Integrated Diagnostic Approach for Suspected Myelodysplastic Syndrome As a Basis for Advancement of Diagnostic Criteria

Abstract Background: Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases diagnosed and classified based on cytomorphology and cytogenetics according to the WHO classification. Flow cytometry and mutation analysis may provide additional diagnostic potential. Aim: To correlate the diagnostic results derived from flow cytometry and mutation analysis with those of cytomorphology and cytogenetics in patients with suspected MDS. To estimate the impact of these findings on the cytomorphologic reevaluation during follow up. Methods: Between February 2008 and July 2016 bone marrow samples from a total of 1681 patients with cytopenias and suspected MDS were prospectively analyzed by a combined diagnostic approach. This included in all cases cytomophology and cytochemistry, cytogenetics based on chromosome banding analysis supplemented by FISH analysis, flow cytometric assessment according to ELN criteria (Westers et al., Leukemia 2012) and mutation analysis for ASXL1, EZH2, RUNX1 and TP53which represent the prognostically most important molecular markers both in the pivotal study on molecular genetics in MDS (Bejar et al. NEJM 2011) and in a large multicenter study (Bejar et al., ASH 2015). Patients diagnosed with non-MDS hematologic malignancies were excluded. Patients´ age ranged from 17 to 95 years (median 72) and male:female ratio was 1.27. Results: 816/1681 (49%) patients were diagnosed with MDS based on cytomorphology. An aberrant karyotype was found in 319/1681 (19%) patients. Flow cytometry was in agreement with MDS in 889/1681 (54%) patients. The number of patients with mutations in the respective genes were 193/1681 (12%) for ASXL1, 37 (2%) for EZH2, 84 (5%) for RUNX1 and 69 (4%) for TP53. At least one of these mutations was present in 318/1681 (19%) patients and one, two and three genes were mutated in 261 (16%), 49 (3%) and 8 (1%) patients, respectively. Comparison between cytomorphology and flow cytometry revealed concordant results in 1300 (77%) patients (both positive for MDS in 667 (40%) and both negative for MDS in 633 (38%) patients). Cytomorphology diagnosed MDS while flow cytometry was negative (C+F-) in 149 (9%) cases and flow cytometry was in agreement with MDS while cytomorphology was negative (F+C-) in 232 (14%) cases. Analyzing genetic results in these discordant cases revealed an aberrant karyotype in 34/149 (23%) of C+F- cases and in 30/232 (13%) of F+C- cases, respectively. At least one of the four analyzed genes was found mutated in 19/149 (13%) of C+F- cases and in 37/232 (15%) of F+C- cases, respectively. Combining these findings, an aberrant karyotype or at least one mutated gene were found in 45/149 (30%) of C+F- cases and in 55/232 (24%) of F+C- cases, respectively. In contrast, in cases rated MDS by both cytomorphology and flow cytometry (C+F+) an aberrant karyotype or at least one mutated gene were found in 354/667 (53%) cases while this was true for 61/633 (10%) C-F- cases only (p<0.001). Follow-up analyses of bone marrow samples by cytomorphology were available for 116 cases initially not diagnosed with MDS by cytomorphology. 40 of them were initially rated in agreement with MDS by flow cytometry. Median follow-up time was 1.0 year. In 29 patients MDS was diagnosed by cytomorphology at follow-up. In the total of 116 patients with follow-up analyses the Kaplan-Meier estimate of probability of MDS was 40% at 2 years. Probability of MDS at 2 years was non-significantly higher in cases initially rated in agreement with MDS by flow cytometry as compared to others (48% vs. 35%). The respective impact of the presence of an aberrant karyotype or at least one mutated gene was even higher (2 year probability of MDS 71% vs. 23%, p<0.001). Combining flow cytometric and genetic results revealed the highest probability of MDS in case of positivity for both (F+G+, 81% at 2 years), followed by G+F- (65%), F+G- (29%) and F-G- (20%, p=0.002). Conclusion: In patients with cytopenia not diagnosed with MDS by cytomorphology the presence of cytogenetic aberrations and molecular mutations typically associated with MDS reveals a high probability of development of MDS, particularly if in parallel flow cytometric evaluation is in agreement with MDS. Further study is warranted aiming at a respective extension of diagnostic criteria. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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SNP/CGH Microarray Analysis in MDS: Correlation with Conventional Cytogenetic, FISH and Flow Cytometry Findings

Blood ◽

10.1182/blood.v124.21.5592.5592 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5592-5592

Author(s):

Barbara Katharina Zehentner ◽

Lisa Eidenschink Brodersen ◽

Christine F Stephenson ◽

Jevon Cutler ◽

Monica E. de Baca ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Microarray Analysis ◽

Cytogenetic Analysis ◽

Fish Analysis ◽

Employment Equity ◽

Conventional Cytogenetic Analysis ◽

Flow Cytometric ◽

Equity Ownership ◽

Gains And Losses

Abstract Background: Single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) microarray analysis is a powerful tool to assess myelodysplastic bone marrow specimens for the presence of genomic gains and losses as well as loss of heterozygosity (LOH) (reviewed by Nybakken & Bagg, JMD 2014). Its application can be a valuable addition to conventional cytogenetic analysis and may be superior to FISH testing for MDS assessment. Currently, microarray analysis does not have widespread use in an MDS work-up. Several groups have demonstrated that flow cytometric analysis can detect phenotypic aberrations in bone marrow aspirates with cytopenias with more abnormalities identified in patients with poor prognosis or with multiple genotypic abnormalities (Loken et al. 2008; Cutler et al. 2011; van de Loosdrecht et al. 2013). In this study SNP microarray results were compared with conventional cytogenetic and MDS panel FISH findings as well as phenotypic abnormalities detected by flow cytometry. Patients and Methods: 185 bone marrow aspirate specimens submitted to our laboratory for MDS work-up were analyzed by SNP/CGH studies. 36 of these (19.5%) were positive by SNP/CGH microarray analysis. 32 of the positive microarray cases (88.9%) were also analyzed by conventional cytogenetic studies, 35 (97.2%) by MDS FISH panel (5p, 7q, +8, -17p, -20q) and 31 (86.1%) were assessed by multidimensional flow cytometry (FCM) and were assigned an FCSS score (Wells et al. 2003). Results: Of the specimens in which the SNP/CGH array demonstrated genotypic abnormalities, 11/32 (34.4%) were negative by conventional cytogenetic analysis while 12/35 (34.3%) showed no abnormalities by MDS FISH panel analysis. SNP/CGH analysis revealed additional chromosomal gains and losses in 18/32 (56%) in comparison to cytogenetic analysis and in 22/35 (63%) in comparison to FISH analysis. Loss of Heterozygosity regions were detected in 28/36 cases (78%) with 96.4% (27/28) of these being larger than 2 Mb and 53% (19/28) spanning a significant chromosomal region (e.g. 1p, 5q, 7q and 17p) with known oncogenic and other MDS related genes. In 10/32 cases (31%), microarray analysis was able to characterize the origin of marker chromosome material, previously reported with unknown identity by conventional cytogenetic analysis. In an additional subset of 10 out of 32 cases (31%), cytogenetic analysis was able to either characterize balanced translocations or low level sub-clonal abnormalities not identified by microarray analysis alone. In 11/36 (31%) microarray analysis was able to detect clonal heterogeneity and evolution. In none of the specimens did FISH analysis detected abnormalities not revealed by microarray analysis. Flow cytometry performed on 31 of the array positive specimens revealed 6 to have >20% abnormal myeloid progenitor cells (classified as AML) while 23 the remaining 25 cases showed phenotypic abnormalities consistent with MDS (FCSS ranging from 1-6). In two specimens with a FCSS of 0, LOH regions on 16q or 1p and 21q were found, respectively, without the presence of numerical aberrations. A FCSS score of 1 with minimal phenotypic abnormalities (n=3), was comprised of one specimen with del(5q), one with LOH of 7q and one with trisomy 8, 1p loss and 1q gain. Specimens with an FCSS of 2 (n=7) showed only one specimen classified as complex (5 or more abnormalities). The two FCSS =3 specimens showed del(5q) with del(12p) and several LOH regions, not complex findings. One of the 4 specimen with FCSS = 4 was classified as complex while the other 3 specimens showed monosomy 7, LOH of 7q or LOH of 1p, respectively. Genotypic abnormalities were also related to phenotypic abnormalities in 4/7 (57%) specimens in the FCSS = 5/6 category which revealed complex microarray findings. Half (3/6) of the AML class had complex findings as well. Conclusions: These results emphasize the additional value that CGH/SNP microarray analysis adds to conventional cytogenetic analysis. Our dataset confirms that FISH studies do not provide additional information for MDS specimens positive by cytogenetic and/or microarray analysis. Most importantly, a high correlation between our phenotypic flow cytometric scoring system for myeloid abnormalities and microarray findings has been identified. Higher flow cytometric abnormality scores correlate with increasing complexity of genomic abnormalities. Disclosures Zehentner: HematoLogics Inc.: Employment, Equity Ownership. Brodersen:Hematologics Inc.: Employment. Stephenson:Hematologics Inc.: Employment. de Baca:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Hammock:Hematologics Inc.: Employment. Johnson:Hematologics Inc.: Employment. Hartmann:Hematologics Inc.: Employment. Loken:Hematologics Inc.: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership.

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Deep Sequencing Approach for Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.1388.1388 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1388-1388

Author(s):

Malek Faham ◽

Jianbiao Zheng ◽

Martin Moorhead ◽

Victoria Carlton ◽

Patricia Lee Stow ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Research Funding ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Clonal Evolution ◽

Employment Equity ◽

Equity Ownership ◽

Minimal Residual

Abstract Abstract 1388 Background: The clinical management of patients with acute lymphoblastic leukemia (ALL) relies on accurate prediction of relapse hazard to determine the intensity of therapy and avoid over- or under-treatment.1 The measurement of minimal residual disease (MRD) during therapy has now emerged as the most important predictor of outcome in ALL.2 We developed the LymphoSIGHT platform, a high-throughput sequencing method, which universally amplifies antigen-receptor gene segments and can identify all leukemia-specific sequences at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In this study, we determined the sensitivity and specificity of this method, delineated the extent of clonal evolution present at diagnosis, and compared its capacity to measure MRD to that of flow cytometry and allele-specific oligonucleotide PCR (ASO-PCR) in follow-up samples from >100 patients with ALL. Methods: Using the sequencing assay, we analyzed diagnostic bone marrow samples from 100 ALL patients for clonal rearrangements of immunoglobulin (IgH@) and T cell receptor (TRB@, TRD@, TRG@) genes, as well as the extent of clonal evolution present at diagnosis. We assessed the capacity of the sequencing assay to detect MRD using diagnostic samples from 12 ALL patients carrying 13 leukemic IgH clonal rearrangements. Serial dilutions were prepared in normal peripheral blood mononucleated cells, at a range between <1 in 1 million to >1 in 1,000 cells. We also assessed MRD in follow-up samples from 106 ALL patients and analyzed concordance between MRD results obtained by the sequencing assay, flow cytometry and ASO-PCR. Results: In diagnostic bone marrow samples, we detected the presence of a high-frequency clonal rearrangement of at least one receptor (“calibrating receptor”) in all the 100 ALL samples; 94 samples had at least 2 calibrating receptors at diagnosis, with 51 having 3 or more. We also detected a variable degree of clonal evolution: the number of evolved clones in each sample ranged from 0 to 6933, with 39 (37%) samples having 1–50 evolved clones and 17 (16%) >50 (Figure 1). In experiments with mixtures of normal and leukemic cells, the sequencing assay unequivocally and accurately detected leukemic signatures in all dilutions up to a concentration of at least one leukemic cell in 1 million leukocytes. In direct comparisons with established MRD assays performed on follow-up samples from patients with B-ALL, sequencing detected MRD in all 28 samples positive by flow cytometry, and in 35 of the 36 positive by ASO-PCR; it also revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively (Figure 2). Conclusions: The sequencing assay is precise, quantitative, and can detect MRD at levels below 1 in 1 million leukocytes (0.0001%), i.e., represents sensitivity 1–2 orders of magnitude higher than standard flow cytometric and ASO-PCR methods. Our assay also allows monitoring of all leukemic rearrangements regardless of their prevalence at diagnosis, which abrogates the risk of false-negative MRD results due to clonal evolution. Finally, the sequencing assay utilizes a set of universal primers and does not require development of patient-specific reagents. These data, together with the results of our comparison with standard MRD assays in clinical samples, strongly support the use of the sequencing assay as a next-generation MRD test for ALL. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Research Funding. Zheng:Sequenta: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta: Employment, Equity Ownership, Research Funding. Carlton:Sequenta: Employment, Equity Ownership, Research Funding.

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A Comparative Assessment of Flow Cytometric Scoring Systems in MDS

Blood ◽

10.1182/blood.v124.21.5589.5589 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5589-5589 ◽

Cited By ~ 2

Author(s):

Lisa Eidenschink Brodersen ◽

Andrew Menssen ◽

Barbara Katharina Zehentner ◽

Christine F Stephenson ◽

Monica E. de Baca ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Sensitivity And Specificity ◽

Scoring Systems ◽

Employment Equity ◽

Flow Cytometric ◽

Specificity And Sensitivity ◽

Equity Ownership ◽

Clonal Abnormalities ◽

Myeloid Progenitors

Abstract Background:Flow cytometric studies are useful in the diagnostic workup of patients with unexplained cytopenias and it has been demonstrated that bone marrow aspirates with immunophenotypic abnormalities by flow cytometry but not diagnostic morphologic or cytogenetic findings frequently evolve into myelodysplastic syndromes (MDS) (Kern 2013). Two flow cytometric scoring systems (FCSSs), the Wells FCSS and the Ogata FCSS, have diagnostic and prognostic utility. The Wells FCSS utilizes a difference from normal algorithm incorporating more than ten phenotypic parameters. The accumulation of these abnormalities is not only useful in diagnosis but is predictive of patient outcome (Wells 2003, Scott 2008, Alhan 2014). The recommended Ogata FCSS has evolved to include four cardinal parameters: (1) CD45 intensity on the myeloid progenitors, (2) frequency of lymphoblasts, (3) frequency of myeloid progenitors, and (4) granularity of the maturing myeloid cells. The Wells FCSS is more comprehensive as it uses more phenotypic characteristics, while the Ogata score is considered straightforward to implement in a routine setting (Della Porta 2012, Ogata 2009). This study compares the Wells FCSS and Ogata FCSS for sensitivity and specificity to detect clonal abnormalities documented by SNP/CGH microarray and conventional cytogenetics. Patients and Methods: The cohort included 99 patients with unexplained cytopenias whose bone marrow aspirates were submitted for SNP/CGH microarray and flow cytometry (HematoLogics). The immunophenotypic data were independently assigned a Wells FCSS (Cutler 2012) and an Ogata FCSS (Della Porta 2012). SNP/CGH microarray was assessed for MDS-associated genetic abnormalities. The findings were further correlated with conventional cytogenetic findings. Results: Of the 99 bone marrow aspirates, 20 exhibited clonal abnormalities associated with MDS. The Wells FCSS identified immunophenotypic abnormalities suggestive of MDS for 18 of 20 CGH positive specimens (sensitivity of 90%) and did not detect phenotypic abnormalities suggestive of MDS in 68 of 79 CGH negative specimens (specificity of 86%). In contrast the Ogata FCSS identified immunophenotypic abnormalities suggestive of MDS for 13 of 20 CGH positive specimens (sensitivity of 65%) and did not detect phenotypic abnormalities suggestive of MDS in 64 of 79 the CGH negative specimens (specificity of 81%). In an attempt to improve the sensitivity and specificity of the Ogata score, the granularity parameter was modified from side scatter channel mode of the granulocytes (compared to the side scatter mode of the lymphocytes) to the side scatter channel at the 15thpercentile of granulocytes (compared to the mean of lymphocytes). This modified parameter detected all specimens defined as hypogranular by the side scatter mode, and detected an additional 11 specimens as hypogranular. All of these specimens were detected as hypogranular by the Wells definition. This modified granularity method was then used along with the other three cardinal parameters to create a modified Ogata FCSS. The granularity modification resulted in improved sensitivity (70% versus 65%); specificity was unchanged. While the modified method outperformed the original, it did not match the performance of the Wells FCSS. Conclusions: In patients with unexplained cytopenias, the Wells FCSS demonstrates superior specificity and sensitivity than the Ogata FCSS for detecting myeloid immunophenotypic clones associated with SNP/CGH array and cytogenetic abnormalities. Modifying the Ogata granularity parameter marginally improves the sensitivity but does not improve the specificity. Implementation of the Wells FCSS requires a comprehensive understanding of phenotypic intensities and relationships in non-clonal hematopoiesis for patients with cytopenias. While the relative ease of implementing the Ogata FCSS is attractive, improvements are essential for diagnostic accuracy; improving the granularity parameter alone is not sufficient. Adding measurements for the maturing myeloid and erythroid compartments may increase the diagnostic utility of the Ogata FCSS but requires further study. Disclosures Brodersen: Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Zehentner:HematoLogics Inc.: Employment, Equity Ownership. Stephenson:Hematologics Inc.: Employment. de Baca:Hematologics Inc.: Employment. Johnson:Hematologics Inc.: Employment. Singleton:Hematologics Inc.: Employment. Hartmann:Hematologics Inc.: Employment. Loken:Hematologics: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership.

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Combined Impact of Aberrant Immunophenotypes, Somatic Mutations and Cytogenetic Aberrations on the Probability of Developing Myelodysplasia in Patients with Cytopenias of Undetermined Significance

Blood ◽

10.1182/blood-2018-99-118244 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 795-795

Author(s):

Wolfgang Kern ◽

Manja Meggendorfer ◽

Constance Baer ◽

Claudia Haferlach ◽

Torsten Haferlach

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Median Time ◽

Somatic Mutations ◽

Employment Equity ◽

Cytogenetic Aberrations ◽

Diagnostic Potential ◽

Equity Ownership ◽

Undetermined Significance

Abstract Background: Myelodysplastic syndromes (MDS) are diagnosed according to WHO based on cytomorphology and cytogenetics. Various conditions without dysplasia have been described which are associated with an increased although low risk of developing myeloid malignancy (Steensma et al. Blood 2015): non-clonal idiopathic cytopenias of undetermined significance (ICUS, cytopenias and no mutations), clonal hematopoiesis of indeterminate potential (CHIP, mutations and no cytopenias) and clonal cytopenias of undetermined significance (CCUS, both cytopenias and mutations). The spectrum of encountered mutations resembles the one of MDS. MDS-typical immunophenotypes have been identified and are recommended by ELN in the diagnostic work-up of patients (pts) with suspected MDS (Malcovati et al., Blood 2013). Similar to mutations described in both MDS and CHIP/CCUS, MDS-typical immunophenotypes are found in cytopenic non-MDS pts. Our study aims at the determination of the diagnostic potential of both of these findings. Aims: To determine the impact of aberrant immunophenotypes, somatic mutations and cytogenetic aberrations in the absence of dysplasia on the later diagnosis of MDS. Patients and methods: Between 2005 and 2018 a total of 2279 bone marrow samples of pts with suspected MDS were analyzed by a combination of cytomorphology and cytogenetics as well as NGS and flow cytometry. NGS targeted the following genes in all pts: ASXL1, CBL, DNMT3A, EZH2, JAK2, RUNX1, SF3B1, SRSF2, TET2, TP53 and U2AF1. Further genes analyzed in subsets of pts only were BCOR, CSF3R, CSNK1A1, ETV6, GATA1, GATA2, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, PTPN11, SETBP1, WT1 and ZRSR2. Flow cytometry was performed according to ELN standards (Westers et al., Leukemia 2012). For 368 pts at least one follow-up bone marrow analysis was available, these pts are the subject of the present study. Patients ages ranged from 20 to 90 years (median 70 years), male:female ratio was 1.9:1. Results: 277/368 (75%) pts were diagnosed with MDS by cytomorphology. In 263/368 (72%) of pts MDS-typical immunophenotypes were present. Findings concordant between cytomorphology and flow cytometry were present in 304/368 (83%) pts reflecting published large series. Focus of further analyses were the 91 pts without diagnosis of MDS by cytomorphology. In 34 of these 91 pts MDS was diagnosed at follow-up, median follow-up time was 16.3 months, median time to MDS was 38.5 months. At initial evaluation, in 25 of these 91 (27%) pts MDS-typical immunophenotypes were present and resulted in a shorter time to MDS as compared to those without MDS-typical immunophenotypes (median 15.9 vs. 39.9 months, p=0.012). Frequencies of mutations with impact on time to MDS were as follows: ASXL1, present in 9/91 (10%), median time to MDS ASXL1 mutated vs non-mutated 14.2 vs. 39.9 months, p=0.010; EZH2, 3/91 (3%), 7.4 vs. 39.9 months, p=0.020; JAK2, 3/91 (3%), 4.9 vs. 39.9 months, p=0.003; RUNX1, 5/91 (5%), 7.4 vs. 39.9 months, n.s.; SRSF2, 7/91 (8%), 11.9 vs. 39.9 months, p=0.008; TET2, 14/91 (15%), 14.2 vs. 57.1 months, p=0.029; TP53, 4/91 (4%), 6.5 vs. 44.1 months, p=0.007. Other genes were less frequently found mutated without an impact on the time to MDS. An aberrant karyotype was present in 9 of 89 evaluable pts and was associated with a shorter time to MDS (median 4.9 vs. 57.1 months, p<0.001). Also age was associated with a shorter time to MDS (HR 1.47 per decade, p=0.029), peripheral blood cell counts were not. Patients with two out of the three parameters 1) aberrant immunophenotype, 2) at least one mutation in the genes ASXL1, EZH2, JAK2, RUNX1, SRSF2, TET2, TP53, 3) aberrant karyotype had a significantly shorter duration to MDS as compared to those without: 1) and 2), 7.4 vs. 44.1 months, p<0.001; 1) and 3), 4.9 vs. 39.9 months, p=0.004; 2) and 3), 16.0 vs. 44.1 months, p=0.041. Patients presenting with any two out of the parameters 1), 2) and 3) had a highly significantly shorter time to MDS as compared to those presenting with only one or none: 6.5 vs. 57.1 months, p<0.0001. Conclusions: In the absence of cytomorphologic signs of dysplasia, findings commonly present in MDS patients, i.e. aberrant immunophenotypes, somatic mutations and cytogenetic aberrations, occur in patients with cytopenias of undetermined significance. The presence of two of these three findings may be considered as a pre-diagnostic state of MDS. Further studies should clarify the diagnostic potential of this approach. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Significance of Flow Cytometric and Mutational Findings in Patients with Cytopenias and Limited or No Signs of Myelodysplasia By Cytomorphology

Blood ◽

10.1182/blood.v126.23.1671.1671 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1671-1671

Author(s):

Wolfgang Kern ◽

Manja Meggendorfer ◽

Claudia Haferlach ◽

Susanne Schnittger ◽

Torsten Haferlach

Keyword(s):

Flow Cytometry ◽

Normal Karyotype ◽

Initial Assessment ◽

Employment Equity ◽

Initial Flow ◽

Flow Cytometric ◽

Equity Ownership ◽

Diagnostic Work ◽

Work Up

Abstract Introduction: The diagnosis of myelodysplastic syndromes (MDS) has been clearly defined by the WHO classification but remains a challenge in a significant number of cases with cytomorphologically borderline findings and normal karyotype. Furthermore, flow cytometry is capable of identifying MDS-specific aberrant antigen expression yet its value in these borderline cases as well as in those even without cytomorphologic findings of myelodysplasia remains to be clarified. Follow-up analyses as well as extension of diagnostic work-up to screening for molecular mutations may give further insight. Aims: Assess the significance of cytomorphologically borderline dysplastic changes and of flow cytometric MDS-related findings in the absence of a clear-cut diagnosis of MDS by screening for molecular mutations and by diagnostic reassessment during follow-up. Patients and methods: Bone marrow samples of 322 patients were assessed for suspected MDS by cytomorphology, flow cytometry and cytogenetics in parallel from 08/2005 to 11/2014 which 1) did not reveal a definite diagnosis of MDS by cytomorphology, 2) had a normal karyotype and 3) had at least one follow-up bone marrow assessment. By cytomorphology, 159 (49%) cases had borderline dysplastic findings while 163 (51%) had no sign of MDS. By flow cytometry, 138 (43%) cases had findings in agreement with MDS according to ELN criteria (Westers et al., Leukemia 2012; at least three aberrantly expressed antigens), 141 (44%) had borderline findings (one or two aberrantly expressed antigens) and 43 (13%) had no signs of MDS. A total of 699 follow-up samples were analyzed (median 2/patient). The median follow-up amounted to 3.0 years. In 147/322 patients (46%) screening for molecular mutations was performed on the initial samples, respectively, targeting a total of 20 genes (median 4 genes/patient, range 1-20). Analyzed genes were ASXL1, TET2, RUNX1, SRSF2, BCOR, DNMT3A, IDH2, NPM1, SF3B1, TP53, ZRSR2, CBL, CSF3R, ETV6, KDM6A, KRAS, MLL, SETBP1, SMC3 and U2AF1. Results: A total of 145 patients (45%) were diagnosed with MDS by cytomorphology during follow-up. The median duration until diagnosis amounted to 3.4 years. Regarding initial cytomorphology, more cases with borderline dysplastic findings were diagnosed MDS at follow-up than those without any dysplastic findings (82/159 (52%) vs 63/163 (39%), p=0.025). However, the duration until diagnosis of MDS did not differ significantly between the two groups (median 2.6 vs 3.4 years). Regarding initial flow cytometry, more cases with findings in agreement with MDS were diagnosed MDS by cytomorphology at follow-up than those without (80/138 (58%) vs 65/184 (35%), p<0.001) while there was no difference between cases with one or two aberrantly expressed antigens at initial assessment vs those with none (51/141 (36%) vs 14/43 (33%), n.s.). The duration until diagnosis of MDS significantly differed between the groups as defined by flow cytometry and was shortest in cases in agreement with MDS at initial assessment and longest in those without any aberrantly expressed antigen (median 1.9 vs 4.1 vs 5.6 years, p<0.001). Overall survival (OS) for all cases was 80% at 5 years. While initial cytomorphologic results revealed no impact on OS, patients with an initial flow cytometric result in agreement with MDS tended to have a shorter OS (5 year OS 70% vs 88%, p=0.12). Molecular screening revealed mutations in 21/147 patients (14%) at initial assessment. Mutated genes included ASXL1 (mutated in 6 patients), TET2 (6), RUNX1 (3), SRSF2 (3), as well as 2 cases each for BCOR, DNMT3A, IDH2, NPM1, SF3B1, TP53 and ZRSR2 and 1 case each for CBL, CSF3R, ETV6, KDM6A, KRAS, MLL, SETBP1, SMC3 and U2AF1. The percentage of patients with at least one mutation did not differ between cases with borderline dysplastic findings by cytomorphology as compared to those without any dysplastic findings. In contrast, significantly more cases with findings in agreement with MDS by flow cytometry had at least one mutation as compared to those with one or two aberrantly expressed antigens as well as to those with none (15/71 (21%) vs 6/58 (10%) vs 0/18, p=0.012). Conclusions: This data strongly supports the need to define the role of flow cytometry in the diagnostic work-up in suspected MDS and argues for an integrated approach with cytomorphology and cytogenetics. Implementation also of molecular data on mutations may further improve the validity of MDS diagnostics. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer: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.

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Use Of Flow Cytometry To Identify Myelodysplasia In Peripheral Blood

Blood ◽

10.1182/blood.v122.21.2774.2774 ◽

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.

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

Blood ◽

10.1182/blood.v126.23.132.132 ◽

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.

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TGFb1 Antagonist Inhibits Fibrosis in a Murine Model of Myelofibrosis

Blood ◽

10.1182/blood.v126.23.605.605 ◽

2015 ◽

Vol 126 (23) ◽

pp. 605-605 ◽

Cited By ~ 1

Author(s):

Rajasekhar NVS Suragani ◽

Pedro A. Martinez ◽

Sharon M Cawley ◽

Robert Li ◽

Robert Scott Pearsall ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Murine Model ◽

Jak2 V617f ◽

Mutant Mice ◽

Wild Type ◽

Employment Equity ◽

Bone Marrow Fibrosis ◽

Equity Ownership ◽

Marrow Fibrosis

Abstract Introduction: Myelofibrosis (MF) is a clonal stem cell disorder that originates from acquired mutations in the hematopoietic stem cells leading to abnormal kinase signaling, cell proliferation, cytokine expression, and splenomegaly and ultimately bone marrow (BM) fibrosis. Primary myelofibrosis (PMF), post-polycythemia vera (PV) MF and post-essential thrombocythemia MF are categorized under MF with overlapping disease phenotypes including progression to BM fibrosis. A genetic mutation in Janus kinase 2 (V617F) was identified as causative in ~95% PV, and ~50% of ET and PMF patients. Currently, treatment of MF patients with a JAK2 inhibitor offers symptomatic benefit, but does not alter the natural history of the disease or improve BM fibrosis. It is known that TGFβ1 is a critical regulator of fibrosis in many disease states. Elevated TGFβ1 levels were reported to be important for fibrosis in patients with MF. We hypothesize that inhibition of TGFβ1 signaling may prevent fibrosis and help reduce secondary morbidities associated with disease in MF patients. Therefore, we evaluated this hypothesis using a TGFβ1 antagonist in a murine model of MF. Methods: Transgenic JAK2 (V617F) mutant mice (MF model) and age-matched wild-type controls were used in the studies. Mice were dosed twice weekly with TGFβ1 antagonist (10 mg/kg). Complete blood counts (CBC), serum TGFβ1, bone metabolism and inflammatory cytokines levels were determined at different ages (2-12 months) during disease progression. Bone marrow and spleen cells were analyzed for different cell lineages by flow cytometry. Tissue sections were stained with H&E and reticulin to determine cellularity or degree of fibrosis respectively. Results: To understand the onset and progression of MF disease in JAK2 (V617F) mice, we initially analyzed the CBC and degree of fibrosis at various ages (2, 3, 4, 5, 8, 10 and 12 months) and compared the data with wild-type mice. These data were then correlated with the levels of TGFβ1 and other cytokines. As expected, red blood cells (RBC) and platelets were elevated in JAK2 mutant mice at all ages compared to wild-type mice, although a trend towards a progressive increase was observed between 2 to 5 months followed by a decrease from 8 to 14 months. Bone marrow fibrosis was detected starting at 5 months and worsened with age. JAK2 mutant mice displayed splenomegaly that increased as the disease progressed. Interestingly, serum levels of TGFβ1, TGFβ3 and bone metabolism cytokines (OPG, OPN, aFGF and Trance) displayed an increase at earlier ages (2-5 months) compared to the latter ages, a trend similar to RBC levels. These levels peaked during the initiation of fibrosis at 5 months. In contrast, inflammatory cytokines (such as IL6, IL-1β, and TNFα) were elevated at later ages consistent with disease progression. We initiated treatment with TGFβ1 antagonist in JAK2 (V617F) mice (N=8/treatment group) at 4 months of age, the age corresponding to elevated serum TGFβ1 levels and prior to the onset of fibrosis (at 5 months of age). Following 6 months of treatment, vehicle (VEH) treated JAK2 mutant mice displayed elevated RBC (+37.1%, P<0.001), platelets (+74.5%, P<0.001) and spleen weights (+9.5 fold, P<0.001) compared to wild-type mice. BM and spleen sections from VEH treated JAK2 mutant mice revealed severe fibrosis. TGFβ1 antagonist treatment of JAK2 mice displayed moderate effect on RBC (-8.4%, N.S) without any effect on platelet counts compared to VEH treatment. Flow-cytometry identified a reduced proportion of Ter119+ erythroid precursors in BM and spleen (-15%, P<0.05) and no change in CD41+ megakaryocytes. TGFβ1 antagonist treated mice displayed reduced spleen weights (-29%, P<0.01), and marked reduction in fibrosis in bone marrow (Figure) and spleen sections compared to VEH. Consistent with the reduction in fibrosis, TGFβ1 antagonist treated JAK2 mice displayed reduced IL-6 levels (-48.9%, P<0.05) compared to VEH treatment. Conclusion: Together, these data demonstrated that TGFβ1 levels were correlated with bone marrow fibrosis in a murine model of MF disease, and its inhibition using TGFβ antagonist reduces fibrosis, splenomegaly and inflammation in this murine model of myelofibrosis. Figure 1. Figure 1. Disclosures Suragani: Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties: No royalties. Martinez:Acceleron Pharma: Employment. Cawley:Acceleron Pharma Inc: Employment. Li:Acceleron Pharma: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties. Kumar:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties.

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Prognostic Molecular Stratification in Relapsed/Refractory Multiple Myeloma - Results of the Pomalidomide Mukseven (NCT02406222) Biomarker Trial

Blood ◽

10.1182/blood-2019-123640 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4327-4327

Author(s):

James Croft ◽

Andrew Hall ◽

Amy L Sherborne ◽

Katrina Walker ◽

Sidra Ellis ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

T Cell ◽

High Risk ◽

Real World ◽

Research Funding ◽

Unmet Need ◽

Employment Equity ◽

Equity Ownership

Background Treatment of relapsed/refractory multiple myeloma (RRMM) remains challenging as durable remissions are achieved in patient sub-groups only. Identifying patients that are likely to benefit prior to or early after starting relapse treatments remains an unmet need. MUKseven is a trial specifically designed to investigate and validate biomarkers for treatment optimization in a 'real-world' RRMM population. Design In the randomized multi-center phase 2 MUKseven trial, RRMM patients (≥2 prior lines of therapy, exposed to proteasome inhibitor and lenalidomide) were randomized 1:1 to cyclophosphamide (500 mg po d1, 8, 15), pomalidomide (4 mg days 1-21) and dexamethasone (40 mg; if ≥75 years 20 mg; d1, 8, 15, 21) (CPomD) or PomD and treated until progression. All patients were asked to undergo bone marrow (BM) and peripheral blood (PB) bio-sampling at baseline, cycle 1 day 14 (C1D14, on-treatment) and relapse. For biomarker discovery and validation, IGH translocations were profiled by qRT-PCR, copy number aberrations by digital MLPA (probemix D006; MRC Holland), GEP by U133plus2.0 array (Affymetrix), PD protein markers by IHC and PB T-cell subsets by flow cytometry for all patients with sufficient material. Primary endpoint was PFS, secondary endpoints included response, OS, safety/toxicity and biomarker validation. Original planned sample size was 250 patients but due to a change in UK standard of care during recruitment with pomalidomide becoming available, a decision was made to stop recruitment early. Results In total, 102 RRMM patients were randomized 1:1 between March 2016 and February 2018. Trial entry criteria were designed to include a real-world RRMM population, permitting transfusions and growth factor support. Median age at randomization was 69 years (range 42-88), 28% of patients had received ≥5 prior lines of therapy (median: 3). Median follow-up for this analysis was 13.4 months (95% CI: 12.0-17.5). 16 patients remained on trial at time of analysis (median number of cycles: 19.5; range 8-28). More patients achieved ≥PR with CPomD compared to PomD: 70.6% (95% CI: 56.2-82.5%) vs. 47.1% (CI: 32.9-61.5%) (P=0.006). Median PFS was 6.9 months (CI: 5.7-10.4) for CPomD vs. 4.6 months (CI: 3.5-7.4) for PomD, which was not significantly different as per pre-defined criteria. Follow-up for OS is ongoing and will be presented at the conference. High-risk genetic aberrations were found at following frequencies: t(4;14): 6%, t(14;16)/t(14;20): 2%, gain(1q): 45%, del(17p): 13%. Non-high risk lesions were present as follows: t(11;14): 22%, hyperdiploidy: 44%. Complete information on all high-risk genetic markers was available for 71/102 patients, of whom 12.7% had double-hit high-risk (≥2 adverse lesions), 46.5% single-hit high-risk (1 adverse lesion) and 40.8% no risk markers, as per our recent meta-analysis in NDMM (Shah V, et al., Leukemia 2018). Median PFS was significantly shorter for double-hit: 3.4 months (CI: 1.0-4.9) vs. single-hit: 5.8 months (CI: 3.7-9.0) or no hit: 14.1 months (CI: 6.9-17.3) (P=0.005) (Figure 1A). GEP was available for 48 patients and the EMC92 high-risk signature, present in 19% of tumors, was associated with significantly shorter PFS: 3.4 months (CI: 2.0-5.7) vs. 7.4 (CI: 3.9-15.1) for EMC92 standard risk (P=0.037). Pharmacodynamic (PD) profiling of cereblon and CRL4CRBN ubiquitination targets (including Aiolos, ZFP91) in BM clots collected at baseline and C1D14 is currently ongoing. Preliminary results for the first 10 patients demonstrate differential change of nuclear Aiolos (Figure 1C), with a major decrease in Aiolos H-scores in 7/10 patients from baseline to C1D14 and reconstitution at relapse. T-cell PB sub-sets were profiled at baseline and C1D14 by flow cytometry. Specific sub-sets increased with therapy from baseline to C1D14, e.g. activated (HLA-DR+) CD4+ T-cells, as reported at last ASH. CD4+ T-cell % at baseline was associated with shorter PFS in these analyses in a multi-variable Cox regression model (P=0.005). PD and T-cell biomarker results will be updated and integrated with molecular tumor characteristics and outcome. Discussion Our results demonstrate that molecular markers validated for NDMM predict treatment outcomes in RRMM, opening the potential for stratified delivery of novel treatment approaches for patients with a particularly high unmet need. Additional immunologic and PD biomarkers are currently being explored. Disclosures Croft: Celgene: Other: Travel expenses. Hall:Celgene, Amgen, Janssen, Karyopharm: Other: Research funding to Institution. Walker:Janssen, Celgene: Other: Research funding to Institution. Pawlyn:Amgen, Janssen, Celgene, Takeda: Other: Travel expenses; Amgen, Celgene, Janssen, Oncopeptides: Honoraria; Amgen, Celgene, Takeda: Consultancy. Flanagan:Amgen, Celgene, Janssen, Karyopharm: Other: Research funding to Institution. Garg:Janssen, Takeda, Novartis: Other: Travel expenses; Novartis, Janssen: Research Funding; Janssen: Honoraria. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Wang:Celgene Corporation: Employment, Equity Ownership. Boyd:Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Pierceall:Celgene: Employment. Thakurta:Celgene: Employment, Equity Ownership. Cook:Celgene, Janssen-Cilag, Takeda: Honoraria, Research Funding; Janssen, Takeda, Sanofi, Karyopharm, Celgene: Consultancy, Honoraria, Speakers Bureau; Amgen, Bristol-Myers Squib, GlycoMimetics, Seattle Genetics, Sanofi: Honoraria. Brown:Amgen, Celgene, Janssen, Karyopharm: Other: Research funding to Institution. Kaiser:Takeda, Janssen, Celgene, Amgen: Honoraria, Other: Travel Expenses; Celgene, Janssen: Research Funding; Abbvie, Celgene, Takeda, Janssen, Amgen, Abbvie, Karyopharm: Consultancy.

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Weekly Inotuzumab Ozogamicin in Adult Patients with Relapsed or Refractory CD22-Positive Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v120.21.2612.2612 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2612-2612 ◽

Cited By ~ 2

Author(s):

Daniel DeAngelo ◽

Wendy Stock ◽

Stephen Petersdorf ◽

Shaw-Ling Wang ◽

Angela Volkert ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Research Funding ◽

Response Rate ◽

Lymphoblastic Leukemia ◽

Hematologic Toxicity ◽

Employment Equity ◽

Inotuzumab Ozogamicin ◽

Equity Ownership ◽

Grade 3

Abstract Abstract 2612 Background: Inotuzumab ozogamicin (INO) is a humanized anti-CD22 antibody conjugated to calicheamicin, a potent cytotoxic antitumor agent. CD22 is expressed on a majority of B-cell acute lymphoblastic leukemia (ALL). An initial study suggested INO efficacy and tolerability in ALL (Lancet Oncol 2012;13:403-11). Objectives: The current phase 1, multicenter, dose-escalation study was performed to optimize the INO dose and schedule (weekly dosing) based on safety, efficacy, and pharmacokinetic data in CD22+ relapsed or refractory ALL. The safety and efficacy of INO at the recommended dose and schedule will subsequently be further evaluated in a 12-patient (pt) expanded cohort. Methods: Eligible pts were aged ≥18 y with CD22+ ALL (defined as ≥20% blasts CD22+ by flow cytometry) refractory to initial induction or in relapse (≥first relapse), with no evidence of central nervous system disease. INO was administered in 28-d cycles (see Table), with a maximum of 6 cycles. The final dose was to be determined based on both toxicity (ie, rate of dose-limiting toxicities [DLT] at each dose level) and evidence of efficacy using the EffTox V2.10 software (Biometrics 2004;60:684–693). Adverse event (AE) severity was assessed per CTCAE V3 with DLTs defined as any of the following INO-related events during Cycle 1: grade ≥4 non-hematologic toxicity; prolonged myelosuppression (absolute neutrophil count [ANC] <500/μL or platelets <25,000/μL in bone marrow) with no evidence of leukemia persisting >45 d from last dose; grade 3 non-hematologic toxicity persisting >7 d from the last dose; grade ≥3 elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST), or bilirubin persisting >7 d; or any toxicity resulting in permanent INO discontinuation. Weekly teleconferences with investigators were used to assess toxicity. Complete response (CR) was defined as <5% bone marrow blasts with absence of peripheral blasts, ANC ≥1,000/μL, platelets >100,000/μL, and no extramedullary disease; incomplete CR (CRi) was similar but permitted ANC <1,000/μL and/or platelets ≤100,000/μL. Results: We report preliminary data for 13 pts (see Table), with a median duration of follow-up of 147 d (range, 30–188 d). Median age was 56 y (range, 23–65 y), and 69% of pts were male. Five (39%) pts were in salvage 1, 2 (15%) were in salvage 2, and 4 (31%) were in salvage ≥3. Two pts had prior allogeneic stem cell transplant. Three (23%) pts were Ph+ and 7 (54%) pts had circulating blasts at baseline; median baseline WBC was 2.01×103/mm3 (range, 0.5–29.11×103/mm3). The single DLT observed to date was transient grade 4 elevated lipase occurring at INO dose level 3. The most frequent (≥10% of pts) treatment-related AEs were thrombocytopenia (31%, all grade 3/4), neutropenia (15%), and elevated ALT (15%). Treatment-related elevated AST and alkaline phosphatase were each reported for 8% of pts. Reported dose delays were due to thrombocytopenia (n = 3), neutropenia (n = 2), elevated LFT (n = 2), bacteremia, increased blood creatinine, periorbital cellulitis, and QTc prolongation (n = 1 each). Fourteen serious AEs were reported for 9 pts, including 2 cases each of febrile neutropenia and septic shock. Responses were observed across all INO doses explored to date (see Table). The preliminary response rate was 82% (9/11 evaluable pts), including 36% of pts with a CR and 45% with a CRi. Median time to response was 43 d (range, 28–56 d). Six of 9 (67%) pts who achieved CR/CRi also achieved minimal residual disease (<1 blast out of 104 mononuclear cells by flow cytometry). Seven pts discontinued treatment, including 1 each due to disease progression and an AE (acute renal failure, not treatment related), and 5 pts who proceeded to transplant. Four deaths were reported, including 1 due to disease progression and 3 due to sepsis occurring within 30 d after stem cell transplantation. Conclusions: INO had a safety profile consistent with prior reports, characterized by hematologic, gastrointestinal, and hepatic events and infection. The remarkable response rate of 82% for single-agent INO in this relapsed/refractory population warrants further exploration in CD22+ ALL. Updated results will be presented at the meeting. Disclosures: Stock: Tau for work done through the CALGB/ALLIANCE: Research Funding. Wang:Pfizer Inc: Employment, Equity Ownership. Volkert:Pfizer Inc: Employment, Equity Ownership. Vandendries:Pfizer Inc: Employment, Equity Ownership. Advani:Pfizer Inc: Consultancy, Honoraria, Research Funding.

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