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

Blood ◽  
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.

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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 299-299 ◽  
Author(s):  
Wolfgang Kern ◽  
Manja Meggendorfer ◽  
Claudia Haferlach ◽  
Torsten Haferlach
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Diagnostic Criteria ◽  
Mutation Analysis ◽  
Diagnostic Approach ◽  
Employment Equity ◽  
Flow Cytometric ◽  
Equity Ownership ◽  
Genetic Results

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.

scholarly journals A Comparative Assessment of Flow Cytometric Scoring Systems in MDS

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5589-5589 ◽  
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.

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.

TGFb1 Antagonist Inhibits Fibrosis in a Murine Model of Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 605-605 ◽  
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.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2612-2612 ◽  
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.

scholarly journals Immune Microenvironment Analysis of Bone Marrow By Mass Cytometry and RNA Sequencing in Multiple Myeloma Patients Treated with Daratumumab and Durvalumab

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3296-3296 ◽  
Author(s):  
Frances Seymour ◽  
Mary H Young ◽  
Mark Tometsko ◽  
Jamie Cavenagh ◽  
Ethan G. Thompson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Research Funding ◽  
Cell Populations ◽  
Mass Cytometry ◽  
Immune Microenvironment ◽  
Employment Equity ◽  
Equity Ownership

Abstract Introduction Relapsed and refractory multiple myeloma (RRMM) remains a challenging disease to treat due to its heterogeneity and complexity. There is an urgent need for novel combination strategies, including immunotherapy. The study of the tumour and immune microenvironment before and after treatment with combination therapy is a crucial part of understanding the underpinning of disease response. Methods Longitudinal samples of bone marrow aspirates and whole blood were collected from a phase II clinical trial, MEDI4736-MM-003 (NCT02807454) where daratumumab and durvalumab naïve patients were exposed simultaneously to both these drugs. A combination of mass cytometry (CyTOF), RNAseq and flow cytometry were performed on a subset of samples from these subjects. Specifically, paired bone marrow mononuclear cells (BMMC) samples from nine patients taken at screening and six weeks post-treatment were analysed by mass cytometry (CyTOF) using a 37-marker pan-immune panel that included both lineage and functional intracellular/extracellular markers. In addition, whole blood sample specimens were collected at screening and on treatment (8, 15, 30, and 45 days after treatment) and analysed by flow cytometry. Flow cytometry panels were designed to allow interrogation of the abundance and activation status of immune cell subsets. Finally, RNA from bone marrow aspirates at screening and C2D15 were analysed by RNA sequencing. Expression profiles from the aspirates were used to estimate cell proportions by computational deconvolution. Individual cell types in these microenvironments were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results In a heavily pre-treated population with RRMM, treatment with durvalumab and daratumumab leads to shifts in a number of key immunological populations when compared to pre-treatment. In the bone marrow, CD8 and CD4 populations rise (by CyTOF and RNAseq), while NK, DC and B cell populations fall (by CyTOF). In the bone marrow within CD8+ T lymphocyte populations, we observed a post-treatment rise in markers of degranulation (granzyme p=0.0195, perforin p=0.0078, Wilcoxon signed-rank test). This is also accompanied by a fall in PD1 expression (p=0.0078) and rise in the co-stimulatory receptor DNAM1 (p=0.0273). These changes are most marked on cells with an effector memory CD45RA+ CD8+ T cell phenotype. In the blood, similar to the bone marrow, CD8+ T cells proliferate over the course of treatment (flow cytometry). A fall in both naïve and active NK cell populations is seen following treatment in bone marrow. NK cells express high levels of CD38 and are therefore depleted by daratumumab. Those NK cells which remain have an active phenotype with increased expression of TNFa (p=0.0039) and IFNg (p=0.0195) following treatment. Across the time points sampled in peripheral blood, NK cells were also decreased and those that remained were proliferating. Dendritic cells with a tolerogenic phenotype can be identified prior to treatment and are seen to fall in abundance following treatment with durvalumab and daratumumab. Conclusions The combination of durvalumab and daratumumab leads to several immune microenvironment changes that biologically portend clinical effect. We see increases in the abundance of cell populations with functional anti-tumour activity, including granzyme B+ CD8 T cells and a reduction in PD1high T cells. Despite the treatment expectedly reducing NK cell numbers, many functionally competent NK cells remain, as evidenced by the presence of anti-tumour cytokines. This combination strategy also reduces immunosuppressive tolerogenic DCs, which suppress CD4 and CD8 T cell activity. Taken together, this suggests that this chemotherapy free, doublet treatment has the potential to up-regulate anti-tumour immunological responses, which may restore immunosurveillance mechanisms critically needed in these highly refractory patients. Disclosures Seymour: Celgene: Research Funding. Young:Celgene Corporation: Employment, Equity Ownership. Tometsko:Celgene Corporation: Employment, Equity Ownership. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Thompson:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Danziger:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Dervan:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Gribben:Acerta Pharma: Honoraria, Research Funding; Cancer Research UK: Research Funding; TG Therapeutics: Honoraria; Roche: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Unum: Equity Ownership.

scholarly journals Development and Validation of Automated Flow Cytometry System for the Diagnosis and Prediction of Molecular Abnormalities in Myelodysplastic Syndrome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1977-1977
Author(s):  
Maher Albitar ◽  
Babak Shahbaba ◽  
Sally Agersborg ◽  
Richard Chang ◽  
Adam Albitar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndrome ◽  
Univariate Analysis ◽  
Support Vector ◽  
State University ◽  
Employment Equity ◽  
Molecular Abnormalities ◽  
Cytogenetic Data ◽  
Equity Ownership

Abstract Background: Diagnosis of myelodysplastic syndrome (MDS) is not clear-cut based on morphology or flow cytometry, especially when blast count is not increased. Cytogenetics and molecular profiling remains the most important means for confirming the diagnosis of MDS. Numerous studies have attempted to use flow cytometry-based scores for the diagnosis of MDS. However, most of these scores involve subjective parameters that are difficult to standardize. We developed a flow cytometry software with a capability to automatically capture additional parameters of each gated cell population and used the generated metadata for developing an algorithm for the diagnosis and prediction of molecular abnormalities in MDS then integrated this algorithm as a feature of the software for routine analysis. Methods and Results: This new smart software automatically captures and saves the following parameters from each quadrant from each gate: percentage of cells, mean intensity, dispersion in this quadrant (variance) for each antibody on the X and Y axis, and the correlation coefficient between the X and Y dispersions. Using a standard 23 antibodies panel for leukemia and lymphoma evaluation and conventional gating leads to capturing on the average a 2623 different data points. Using this smart software, we analyzed 294 bone marrow samples referred for suspected diagnosis of MDS due to cytopenia and captured the metadata. All samples had molecular evaluation by NGS using 54 gene panel and majority had cytogenetic data. Patients classified as having MDS if molecular studies or cytogenetic data showed one or more abnormality associated with MDS. Univariate analysis showed that 103 variables to be statistically significant in distinguishing MDS with adjusted P-values less than 0.05 after controlling for false discovery rate (FDR). In multivariate analysis we first used a lasso logistic regression model and selected 40 variables. Using these variables, we developed a predictive model using a support vector machine (SVM) to identify MDS. Upon testing this model using the leave-one-out procedure, the area under the ROC curve was 91.6%. For further validation of this algorithm after integration into the software, we tested blindly additional cohort of 115 patients that had bone marrow submitted for ruling out MDS. The algorithm correctly distinguished between MDS and non-MDS in 104 (90.4%) of these patients using a cut-of point at 0.55 and predicted the presence of cytogenetic abnormality or the presence of one or more genes mutated. Mutations at allele frequency ≥20% are considered adequate for the diagnosis of MDS. Upon correlating the algorithm score with the number of mutated genes as a reflection of the severity of the disease, there was statistically significant (P< 0.0001) correlation between the score and the number of mutated genes (figure). Conclusion: We developed a system in flow cytometry analysis that captures new parameters reflecting dispersion of staining in each gated subpopulation and the correlation between the dispersion of staining antibodies. These new parameters have been proven to be very useful in the diagnosis and prediction of the diagnosis of MDS allowing us to develop automated and reliable algorithm for the diagnosis of MDS and the prediction of level of molecular abnormalities. Figure Figure. Disclosures Albitar: Neogenomics Laboratories: Employment, Equity Ownership. Shahbaba:University of California, Irvine: Employment. Agersborg:Neogenomics Laboratories: Employment, Equity Ownership. Chang:Neogenomics Laboratories: Employment. Albitar:Neogenomics Laboratories: Employment. Uyeji:Neogenomics Laboratories: Employment. Luchetta:Neogenomics Laboratories: Employment. Su:Armstrong State University: Employment. Zhang:Armstrong State University: Employment.

Megakaryocytes Do Not Express Erythropoietin Receptor (EpoR) and Do Not Respond to Recombinant Human Erythropoietin (rHuEpo) Stimulation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4783-4783
Author(s):  
Robert D Loberg ◽  
John M Rossi ◽  
Raffi Manoukian ◽  
Shen-Wu Wang ◽  
Katherine L Paweletz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Negative Control ◽  
Employment Equity ◽  
Downstream Signaling ◽  
Human Erythropoietin ◽  
Equity Ownership ◽  
And Function ◽  
No Activation ◽  
Epor Expression

Abstract Abstract 4783 Recently, clinical studies were conducted in chronic kidney disease of type 2 diabetes patients that suggested an increased risk of stroke in patients receiving Erythropoiesis Stimulating Agents (ESAs) compared to placebo. One proposed explanation is that megakaryocytes and/or platelets express EpoR and respond to ESA challenge. We evaluated the underlying and untested assumption that megakaryocytes or platelets express a functional EpoR using flow cytometry assays developed to detect cell surface EpoR using validated EpoR-specific antibodies. Additionally, in order to investigate whether megakaryocytes/platelets are responsive to ESAs, the activation status of downstream signaling proteins was evaluated after stimulation with ESAs or vehicle control. Whole bone marrow aspirates were collected from healthy volunteers and bone marrow mononuclear cells (BMMCs) isolated by Ficoll separation (n=25). Megakaryocytes were isolated from BMMCs by using CD61 immuno-magnetic bead capture and analyzed by flow cytometry, gating for megakaryocytes using viability, CD41a expression and polyploidy. Platelets were analyzed in peripheral blood from healthy volunteers (n=10) using CD41a as marker. To measure EpoR expression an EpoR-specific monoclonal antibody was used for flow cytometry analysis in both platelets and megakaryocytes that were isolated and gated as described above. Megakaryocytes and platelets were stimulated with rHuEpo (1, 10, and 300 U/mL for 5 and 30 min) and induction of pSTAT5 was measured by intracellular flow cytometry using phospho-specific antibodies. In addition, platelets (CD41a+) were assessed for EpoR expression and functional response to recombinant human erythropoietin (rHuEpo) challenge. Stimulation with thrombopoietin (TPO) was used as a positive control for pSTAT5 induction in megakaryocytes and platelets. While robust EpoR expression was, as expected, observed consistently in UT7/Epo control cells and absent in CMK negative control cells (fold over control – UT7/Epo 12.78 ± 5.03; CMK 0.77 ± 0.22 mean ± 95%CI), no significant expression of EpoR was observed on CD41a+/polyploid megakaryocytes (fold over control − 1.006 ± 0.03; mean ± 95%CI). No activation of downstream signaling (pSTAT5) was detected in megakaryocytes in any of the samples stimulated with rHuEpo at any of the concentrations or timepoints analyzed, while robust pSTAT5 was detected in the UT7/Epo control cell lines. CMK cells were included as a negative control and demonstrated an absence of pSTAT5 induction in response to rHuEpo while a robust induction to TPO was observed (fold over control – UT7/Epo 12.11 ± 7.86; CMK 1.05 ± 0.03; mean ± 95%CI). Similarly, no significant EpoR expression was detected on CD41a+ platelets while the control, c-Mpl expression, was significant (fold over control – EpoR 1.11 ± 0.019; c-Mpl 2.936 ± 0.71; mean ± 95%CI) and no activation of downstream signaling (pSTAT5) was detected in platelets in any of the samples stimulated with rHuEpo at all concentrations and timepoints analyzed. The lack of EpoR expression on megakaryocytes was confirmed by laser scanning cytometric analysis in patient samples. A robust/sensitive platform was developed to profile biologically relevant EpoR expression and function in megakaryocytes and platelets. The assays for EpoR expression and function were sufficiently sensitive to allow characterization during in-vitro differentiation of erythroid progenitors. Using this platform we demonstrated a lack of EpoR expression and functional response to rHuEpo on megakaryocytes and platelets isolated from human bone marrow (CD41a+/CD61+/polyploid). Disclosures: Loberg: Amgen: Employment, Equity Ownership. Rossi:Amgen: Employment, Equity Ownership. Manoukian:Amgen: Employment, Equity Ownership. Wang:Amgen: Employment, Equity Ownership. Paweletz:Amgen: Employment, Equity Ownership. Sable:Amgen: Employment, Equity Ownership. Tudor:Amgen: Employment, Equity Ownership. Patterson:Amgen: Employment, Equity Ownership. McCaffery:Amgen: Employment, Equity Ownership.

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

Blood ◽  
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.

Use Of Rearranged Immune Receptor Sequencing To Measure Minimal Residual Disease (MRD) In Bone Marrow, Cerebrospinal Fluid and Testes In Relapsed Childhood Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4984-4984
Author(s):  
Norman J. Lacayo ◽  
Li Weng ◽  
Charles Gawad ◽  
Malek Faham ◽  
Gary V Dahl
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cerebrospinal Fluid ◽  
Acute Lymphoblastic Leukemia ◽  
Deep Sequencing ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Employment Equity ◽  
Equity Ownership ◽  
Minimal Residual

Abstract Background Detection of minimal residual disease (MRD) in pediatric acute lymphoblastic leukemia (ALL) is a strong predictor of outcome. In addition, MRD testing prior to stem cell transplant for ALL can inform on the risk of relapse. The ClonoSIGHT test uses deep sequencing of immunoglobulin and T-cell receptors to identify and monitor MRD. In retrospective cohorts, we have previously shown this technology is highly correlated with flow cytometry and PCR-based MRD methods, but has even greater sensitivity than both technologies (Faham et al, Blood 2012; Gawad et al, Blood 2012).  Here we report on four clinical cases where we used the ClonoSIGHT assay to prospectively monitor MRD, in both the medullary and extramedullary compartments, to demonstrate the feasibility of this technology for MRD monitoring of children with relapsed ALL. Methods Universal primer sets were used to amplify rearranged variable (V), diversity (D), and joining (J) gene segments from the immunoglobulin heavy and kappa chain (IGH and IGK), as well as T-cell receptor beta, delta and gamma.  The assay was performed on genomic DNA isolated from cells from the bone marrow, cerebrospinal fluid, or testes.  The test was first done at the time of relapse to identify the malignant clonotype, which was monitored at subsequent time points. The patients were ineligible for clinical trials and concurrently underwent MRD testing using flow cytometry. The sequencing assays were performed to show feasibility of the approach. Results  Patient one was a 14 y/o ALL relapse patient who was not in morphologic remission after standard re-induction therapy. The malignant clonotype was identified on a bone marrow aspirate from relapse; follow-up MRD tests were done using both flow cytometry and deep sequencing five times throughout salvage therapy with 5-aza-2'-deoxycytidine, suberoylanilide hydroxamic acid and high dose cytarabine over 75 days; the last two MRD data points showed 0.6% and 6% by ClonoSIGHT MRD and 0.4% and 1.3% by flow cytometry MRD. Morphologic remission with count recovery was used as the criteria to direct this patient to SCT. Patient two was a 9 y/o with ALL, for whom MRD was used to test for relapsed disease in multiple tissues.  This patient experienced three isolated testicular relapses (M1 marrow and no CNS involvement) at the time of each relapse. The ClonoSIGHT assay was used on tissue from a testicular biopsy to identify the malignant clone(s).  Testing of the bone marrow and cerebrospinal fluid did not detect the malignant clones in those sites. This patient underwent therapeutic orchiectomy and 4-week systemic re-induction resulting in a fourth complete remission and now is under evaluation for consolidation therapy with a SCT. A third patient was an 8 y/o with a combined bone marrow and testicular ALL relapse, who was in morphologic remission in the marrow after re-induction therapy and testicular radiotherapy. Prior to undergoing SCT the patient had negative MRD by flow cytometry but had 0.008% MRD using the ClonoSIGHT MRD assay.  The fourth patient was a 15-yo with ALL relapse at 9 years from first remission, treated with a four-drug re-induction and Berlin-Frankfurt-Münster based consolidation and maintenance therapy.  This patient was MRD negative by both flow cytometry and ClonoSIGHT MRD at end of re-induction as well as end of consolidation and remains in remission. Conclusions We have shown the feasibility of using sequencing-based tests for monitoring MRD in children with relapsed ALL in medullary (bone marrow) and extramedullary compartments (testes and CSF).  Further studies are needed to establish the prognostic value of MRD detected by the ClonoSIGHT assay in both medullary and extramedullary sites that are below the limit of detection of PCR and flow cytometry. These sequencing-based tests may provide a useful tool to develop risk stratification schemas for drug development in relapsed childhood ALL. Disclosures: Weng: Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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