Detection of the Malignant B Cell Clone in Multiple Myeloma Via High Throughput Sequencing Is Robust to Significant Levels of Somatic Hypermutation

Abstract Background: High-throughput sequencing (HTS) of antibody gene rearrangements is an emerging tool for minimal residual disease (MRD) monitoring in B cell malignancies in which the malignant clone harbors a monoclonal Ig heavy chain (IgH) and/or light chain (κ or λ) rearrangement. This approach has shown promise in B-ALL and CLL, but experience with this technique applied to samples from multiple myeloma patients is limited. Approach: We conducted HTS of PCR-amplified IgH (VDJ and DJ) rearrangements from bone marrow aspirates of 21 patients with various plasma cell dyscrasias (MM, MGUS, LPL) and peripheral blood of a patient with plasma cell leukemia. In 17/21 samples, an aliquot was enriched for CD138+ cells by immunomagnetic separation and analyzed separately. Dominant clones from enriched and un-enriched aliquots were compared to verify the malignant clonotype sequence(s). Disease burden in un-enriched samples was also evaluated by microscopy of the bone marrow aspirate smear and ranged from 0 (hemodilute) to 37% plasma cells. Results: In 19 out of 21 samples, a clearly dominant IgH gene rearrangement (>2.5% of total sequences, range 2.9-99.9%) was identified with clear separation from background frequency (at least 2.7-fold higher frequency than next most common clone). In 17/17 cases with paired CD138-enriched samples, the dominant sequences in the enriched and un-enriched samples were identical, indicating successful identification of the malignant clonal Ig rearrangements in the un-enriched sample. More than one IgH rearrangement suitable for longitudinally tracking the malignant clone was identified in 8 of 21 cases. The two cases without an expected, productive IgH rearrangement were IgG-κ and IgG-λ. This suggested that somatic hypermutation (SHM) in the primer binding sites might interfere with some clonal amplifications, so we investigated the degree of SHM in the VH segment of the 19 cases with at least one detected dominant Ig rearrangement. A total of 18 productive VDJ rearrangements were identified, and had SHM frequencies ranging from 2% to 19% in the sequenced portions of the rearranged VH gene. 8 myeloma clones harbored an identifiable DJ rearrangement, none of which showed evidence of SHM. Finally, 3 myeloma clones harbored nonproductive VDJ rearrangements, two with no SHM, and one with 2 SHM in 84 bp of the sequenced VH gene. Conclusion: HTS of Ig heavy and light chain rearrangements can successfully identify the malignant plasma cell clone in clinical specimens, including those with low disease burden and significant SHM. Application of this technique to MRD evaluation in multiple myeloma warrants further development. Disclosures Carlson: Adaptive Biotechnologies: Consultancy, Equity Ownership. Vogl:Celgene Corporation: Consultancy; Amgen: Consultancy; Millennium/Takeda: Research Funding; GSK: Research Funding; Acetylon: Research Funding. Stadtmauer:Janssen: Consultancy.

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Ig VH gene mutational patterns indicate different tumor cell status in human myeloma and monoclonal gammopathy of undetermined significance

Blood ◽

10.1182/blood.v87.2.746.bloodjournal872746 ◽

1996 ◽

Vol 87 (2) ◽

pp. 746-755 ◽

Cited By ~ 71

Author(s):

SS Sahota ◽

R Leo ◽

TJ Hamblin ◽

FK Stevenson

Keyword(s):

Multiple Myeloma ◽

Tumor Cell ◽

Plasma Cell ◽

Monoclonal Gammopathy ◽

Somatic Hypermutation ◽

Wide Spectrum ◽

Malignant Potential ◽

Gene Sequences ◽

Vh Gene ◽

Undetermined Significance

Plasma cell tumors display a wide spectrum of clinical progression, ranging from aggressive multiple myeloma to a benign form known as monoclonal gammopathy of undetermined significance (MGUS), which requires no treatment. Because both diseases involve mature Ig- secreting plasma cells, the reason for this variation in malignant behavior is unclear. However, assessment of malignant potential is desirable for choice of treatment protocols. Ig variable (VH) gene sequences analysis has previously shown the tumor cell of multiple myeloma to be postfollicular, with mutated homogeneous clonal sequences indicating no continuing exposure to the somatic hypermutation mechanism, and this was confirmed in 7 of 7 patients. Comparison of the VH gene sequences in the monoclonal cells in MGUS yielded a different result, with 3 of 7 patients demonstrating mutated heterogeneous sequences consistent with the tumor cells remaining under the influence of the mutator. In 1 of 3 of these patients, an IgM-positive precursor cell was identified that expressed heterogeneous VH sequences similar to those of the isotype-switched plasma cell. These results indicate that the clonal cells in MGUS differ from those in myeloma and suggest that the difference may reflect malignant potential.

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Secretome Analyses of Primary Bone Marrow Fibroblasts Isolated From MGUS and Multiple Myeloma Show a Stepwise Occurrence of Alterations.

Blood ◽

10.1182/blood.v114.22.1801.1801 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1801-1801

Author(s):

Johannes Drach ◽

Astrid Slany ◽

Thomas Mohr ◽

Johannes Griss ◽

Christoph C Zielinski ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Growth Factor ◽

Plasma Cell ◽

Conflicts Of Interest ◽

Cell Clone ◽

Serum Levels ◽

Plasma Cell Dyscrasia ◽

Healthy Donors ◽

Bone Marrow Fibroblasts

Abstract Abstract 1801 Poster Board I-827 The microenvironment of tumor cells in the bone marrow was demonstrated to contribute to tumor promotion and survival. The role of bone marrow fibroblasts (BMFs) in supporting the malignant plasma cell clone in multiple myeloma (MM) has been established, but it remains unclear to which extent the BM microenvironment in general and BMFs in particular are involved in the progression of monoclonal gammopathy of undetermined significance (MGUS) to MM. Therefore we performed proteomics studies on the secretome of BMFs isolated from healthy donors, patients suffering from MGUS and patients suffering from MM. Compared to normal background, BMFs derived from MGUS secreted elevated levels of proteins indicating mitogenic activity and moderate inflammation. These proteins included periostin, IL-6, CXCL5 and CSF-1. Insulin-like growth factor II, which is normally not expressed by normal BMFs, was secreted by BMF cells derived from MGUS as well as from MM. In addition to those and other proteins, BMF cells derived from MM were found to specifically secrete stem cell growth factor, MMP-28 and stanniocalcin-1. These data indicate a step-wise alteration of BMF secretion activity related to the stage of the underlying plasma cell dyscrasia. Therefore BMF might support the progression from MGUS to MM. In order to correlate the secretion performance of BMF with blood serum levels of candidate marker proteins, Luminex assays are employed. Based upon these results, it is our aim to identify serum biomarkers which allow to assess the functional state of BMF and thus the risk for the progression of MGUS to MM. Disclosures No relevant conflicts of interest to declare.

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A Novel Role For Histone Deacetylase 11 (HDAC11) In Plasma Cell Differentation and Survival

Blood ◽

10.1182/blood.v122.21.1907.1907 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1907-1907

Author(s):

Eva Sahakian ◽

Jason B. Brayer ◽

John Powers ◽

Mark Meads ◽

Allison Distler ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

B Cells ◽

Plasma Cell ◽

Research Funding ◽

Plasma Cells ◽

Myeloma Cells ◽

Peripheral Blood Plasma

Abstract The role of HDACs in cellular biology, initially limited to their effects upon histones, is now appreciated to encompass more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution, and the stage of cellular differentiation. Recently, our group has demonstrated that the newest member of the HDAC family of enzymes, HDAC11, is an important regulator of IL-10 gene expression in myeloid cells (Villagra A Nat Immunol. 2009). The role of this specific HDAC in B-cell development and differentiation is however unknown. To answer this question, we have utilized a HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allows the monitoring of the dynamic changes in HDAC11 gene expression/promoter activity in B-cells at different maturation stages (Heinz, N Nat. Rev. Neuroscience 2001). First, common lymphoid progenitors are devoid of HDAC11 transcriptional activation as indicated by eGFP expression. In the bone marrow, expression of eGFP moderately increases in Pro-B-cells and transitions to the Pre- and Immature B-cells respectively. Expression of eGFP doubles in the B-1 stage of differentiation in the periphery. Of note, examination of both the bone marrow and peripheral blood plasma cell compartment demonstrated increased expression of eGFP/HDAC11 mRNA at the steady-state. These results were confirmed in plasma cells isolated from normal human subjects in which HDAC11 mRNA expression was demonstrated. Strikingly, analysis of primary human multiple myeloma cells demonstrated a significantly higher HDAC11 mRNA expression in malignant cells as compared to normal plasma cells. Similar results were observed in 4/5 myeloma cell lines suggesting that perhaps HDAC11 expression might provide survival advantage to malignant plasma cells. Support to this hypothesis was further provided by studies in HDAC11KO mice in which we observed a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. Taken together, we have unveiled a previously unknown role for HDAC11 in plasma cell differentiation and survival. The additional demonstration that HDAC11 is overexpressed in primary human myeloma cells provide the framework for specifically targeting this HDAC in multiple myeloma. Disclosures: Alsina: Millennium: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Baz:Celgene Corporation: Research Funding; Millenium: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding.

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Gene Expression Profiling (GEP) in MGUS and AMM: Predictors of Progression.

Blood ◽

10.1182/blood.v120.21.2933.2933 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2933-2933

Author(s):

Sarah Waheed ◽

Christoph Heuck ◽

Pingping Qu ◽

Frits van Rhee ◽

Saad Z Usmani ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cell ◽

Research Funding ◽

Immunoglobulin M ◽

Clinical Variable ◽

Normal Donor ◽

Average Risk ◽

Validation Data ◽

M Spike

Abstract Abstract 2933 Monoclonal gammopathy of undetermined significance (MGUS), a premalignant plasma cell dyscrasia, is differentiated from asymptomatic multiple myeloma (AMM), based on the level of monoclonal immunoglobulin (M spike) and bone marrow plasma cell infiltration (M spike > 3 g/dl and/or marrow plasmacytosis >10% in AMM). MGUS progresses to active Multiple Myeloma (MM) at a rate of 1–2% per year, thus imparting an average risk of 25% of progression over a lifetime. No single clinical variable, imaging modality or molecular study has yet been identified to predict progression. As a part of SWOG observational trial S0120, 262 patients were enrolled at MIRT and their GEP of purified plasma cells (PC) prospectively obtained. Here we report the results of GEP as predictor of progression. Among those with baseline GEP data available, 127 were non-IgM, AMM or MGUS and were otherwise eligible for the study. Baseline samples were restricted to those samples taken no more than 6 months prior to or 14 days following registration to S0120. If a patient had multiple baseline samples, then the sample taken closest to the patient's registration date was used. GEP studies were performed to assign molecular classification and risk scores, along with GEP-derived amp1q21, delTP53, DKK1 and other variables. With a follow-up of 36 months in the subset with evaluable GEP baseline data in 40 MGUS and 87 AMM cases, GEP characteristics were compared with those of untreated MM patients treated with TT2, TT3, TT4, and TT5 protocols, in the context of normal donor PC signatures. Comparing MGUS with AMM, CD-2 molecular subgroup was over-represented in MGUS and HY in AMM. PSMD4 on chromosome 1q21 was also linked to AMM. In terms of GEP-70 risk model, 36% of AMM and 15% of MGUS had scores exceeding -0.26. Univariate predictors for treatment-requiring MM included the well-known serum-M and bone marrow PC variables, and GEP-70 score >-0.26 (HR=8.82, p<0.001). Whole bone marrow biopsy (BMB) GEP samples from MGUS/AMM were available in 26 samples. Applying a BMB-65 model distinguishing MM-BMB from NL-BMB (normal donors) and MGUS/AMM-BMB, we observed that there was a strong trend for delayed MM progression when MGUS/AMM-BMB scores were in the top tertile (most NL-like). Independent validation data will be presented. Disclosures: Dhodapkar: Celgene: Research Funding; KHK: Research Funding.

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Pilot Study To Evaluate The Prevalence Of Actionable Oncogenic Mutations In Patients With Relapsed Refractory Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.755.755 ◽

2013 ◽

Vol 122 (21) ◽

pp. 755-755 ◽

Cited By ~ 1

Author(s):

Alexander Lesokhin ◽

Mithat Gonen ◽

Kaitlyn Redling ◽

Nikoletta Lendvai ◽

Hani Hassoun ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Pilot Study ◽

High Risk ◽

Plasma Cell ◽

Proteasome Inhibitor ◽

Research Funding ◽

Small Sample ◽

Braf V600e ◽

Oncogenic Mutations

Abstract Background Patients with multiple myeloma (MM) have realized improved survival with the development of multi-drug combinations using immunomodulatory drugs (IMiDs), proteasome inhibitors, and alkylating agents. Nevertheless, all MM patients eventually become refractory to available therapies, underscoring the importance of identifying additional rational therapeutic targets. Recent genomic studies using exome/copy number analysis have demonstrated that, at presentation, multiple myeloma is characterized by a dominant plasma cell clone and a heterogeneous group of subclones, with resistance emerging due to altered clonal dominance driven by therapeutic selective pressure or clonal evolution through the acquisition of additional mutational events. This suggests oncogenic mutations in dominant plasma cell clones in multiply relapsed disease may not only be involved in resistance, but should also be prioritized for further clinical development. Methods We performed a pilot study by sequencing DNA from cryopreserved whole bone marrow aspirate samples obtained pre-treatment from 28 patients with newly diagnosed myeloma (Cohort A) and 27 heavily pre-treated patients enrolled on a phase II clinical study of infusional carfilzomib (NCT01351623), a selective 2nd generation proteasome inhibitor (Cohort B). Genomic DNA and total RNA was isolated from all patient samples. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 712X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. Results Median follow-up for both cohorts was 21 months (26.3m for A; 15.6m for B). Cohort B patients were treated with a median of 5 prior therapies, with 74% refractory to the non-selective 1st generation proteasome inhibitor bortezomib, 70% refractory to IMiD therapy, and 55% refractory to both therapies. 44% had high-risk cytogenetics. Responses to initial therapy in Cohort A demonstrated that 21%, 7%, and 7%, respectively harbored bortezomib--resistant, IMiD-resistant, or double-resistant myeloma at presentation. 28% of cohort A patients had high risk cytogenetics. We obtained high coverage, high quality sequence data for 54/55 cases and examined alteration prevalence in the 35 samples with sufficient plasma cell content. We observed a high frequency of mutations in the MAPK pathway, including mutually exclusive mutations in NRAS and KRAS in 48% of cases and BRAF V600E mutation in 3%. 14% of cases had TET2 frameshift/nonsense mutations or IDH2 mutations, suggesting the DNA hydroxymethylation pathway is targeted by recurrent somatic mutations in MM. Given that MEK/RAF inhibition has demonstrated efficacy in a spectrum of human tumors and that there are emerging data that epigenetic (decitabine and 5-azacytadine) and targeted (IDH2) therapies offer significant benefit in patients with TET2/IDH mutations, these data demonstrate that mutational profiling can identify patients with actionable mutations that can lead to novel therapies, including mechanism-based clinical trials. Taken together, we identified mutations in epigenetic modifiers in 41% of the patients in our cohort, including mutations in TET2/IDH, in chromatin modifying enzymes/scaffolds (ARID1A, ASXL1), and DNA methyltransferases (DNMT3A). Moreover, we identified novel mutations in DNA repair pathways (ATM, FANCA, FANCD2) and in FAT3, suggesting there are novel disease alleles, which require functional investigation for their role in MM pathogenesis. No differences in mutation frequency were found between bortezomib sensitive vs resistant MM cases present in either cohort. We did not identify mutations, which impacted progression free and overall survival in this small sample set. Conclusions We demonstrate next generation sequencing of unsorted bone marrow samples is feasible in MM and can rapidly identify actionable mutations based on genetic profiling of limited clinical isolates. These include the identification of mutations, which can guide therapeutic trials of clinically targeting specific oncogenic pathways (ex, MAPK or TET2/IDH) on an individual patient level. Disclosures: Lesokhin: Janssen Pharmaceuticals, Inc: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Foundation Medicine, inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Wang:Foundation Medicine, Inc: Employment. Sanford:Foundation Medicine, Inc: Employment. Brennan:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy.

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Monoclonal gammopathy of undetermined significance

Blood ◽

10.1182/blood.2019846782 ◽

2019 ◽

Vol 133 (23) ◽

pp. 2484-2494 ◽

Cited By ~ 11

Author(s):

Tarek H. Mouhieddine ◽

Lachelle D. Weeks ◽

Irene M. Ghobrial

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cell ◽

Monoclonal Gammopathy ◽

Cell Clone ◽

Plasma Cell Dyscrasia ◽

Bone Marrow Niche ◽

Immunologic Factors ◽

Risk Of Progression ◽

Undetermined Significance

Abstract Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant plasma cell dyscrasia that consistently precedes multiple myeloma (MM) with a 1% risk of progression per year. Recent advances have improved understanding of the complex genetic and immunologic factors that permit progression from the aberrant plasma cell clone to MGUS and overt MM. Additional evidence supports bidirectional interaction of MGUS cells with surrounding cells in the bone marrow niche that regulates malignant transformation. However, there are no robust prognostic biomarkers. Herein we review the current body of literature on the biology of MGUS and provide a rationale for the improved identification of high-risk MGUS patients who may be appropriate for novel clinical interventions to prevent progression or eradicate premalignant clones prior to the development of overt MM.

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Molecular Characterization of Bone Marrow and Peripheral Blood Compartments from Patients with Plasma Cell Leukemia in the Mmrf Commpass Study

Blood ◽

10.1182/blood-2019-130697 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1782-1782

Author(s):

Sheri Skerget ◽

Austin Christofferson ◽

Sara Nasser ◽

Christophe Legendre ◽

The MMRF CoMMpass Network ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Plasma Cell ◽

Peripheral Blood ◽

Research Funding ◽

Plasma Cells ◽

Cell Cycle Control ◽

Cell Leukemia

Plasma cell leukemia (PCL) is rare but represents an aggressive, advanced form of multiple myeloma (MM) where neoplastic plasma cells (PCs) escape the bone marrow (BM) and circulate in the peripheral blood (PB). Traditionally, PCL is defined by the presence of >20% circulating plasma cells (CPCs), however, recent studies have suggested that PCL be redefined as the presence of >5% CPCs. The Multiple Myeloma Research Foundation CoMMpass study (NCT01454297) is a longitudinal, observational clinical study with 1143 newly diagnosed MM patients. BM-derived MM samples were characterized using whole genome (WGS), exome (WES), and RNA (RNAseq) sequencing at diagnosis and each progression event. When >5% CPCs were detected by flow cytometry, PCs were enriched independently from both compartments, and T-cells were selected from the PB as a control for WGS and WES. This substudy within CoMMpass provides the largest, most comprehensively characterized dataset of matched MM and PCL samples to date, which can be leveraged to better understand the molecular drivers of PCL. At diagnosis, 813/1143 CoMMpass patients had flow cytometry data reporting the percent PCs in PB, of which 790 had <5%, 17 had 5-20%, and 6 had >20% CPCs. Survival analyses revealed that patients with 5-20% CPCs (median = 20 months) had poor overall survival (OS) outcomes compared to patients with <5% CPCs (median = 74 months, p < 0.001), and no significant difference in outcome was observed between patients with 5-20% and >20% (median = 38 months) CPCs. Patients with 1-5% CPCs (median = 50 months, HR = 2.45, 95% CI = 1.64 - 3.69, p < 0.001) also exhibited poor OS outcomes compared to patients with <1% CPCs (median = 74 months), suggesting that patients with >1% CPCs are a higher risk population, even if they do not meet the PCL threshold. Using a cutoff of >5% CPCs, 23/813 (2.8%) patients presented with primary PCL (pPCL) at diagnosis. Of these patients, 7 (30%) were hyperdiploid (HRD), of whom 1 had a CCND1 and 1 had a MYC translocation; while 16 (70%) were nonhyperdiploid (NHRD), all of whom had a canonical immunoglobulin translocation (6 CCND1, 5 WHSC1, 3 MAF, 1 MAFA, and 1 MAFB). Of 124 patients with serial sample collections, 5 (4%) patients without pPCL had >5% CPCs at progression, and thus relapsed with secondary PCL (sPCL). Of the 5 sPCL patients, 2 (40%) were NHRD with a CCND1 or MAF translocation; while 3 (60%) were HRD, 1 with a WHSC1 translocation. Median time to diagnosis of sPCL was 22 months (range = 2 - 31 months), and patients with sPCL (median = 22 months) and pPCL (median = 30 months) exhibited poor OS outcomes as compared to MM patients (74 months, p < 0.001). Sequencing data was available for 15 pPCL and 5 sPCL samples. For 12 patients with WES, WGS, and RNAseq performed on their PCL tumor sample, an integrated analysis identified recurrent, complete loss-of-function (LOF) events in only CDKN2C/FAF1, SETD2, and TRAF3. Five pPCL patients had complete LOF of a gene involved in G1/S cell cycle control, including CDKN2C, CDKN2A, CDKN1C, and ATM. These LOF events were not observed in NHRD t(11;14) PCL patients, suggesting that CCND1 overexpression and LOF of genes involved in G1/S cell cycle control may represent independent drivers of PCL. Comparing WES and WGS data between matched MM and PCL tumor samples revealed a high degree of similarity in mutation and copy number profile. However, differential expression analysis performed for 13 patients with RNAseq data comparing their MM and PCL tumors revealed 27 up- and 39 downregulated genes (padj < 0.01, FDR = 0.1) in PCL versus MM. Pathway analysis revealed an enrichment (p < 0.001) for genes involved in adhesion and diapedesis, including upregulation of ITGB2, PF4, and PPBP, and downregulation of CCL8, CXCL12, MMP19, and VCAM1. The most significantly downregulated gene in PCL (log2FC = -6.98) was VCAM1, which plays a role in cell adhesion, and where loss of expression (TPM < 0.01) was observed across all PCL samples. Upregulation of four S100 genes including S100A8, S100A9, S100A12, and S100P, which have been implicated in tumor growth, metastasis, and immune evasion, was also observed in PCL. Interestingly, a S100A9 inhibitor has been developed and may represent a novel treatment option for PCL patients. In summary, PCL was found to be associated with molecular events dysregulating G1/S cell cycle control coupled with subtle changes in transcription that likely occur in a subclonal population of the MM tumor. Disclosures Lonial: Genentech: Consultancy; GSK: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; Karyopharm: Consultancy; Takeda: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Amgen: Consultancy.

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Cytokine Interleukin-16 Supports Progression Of Multiple Myeloma Through Different Molecular Mechanisms

Blood ◽

10.1182/blood.v122.21.5367.5367 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5367-5367

Author(s):

Julia Templin ◽

Tim Luetkens ◽

Djordje Atanackovic

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Extracellular Matrix ◽

Cell Proliferation ◽

Plasma Cell ◽

Cell Clone ◽

Myeloma Cell ◽

Gene Activity ◽

Myeloma Cells ◽

Malignant Plasma Cell

Abstract Background Multiple Myeloma (MM) is an incurable hematologic malignancy which emerges from a malignant plasma cell clone expanding within the patient’s bone marrow (BM). Plasma cell proliferation typically results in BM failure with anemia, skeletal involvement with lytic bone lesions, and consecutive hypercalcemia. The excessive production of paraprotein can lead to renal failure and recurrent bacterial infections due to a decrease in polyclonal immunoglobulins. It has previously been indicated that increased levels of Interleukin-(IL)-16 in the peripheral blood of MM patients are related to a poorer clinical outcome. We have recently demonstrated that this might be due to the fact that IL-16 is an autocrine factor used by myeloma cells to support proliferation and progression of the malignancy [1]. Here, we have investigated in detail in which way the molecular function of IL-16 is associated with the progression of MM. Methods We investigated IL-16 expression kinetics by quantitative real-time polymerase chain reaction and live-cell reporter systems for intracellular trafficking of protein expression. For functional exploration we employed a system based on the modular use of stable or inducible IL-16 knockdown by micro-RNA adapted shRNA that is expressed lentivirally. Trizium-Thymidin incorporation, flow cytometry, mRNA-based pathway analysis, investigation of metabolism of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) and fluorescence-based binding studies were performed to examine different molecular mechanism of tumor progression. Results Exploring the expression kinetics of IL-16 in myeloma cell lines we observed an energy-expensive, cyclical regulation of IL-16 gene activity leading to oscillating mRNA and protein levels. Analyzing the impact of this cyclical IL-16 production on the proliferative behavior of myeloma cells we could identify a significant decrease of proliferative activity, but not in apoptosis, in myeloma cells after IL-16 knockdown. Moreover, the ability to form colonies was strongly reduced compared to myeloma cells with unmodified IL-16 expression. Analysis of proliferative pathways resulted in the identification of different key factors for myeloma cell proliferation that show a reduced gene activity when IL-16 is missing. After induction of IL-16 knockdown, myeloma cells evidenced a declined metabolic activity and, importantly, a decreased ability to adhere to different components of the extracellular matrix. Conclusions Our findings demonstrate that IL-16, produced in a pulsatile fashion, promotes the malignant phenotype of myeloma cells through different mechanisms. It not only promotes the proliferation of the malignant plasma cell clone but it also seems to support adhesion of the tumor cells to the extracellular matrix, which is critical for settling and expanding of the malignancy within the bone marrow niche. These findings open the perspective for novel therapeutic approaches targeting IL-16 in patients with MM. References 1. Atanackovic, D., et al., Role of interleukin 16 in multiple myeloma. J Natl Cancer Inst, 2012. 104(13): p. 1005-20. Disclosures: No relevant conflicts of interest to declare.

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Novel Alkylating Agent Melflufen Displays Potent Efficacy in Plasma Cell Leukemia Samples and Other High-Risk Subtypes of Multiple Myeloma

Blood ◽

10.1182/blood-2020-138762 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-13

Author(s):

Beau M Idler ◽

Olivia Perez De Acha ◽

Owen Lockerbie ◽

Ken Flanagan ◽

Fredrik Lehmann ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

High Risk ◽

Plasma Cell ◽

Research Funding ◽

Ex Vivo ◽

Plasma Cell Leukemia ◽

Peptide Drug ◽

Cell Leukemia ◽

Plasma Cell Disorders

Introduction: Despite the progress that has been made for standard risk multiple myeloma (MM), subsets of patients with the most advanced and aggressive plasma cell dyscrasias still suffer comparatively poor outcomes. One example is plasma cell leukemia (PCL), which carries a median overall survival of under two years. For patients with PCL, response to front line therapy occurs but is often short-lived, ultimately giving way to aggressive multi-drug resistant disease and patient mortality. Thus, there is a need for the development of new strategies that improve the prognoses for these patients. Melflufen (melphalan flufenamide) is a first-in-class peptide-drug conjugate that is currently in late-phase clinical trials for multiple myeloma. This highly lipophilic agent is preferentially retained in malignant plasma cells (MPCs), where overexpressed aminopeptidases lead to trapping of the alkylator melphalan. We evaluated the anti-myeloma effects of melflufen on patient samples treated ex vivo, and found pronounced sensitivity to melflufen in most samples, with particularly potent efficacy in PCL samples. Methods: Bone marrow aspirate or peripheral blood samples were obtained from patients with plasma cell disorders after IRB approval and informed consent. Ex vivo efficacy of melflufen and melphalan were compared using our Myeloma Drug Sensitivity Testing (My-DST) platform that optimizes viability and tests the malignant cells in the context of the normal cells from their microenvironment (Walker et al, Blood Advances, 2020). In brief, mononuclear cells from patients with plasma cell dyscrasia, including MM and PCL, were isolated and cultured in triplicate wells with titrations of melphalan, melflufen or untreated controls for 48 hours. Post-treatment survival was measured by high-throughput flow cytometry with antibodies for CD138, CD38, CD45 and CD19, and a live/dead dye to discriminate viable MPCs from normal bone marrow cells. EC50 values were determined from these titrations using nonlinear regression curve fits. When the EC50 for melflufen was established in My-DST, a single dose concentration of 10 nM was used to screen patient samples and distinguish relative sensitivity or resistance. Results: Using the My-DST approach with 48 hour drug treatments, melflufen significantly decreased the viable MPC populations, whereas melphalan had little effect (Fig 1A). Concurrent titrations revealed significantly higher MPC sensitivity to melfufen (mean melphalan EC50 = not reached, mean melflufen EC50 = 22.9 nM) (Fig 1B). By comparison to another alkylator, cyclophosphamide's active metabolite has an EC50 of 3.75 µM in this assay. Response to melflufen was accentuated in 2/3 PCL samples tested (HTB-1802.1, HTB-1389.1), with the EC50 < 1nM (Fig 1B). Melflufen demonstrated toxicity in CD45 positive white blood cells, which is consistent with neutropenia observed in clinical trials (data not shown). In single dose screening studies in additional MM patient samples, 4/8 (50%) showed >20% decrease in viable MPCs after incubation with melflufen at 10 nM (Fig 1C). Overall, using those parameters for ex vivo "response" to meflufen, 3/3 patients with PCL responded, 5/6 patients with del(17p) responded, and 3/3 patients with c-MYC translocations responded (Fig 1C, italics). In addition, 3/5 samples from patients that were clinically daratumumab-refractory displayed sensitivity to melflufen. Of five samples from patients with prior exposure to alkylators, four were sensitive to melflufen. Conclusion: Overall, these data support that the peptide-drug-conjugate melflufen shows a broad efficacy across samples from patients with plasma cell disorders. Patients facing poor prognoses, including those with PCL, high-risk cytogenetics and daratumumab-refractory disease, have a great need for new treatments. Thus, the encouraging ex vivo results with melflufen in samples from these aggressive subsets support further clinical exploration. In particular, our preliminary data suggest that plasma cell leukemia patients may be exquisitely sensitive to melflufen. To follow-up these findings, we will expand the number of samples tested from PCL and other forms of high-risk MM samples. Ultimately, if the trend for accentuated sensitivity in plasma cell leukemia holds, a clinical approach for melflufen in these patients may improve outcomes for this group. Figure 1 Disclosures Lockerbie: Oncopeptides AB: Current Employment. Flanagan:Oncopeptides AB: Current Employment. Lehmann:Oncopeptides AB: Current Employment. Forsberg:Celgene: Speakers Bureau; Genentech, Inc., Sanofi, Karyopharm, Abbvie: Research Funding. Mark:Takeda: Consultancy; Kayopharm: Consultancy; Bristol-Myers Squibb: Research Funding; Janssen: Research Funding; Celgene: Consultancy; Amgen: Consultancy; Sanofi: Consultancy; Janssen: Consultancy. Sherbenou:Oncopeptides Inc.: Research Funding.

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Capture Rate of the Adaptive Next Generation Sequencing VDJ Assay in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-118768 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3184-3184 ◽

Cited By ~ 2

Author(s):

Malin Hultcrantz ◽

Even H Rustad ◽

Venkata Yellapantula ◽

Theresia Akhlaghi ◽

Allison Jacob ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Light Chain ◽

Research Funding ◽

Plasma Cells ◽

Somatic Hypermutation ◽

Capture Rate ◽

Genomic Aberration ◽

Advisory Committees

Abstract Background Minimal residual disease (MRD) negativity is a strong predictor for outcome in multiple myeloma. Next generation sequencing (NGS) for immunoglobulin heavy chain and kappa light chain VDJ rearrangements has become increasingly more common for MRD assessment. One of the known challenges with NGS for VDJ rearrangements is the vast diversity of sequences that are present, resulting in a need for a multiplex approach as common primers cannot be used to amplify all rearrangements. Also, somatic hypermutation may affect the annealing of primers and decrease the capture rate. The NGS VDJ assay developed by Adaptive Biotechnologies targets all theoretical combinations of VDJ sequences and has been used in several recent large randomized trials in multiple myeloma. The reported ~80% capture rate of the first version of the Sequenta/Adaptive 1.3 assay limited the ability to track MRD status post therapy. The assay has recently been updated and validated to increase resilience to somatic hypermutation. As there is no published reference data using this assay, we were motivated to assess VDJ capture in the clinical setting. Methods In total, 147 patients with newly diagnosed multiple myeloma (NDMM, n=101) or relapse/refractory multiple myeloma (RRMM, n=46) seen at Memorial Sloan Kettering Cancer Center were identified and included in the study. At bone marrow collection, patient samples were sorted for mononuclear cells and a subset of samples were sorted for CD138+ plasma cells. Stored bone marrow samples from these patients underwent DNA extraction and were sequenced with the Adaptive NGS VDJ assay. The same samples were also sequenced for genomic events using our internal NGS panel myTYPE. myTYPE is a custom capture panel targeting the most frequent multiple myeloma associated-somatic mutations, copy number alterations, and IGH translocations. Logistic regression was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs) of detection success in relation to clinical parameters such as age, gender, percent bone marrow plasma cells, as well as immunoglobulin heavy and light chain types, and myTYPE positivity. Results There overall capture rate for a unique VDJ sequence was 80%, 75% in NDMM samples and 89% in RRMM samples, respectively. The VDJ capture rate in samples that were myTYPE positive, e.g. samples with at least one genomic aberration detected by myTYPE, was 94%. In univariate analysis, the ORs of detecting a clonal VDJ sequence was 1.8 (95% CI 1.3-2.5) and 1.5 (1.2-1.9) for every 10% increase in plasma cells on bone marrow aspirate and biopsy, respectively. For every 1g/dL increase in M-spike, the OR of VDJ capture was 1.6 (1.2-2.2). Samples with at least one genomic aberration detected by myTYPE had a significantly higher detection rate of VDJ sequence, the OR of VDJ capture in myTYPE positive samples was 8.8 (3.2-31.3). Conversely, age, gender, type of immunoglobulin heavy chain (IgG or IgA), or light chain type (kappa or lambda) had no significant effect on the VDJ detection rate (Table). In multivariate analysis, myTYPE positivity was found to be an independent predictor of VDJ capture, with an OR of 4.9 (1.6-18.4, p=0.009) for myTYPE positive samples. The ORs were 1.4 (1.1-2.2, p=0.052) for an increase in 10% plasma cells on bone marrow aspirate and 1.5 (0.97-2.3, p=0.083) every 1g/dL increase in M-spike. Conclusion The VDJ capture rate using the updated Adaptive NGS VDJ assay was 94% in multiple myeloma samples of high quality as indicated by myTYPE positivity. The capture success rate was higher in samples with a greater disease burden. As expected, the assay was less sensitive in samples with insufficient DNA content. Our results are supportive of the use of this NGS VDJ in multiple myeloma, but also illustrate the importance of optimal sample ascertainment and processing. Disclosures Jacob: Adaptive Biotechnologies: Employment, Equity Ownership. Korde:Amgen: Research Funding. Mailankody:Juno: Research Funding; Physician Education Resource: Honoraria; Janssen: Research Funding; Takeda: Research Funding. Lesokhin:Serametrix, inc.: Patents & Royalties: Royalties; Squibb: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Research Funding; Genentech: Research Funding. Hassoun:Oncopeptides AB: Research Funding. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Landgren:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding.

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