Malignant Plasma Cells In Multiple Myeloma Research Articles

Updated results of a phase 1, first-in-human study of talquetamab, a G protein-coupled receptor family C group 5 member D (GPRC5D) × CD3 bispecific antibody, in relapsed/refractory multiple myeloma (MM).

8008 Background: New immunotherapy targets in MM are needed as patients (pts) continue to relapse. The orphan receptor GPRC5D is expressed on malignant plasma cells in MM. Talquetamab (JNJ-64407564) is a bispecific IgG4 antibody that redirects T cell killing to MM cells by binding to the novel target, GPRC5D, and CD3. We present updated results of talquetamab at the recommended phase 2 dose (RP2D) from a phase 1 trial in relapsed/refractory MM. Methods: Eligible pts with MM who had relapsed or refractory disease or were intolerant to standard therapies received talquetamab intravenously (IV; range 0.5–180 µg/kg) or subcutaneously (SC; range 5.0–800 µg/kg) weekly or biweekly. Primary objectives were identification of the RP2D (part 1) and talquetamab safety and tolerability at the RP2D (part 2). Adverse events (AEs) were graded by CTCAE v4.03 (cytokine release syndrome [CRS] per Lee 2014). Response was assessed per IMWG criteria. Results: As of Feb 8, 2021, 174 pts received talquetamab, 102 by IV and 72 by SC; in parts 1 and 2, 28 pts were treated at the RP2D, identified as weekly SC 405 µg/kg, with 10.0 and 60.0 µg/kg step-up doses. Pts treated at the RP2D had a median age of 61.5 y (range 46–80) and a median of 5.5 prior lines of therapy (range 2–14; 100%/79% triple-class/penta-drug exposed; 71%/18% triple-class/penta-drug refractory; 86% refractory to last line of therapy; 21% with prior B-cell maturation antigen–directed therapy). No dose-limiting toxicities occurred at the RP2D in part 1. Most common AEs at the RP2D were CRS (79%; grade 3 4%; median time to onset: day after SC injection), neutropenia (64%; grade 3/4 54%), anemia (57%; grade 3/4 29%) and dysgeusia (57%; all grade 1/2); infections were reported in 32% of pts (grade 3/4 4%) and neurotoxicity in 7% (grade 3/4 0). In all, 75% of pts dosed at the RP2D had skin-related AEs (grade 3/4 0), including 18% with nail disorders. The overall response rate at the RP2D in response-evaluable pts (n = 24) was 63%, with 50% reaching very good partial response or better; 9/17 (53%) evaluable triple-class refractory pts and 3/3 (100%) penta-drug refractory pts had a response. Median time to first confirmed response at the RP2D was 1.0 mo (range 0.2–3.8); responses were durable and deepened over time (median follow-up 6.2 mo [range 2.7–9.7+] for responders at the RP2D). At the RP2D, exposure was maintained over the maximum EC90 target level from an ex vivo cytotoxicity assay, and consistent T cell activation was seen. Conclusions: At the RP2D of weekly 405 µg/kg SC, talquetamab showed a high clinical response rate and was well-tolerated in pts with relapsed/refractory MM; based on pharmacokinetic data, other SC dosing strategies are being explored. The promising efficacy, safety profile and convenience of SC dosing support monotherapy development and combination approaches with this novel agent. Clinical trial information: NCT03399799.

Treatment of Lymphoid and Myeloid Malignancies by Immunomodulatory Drugs.

Thalidomide and its derivatives (lenalidomide, pomalidomide, avadomide, iberdomide hydrochoride, CC-885 and CC-90009) form the family of immunomodulatory drugs (IMiDs). Lenalidomide (CC5013, Revlimid®) was approved by the US FDA and the EMA for the treatment of multiple myeloma (MM) patients, low or intermediate-1 risk transfusion-dependent myelodysplastic syndrome (MDS) with chromosome 5q deletion [del(5q)] and relapsed and/or refractory mantle cell lymphoma following bortezomib. Lenalidomide has also been studied in clinical trials and has shown promising activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). Lenalidomide has anti-inflammatory effects and inhibits angiogenesis. Pomalidomide (CC4047, Imnovid® [EU], Pomalyst® [USA]) was approved for advanced MM insensitive to bortezomib and lenalidomide. Other IMiDs are in phases 1 and 2 of clinical trials. Cereblon (CRBN) seems to have an important role in IMiDs action in both lymphoid and myeloid hematological malignancies. Cereblon acts as the substrate receptor of a cullin-4 really interesting new gene (RING) E3 ubiquitin ligase CRL4CRBN. This E3 ubiquitin ligase in the absence of lenalidomide ubiquitinates CRBN itself and the other components of CRL4CRBN complex. Presence of lenalidomide changes specificity of CRL4CRBN which ubiquitinates two transcription factors, IKZF1 (Ikaros) and IKZF3 (Aiolos), and casein kinase 1α (CK1α) and marks them for degradation in proteasomes. Both these transcription factors (IKZF1 and IKZF3) stimulate proliferation of MM cells and inhibit T cells. Low CRBN level was connected with insensitivity of MM cells to lenalidomide. Lenalidomide decreases expression of protein argonaute-2, which binds to cereblon. Argonaute-2 seems to be an important drug target against IMiDs resistance in MM cells. Lenalidomide decreases also basigin and monocarboxylate transporter 1 in MM cells. MM cells with low expression of Ikaros, Aiolos and basigin are more sensitive to lenalidomide treatment. The CK1α gene (CSNK1A1) is located on 5q32 in commonly deleted region (CDR) in del(5q) MDS. Inhibition of CK1α sensitizes del(5q) MDS cells to lenalidomide. CK1α mediates also survival of malignant plasma cells in MM. Though, inhibition of CK1α is a potential novel therapy not only in del(5q) MDS but also in MM. High level of full length CRBN mRNA in mononuclear cells of bone marrow and of peripheral blood seems to be necessary for successful therapy of del(5q) MDS with lenalidomide. While transfusion independence (TI) after lenalidomide treatment is more than 60% in MDS patients with del(5q), only 25% TI and substantially shorter duration of response with occurrence of neutropenia and thrombocytopenia were achieved in lower risk MDS patients with normal karyotype treated with lenalidomide. Shortage of the biomarkers for lenalidomide response in these MDS patients is the main problem up to now.

Altering Glycosphingolipid Composition to Improve Multiple Myeloma Bone Complication

Multiple myeloma (MM) is an incurable cancer of plasma cells (PC), with a median survival of 5-7 years. Osteolytic bone disease and skeletal complications occur in more than 80% of MM patients and significantly contribute to the morbidity and mortality of these patients. Glycosphingolipid (GSL), an essential constituent of the outer leaflet of the cellular membrane, is altered in MM and other hematological cancers. We previously reported that GM3, a subtype of GSL promotes osteoclastogenesis. On the other hand, the GSL synthase inhibitor N-butyl-deoxynojirimycin (NB-DNJ) reduces myeloma bone disease in the 5TGM1 mouse model of MM. Mechanistically, NB-DNJ prevents osteoclast (OC) development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of the transcriptional activator NFATc1.Although NB-DNJ is an FDA-approved drug treating Gaucher's disease, it has many undesired off-target effects, such as inhibiting lysosomal and plasma membrane Beta-glucocerebrosidase and interfering with intestinal glucosidases which leads to gastrointestinal toxicities and severe weight loss. Therefore, more specific GSL inhibitors are required to minimize the side effects.Here we report a novel GSL inhibitor called Genz112638 with comparable effects as NB-DNJ but reduced side effects. Genz112638 inhibits both OC formation (p < 0.01) and MM cell growth (p < 0.0001) in vitro in a dose-dependent manner. Moreover, compared to NB-DNJ, Genz112638 more significantly improved bone condition and potentially reduced MM burden, as evidenced by the amelioration of bone loss in the 5TGM1 model of myeloma, and a reduction in the proportion of MM within bone marrow and spleen without obvious adverse effects (n=6) (p < 0.01).As excessive malignant PC in MM normally arise from germinal centre, we also checked the effects of Genz112638 on germinal centre reactions in wildtype mice. We found that Genz112638 suppresses the formation of germinal centre B cells in mouse spleen induced by sheep red blood cells (n=7). Thus, Genz112638 may affect the pathogenesis of MM disease at the initial stage.Taken together, our data elucidate a novel specific GSL inhibitor as a promising candidate drug relieving two main features of MM: bone destruction and tumour burden with negligible side effects. In vitro, it decreases OC differentiation and proliferation, and meanwhile decreases MM viability and proliferation. In vivo, it may suppress B cell formation in germinal centre, ameliorate bone destruction, and potentially interfere with the vicious cycle between increased OC and susceptibility to MM. In short, we provide a preclinical platform for GSL inhibition as a new tool against MM and its related complications. [Display omitted] DisclosuresHorwood:Genzyme: Research Funding.

Role and Regulation of Pro-survival BCL-2 Proteins in Multiple Myeloma.

Apoptosis plays a key role in protection against genomic instability and maintaining tissue homeostasis, and also shapes humoral immune responses. During generation of an antibody response, multiple rounds of B-cell expansion and selection take place in germinal centers (GC) before high antigen affinity memory B-cells and long-lived plasma cells (PC) are produced. These processes are tightly regulated by the intrinsic apoptosis pathway, and malignant transformation throughout and following the GC reaction is often characterized by apoptosis resistance. Expression of pro-survival BCL-2 family protein MCL-1 is essential for survival of malignant PC in multiple myeloma (MM). In addition, BCL-2 and BCL-XL contribute to apoptosis resistance. MCL-1, BCL-2, and BCL-XL expression is induced and maintained by signals from the bone marrow microenvironment, but overexpression can also result from genetic lesions. Since MM PC depend on these proteins for survival, inhibiting pro-survival BCL-2 proteins using novel and highly specific BH3-mimetic inhibitors is a promising strategy for treatment. This review addresses the role and regulation of pro-survival BCL-2 family proteins during healthy PC differentiation and in MM, as well as their potential as therapeutic targets.

Inhibitors of the Cyclin-Dependent Kinase and PIM Kinase Pathways in the Treatment of Myeloma.

Multiple myeloma (MM) cells are characterized by genomic alternations that lead to increased cell proliferation and resistance to therapeutic interventions. Up-regulation of cyclins is a characteristic finding in a significant proportion of myeloma patients, mediated through a variety of mechanisms including chromosomal translocations. Cyclins and the cyclin-dependent kinases (CDKs) play a critical role in the cell proliferation seen in MM, especially in the high-risk disease. Given this, CDK inhibitors have been evaluated in this disease, and studies so far have led to a mixed picture. Recent studies with targeted CDK inhibitors have shown early promise, and trials of these drugs in combination with other myeloma drugs are ongoing. The malignant plasma cells in MM are highly dependent on the microenvironment for their growth and survival. Multiple signaling pathways have been found to mediate the interactions between the microenvironment and the plasma cells, whether mediated through cytokines or adhesion molecules. The PIM kinase pathway appears to play a major role in the myeloma cell survival, and PIM kinase inhibitors have shown efficacy in the laboratory, and a recent clinical trial also demonstrates important clinical activity.

Interrelationship between angiogenesis, inflammation and oxidative stress in Indian patients with multiple myeloma.

Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of clonal population of plasma cells in the bone marrow (BM). A variety of angiogenic factors, proteases, reactive oxygen species and inflammatory cytokines induce the formation of an extensive and suitable BM microenvironment. Previous studies have established the importance of angiogenic factors, inflammatory molecules and oxidative stress in MM but their interplay and effect on each other are not being taken together. Circulatory levels of VEGF, angiopoietin-2 (Ang-2), IL-6 and TNF-α along with the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were investigated in 112 subjects including 62 MM patients and 50 healthy controls. Inter-stage analysis was done to evaluate the association of these molecules with the severity of disease. Pearson correlation was determined to find interrelationship, if any, between these molecules. We have observed elevated levels of VEGF, Ang-2, IL-6, TNF-α and decreased activity of SOD, GPx in MM patients in comparison to controls. All these molecules also showed a trend with the severity of disease. We have found strong association between these factors upon their correlation and regression analysis. This study is a step toward understanding the indepth contribution of angiogenesis, inflammation and oxidative stress together in making BM microenvironment suitable for growth, survival and proliferation of malignant plasma cells in MM.

Anti-Myeloma Activity by the Combination of the JAK2 Inhibitor Ruxolitinib with Lenalidomide and Corticosteroids

Introduction: The JAK2 inhibitor ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) that is effective for the treatment of myeloproliferative diseases. Immunomodulatory drugs (IMiDs) including lenalidomide (LEN) and corticosteroids have shown efficacy for the treatment of multiple myeloma (MM). The JAK-STAT signaling pathway plays key roles in the growth and survival of malignant plasma cells in MM. In this study, we evaluated the preclinical anti-MM effects of RUX in combination with LEN and corticosteroids, both in vitro and in vivo, and in a patient with MM and polycythemia rubra vera (PRV).Methods: The human MM cell lines U266, RPMI8226 and MM1S cells were derived from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow aspirates. The cells were seeded at105 cells/100ul/well in 96-well plates and incubated for 24 h in the presence of vehicle, RUX, LEN or dexamethasone (DEX) alone, RUX + LEN, RUX + DEX, or all three drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. In vitro, synergy between ruxolitinib and lenalidomide or dexamethasone was assessed using the median effect method of Chou and Talalay. For the in vivo studies, the human myeloma tumors (LAGκ-1A or LAGκ-2) were surgically implanted into the left superficial gluteal muscle of anaesthetized naive SCID mice. Mice were blindly assigned to one of the experimental groups, and treatment was initiated 7–21 d after tumor implantation. LEN was administered via oral gavage daily (30 mg/kg). RUX (3 mg/kg) was given via intraperitoneal (IP) injection twice daily. Dexamethasone was administered daily (1.5mg/kg) via IP injection. An 88 year old MM patient with PRV who developed MM on RUX alone and then progressed on LEN+DEX was treated with the combination of all three drugs.Results:In vitro, RUX induced concentration-dependent inhibition of viability in all three MM cell lines (U266, RPMI8226 and MM1S) at RUX 50 mM and inhibition of primary MM tumor cells at a higher concentration (100 mM). In contrast, RUX had negligible cytotoxic effects on normal peripheral blood mononuclear cells (PBMCs). We next examined cell viability in the presence of RUX plus LEN or DEX. First, U266 cells were incubated with a fixed concentration of LEN (30 mM) or DEX (40 mM) with increasing concentrations of RUX (0.1–100 mM) for 48 h. At RUX 50 mM, the cytotoxic effects of LEN were enhanced and at RUX 1 mM, the anti-myeloma effect of DEX was increased. Moreover, the cytotoxic effects of RUX, LEN and DEX were greater than RUX in combination with either LEN or DEX in U266 cells. Similar results were obtained using the RPMI8226 and MM1S cell lines as well as primary MM tumor cells. Next, we evaluated RUX in combination with lenalidomide and dexamethasone in vivo using SCID mice bearing either the human LAGκ-1A or LAGκ-2 MM xenografts. RUX (3mg/kg), LEN (15mg/kg) or DEX (1mg/kg) alone did not inhibit tumor growth in either mice bearing LAGκ-1A or LAGκ-2. In contrast, the combination of RUX with DEX but not LEN slightly decreased tumor volume. However, the combination of all three drugs at the same doses showed a marked reduction of tumor size and delay of tumor growth in both human MM xenograft models. In addition, a patient with MM and PRV experienced sustained and ongoing reductions in his serum M-protein, IgG, and 24-urine M-protein with achievement of a partial response on low doses of RUX (2.5 mg twice daily), LEN (2.5 mg daily), and methylprednisolone (20 mg daily) that has been ongoing for more than 12 months after developing MM on RUX alone and then progressing on the combination of LEN and methylprednisolone.Conclusion: This study illustrates that the combination of the JAK2 inhibitor RUX, LEN and corticosteroids shows both preclinical and promising clinical results for the treatment of MM. DisclosuresNo relevant conflicts of interest to declare.

The Significance Of Abnormal Plasma Cell Clone In Bone Marrow Of Primary Systemic Light Chain Amyloidosis Patients

In this study we analyzed the immunophenotype characteristics of the plasma cells and evaluated the significance of the abnormal plasma cell clone in bone marrow in primary systemic light chain amyloidosis (AL) patients. Fresh bone marrow samples were collected from 74 cases of plasma cell disease (PCD), including 51 cases of AL, 21 cases of multiple myeloma (MM), 2 cases of Waldenström's macroglobulinemia (WM). All patients diagnosed according to WHO 2008 diagnostic criteria. The diagnosis of AL was confirmed by the presence of monoclonal immunoglobulin or free light chain in blood or urine, and/or amyloidosis in fat tissues or biopsies by Congo red staining. Ten healthy donors were also collected as controls. Their clinical characteristics and immunophenotype of bone marrow cells were compared and analyzed. The immunophenotype were analyzed with a panel of antibodies including CD45, CD38, CD138, CD117, CD56, CD19, CD20, Igκ, Igλ, CD7, CD22, CD3, CD34 and CD27 by flow cytometry (FCM). The results were for statistical processing.The prominent feature of AL patients was multi-organ and multi-system involvement. Kidney was the major organ involvement (82.4%), followed by cardiovascular system (58.8%); MM mainly had the clinical manifestations of bone lesions (85.7%) and renal involvement (47.6%). The serum immunoglobulin of AL mainly manifested as λ light chain (74.5%), while the majority of MM manifested as κ (61.9%). In the 51 patients of AL, the ratio of plasma cellsin bone marrow was mean 3.87% (0.17∼9.34%) by FCM, and 4.47% (0∼14.5%) by morphological examination. In MM, the ratio of plasma cells was mean 13.17% (1.30∼48.91%) by FCM and 33.55% (3.0∼81.5%) by morphological examination. The plasma cells proportion between AL and MM had significant difference (P< 0.05). The κ or λ light chain restriction can be used for the detection of abnormal plasma cell clones in AL patients. The κ/λ ratio>4.0 or <0.5 can be used as the criteria to identify light chain restriction in plasma cells in AL patients. The 31/51 cases of AL could detected abnormal plasma cell clone that used κ/λ light chain restriction and were mainly expressed λ light chain (24/31, 77.4%). The 21 cases of MM had light chain restriction, mainly expressed κ light chain (13/21, 61.9%) (P<0.05). In CD45/SSC scattergram, the position of abnormal plasma cells of AL patients varied in a wider range. According to the features of CD38+/CD138+ as the basic markers for plasma cells, abnormal plasma cells were CD45 negative or weak positive in AL patients, similar to the CD45 level distribution in malignant plasma cells in MM. In WM, the proliferated cells were plasmacytoid lymphocytes with CD45 weakly or strong positive. FCM can identify abnormal plasma cell clone in bone marrow of AL patients. In 51cases of AL, 78.4% of bone marrow plasma cells were CD56+, 68.6% were CD117+, and 88.2% were CD19-. In 21 of MM, 66.7% were CD56+, 38.1% were CD117+, and 90.4% were CD19-. These results manifested significant difference compared with those of normal plasma cells (P< 0.05). In 2 cases of WM, these plasmacytoid lymphocytes were CD19+ and CD56-, CD117-.The ratios of CD56+, CD117+, CD19-, and CD45-/dim in bone marrow plasma cells were significantly higher in AL patients than in WM patients and healthy individuals (P<0.05), but were similar to those in MM patients (P>0.05). The main difference between AL and MM was the larger size of plasma cell group in MM (P＜0.05).In summary, according to light chain restricted expression and abnormal immunephenotype by FCM analysis we can determine abnormal plasma cell clone in bone marrow of AL patients and the abnormal plasma cells clone can be used as an important diagnostic marker of AL. Disclosures:No relevant conflicts of interest to declare.

Macrophages and mesenchymal stromal cells support survival and proliferation of multiple myeloma cells

Multiple myeloma (MM) is characterized by almost exclusive tropism of malignant cells for the bone marrow (BM) milieu. The survival and proliferation of malignant plasma cells have been shown to rely on interactions with nonmalignant stromal cells, in particular mesenchymal stromal cells (MSCs), in the BM microenvironment. However, the BM microenvironment is composed of a diverse array of cell types. This study examined the role of macrophages, an abundant component of BM stroma, as a potential niche component that supports malignant plasma cells. We investigated the proliferation of MM tumour cell lines when cultured alone or together with MSCs, macrophages, or a combination of MSCs and macrophages, using the carboxyfluorescein succinimidyl ester assay. Consistently, we observed increased proliferation of MM cell lines in the presence of either MSCs or macrophages compared to cell line-only control. Furthermore, the combined co-culture of MSCs plus macrophages induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, MSCs and macrophages decreased the rate of apoptosis of myeloma cells. Our in vitro studies provide evidence that highlights the role of macrophages as a key component of the BM microenvironment facilitating the growth of malignant plasma cells in MM.

Comprehensive Assessment of Potential Multiple Myeloma Immunoglobulin Heavy Chain V-D-J Intraclonal Variation Using Massively Parallel Pyrosequencing

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). MM is viewed as a clonal disorder due to lack of verified intraclonal sequence diversity in the immunoglobulin heavy chain variable region gene (IGHV). However, this conclusion is based on analysis of a very limited number of IGHV subclones and the methodology employed did not permit simultaneous analysis of the IGHV repertoire of non-malignant PCs in the same samples. Here we generated genomic DNA and cDNA libraries from purified MM BMPCs and performed massively parallel pyrosequencing to determine the frequency of cells expressing identical IGHV sequences. This method provided an unprecedented opportunity to interrogate the presence of clonally related MM cells and evaluate the IGHV repertoire of non-MM PCs. Within the MM sample, 37 IGHV genes were expressed, with 98.9% of all immunoglobulin sequences using the same IGHV gene as the MM clone and 83.0% exhibiting exact nucleotide sequence identity in the IGHV and heavy chain complementarity determining region 3 (HCDR3). Of interest, we observed in both genomic DNA and cDNA libraries 48 sets of identical sequences with single point mutations in the MM clonal IGHV or HCDR3 regions. These nucleotide changes were suggestive of putative subclones and therefore were subjected to detailed analysis to interpret: 1) their legitimacy as true subclones; and 2) their significance in the context of MM. Finally, we report for the first time the IGHV repertoire of normal human BMPCs and our data demonstrate the extent of IGHV repertoire diversity as well as the frequency of clonally-related normal BMPCs. This study demonstrates the power and potential weaknesses of in-depth sequencing as a tool to thoroughly investigate the phylogeny of malignant PCs in MM and the IGHV repertoire of normal BMPCs.

Reduction In Proliferative Rate of Myeloma Plasma Cells Measured by the Plasma Cell Labeling Index Predicts Improved Survival In Multiple Myeloma

AbstractAbstract 4048 Background:The plasma cell labeling index (PCLI) is measured as the percentage of bone marrow plasma cells (PC) in the S phase of the cell cycle and provides an estimate of the proliferative capacity of malignant PC in multiple myeloma (MM). The International Myeloma Working Group (IMWG) uniform response criteria measure the monoclonal (M) protein and serum free light chain (FLC) assay to evaluate clinical response. An elevated PCLI is an established adverse prognostic risk factor for survival, however it is unknown if the PCLI response to therapy offers further prognostic value. Methods:Data from 176 newly diagnosed MM patients from 1985 to 2005 were collected from the Mayo Clinic dysproteinemia database. Inclusion criteria for the study required a minimum initial PCLI of 0.5% and a subsequent PCLI 4 months after initiation of therapy. Baseline and subsequent serum M-protein, progression date, and overall survival were recorded. Clinical responses were assessed by the IMWG uniform response criteria. The association of PCLI response and treatment response (CR, VGPR, or PR) was assessed univariately. Overall survival based on PCLI response, M-spike reduction, and treatment response by IMWG criteria was estimated using the Kaplan-Meier method. Results:The median overall survival (OS) was 47 months (range: 4 – 165). Ninety-four patients (53%) demonstrated a ≥60% reduction in PCLI after initial therapy (responders) and this conferred a significant OS advantage of 51 months vs. 34 months (P<0.01; Figure 1) compared to those who achieved <60% PCLI reduction (non-responders) after initial therapy. The median PCLI percent difference following 4 months of therapy was -67% (+/− 137%; range: -100 to 953%). When patients were stratified into high (PCLI of ≥3%), intermediate (PCLI ≥1%) and low (PCLI <1%) risk groups, OS was significantly improved in the high and intermediate PCLI groups if they achieved ≥60% reduction in PCLI after initial therapy, 31 vs. 7 months (P<0.0022; Figure 2) and 63 vs. 25 months (P<0.0005; Figure 3), respectively. As observed in prior studies, patients with PCLI of ≥3% at diagnosis had shorter overall survival (17 months) when compared to patients with an initial PCLI of <1% (61 months; P < 0.0001). Of the 94 responders, 39% (n=37) did not achieve a response to therapy as measured by M-protein. Thirteen patients (14%) had no measurable serum M-protein at baseline. Conversely, 35% (n=31) of patients that responded by M-protein criteria did not achieve a PCLI response. In patients who achieved CR/PR after 4 months of therapy, the median PCLI reduction was 83.7% vs. 22.5% (P <0.0004) compared to patients with stable disease (not meeting criteria for CR or PR) or progressive disease (as specified by IMWG response criteria). This reflects a significant reduction in disease burden and as expected, median OS was better in the CR/PR group (53 vs. 31 months, P <0.0016). [Display omitted] Conclusion:Our study demonstrates that the degree of PCLI reduction after therapy is important in predicting overall survival and response to therapy. This is an important finding in the current setting of effective MM treatment options, whereby patients who do not achieve an adequate response as defined as ≥60% reduction in PCLI in our study, may require change in therapy. Our data confirm that reduction of M-protein is an important surrogate endpoint, as patients who achieved PR and CR lived significantly longer. A significant portion of individuals 39% had a measurable response by PCLI that was not detected by M-protein. Further studies are needed to evaluate the predictive value of the addition a criterion that looks at reduction in proliferative rate as measured by PCLI or other methods such as flow cytometry. Disclosures:Greipp:Mayo Clinic: Patents & Royalties.

MiRNA Analysis Identifies a Unique Expression in Waldenström Macroglobulinemia B Cells and Plasma Cells

MicroRNAs (miRNAs) are small noncoding RNAs that are approximately 20–22 nucleotides with critical functions in cell growth, survival, and differentiation. These conserved sequences can regulate expression of multiple genes and are often tissue specific and dysregulated in malignancies. Thus, miRNA profiling has been used to create signatures for many solid tumors. These profiles have been used to classify tumors and to help predict survival and outcome. In the present study, we utilized the DiscovArray miRNA profiling service (Asuragen Services, Austin, TX) which utilizes a custom-manufactured Affymetrix GeneChip® from Ambion that covers miRNAs derived from the Sanger miRBase (http://microrna.sanger.ac.uk/sequences/index.shtml) and over 11,000 predicted miRNAs derived from published reports. The signal processing implemented was a multi-step process involving probe-specific signal detection calls, background estimation and correction, constant variance stabilization and global normalization. For each probe, an estimated background value was subtracted derived from the median signal of a set of G-C-matched anti-genomic controls. Arrays within a specific experiment were normalized together according to variance stabilization method. Detection calls were based on a Wilcoxon rank-sum test of the miRNA probe signal compared to the distribution of signals from GC-content matched anti-genomic probes. For statistical hypothesis testing, a two-sample t-test, with assumption of equal variance, was applied. One-way ANOVA was used for multiple group comparison. Probes were considered to be differentially expressed based on two criteria: a p-value of < 0.001 and glog2 difference > 1. miRNA expression was analyzed in all malignant B lineage cells (CD19+ CD138+) (n=8), malignant B cells alone (CD19+) (n=6) and plasma cells alone (CD138+) (n=3) from Waldenström macroglobulinemia (WM) patients. The expression was compared to malignant CD19+ B cells from chronic lymphocytic leukemia (CLL) patients (n=5), malignant plasma cells (CD138+) from multiple myeloma (MM) patients (n=5) and to B lineage cells (CD19+ CD138+) (n=4), CD19+ B-lymphocytes (n=3) and CD138+ plasma cells (n=6) from healthy donors. Data analysis based on a total of approximately 11,000 miRNAs analyzed shows that CD19+ CD138+ cells (double sorting) from WM patients did not cluster as a unique group. Some samples had a pattern similar to CLL, some similar to MM and others similar to CD19+ CD138+ cells from healthy controls. This lack of clear signature was observed by others in gene expression profiling and CGH arrays. We therefore hypothesized this lack of clustering was due to the lymphoplasmacytic nature of WM cells and therefore we analyzed B cells (CD19+) and plasma cells (CD138+) separately. miRNA expression in B cells (CD19+) identifies a signature in normal B cells that is absent in both WM B cells (CD19+) and CLL cells. There is also a set of miRNAs that are absent in normal B cells that are expressed in WM B cells and CLL. In addition, WM B cells had a unique miRNA signature that is unique compared to CLL and normal B cells. An additional set of miRNAs were expressed and clustered only in CLL patients. Similar to B cells, plasma cell (CD138+) analysis in WM, MM and healthy donors shows a clustering pattern that identifies normal plasma cells from MM plasma cells. WM plasma cells had a miRNA signature that is unique only to WM patients, however, a subset of miRNAs shared an expression pattern with MM plasma cells. While miRNAs can target multiple genes, some of the genes that are targets of the miRNAs identified in this analysis include XBP-1, Blimp-1, IRF-4, Bcl-6 and TACI. These target genes are known to be important in B cell and plasma cell development. In summary, we have analyzed miRNA expression in malignant B cells (CD19+) and malignant plasma cells (CD138+) from WM patients and compared their expression pattern to their normal counterpart as well as malignant counterpart in CLL B cells and MM plasma cells. Our analysis shows that WM B cells have a miRNA signature unique to WM only and one that is shared by CLL cells. Similarly, WM plasma cells have a unique miRNA signature but also has some miRNAs that are shared by malignant plasma cells in MM. These miRNAs target genes involved in B cell differentiation. Analysis of the functional roles of these miRNAs will and their regulation will further our understanding of the regulation of B cells development in normal and malignant conditions.

Potential of bone marrow biopsy/aspirate for classification, staging and monitoring of multiple myeloma. A prospective study of the German Myeloma Treatment Group

Potential of bone marrow biopsy/aspirate for classification, staging and monitoring of multiple myeloma. A prospective study of the German Myeloma Treatment Group