PB2112 COEXISTENCE OF MONOCLONAL B- CELL POPULATION IN PATIENTS WITH NEWLY DIAGNOSED MULTIPLE MYELOMA; INCIDENCE, IMMUNOPHENOTYPING AND CLINICAL CHARACTERIZATION

Y. Cohen; I. Cohen; C. Perry; I. Avivi; S. Kay; Y. Herishanu

doi:10.1097/01.hs9.0000566932.71036.62

Evaluation of Immune Profile in Patients with Multiple Myeloma Using Cytof Technology

Blood ◽

10.1182/blood.v124.21.3404.3404 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3404-3404

Author(s):

Jana Jakubikova ◽

Efstathios Kastritis ◽

Danka Cholujova ◽

Teru Hideshima ◽

Ludmila M Flores ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Natural Killer ◽

Plasma Cells ◽

Memory B Cells ◽

High Dimensional ◽

Newly Diagnosed ◽

D Cells

Abstract Introduction: CyTOF (time-of-flight mass cytometry) is a novel high-dimensional technology which permits immunophenotyping and analysis of signaling in single cells. This approach enables simultaneous evaluation of up to 40 parameters using antibodies tagged with distinct elemental isotopes, by combining flow cytometry with atomic mass spectrometry. Since multiple myeloma (MM) is characterized by immune dysfunction, we used CyTOF technology to define the complex immune profile in MM patient bone marrow (BM) samples. Methods: We used 40 different markers to define various B, T, natural killer (NK) subsets, as well as cells of monocytic, granulocytic, erythroid and platelet lineages. Our preliminary data are results from 10 patients with MGUS/ smoldering MM (SMM); 10 newly diagnosed MM; 20 relapsed/refractory MM; and 15 WM patients (5 newly diagnosed and 10 receiving treatment) in comparison with age-matched healthy donors’ BM (HD). A significantly larger cohort of MM (N=150) and WM (N=50) patients is being similarly analyzed and will be presented. To evaluate phenotypic abnormalities in various B cell subsets, we used B lineage markers CD10, CD19, CD20, CD22, CD27, CD34, CD38, CD45, IgA, IgD, IgG and IgM to define B cells maturation stages from hematopoietic stem cells (HSC) to naïve to mature B lymphocytes (pro-B; pre-B-I; pre-B-II; immature B; and mature (naïve) B cells), as well as memory non-switched and memory switched B cells, plasmablasts, normal (CD138+CD38+CD19+CD45+) and clonal plasma cells (CD138+CD38+CD19-CD45-/low), which reside in the specific BM niche. Furthermore, natural killer (NK) subsets (such as NK and NKT cells) and T cells (such as memory CD4T, naive CD4T, memory CD8T, naive CD8T, T regulatory cells and Tg/d cells) were examined. High-dimensional data was obtained using CyTOF technology and analyzed by SPADE and viSNE software. Results: Our data showed significantly decreased HSC in patients with newly diagnosed and relapsed/refractory MM compared to HD (P<0.025). A significant increase in pre-B-I cells was detected in relapsed/refractory MM vs. MGUS/SMM (P<0.028), but the opposite trend was observed in the pre-B-II subpopulation (P<0.005). No differences in immature B cell populations were observed in different stages of MM. However, a significantly higher percentage of immature B cells was present in relapsed/refractory MM compared to HD (P=0.008), and transitional B cells were significantly decreased in newly diagnosed MM compared to HD (P<0.001). Moreover, memory B cells were significant decreased in all MM stages compared to HD (P<0.003). Non-switched memory B cells were significantly increased in MGUS and SMM compared to newly diagnosed MM, while a significant increase of switched memory B cells was present in newly diagnosed MM compared to relapsed/refractory MM. A significant increase in plasmablasts was seen in relapsed/refractory MM in comparison with other MM stages (P<0.011) by CyTOF analyses. Malignant plasma cells (PC) were defined as CD19-, CD38++, CD45-/dim, CD138+ and either cyk or cyl positive. Importantly, a significant increase in clonal PC was found in all MM stages vs. HD, as well as in newly diagnosed MM compared to relapsed/refractory MM (P<0.01). The percentage of PC from CyTOF analyses correlated with % of PC obtained using flow cytometry by Bland-Altman method comparison. We also observed significant differences in T cell subsets including naïve, central memory, effector, and effector memory CD4+ and CD8+T populations between MGUS and newly diagnosed MM, but no significant changes in T regulatory and Tg/d cells. Furthermore, plasmacytoid dendritic (pDC) cells were significantly increased in newly diagnosed MM, and PD-1 expressed on pDC was significantly decreased in newly diagnosed MM compared to MGUS (P=0.007). Interestingly, PD-1 and its ligand PD-L1 were variably expressed on B cells (2-9% and 3-27%) and PC (0.5-46% and 3-41%) from MM BM samples. Other surface molecules including CD269 (4-32%), CD289 (1-8%), CD362 (0.5-1%) and CD329 (1-4%), were variably expressed in PC. Conclusion: A better understanding of the neoplastic BM milieu will provide the framework for identifying and validating novel targeted therapies directed against MM. CyTOF technology represents a novel diagnostic tool to assess the status not only of MM, but also of host immunity, and may allow for the development of rational personalized therapies. Disclosures No relevant conflicts of interest to declare.

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Multiple Myeloma-Specific Targeted Therapy: Use of the Tumor Specific Immunoglobulin Gene Sequence to Activate Drugs Only within Tumor Cells.

Blood ◽

10.1182/blood.v114.22.1851.1851 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1851-1851

Author(s):

Zhi-Wei Li ◽

Dror Shalitin ◽

Jennifer Finefield ◽

Crystal Leung ◽

Mengyin Hu ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cell ◽

Cell Line ◽

Tumor Cells ◽

Cell Population ◽

Gene Sequence ◽

Conflicts Of Interest ◽

Immunoglobulin Gene ◽

B Cell Malignancies ◽

Cytotoxic Effects

Abstract Abstract 1851 Poster Board I-877 Currently available drugs for multiple myeloma (MM) treatment show very little ability to distinguish MM cells from normal cells. This limits the dose of drugs with anti-MM activity that can be administered safely; and, thus, reduces their efficacy in eliminating the malignant plasma cell population. Therefore, novel strategies are needed that allow concentrations of higher levels of active drugs within tumor cells than in other healthy cells. One approach is to allow release of active drugs only within the tumor cells in these patients without affecting nonmalignant cells. MM is a malignancy of clonal antibody-secreting plasma cells with specific rearrangement of DNA that is transcribed into a unique mRNA sequence that is translated into the tumor specific monoclonal antibody. These tumor specific transcripts are abundant in all of the malignant cells in these patients. We have explored the possibility of using a unique sequence from this tumor specific transcript, the complementarity determining region (CDR) gene sequence, to direct drug release only within MM cells. First, we determined whether this type of tumor specific oligonucleotide could specifically recognize the tumor cell population. Using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from the MM cell line RPMI8226 as a probe (designated as molecular beacon [MB] 8226), we demonstrated that this oligonucleotide specifically distinguished the RPMI8226 from other cell lines, including U266, another MM cell line. Second, in order to translate this approach into potentially therapeutically active anti-MM agents, we synthesized naphthyridine-modified (N) vorinostat, a FDA-approved histone deacetylase inhibitor, and N-melphalan. The modification allowed us to conjugate vorinostat or melphalan, two drugs currently used in MM treatment, with MB8226. Histone acetylation analysis demonstrated that the modification of vorinostat with naphthyridine did not change its function compared to the unmodified compound. Moreover, using cell viability and apoptosis assays, there was no reduction in the cytotoxic effects of N-vorinostat or N-melphalan compared to the parent drugs. In addition, induction of cell death occurred in a cell specific matter with N-melphalan conjugated to its tumor specific oligonucleotide. Upon transfection with N-melphalan-conjugated MB8226, only RPMI8226 but not U266 cells showed cytotoxic effects from exposure to this tumor specific cytotoxic construct. We are currently evaluating the specific efficacy of N-vorinostat-conjugated MB8226 as well as an oligonucleotide that specifically recognizes the CDR3 sequence of U266 cells conjugated with N-vorinostat or N-melphalan. We are also determining the efficacy of this approach in vivo using our severe combined immunodeficiency-hu mouse models of human MM. Our studies provide a novel targeted therapeutic approach for the treatment MM as well as other B cell malignancies that should specifically be active only within the malignant cell population and not impact nonmalignant cells. This type of treatment should allow delivery of higher concentrations of drugs that will be active only within tumor cells ultimately leading to both much more effective and better tolerated therapies for patients with MM and other B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

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Immunological Consequences of Lenalidomide with and without Dexamethasone in Newly Diagnosed Multiple Myeloma

Blood ◽

10.1182/blood-2019-128335 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3070-3070

Author(s):

Jake A. Kloeber ◽

Teresa K. Kimlinger ◽

Jessica L. Haug ◽

Kimberly J. Henderson ◽

S.Vincent Rajkumar ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Research Funding ◽

Nk Cell ◽

Single Agent ◽

Cell Populations ◽

Newly Diagnosed

Introduction: Recent advancements in the treatment of multiple myeloma (MM) have centered on engaging the immune system to target multiple myeloma cells. Although these therapies are being combined with immunomodulatory imide drugs (IMiDs) and corticosteroids, the individual contributions of these drugs on the immune system of MM patients has not been examined in the upfront setting. In this study, we examined the peripheral blood immunophenotypes of newly diagnosed multiple myeloma (NDMM) patients receiving the IMiD lenalidomide with or without the corticosteroid dexamethasone. Methods: To characterize immunophenotypes, we utilized flow cytometry to profile white blood cell populations from 35 patients enrolled in a clinical trial testing the efficacy of lenalidomide with and without dexamethasone in NDMM (NCT00772915). In this trial, all patients were initiated on single-agent lenalidomide. Dexamethasone was initiated in patients that did not meet desirable responses or for disease progression. At each cycle, peripheral blood was stained with a 17-marker antibody panel against several immune lineages and functional surface markers. We grouped patients into two groups: 1) lenalidomide alone or 2) lenalidomide with dexamethasone according to their treatment regimen at each cycle timepoint. Results: First we confirmed anti-myeloma cell activity for both groups by measuring a steady decline in circulating plasma cells in both groups. Examining peripheral blood immunophenotypes showed an expected decrease in T cells and a smaller decrease in B cells in both groups of patients. Closer inspection of B cell populations revealed a switch towards a more immature B cell phenotype in both treatment groups. This was measured as a switch from CD19-CD20+ cells to CD19+CD20- B cells. Inspection of T cell subsets revealed that patients receiving single-agent lenalidomide had a sustained decrease in the levels of CD4+ T cells and increase in the levels of CD8+ T cells. This was seen in both naïve and regulatory T cells evidenced by a decrease in the CD4/CD8 ratio among CD28+ T cells as well as CD25+ T cells. Importantly, this alteration did not lead to sustained alterations in the overall level of CD25+ or CD28+ T cells, and the addition of dexamethasone reverses this trend. In addition to the effects seen on T and B cell numbers, we detected expansions of NK cell populations in patients receiving lenalidomide alone. This expansion is detected as an overall increase in CD56+ mononuclear cells with the majority of cells being CD56+CD3- cells. Conclusions: Our data show that lenalidomide and dexamethasone therapy have shared but distinct effects on peripheral blood immunophenotypes in NDMM. Both drugs alter B cells numbers and populations leading to an expansion of CD19+CD20- B cells. However, lenalidomide alone decreases the CD4/CD8 T cell ratio; and, lenalidomide more strongly expands NK cell populations. The addition of dexamethasone reverses this trend and leads to a restoration of the CD4/CD8 ratio. This suggests that lenalidomide without dexamethasone might be counterproductive in immunotherapies intended to recruit CD4+ T cells. Conversely, lenalidomide alone could increase the efficacy of immunotherapies dependent on NK cell recruitment such as antibody-dependent cellular cytotoxicity (ADCC). This information may benefit future investigations of immune responses in MM patients and improve the adoption of immunotherapies to MM patients. Figure Disclosures Kumar: Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding.

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Atypical Serum Immunofixation Pattern (ASIP) Development during Induction Therapy with BiRD for Newly Diagnosed Multiple Myeloma Correlates with a High Rate of Complete Remission.

Blood ◽

10.1182/blood.v110.11.2737.2737 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2737-2737

Author(s):

Tomer Mark ◽

David Jayabalan ◽

Roger N. Pearse ◽

Y. Lynn Wang ◽

Richard Lent ◽

...

Keyword(s):

Multiple Myeloma ◽

Stem Cell ◽

Complete Remission ◽

B Cell ◽

Plasma Cell ◽

Induction Therapy ◽

Light Chain ◽

Newly Diagnosed ◽

Disease Response ◽

Monoclonal Immunoglobulins

Abstract The excess production of monoclonal immunoglobulin is the hallmark of multiple myeloma (MM) diagnosis and is an essential element of the determination of disease response to therapy. The development of new monoclonal immunoglobulins that are distinct from the baseline diagnostic paraprotein during the course of MM therapy can therefore pose a challenge in assessing disease response or potential relapse. There are prior reports of the development of abnormal protein banding (APB) after autologous stem cell transplantation for MM comprised of transient non-myeloma related mono- or oligoclonal protein bands that have been correlated with higher rates of event-free and overall survival. We now report the development of atypical serum immunofixation patterns (ASIPs) for the first time outside of the setting of stem cell transplant during the course of MM induction therapy with the BiRD (Biaxin® [clarithromycin], Revlimid® [lenalidomide], Dexamethasone) regimen. 72 patients with newly diagnosed Stage II and III MM (Salmon-Durie Criteria) were treated in a phase 2 clinical study of BiRD. The BiRD treatment regimen was given in 28-day cycles as follows: Clarithromycin 500mg po BID for days 1 – 28, Lenalidomide 25mg po daily for days 1 – 21, and Dexamethasone 40mg po weekly on days 1, 8, 15, and 21. The median age of the patients was 63 (range 36–83), and the baseline monoclonal immunoglobulins detected on serum immunofixation were as follows: 61% IgG, 22% IgA, 17% light chain only. 24 patients (33%) developed one or more ASIPs with either a mono- or oligoclonal banding pattern during the course of BiRD therapy. The new protein bandsvaried widely in duration of appearance (range 26–315 days) as well as isotype with IgM-κ, IgM-λ, IgG-κ, IgG-λ, IgA-λ, free λ light chain, and free IgM heavy chain all observed in one or more instances. The extent of therapy prior to first ASIP development was variable as well, (range 27– 724 days, median of 178 days). Patients with ASIPs had significantly better response to BiRD vs. non-ASIP patients (p = .00002), with CR+sCR rate of 71% vs. 23%, and a VGPR or better rate of 96% vs. 61%. The extent of BiRD therapy prior to development of first ASIP did not correlate with response rate (p = .7284). Bone marrow examination by histology, karyotype, FISH, and PCR IgH / IgK clonality analysis confirmed the disease response and furthermore showed no evidence for newly emergent plasma cell or other B-cell malignancy to account for ASIP generation suggesting molecular remission in some of the patients. Peripheral blood analysis by flow cytometry also showed no evidence for a circulating B-cell neoplasm to account for the new immunoglobulin production. We propose that ASIP development during myeloma therapy with a lenalidomide-based regimen heralds a robust tumor reduction with a hitherto unprecedented rate of complete remission. The immune phenomena that contribute to ASIP generation are unclear at present, however it is neither due to a new clonal B-cell population or transformation of the original MM plasma cell clone.

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Newly Diagnosed Childhood AML Patients Treated with Bortezomib Show Superior Survival If CD74 Is Expressed: A Report of 991 Patients from the Children's Oncology Group AAML1031 Protocol

Blood ◽

10.1182/blood-2020-142659 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 39-39

Author(s):

Lisa Eidenschink Brodersen ◽

Chad A. Hudson ◽

Todd A. Alonzo ◽

Robert B. Gerbing ◽

Laura Pardo ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

B Cell ◽

Clinical Characteristics ◽

De Novo ◽

Newly Diagnosed ◽

Oncology Group ◽

Standard Chemotherapy ◽

Childhood Aml ◽

De Novo Aml

Introduction: CD74 is a type II transmembrane protein expressed on antigen-presenting cells and an MHC class II chaperone. It has been associated with tumor progression and metastasis in solid tumors, and its expression has been suggested to serve as a prognostic factor in many cancers, with higher relative expression associated with oncogenesis. As the expression of CD74 has been associated with response to bortezomib in multiple myeloma, we inquired whether such correlation might be seen in pediatric AML. We prospectively evaluated CD74 expression by difference from normal (ΔN) flow cytometry and correlated expression with clinical characteristics and outcome as part of Children's Oncology Group protocol AAML1031 that randomized patients younger than 30 years of age with de novo AML to standard treatment with (Arm B) or without (Arm A) bortezomib. The addition of bortezomib to standard chemotherapy increased toxicity but did not improve survival. Given the association of CD74 with B-lymphoid neoplasms and bortezomib's known efficacy in B-cell neoplasms and multiple myeloma, we hypothesized that within Arm B, the patients with CD74 expression would have a more favorable outcome. Methods: In total, 1,139 newly diagnosed pediatric patients with de novo AML were randomized to standard chemotherapy (n=561, Arm A) or standard chemotherapy with bortezomib (n=578, Arm B). All patients received the identical chemotherapy backbone with either four intensive chemotherapy courses or three courses followed by allogeneic hematopoietic stem cell transplantation for high-risk patients. To qualify for this correlative study, 991 patients satisfied 2 criteria: (1) submitting a bone marrow aspirate for ΔN at diagnosis and (2) providing consent for correlative biology studies. All diagnostic specimens were centrally and prospectively evaluated for the expression of CD74 by ΔN. AML was considered to be CD74-positive if the MFI was more than two times above background autofluorescence and more than 40% of the leukemia was above background autofluorescence. Results: Among 991 patients, 263 were CD74-positive (26.5%) by ΔN, with similar prevalence in Arm A (27.9%) and Arm B (25.2%). Correlation of CD74 expression with clinical characteristics showed that those with CD74 expression had higher median age (p<0.001), lower median WBC (p<0.001), higher prevalence of low risk protocol status (p=0.039), lower frequency of CEBPA mutation (p=0.039), inv(16) (p=0.001), and KMT2A rearrangements (p=0.002), and were enriched for t(8;21) (p<0.001) and t(6;9) (p=0.014) fusions. All these features retained significance when patients were sub analyzed by respective treatment arms. CD74-positive patients had a higher morphologic CR rate (p=0.016), however, measurable residual disease by flow cytometry was not significant in the entire cohort or in the sub analysis of treatment arms (p=0.155). CD74-positive patients showed superior 5-year OS (70.6% vs 61.8%, p=0.003) and EFS survival (51.2% vs 43.1%, p=0.007) compared to those who were CD74-negative. For patients in Arm A (no bortezomib), the differences in OS (66.1% vs 61.0%, p=0.138) and EFS (48.9% vs 41.7% p=0.088) were not significant between those that were CD74-positive and those that were CD74-negative (Figure 1). However, patients in Arm B receiving bortezomib that were CD74-positive showed a significant improvement in OS (75.3% vs 62.5%, p=0.006) and EFS (53.6% vs 44.3%, p=0.028) compared to those who were CD74-negative (Figure 1). Comparison of the outcomes for CD74-positive patients with and without bortezomib exposure showed a difference in OS of 66.1% vs. 75.3% for those in Arm A vs. Arm B but did not reach significance (p=0.155). Multivariable analysis for OS yielded a hazard ratio of 0.67 (95% CI: 0.44 - 1.02) and p=0.061, approaching, but not reaching, statistical significance. Conclusions: These data demonstrate that CD74 expression is associated with more favorable disease characteristics and survival. Patients receiving bortezomib that were CD74-positive showed a superior response to therapy compared to patients who did not express CD74, by both OS and EFS, suggesting that CD74-positive childhood AML patients stand to benefit from bortezomib therapy. Bortezomib may induce a mechanistic response in CD74-positive AMLs similar to that in bortezomib-treated B-cell neoplasms and/or multiple myeloma, where bortezomib has proven to be beneficial. Disclosures Cooper: Celgene: Other: Spouse was an employee of Celgene (through August 2019). Pollard:Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

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