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.

Single Cell Network Profiling (SCNP) Functionally Characterizes FLT3 Pathway Deregulation in Non-M3 Acute Myeloid Leukemia (AML) and Provides Prognostic Value Independent From Mutational Status

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2512-2512
Author(s):  
Alessandra Cesano ◽  
Santosh Putta ◽  
Kavita Mathi ◽  
David B. Rosen ◽  
Urte Gayko ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Induction Therapy ◽  
Mutational Load ◽  
Receptor Signaling ◽  
Employment Equity ◽  
Cell Network ◽  
Mutational Status ◽  
Equity Ownership

Abstract Abstract 2512 Background: FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations (FLT3 ITD+) result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients (pts) with AML; however, substantial heterogeneity in clinical outcomes still exists within both the FLT3 ITD+ and FLT3 ITD- AML subgroups, suggesting alternative mechanisms of disease relapse not accounted for by FLT3 mutational status. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). Previously, we reported the use of this assay to functionally characterize FLT3 receptor signaling in healthy bone marrow and AML samples (Rosen et al. PLoS One 2010). By applying it to a separate cohort of samples collected from elderly non-M3 AML pts at diagnosis, a subclassification of AML samples beyond their “static” molecular FLT3 ITD status was generated (Rosen et al. ASH 2010 Abstr 2739). Specifically, FLT3 ITD- AML samples displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD+ AML samples. Conversely, FLT3 ITD+ AML samples displayed more homogeneous induced signaling, with the exception of those with low mutational load, which had profiles more analogous to FLT3 ITD- AML samples. Due to the small numbers of pts in that exploratory study (n=44 [38 FLT3 ITD- and 6 FLT3 ITD+ pts]), an independent study was undertaken to confirm the observations, as well as to evaluate their clinical relevance (i.e., association with disease free survival (DFS) following anthracycline/cytarabine-based induction therapy). Methods: SCNP was performed as previously described on cryopreserved bone marrow or peripheral blood samples collected prior to anthracycline/cytarabine-based induction therapy from 104 elderly (>60y) non-M3 AML pts enrolled on ECOG trial 3999 or 3993 for whom ITD mutational status (including % mutational load), response and DFS data were available. Samples included 85 FLT3 ITD- and 19 FLT3 ITD+ AML, 30 and 8 of which, respectively, were collected from patients who achieved complete remission (CR). Objectives: The primary study objective was to confirm that levels of FLT3 ligand (FLT3L)-induced signaling (as measured by changes in intracellular phospho-S6 level) are more homogeneous in FLT3 ITD+ than in FLT3 ITD- myeloblasts. Four FLT3 ITD+ groups were pre-defined based on % mutation load (>0, 30%, 40%, or 50%). In addition, FLT3 ITD mutational status and signaling data from the SCNP assay (FLT3L and stem cell factor-induced phospho-S6 signaling and cytarabine/daunorubicin-induced apoptosis [cleaved PARP]) were combined to mathematically model their association with DFS among pts who achieved CR. DFS was defined as time from date of confirmed CR to date of relapse or death. Results: As shown in Figure 1a, our previous observations that variance in FLT3L-induced signaling is higher in FLT3 ITD- AML samples than in FLT3 ITD+ ones and that variance is decreased with increasing mutational load were verified in this study (Levene Test for FLT3 ITD- vs FLT3 ITD+ 50 p value=0.023). Further, when the association of DFS with FLT3 ITD mutational status and signaling data from the SCNP assay was measured using a Cox Proportional-Hazards model, the SCNP data were shown to provide independent information from FLT3 ITD mutational status (p =0.0115 for FLT3L-induced phospho-S6 signaling, Figure 1b). Conclusions: These data add to the growing body of evidence that, even within currently accepted risk stratification groups, AML is a heterogeneous disease. Functional characterization of FLT3 receptor signaling deregulation using SCNP provides prognostic information independent from FLT3 ITD mutational status and allows for more accurate pt stratification by functionally defining DFS risk sub-groups. Characterization of FLT3 signaling deregulation by SCNP could ultimately aid in the improved clinical management of AML pts and help identify candidates for FLT3 receptor inhibitor studies. Disclosures: Cesano: Nodality: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Mathi:Nodality: Employment. Rosen:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Hawtin:Nodality: 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.

scholarly journals Partial Reconstitution of Humoral and Cellular Immunity in Patients with Chronic Lymphocytic Leukemia Treated with Acalabrutinib

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1874-1874 ◽  
Author(s):  
Christopher Pleyer ◽  
Clare C. Sun ◽  
Pia Niermann ◽  
Xin Tian ◽  
Inhye E. Ahn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Lymphocytic Leukemia ◽  
Light Chains ◽  
Free Light Chains ◽  
Employment Equity ◽  
Cell Numbers ◽  
Serum Iga ◽  
Btk Inhibitor ◽  
Equity Ownership

Abstract Introduction Immune dysregulation in chronic lymphocytic leukemia (CLL) contributes to a high rate of infections and morbidity. We previously reported that treatment of CLL with ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, leads to partial reconstitution of humoral immunity and fewer infections, especially in patients who achieved a ≥50% increase in serum IgA levels. Acalabrutinib is also an irreversible BTK inhibitor that is more selective than ibrutinib and has demonstrated safety and efficacy in the treatment of relapsed or refractory CLL. It is currently unclear how the increased specificity of acalabrutinib affects immune reconstitution and infection rates. We assessed the immunological impact of acalabrutinib in patients with CLL treated with single-agent acalabrutinib. Methods Samples originated from a phase 2, single-center trial studying acalabrutinib 100 mg twice daily (BID) or acalabrutinib 200 mg once daily (QD) in patients with relapsed/refractory (RR) CLL or high-risk, treatment naïve (TN) CLL (chromosome 17p deletion or mutation in TP53 or NOTCH1) (NCT02337829). Patients who received at least 6 months of acalabrutinib and had paired longitudinal data available were included in the analyses. Patients receiving IV immunoglobulin replacement were excluded from analysis of IgG levels. Additionally, patients with detectable monoclonal IgG, IgA and/or IgM proteins on serum immunofixation were excluded from analysis of the corresponding immunoglobulin isotype. The analysis of free light chains was stratified based on k or λ restriction of CLL cells determined by flow cytometry. Immunohistochemical staining of bone marrow biopsies was performed: T cell numbers were estimated by CD3 staining and the degree of CD68-positive macrophage extensions in contact with CLL cells were semi-quantitatively assessed on a scale from 0 (no extensions) to 4 (maximum number of extensions). The Wilcoxon signed-rank test and the Mann-Whitney U test were used to compare paired and unpaired data, respectively. Differences in the rate of infection between groups were examined using the Cox regression model for recurrent events. Results Serum IgA levels increased as early as 3 months after the initiation of acalabrutinib (median increase 35.7%, P = .0001), with levels sustained up to 24 months (Figure 1), whereas serum IgG and IgM levels were not affected by acalabrutinib. There was no difference (P > .05) in IgA, IgG, IgM trend between TN or RR CLL. Furthermore, there was no difference (P > .05) in IgA, IgG and IgM trends between patients treated with QD compared to BID dosing of acalabrutinib. Among 20 k-restricted and 18 λ-restricted CLL cases, the involved (tumor-derived) free light chain was elevated at baseline and trended toward the normal range after 3 months of acalabrutinib therapy consistent with an anti-tumor effect (k: median decrease 55.4%; P < .0001 and λ: median decrease 49.1%; P = .0003). The uninvolved free light chain did not change (P > .05). Peripheral blood CD3+, CD4+ and CD8+ T cell counts were elevated above the laboratory reference range at baseline and normalized after 6 months (CD4+ median decrease 49.2%; P = .0074 and CD8+ median decrease 54.8%; P = .003). T cell numbers in the bone marrow did not appreciably change. However, treatment-induced changes of the immune microenvironment were apparent in tumor-macrophage interactions. At baseline, macrophages tightly interacted with CLL cells, often with multiple podocytes making contact with CLL cells. On acalabrutinib, we observed a decrease in these macrophage podocyte interactions (P = .0007). At a median follow-up of 20 months, 31 (68.9%) patients developed a total of 68 infections, including 7 (10.3%) grade 3 and 1 (1.5%) grade 4 infections. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥ a median of 36% from baseline to 3 months, had a significantly lower rate of infections (risk ratio = 0.52, P = 0.029). Conclusions These data indicate that acalabrutinib allows for partial reconstitution of humoral and cell-mediated immunity and disrupts macrophage-CLL cell interaction in the bone marrow microenvironment in patients with CLL. Furthermore, acalabrutinib did not interfere with uninvolved free light chains, suggesting that acalabrutinib selectively inhibits CLL B-cells and does not impair normal B-cell function. Disclosures Izumi: Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Hamdy:Acerta Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Multiparameter Flow Cytometry in Patients with Suspected Myelodysplastic Syndromes Adds Significant Prognostic Information: A Study on 1,013 Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1722-1722
Author(s):  
Wolfgang Kern ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Continuous Variable ◽  
The Other ◽  
Multiparameter Flow Cytometry ◽  
Prognostic Information ◽  
Employment Equity ◽  
Aberrant Expression ◽  
Equity Ownership ◽  
Cox Analysis

Abstract Abstract 1722 Backgroundn: Multiparameter flow cytometry (MFC) has been demonstrated capable of identifying aberrant antigen expression related to myelodysplastic syndromes (MDS). The exact role and place of MFC in the diagnostic work-up of patients with suspected MDS, however, remains to be defined. Aim: Evaluation of the diagnostic impact of MFC in relation to cytomorphology (CM) and cytogenetic (CG) by determining the association of MFC results to overall survival (OS). Patients and Methods: In 1,013 patients with suspected MDS bone marrow samples had been analyzed in parallel by MFC, CM, and CG. CM confirmed MDS in 511 patients, excluded MDS in 277 patients, and showed dysplastic features but not sufficient to unequivocally diagnose MDS by CM in 225. The MFC diagnostic result was in agreement with MDS (“MDS by MFC”) in 446 patients including 382/511 patients with MDS proven by CM. CG revealed an aberrant karyotype in 245/1,013 patients. The median follow-up time amounted to 14.8 months, a total of 156 deaths was recorded. Results: The first set of analyses was performed on the cohort of 511 patients with MDS confirmed by CM. The median total number of aberrantly expressed antigens amounted to 3 (range, 0–11) and included expression of mature antigens in myeloid progenitors; abnormal CD13-CD16- and CD11b-CD16-expression patterns, aberrant expression of myeloid markers and reduced side scatter signal (SSC) in granulocytes; reduced expression of myelomonocytic markers in monocytes; aberrant expression of CD71 in erythroid cells; as well as expression of lymphoid markers in all myeloid cell lines. A higher total number of aberrantly expressed antigens as a continuous variable correlated with a shorter OS (Cox analysis, p=0.008). Next, patients were categorized based on the three parameters i) at least 3 aberrantly expressed antigens, ii) significantly reduced SSC in granulocytes, and iii) >5% myeloid progenitor cells in MFC. Patients with at least one of these criteria had a significantly shorter OS than those without (median 48.5 months vs. not reached (n.r.), p<0.001). Overall, the global diagnostic rating of “MDS by MFC” was the strongest MFC parameter: Patients with “MDS by MFC” had a shorter OS as compared to patients without (median 56.8 months vs. n.r., p=0.001). Non-MFC parameters related to OS in univariable Cox analysis included WBC count, thrombocyte count, CG (grouped according to IPSS), % blasts by CM (p<0.001 each), Hb level (p=0.001), and age (p=0.002). In order to determine the clinical relevance of “MDS by MFC” a multivariable analysis for OS was performed on this parameter together with non-MFC parameters (blood counts excluded due to incomplete data sets). It revealed an independent relation between “MDS by MFC” and OS (p=0.045). This was also true for relation of OS to the other parameters (CG, p<0.001; age, p=0.001, % blasts by CM p=0.014). Given this strong prognostic value of “MDS by MFC” in cases with MDS proven by CM a second set of analyses on the relation between MFC findings and OS were performed for the complete cohort of 1,013 patients, i.e. additionally including all cases with a diagnostic result by CM of “no MDS” or “dysplastic features not sufficient to diagnose MDS”. Again, significant relations to OS was found for the total number of aberrantly expressed antigens as a continuous variable (Cox analysis, p<0.001), for at least one of the above mentioned criteria i), ii) or iii) (median 75.6 months vs. n.r., p<0.001), as well as for “MDS by MFC” (median 60.5 months vs. n.r., p<0.001). Again, “MDS by MFC” proved to be the most relevant MFC parameter. Multivariable Cox analysis for OS including “MDS by MFC” and non-MFC parameters revealed a trend only for “MDS by MFC” (p=0.135) and significance for the other parameters (age, p<0.001; CG, p<0.001; blasts by CM, p=0.045). Conclusions: 1) The present data indicates the diagnostic use of MFC for MDS results in independent prognostic information for cases with MDS as proven by CM. 2) Furthermore, the diagnosis of MDS by MFC has a strong prognostic impact even without prove of MDS by CM which strengthens the diagnostic value of MFC even more. 3) This analysis therefore argues in favour of diagnosing MDS not only based on a combination of CM and CG but of adding also MFC for better classification and even prognostication in the future. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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.

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

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

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

Molecular Screening In Blastic Plasmacytoid Dendritic Cell Neoplasms Reveals Mutations In TET2 and ASXL1 In The Majority Of Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2581-2581 ◽  
Author(s):  
Wolfgang Kern ◽  
Sabrina Kuznia ◽  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cell ◽  
Malignant Disease ◽  
Plasmacytoid Dendritic Cell ◽  
Multiparameter Flow Cytometry ◽  
Missense Mutations ◽  
Employment Equity ◽  
Cell Neoplasm ◽  
Equity Ownership ◽  
Generation Sequencing

Abstract Background Blastic plasmacytoid dendritic cell neoplasm is a rare and clinically aggressive disease with frequent involvement of skin and bone marrow. It derives from precursors of plasmacytoid dendritic cells and is classified according to WHO 2008 into the group of “Acute myeloid leukemia and related precursor neoplasms”. Cytogenetics frequently reveals complex karyotypes data on molecular mutations is scarce. Aims To characterize blastic plasmacytoid dendritic cell neoplasm on the molecular level applying a comprehensive next-generation sequencing panel targeting 26 genes commonly mutated in myeloid neoplasms. Patients and Methods We studied six patients with blastic plasmacytoid dendritic cell neoplasm (5 bone marrow, 1 peripheral blood; 1 female, 5 male). The median age was 71.7 yrs, (range: 58.0-81.9). Cytomorphologic assessment revealed the findings characteristic for blastic plasmacytoid dendritic cell neoplasm in all cases. Furthermore, by multiparameter flow cytometry all cases had the typical immunophenotype with strong expression of CD56, expression of CD4 and CD123 and lack of expression of most myeloid and lymphoid markers. The degree of bone marrow infiltration as quantified by multiparameter flow cytometry ranged from 3% to 83% (median: 41%). One patient had a complex karyotype (44,XY,der(2)t(1;2)(q12;p22),del(5)(q14q35),del(7)(q11q22),-13,-15), four had other aberrations (46,XX,t(1;9)(q23;q33); 46,XY,t(3;8)(p12;q21),der(17)t(1;17)(q24;p13); 46,XY,i(7)(q10); 46,XY,del(13)(q14q31)), and one had a normal karyotype. None of the cases had received anti-neoplastic therapy. Mutational analysis was based on sensitive next-generation sequencing assays comprising in total 26 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. Targets of interest included either complete coding gene regions or hotspots. With the exception of RUNX1, which was sequenced on the 454 Life Sciences NGS platform (Branford, CT), all remainder genes were studied using a combination of a microdroplet-based assay (RainDance, Lexington, MA) and the MiSeq sequencing instrument (Illumina, San Diego, CA). Results Strikingly, all six patients had TET2 mutations, two had one and the other four cases had two different TET2 mutations. As described for other hematologic malignancies, there was no hotspot observed and all cases displayed different mutations (p.Leu615Serfs*24 and p.Ser1648Tyrfs*35, p.Asn767Metfs*46, p.Cys1193Tyr and p.Cys1811*, p.Gln1138*, p.Ser657Hisfs*43 and p.Glu798*, p.Ala1344Glyfs*3 and p.His1380Tyr). Of the ten TET2 mutations five were frame-shift mutations, three were nonsense mutations and two were missense mutations. Missense mutations were located in the same conserved region. The mutational load ranged from 12% to 50% (median, 32%). In one case a follow-up analysis after three weeks revealed the same two TET2 mutations with increases of the mutational load from 23% to 44% and from 24% to 39%. Although the sample size of the present series is rather limited, this is the first time that at least almost all cases of a distinct malignant disease entity are reported to carry TET2 mutations. Furthermore, there was also a very high incidence of ASXL1 mutations with 3/6 patients being affected. All cases carried the common mutation p.Gly646Trpfs*12, with mutational loads of 23%, 16% and 29%. Further mutations included SRSF2 (p.Pro95Ala) and ETV6 (p.Ile140Tyrfs*14) in one case and SRSF2 (p.Pro95Arg) with no further mutation in another case, PHF6 (p.Glu293Lys) in one case, and WT1(p.Arg596His) in one case. No mutations in any of the other analyzed genes were found. Conclusions The pattern of mutations in genes that have been described in other myeloid malignancies clearly underlines the correct classification of blastic plasmacytoid dendritic cell neoplasm into a myeloid disease category. Based on the present series with TET2 mutations in all (6/6) patients with blastic plasmacytoid dendritic cell neoplasm, TET2 mutations have to be considered to play a central role in the pathogenesis of this malignant disease. Additional mutational analyses on extended patient cohorts including ASXL1 should aim at clarifying the frequencies of these mutations and their potential impact on diagnostic and possible therapeutic interventions. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kuznia:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

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&lt;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&lt;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 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.

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