases were considered with signs of dysplasia by CM, which are not sufficient to diagnose MDS, multivariable analysis also revealed an independent impact of the MFC score on OS (p=0.016, HR 1.3). Conclusions: The present data indicates that MDS-related findings by MFC provide prognostic information not just in pts with MDS proven by CM but also in a comprehensive cohort of pts being diagnosed for suspected MDS. Furthermore, even in pts with evidence of dysplasia not sufficient to diagnose MDS by CM a prognostic impact of MFC was demonstrated. This data thus suggests to integrate MFC into the diagnostic work-up of pts with suspected MDS. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Prognosis of MDS Subtypes RARS, RCMD and RCMD-RS Does Not Differ by Cytomorphologic Criteria but Cytogenetics Allows to Delineate a Subgroup with Inferior Clinical Course,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3796-3796
Author(s):  
Ulrike Bacher ◽  
Wolfgang Kern ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
Multivariable Analysis ◽  
Risk Groups ◽  
Low Risk ◽  
Refractory Anemia ◽  
Employment Equity ◽  
P Values ◽  
Multilineage Dysplasia ◽  
Ring Sideroblasts ◽  
Equity Ownership ◽  
Wbc Count

Abstract Abstract 3796 Background: In 2008 the WHO classification combined the former categories RCMD (refractory cytopenia with multilineage dysplasia) and RCMD-RS (with ring sideroblasts ≥15%) thus not separating according to ring sideroblasts anymore in MDS with multilineage dysplasia, while the category refractory anemia with ring sideroblasts (RARS) was maintained separately. One aim of this study was to evaluate whether or not a separation with respect to ring sideroblasts is reasonable. Study Design: To investigate the clinical impact and genetic background of these MDS subtypes, we studied outcomes, cytogenetics, and molecular genetics in 1082 de novo MDS pts (153 RARS, 606 RCMD, 323 RCMD with ring sideroblasts ≥15% termed “RCMD-RS“): 703 m/379 f; median age, 73.1 yrs; 21.0–90.4 yrs. Cytogenetic risk groups were defined according to the International Prognostic Scoring System (IPSS; Greenberg et al., 1997). Results: Sex ratio (male preponderance in all subtypes; male/female ratio 1.9 in the whole cohort) did not differ significantly between the 3 MDS subgroups. Mean age (RARS: 71.8; RCMD: 70.1; RCMD-RS: 72.6 yrs) reached significant difference between RARS vs RCMD (p=0.020) and between RCMD vs RCMD-RS (p=0.004). Mean WBC count differed between all subgroups (RARS: 6.1; RCMD: 4.4; RCMD-RS: 5.3×10(9)/l; RARS vs RCMD p<0.001; RARS vs RCMD-RS p=0.039; RCMD vs RCMD-RS p<0.001) whereas mean platelet count (RARS: 232; RCMD: 144; RCMD-RS: 218 × 10(9)/l) and Hb level (RARS: 96; RCMD: 105; RCMD-RS: 98 g/l) differed between RARS vs RCMD and RCMD vs RCMD-RS (all p-values <0.001). IPSS categories were available in 854/1082 pts as follows: 514 pts (60.2%) low-risk, 312 (36.5%) intermediate-1, and only 28 (3.3%) intermediate-2 risk (no pt was assigned to high risk IPSS). Cytogenetics was available in all 1082 pts: The majority (918/1082; 84.4%) had good karyotypes (KTs) according to IPSS mainly due to high rates of normal KTs (821/1082, 75.9%); 112 pts had intermediate (10.4%); and only 52 (4.8%) had poor KTs. In detail, good KTs were equally distributed in RARS: 123/153; 80.4%; in RCMD: 530/606; 87.5%; and in RCMD-RS: 265/323; 82.0%. 3-yr overall survival (OS) rates did not differ significantly between the three MDS subtypes (RARS: 78.1%; RCMD: 86.7%; RCMD-RS: 80.6%). Also when entities with ≥15% ring sideroblasts (RARS + RCMD-RS) were compared to RCMD (<15% ring sideroblasts), 3-yr OS rate was similar (80.0% vs 86.7%; n.s.). Further, multilineage dysplasia per se did not impact on 3-yr OS rate (RCMD + RCMD-RS: 83.8% vs 78.1% in RARS; n.s.). In contrast, 3-year OS rate was better in good KTs compared to intermediate or poor KTs (91.4% vs 60.0% vs 29.3%; p<0.001) in the total cohort. Also in the different MDS subtypes, good karyotypes showed better 3-year OS rate than intermediate/poor karyotypes (p-values for comparison of cytogenetic risk groups: RARS: p<0.001; RCMD: p=0.032; RCMD-RS: p=0.007). In subcohorts genes were analysed and were found to be mutated only with low frequencies: RUNX1 mut: 14/213 (6.6%), NRAS mut: 1/283 (0.4%), MLL -PTD: 4/294 (1.4%), FLT3 -ITD: 0/285. This underlines the low risk profile of the cohort. In univariable analysis, good + intermediate vs poor KTs (p<0.001), aberrant vs normal KT (p<0.001), age (p=0.080), and Hb level (both continuous; p=0.007) had a significant impact on OS, while WBC count, platelets, and percentage of ring sideroblasts (all as continuous variables) were not significant. In multivariable analysis, IPSS cytogenetic risk groups (p=0.005) and Hb (p=0.008) only remained significant. Conclusions: As investigated here in 1082 pts, RARS, RCMD, and RCMD-RS all show high rates of good karyotypes as defined by IPSS and show similar clinical outcomes which clearly supports to skip the RCMD-RS category as done by the WHO in 2008. The karyotype and Hb level were the only independent prognostically relevant parameters in multivariable analysis. However, >80% of patients show a good risk cytogenetic profile making prognostication according to karyotype relevant only in a small subset of patients. To overcome this shortcoming only an increasing panel of new molecular markers in MDS can pave future investigations which presently becomes available by the advanced sequencing techniques. After diagnosed by morphology and cytogenetics especially molecular genetic information may therefore guide treatment in the near future for this low risk subgroup of MDS patients as investigated here. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Mutations of TET2 and JAK2 but Not CBL Are Detectable in a High Portion of Patients with Refractory Anemia with Ring Sideroblasts and Thrombocytosis (RARS-T).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1766-1766
Author(s):  
Johanna Flach ◽  
Sonja Schindela ◽  
Frank Dicker ◽  
Susanne Schnittger ◽  
Alexander Kohlmann ◽  
...  
Keyword(s):  
Microarray Analysis ◽  
Stop Codon ◽  
Myeloproliferative Neoplasms ◽  
Premature Stop Codon ◽  
Jak2v617f Mutation ◽  
Refractory Anemia ◽  
Missense Mutations ◽  
Myeloid Malignancies ◽  
Ring Sideroblasts ◽  
Equity Ownership

Abstract Abstract 1766 Poster Board I-792 Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) forms a provisional entity within the category of MDS/MPN-U in the 2008 WHO classification. Although the identification of the JAK2V617F mutation was an important first step in distinguishing this entity from other hematological diseases, further genetic characterization is necessary. We performed comprehensive cytogenetic and molecular genetic investigations including targeted analysis of JAK2V617F, TET2, MPLW515 and CBL, markers known to be altered in MPN, as well as genome-wide single nucleotide polymorphism microarray analysis (SNP-A) in 23 RARS-T patients who fulfilled WHO 2008 diagnostic criteria. The JAK2V617F mutation was detectable in 15 out of 19 analyzed patients (78.9%), four of which were homozygous. However, our patients neither carried a MPLW515 mutation nor mutations in exons 8 or 9 of CBL genes. These genes were recently described to be mainly mutated in myeloproliferative neoplasms. In addition, conventional cytogenetic analysis did not reveal any recurrent cytogenetic abnormalities in RARS-T patients. We also performed SNP microarray analysis in a subset of 10 RARS-T patients. Although we did neither observe recurrent chromosomal gains or losses nor recurring regions of UPD, one patient showed a deletion spanning a 1.3 Mb region on the long arm of chromosome 4 (start: 105,497,200 bp from pter; end: 106,825,780 bp from pter). The deleted region contained TET2, a gene recently found to be altered in many subtypes of myeloid malignancies. To further clarify the 4q24 deletion detected by SNP-A analysis we performed fluorescence in situ hybridization (FISH). 20 out of 100 analyzed interphase nuclei and three metaphases showed only one signal for the probe spanning the TET2 gene in this patient. Interphase FISH with the TET2 probe was performed in nine additional cases not analyzed by SNP arrays due to a lack of material, but no additional case showing a deletion was detected. In addition to FISH, we performed TET2 sequencing in 19/23 RARS-T patients. TET2 mutations were detected in 5/19 patients (26%), of which 3/5 also presented the JAK2V617F mutation, whereas the remaining 2/5 did neither show JAK2V617F nor MPL nor CBL mutations. The five patients showed 6 individually different TET2 mutations. Three were nonsense and two missense mutations. One patient displayed a frameshift mutation leading to a premature stop codon. In summary, RARS-T patients demonstrated a high frequency of both JAK2 and TET2 mutations. Together with the less common MPL mutations described by others RARS-T presents a variety of mutations that overlap with the spectrum of mutations seen in MPN and other myeloid malignancies. Thus, a combination of molecular markers including JAK2 and TET2 should be investigated to more precisely describe RARS-T as an independent disease entity. Disclosures Flach: MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndrome: A Retrospective Validation From a Single Centre.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4846-4846
Author(s):  
Pervin Topcuoglu ◽  
Klara Dalva ◽  
Sule Mine Bakanay ◽  
Sinem Civriz Bozdag ◽  
Onder Arslan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndrome ◽  
Antigen Expression ◽  
Pathological Examination ◽  
Light Scatter ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Altered Expression ◽  
Flow Cytometric

Abstract Abstract 4846 Myelodysplastic syndrome (MDS) is heterogeneous clonal hematopoietic stem cell disorder characterized by cytopenia(s) and dysplasia in one or more cell lineage. Though flow cytometry (FCM) is an important diagnostic tool in hematopoietic cell disorders, a prominent immunophenotyping feature in MDS may not be determined. In this study, we retrospectively evaluated flow cytometric features of bone marrow samples diagnosed as MDS with clinical and hematological findings. Patients-Method Between Feb 2004 and March 2009, flow cytometric parameters of 73 patients (M/F: 50/23) with MDS were re-analyzed. Median age was 59 years (17-89 ys). Our general principles are to evaluate quality of bone marrow samples, to determine proportion of the cells and features of their light scatter, and to give percentage of the blast. When detected a finding of dysplasia in the first analysis, the second step includes the determination of the maturation of the cells and the presence of the aberrant antigen expression. Results The samples were interpreted as MDS in % 76.7, MDS-RAEB-1 or RAEB-2 in %16.4, myeloproliferative disorder in %1.4 and non-diagnostic in %6.8 of the cases by flow cytometric examination. We detected variable degrees of hypogranulation in myeloid lineage in %82.2 of the samples by the light scatter features of the cells: 85% of severe and 15% of moderate or mild hypogranulation. The ratio of myeloid and lymphoid was changing from 0.3 to 17.5 (median 2). The decreasing of this ratio (<1) was observed in 19.4% of the samples. We detected altered expression of mature granulocyte. These included decreasing or lack of expression in CD15 45/73 (61.4%), CD13 38/70 (54.3%), CD16 53/67 (79.1%), CD11b 51/71 (71.8%), CD24 44/69 (65.2%), CD10 23/72 (31.9%) and MPO 14/72 (19.4%). Besides, bright expression of CD33 in 53.5% of the samples was observed. CD36 and CD56 in myeloid lineage were co-expressed in about 50 % of the samples. In 80.8 % of the samples dysplasia in erythroid compartment could be evaluated: Expression of CD71 according to glycophorin A (ratio <1) was decreased in 23.7 %. When we made similar analysis in the samples without RAEB-1 and -2 as pathological examination of bone marrow, 13.4 % of the samples could not be evaluated in favor of dysplasia. Of the samples with dysplasia hypogranulation, aberrant antigen expression of myeloid lineage and eryhtroid dysplasia were observed in 92.1%, 34.1% and 31.5%, respectively. In conclusion, FCM events may help to the differantial diagnosis of MDS especially when combining with clinical events. Improving of the analysis by focusing on the blast characteristics may be a standard approach to evaluate for low risk MDS. Disclosures No relevant conflicts of interest to declare.

In Myelodysplastic/Myeloproliferative Neoplasms Aberrant Antigen Expression As Assessed by Multiparameter Flow Cytometry Is Related to Molecular Genetic Mutations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5152-5152
Author(s):  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
Tamara Alpermann ◽  
Claudia Haferlach ◽  
Torsten Haferlach
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Molecular Genetic ◽  
Antigen Expression ◽  
Multiparameter Flow Cytometry ◽  
Employment Equity ◽  
Aberrant Expression ◽  
Equity Ownership ◽  
Diagnostic Work ◽  
Work Up ◽  
Diagnostic Work Up

Abstract Abstract 5152 Background: Immunophenotyping by multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of patients with cytopenias and suspected myelodysplastic syndromes (MDS). Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) comprise a group of diseases with some features of MDS and is separately classified in the current WHO system. While the immunophenotype of chronic myelomonocytic leukemia has been described in detail, data is scarce on the use of MFC in myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPNu) as well as on refractory anemia with ring sideroblasts and thrombocytosis (RARS-T), which is a provisional entity in the current WHO classification. Aim: To assess patients with MDS/MPNu and RARS-T for MDS-related aberrant immunophenotypes in the context of a comprehensive diagnostic work-up including cytomorphology, cytogenetics, and molecular genetics. Patients and Methods: A total of 91 patients were analyzed in parallel by cytomorphology, cytogenetics, and MFC applying an antibody panel designed to diagnose MDS. MFC was used to detect expression of mature antigens in myeloid progenitors; abnormal CD13-CD16- and CD11b-CD16-expression patterns, aberrant expression of myeloid markers and reduced side scatter signal in granulocytes; reduced expression of myelomonocytic markers in monocytes; aberrant expression of CD71 in erythroid cells; as well as expression of lymphoid markers in all myeloid cell lines. In 77/91 patients molecular genetic markers were investigated. The median age of the patients was 75.1 years (range, 35.3–87.4). The male/female ratio was 60/31. Six patients had RARS-T and 85 had MDS/MPNu. Results: In 54/91 (59.3%) patients MFC identified an MDS-immunophenotype. This was true in 4/6 (66.7%) RARS-T and in 50/85 (58.8%) MDS/MPNu (n.s.). Cases with MDS-immunophenotype displayed aberrancies significantly more frequently than those without as follows: in myeloid progenitor cells (number of aberrantly expressed antigens, mean±SD: 0.5±0.6 vs. 0.2±0.4, p=0.002), granulocytes (2.7±1.3 vs. 1.2±1.1, p<0.001), and monocytes (1.7±1.2 vs. 0.5±0.7, p<0.001). Accordingly, there was a significant difference in the total number of aberrantly expressed antigens (4.9±2.4 vs. 2.0±1.4, p<0.001). The presence of an aberrant karyotype was not related to an MDS-immunophenotype which was observed in 11/18 (61.1%) cases with aberrant karyotype and in 43/73 (58.9%) with normal karyotype (n.s.). Mutations in RUNX1 and TET2 as well as FLT3-ITD were predominantly present in cases with an MDS-immunophenotype (10/33, 30.3%) and occurred less frequently in cases without (1/7, 9.1%, n.s.). In detail, RUNX1 mutations were present in 4/26 (10.3%) vs. 0/2, TET2 mutations were present in 4/6 (66.7%) vs. 1/2 (50%), and FLT3-ITD was present in 3/29 (10.3%) vs. 0/5. Accordingly, in cases with RUNX1 or TET2 mutations or with FLT3-ITD a significantly higher number of aberrantly expressed antigens was observed as compared to cases with none of these mutations (mean±SD, 6.4±2.0 vs. 4.4±2.5, p=0.024). In contrast, JAK2V617F mutations occurred at identical frequencies in patients with and without MDS-immunophenotype (11/38, 28.9% vs. 9/31, 29.0%). Regarding prognosis, the presence of an MDS-immunophenotype had no impact on overall survival. Conclusions: These data demonstrates that MDS-related aberrant antigen expression is present in the majority of patients with RARS-T and MDS/MPNu. While there is no association between the presence of an MDS-immunophenotype and the detection of JAK2 mutations cases with an MDS-immunophenotype tended to more frequently carry mutations in RUNX1 and TET2 as well as FLT3-ITDs. These data therefore suggests that MDS/MPNu may be subdivided based on molecular genetics and on the immunophenotype into cases with MDS-related features and those without. Further analyses are needed to validate these findings and their potential significance in RARS-T. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Use Of Flow Cytometry To Identify Myelodysplasia In Peripheral Blood

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2774-2774
Author(s):  
Wolfgang Kern ◽  
Richard Schabath ◽  
Tamara Alpermann ◽  
Claudia Haferlach ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Antigen Expression ◽  
Employment Equity ◽  
Aberrant Expression ◽  
Equity Ownership ◽  
Diagnostic Work ◽  
Work Up ◽  
Diagnostic Work Up

Abstract Background Flow cytometry (FC) is increasingly used in diagnostic work-up of bone marrow (BM) from patients with suspected or proven myelodysplastic syndrome (MDS). Data on FC in peripheral blood (PB) is scarce. Aims Evaluate the use of FC for PB in suspected or proven MDS by comparison to BM analyzed during follow-up. Methods PB of 157 patients (pts) with suspected MDS was analyzed by FC applying ELN criteria defined recently for diagnosis of MDS in BM (Westers et al., Leukemia 2012). For all pts during follow-up at least one BM sample was evaluable by morphology, cytogenetics, and FC in parallel to confirm or exclude MDS (according to WHO 2008 criteria). Pts were then grouped according to results obtained from BM analysis during follow-up time points into 1) proven MDS (n=96), 2) no MDS (n=32), and 3) MPN, MDS/MPN, or “MDS possible” (presence of dysplastic features by morphology but not sufficient to diagnose MDS) (n=29) (median time to MDS confirmation, 0.9 months, range, 0.1-53.0; median time to last BM assessment without confirmation of MDS; 0.8 months, range, 0.2-23.0). Results First, results of FC on PB were compared between pts with finally proven MDS (n=96) by BM vs. those with no MDS by BM as diagnosed during follow-up. All 34 pts with myeloid progenitor cells (MPC) by FC in PB had finally proven MDS. However, in addition 62/94 (66.0%) of those without MPC (p<0.0001) also had proven MDS. Thus, the presence of MPC in PB was at least strongly indicative of MDS while there were also cases with MDS without MPC in PB. Moreover, besides the presence of MPC in PB, 17 of these 34 cases in addition displayed an aberrant antigen expression on MPC. Focusing on granulocytes we first analyzed side-scatter (SSC) signals in granulocytes as ratio of mean SSC signals granulocytes/lymphocytes (G/L). While for BM samples a reduced SSC ratio G/L had been described which reflects hypogranulation, we indeed found similar data for PB with a significantly lower SSC ratio G/L in pts with proven MDS as compared to those without (mean±SD 5.7±1.1 vs. 6.3±1.0, p=0.015). More strict, a mean SSC ratio G/L of 3.9 was found to most specifically identify pts with MDS: all 6 cases with a ratio <3.9 had MDS. Regarding aberrant antigen expression in granulocytes, MDS was more frequently diagnosed among cases with vs. without the following features: aberrant CD11b/CD16 expression pattern (43/46 investigated, 93.5% vs. 53/82, 64.6%; p=0.0002), lack of CD10 expression (37/43, 86.0% vs. 59/85, 69.4%; p=0.052), CD56 expression (19/21, 90.5% vs. 77/107, 72.0%; p=0.098). Cumulating this data, ≥2 aberrantly expressed antigens on granulocytes were found indicative of MDS: 42/45 (93.3%) of pts with aberrant expression of ≥2 antigens had MDS while only 54/83 (65.1%) of those with 0 or 1 aberrantly expressed antigen had finally proven MDS (p=0.0003). Regarding aberrant antigen expression in monocytes, pts with the following features more frequently had MDS as compared to those without: reduced expression of HLA-DR, CD13, CD11b, or CD15, aberrant expression of CD2 or CD34 (as single makers all n.s.). However, cumulating this data also resulted in a significant relation to a diagnosis of MDS during follow-up: 31/36 (86.1%) of pts with aberrant expression of ≥2 antigens on monocytes were diagnosed MDS vs. 65/92 (70.7%) of those without (p=0.052). Integrating the data for the different cell compartments, pts were separated according to the presence of the following 4 criteria: 1) presence of MPC in PB by FC, 2) aberrant expression of ≥1 antigen in MPC in PB, 3) aberrant expression of ≥2 antigens in granulocytes in PB, and 4) aberrant expression of ≥2 antigens in monocytes in PB: 68/76 (89.5%) of pts with ≥1 of these criteria had MDS, which was the case in 28/52 (53.8%) of cases fulfilling none of these criteria (p<0.0001). Strengthening the selection to presence of ≥2 of the criteria, all such 36 cases had MDS which was true for 60/92 (65.2%) of those with ≤1 criterion (p<0.0001). Applying these criteria to the set of remaining 29 pts with MPN, MDS/MPN, or possible MDS, 17 (58.6%) of them fulfilled ≥1 criterion which was true for 8/32 (25.0%) of pts not diagnosed MDS (p=0.010). Conclusions FC reveals MDS-related findings in PB samples using a specific panel targeting 10 antigens and may be used to identify pts with a high probability of MDS. Further studies with direct comparison of PB and BM should clarify the role of PB analysis by FC in the diagnostic work-up of pts with suspected MDS. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schabath:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Characterization of MYD88 mutated Lymphoplasmacytic Lymphoma in Comparison to MYD88 mutated Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 132-132
Author(s):  
Constance Regina Baer ◽  
Frank Dicker ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Claudia Haferlach
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Lymphocytic Leukemia ◽  
Bone Marrow Infiltration ◽  
Multiparameter Flow Cytometry ◽  
Employment Equity ◽  
Cytogenetic Aberration ◽  
Highly Sensitive ◽  
Equity Ownership

Abstract Introduction: MYD88 (Myeloid Differentiation Primary Response 88) mutations are the most common genetic aberration in Waldenström's macroglobulinemia/lymphoplasmacytic lymphoma (LPL). Since the initial description of MYD88 mutations in LPL, the detection has gained great importance in diagnosing the disease. However, in some patients with other B cell malignancies, including chronic lymphocytic leukemia (CLL), MYD88 mutations are detectable. Aim: We describe the molecular and cytogenetic profile of MYD88 mutated LPL in comparison to CLL, in order to identify aberration patterns potentially useful for diagnostic purposes. Patients and Methods: We analyzed bone marrow samples of 78 LPL patients for MYD88 by highly sensitive allele specific PCR (ASP) for the L265P mutation and by next-generation sequencing (NGS) for MYD88 and CXCR4 (Chemokine (C-X-C Motif) Receptor 4) mutations. For CLL, 784 blood or bone marrow samples were sequenced for MYD88 (by NGS), IGHV, TP53, NOTCH1 and SF3B1 by Sanger or NGS as well as the MYD88 mutated CLL cases for CXCR4. For all samples, cytogenetic and multiparameter flow cytometry data was available. Results: In LPL, 68/78 patients (87%) harbored a MYD88 mutation. In 13 cases with low bone marrow infiltration (median: 3%; range: 1-6%), the MYD88 mutation was detected by ASP only and not by NGS. However, one case was identified by NGS only because of a non-L265P mutation, which cannot be detected by ASP (1/68; 1%). In contrast, in CLL only 17/784 (2%) carried a MYD88 mutation. Interestingly, 5/17 (29%) were non-L265P mutations. Of the MYD88 mutated LPL, 17/68 (25%) carried a genetic lesion in the C-terminal domain of CXCR4. In contrast to MYD88, the mutation spectrum of CXCR4 was much broader including non-sense mutations at amino acid S338 (10/18) but also frame shifts resulting in loss of regulatory serine residues. One patient had two independent CXCR4 mutations (S338* and S341Pfs*25). The mean bone marrow infiltration by flow cytometry was 14% and 9% in the CXCR4 mutated and unmuted subsets, respectively (p=0.17). Besides molecular genetic aberrations, 25% (17/68) of MYD88 mutated LPL cases carried cytogenetic aberration. The most frequent cytogenetic aberration in the MYD88 positive LPL was the deletion of 6q (10/68; 15%). Other recurrent cytogenetic abnormalities were gains of 4q (n=3), 8q (n=2), and 12q (n=4), as well as loss of 11q (n=4), 13q (n=2) and 17p (n=3). In the MYD88 unmutated group, we did neither identify any CXCR4 mutation nor any del(6q), suggesting different genetic driver events in this LPL subcohort. Importantly, in the MYD88 positive CLL cohort, cytogenetic analysis did not reveal any patient with del(6q). Instead, del(13q)(q14) was the most prevalent cytogenetic aberration (12/17; 71%). Neither 11q deletions nor 17p deletions were detected. All MYD88 positive CLL had a mutated IGHV status (MYD88 unmutated CLL: 453/767; 59%; P<0.001). The TP53, NOTCH1 and SF3B1 mutational landscape did not reveal any differences between the MYD88 mutated and unmutated cohort. Finally, CXCR4 mutations were present in none of 15 analyzed MYD88 mutated CLL cases. Conclusion: Besides multiparameter flow cytometry, MYD88 mutations are the most powerful tool in the diagnosis of LPL. MYD88 mutated LPL are characterized by a high frequency of CXCR4 mutations and del(6q), while MYD88 unmutated LPLs are associated with a different pattern of genetic abnormalities. MYD88 mutated CLL is a distinct CLL subset associated with mutated IGHV status, a high frequency of 13q deletions and low frequencies of 11q and 17p deletions. MYD88 mutated CLL differs from MYD88 mutated LPL with respect to the pattern of MYD88 mutations, cytogenetic aberrations and the absence of CXCR4 mutations. Highly sensitive ASP allows the L265P mutation detection even in LPL cases with very low bone marrow infiltration; whereas highly sensitive NGS assay are best applicable for detection of more heterogenic MYD88 mutations in CLL or CXCR mutations in LPL. Thus, an integrated molecular and cytogenetic approach allows the characterization of disease specific genetic patterns and should be analyzed for its clinical impact. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals Multiparameter flow cytometry reveals myelodysplasia-related aberrant antigen expression in myelodysplastic/myeloproliferative neoplasms

2013 ◽  
Vol 84B (3) ◽  
pp. 194-197 ◽  
Author(s):  
Wolfgang Kern ◽  
Ulrike Bacher ◽  
Susanne Schnittger ◽  
Tamara Alpermann ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Myeloproliferative Neoplasms ◽  
Antigen Expression ◽  
Multiparameter Flow Cytometry

The Recently Suggested MDS/MPN Subgroup “Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis (RARS-T)” Demonstrates Unique Clinical, Cytogenetic, and Molecular Aspects: A Comprehensive Analysis of 57 Cases Strictly Defined According to WHO Criteria

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3081-3081
Author(s):  
Ulrike Bacher ◽  
Johanna Flach ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Who Classification ◽  
Normal Karyotype ◽  
Favorable Outcome ◽  
Refractory Anemia ◽  
Employment Equity ◽  
Who Criteria ◽  
Ring Sideroblasts ◽  
Equity Ownership ◽  
Wbc Count ◽  
Who 2008

Abstract Abstract 3081 Introduction: In the new WHO 2008 classification, “refractory anemia with ring sideroblasts associated with marked thrombocytosis” (RARS-T) represents a provisional entity defined by platelets ≥450 ×109/l (being lowered from 600 ×109/l; WHO 2001), proliferation of large megakaryocytes, bone marrow (BM) blasts <5%, and ring sideroblasts ≥15% of nucleated erythropoiesis. The separation of RARS-T from other myeloproliferative/myelodysplastic neoplasms is still under debate. Patients: To further characterize this subtype and to evaluate whether its separate position in the WHO classification is justified from biologic/genetic aspects, we analyzed 57 patients with a diagnosis of RARS-T (strictly defined according to WHO 2008 criteria) for peripheral blood parameters, BM morphology, cyto-/molecular genetics, and clinical profiles. The study cohort consisted of 34 females and 23 males (median age, 76 years, range, 51–92 yrs; 52 de novo; 5 therapy-associated). At the time of analysis, all pts were at diagnosis or therapy naïve. Patients with a sole del(5q) or >5% of blasts were excluded according to WHO criteria. Methods: All BM samples underwent May Giemsa Gruenwald and iron stainings. Chromosomal banding analysis (and FISH if needed) were performed in 56/57 cases. PCR was done for the following markers: JAK2V617F (investigated: n=47), MPLW515 (n=46), NRAS (n=24), TET2 mutations (TET2mut, n=14), MLL-PTD (n=13), FLT3-ITD (n=12), and CBL (n=16). Result: Median WBC count was 7.9 ×109/l (range, 3.1–60.0 ×109/l), median hemoglobin (Hb) level was 10 g/dl (range, 6–13 g/dl), and median platelet count was 572 ×109/l (range, 454-1, 737 ×109/l). The median ring sideroblast count was 60% (range, 18–92%). Karyotypes (KT) were as follows: normal KT: n=52 (52/56; 93%); +8: n=2; -Y: n=1. The most frequent mutation was the JAK2V617F (18/47; 38%); an MPLW515 mutation was detected in 3/46 (7%). From the 46 pts being analyzed both for the JAK2 as for the MPLmut, 21 (45.6%) were observed with one of both markers; there was no coincidence of the JAK2 and the MPL mutations. Furtheron, 5/14 (36%) had a TET2 mutation. Coincidences of molecular markers were observed in 3 pts who had a JAK2V617F and a TET2mut in parallel (TET2mut: 3/10; 30% in JAK2mut pts; vs. 2/4 in JAK2 wildtype pts; n.s.). No patient had a JAK2V617F and MPLW515 in parallel. There was no mutation of the NRAS, MLL-PTD, FLT3, or CBL genes in pts investigated for these markers. A positive JAK2V617F mutated status correlated significantly with higher platelets (p=0.038; T-test), whereas no significant correlations were observed for the respective medians taken as thresholds for leukocytes (≥7.9 ×109/l vs. <7.9 ×109/l vs.), Hb (≥10.0 g/dl vs. <10.0 g/dl), or ring sideroblast percentages (≥60% vs. <60%). All 3 pts with MPLW515mut had platelets ≥600×109/l. Cytogenetic aberrations were independent from the JAK2mut status (normal karyotype: 17/45 JAK2mut; 38%; vs. aberrant KT: 1/2 JAK2mut; n.s.) and the MPLmut status (normal KT: 3/44 MPLmut; 7%; vs. aberrant KT: 0/2; n.s.). Higher WBC count (≥7.9 ×109/l) was correlated to a higher Hb level (≥10 g/dl) (p=0.47) and to higher platelets (≥600 ×109/l) (p=0.011). The patients with RARS-T had a favorable outcome with 84.6% being alive at 2.5 years. Conclusion: Investigation of 57 patients strictly fulfilling the criteria of the WHO 2008 classification was able to confirm the unique profile of RARS-T in all aspects: patients with the RARS-T had a normal karyotype (>90% of all cases), had no prognostically adverse cytogenetic alterations, and frequently showed mutations of the JAK2 (V617F) or MPL (W515K/L) genes (45.6% in total). The molecular profile was even more homogeneous in RARS-T cases with ≥600 ×109/l platelets (the WHO threshold from 2001) due to significantly higher proportions of JAK2V617F positive cases when compared to cases with platelets between 450 and <600 ×109/l. However, from clinical aspects, patients with RARS-T had a favorable outcome in our study independent of the molecular state or the number of platelets. These data support to include RARS-T as definite subtype in the next edition of the WHO classification. The frequent occurrence of TET2 mutations in our cohort has to be noted for future diagnostic and classification approaches. Therefore, in cases suspicious for RARS-T but without evidence of a JAK2V617F, molecular screening should be performed including analysis for alterations of the TET2 and MPL genes. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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