Clinical and Biological Correlates of CD45 Expression on Bone Marrow Plasma Cells from Patients with Multiple Myeloma.

Abstract Background: Multiple myeloma (MM) is characterized by accumulation of clonal malignant plasma cells in the bone marrow (BM). CD45 was initially characterized as a leucocyte common antigen and can be found on all hematopoietic cells and is a key regulator of antigen mediated signaling and activation in B and T-lymphocytes. The expression of CD45 appears to be heterogeneous among the plasma cells in MM and this characteristic has been suggested to have biological and prognostic significance. Goals: To study CD45 expression by flow cytometry (FC) on BM plasma cells from patients with different stages of MM, and compared CD45 expression to key clinical and biological characteristics including clinical outcome, labeling index (%S-phase), bone marrow angiogenesis, and bone disease. In addition, the expression profile of various adhesion molecules on CD45+ vs CD45− plasma cells was studied. Results: BM from seventy-five patients seen at the Mayo Clinic during 1995 and diagnosed with newly diagnosed MM (29), relapsed MM (17), smoldering MM (12), and MGUS (17) were studied for CD45 expression.. CD45+ was defined as >20% of gated plasma cells expressing CD45. A mean of 30% of the plasma cells expressed CD45 by flow cytometry (median, 14%; range, 0.1 to 100%). Among the entire cohort of patients, 33 (44%) of the patients were CD45+. Patients with early disease (MGUS and SMM) had a higher percentage of plasma cells expressing CD45 compared with patients with advanced disease (new or relapsed MM) (43% vs. 22%; P = 0.005). Among those with early disease 69% were CD45 positive compared to 28% among those with advanced disease (P = 0.0008). Among the 46 patients with advanced disease, the mean percentage of CD45+ plasma cells was 14% (range, 0.1 – 85) for those with bone lesions compared to 34% (2 – 99) for those with none; P = 0.02. Also, those with high labeling index (>=1%) had a higher percentage of CD45 expressing plasma cells than those with a low labeling index (30% for high LI vs. 15% for low LI; (P = 0.08). Among those with advanced disease, patients with low-grade angiogenesis had a higher percentage of CD45+ plasma cells; 31% vs. 13% (P = 0.03). The median overall survival for the CD45 positive group was 39 mos vs. 18 mos for the CD45 negative group, but the difference was not statistically significant (P=0.1). The expression of CD138 (syndecan-1), CD56 (NCAM) and CD54 (ICAM-1) were higher among the CD45 neg plasma cells. Other adhesion molecules, especially LFA-1, Mac-1, VLA-5, and CD44 were more often expressed on the CD45 + plasma cells. Conclusions: This study demonstrates that CD45 expression is more common in early disease and is lost with disease progression. There are important immunophenotypic and biologic differences in the CD45+ vs CD45− plasma cells that may have implications for the design of immunotherapy for MM.

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The Prognostic Significance of Polyclonal Bone Marrow Plasma Cells in Patients with Actively Relapsing Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.1194.1194 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1194-1194

Author(s):

Toshi Ghosh ◽

Wilson I Gonsalves ◽

Dragan Jevremovic ◽

S. Vincent Rajkumar ◽

Michael M. Timm ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

High Risk ◽

Research Funding ◽

Plasma Cells ◽

Labeling Index ◽

Light Chains ◽

Fish Cytogenetics ◽

Kaplan Meier

Abstract Background: Prior studies suggest that the presence of >5% polyclonal plasma cells (pPCs) among total plasma cells (PCs) within the bone marrow (BM) is associated with a longer progression-free survival, higher response rates, and lower frequency of high-risk cytogenetic abnormalities in patients with newly diagnosed multiple myeloma (MM). However, the incidence and prognostic utility of this factor in patients with relapsed and/or refractory MM has not been previously evaluated. Thus, we evaluated the prognostic value of quantifying the percentage of pPCs among the total PCs in the BM of patients with actively relapsing MM. Methods: We evaluated all MM patients with actively relapsing disease (biochemical and/or symptomatic) seen at the Mayo Clinic, Rochester, from 2012 to 2013, who had BM samples evaluated by seven-color multiparametric flow cytometry. All patients had at least 24 months of follow-up from the date of flow evaluation. Cell surface antigens were assessed by direct immunofluorescence antibodies for CD45, CD19, CD38, CD138, cytoplasmic Kappa and Lambda Ig light chains, and DAPI nuclear stain. The flow cytometry data was collected using the Becton Dickinson FACSCanto II instruments that analyzed 150,000 events (cells); this data was then analyzed by multi-parameter analysis using the BD FACS DIVA Software. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Clonal PCs were separated from pPCs based on the differential expression of CD45, CD19, DAPI (in non-diploid cases), and immunoglobulin light chains. The percentage of pPCs was calculated in total PCs detected. Survival analysis was performed by the Kaplan-Meier method and differences were assessed using the log rank test. Results: There were 180 consecutive patients with actively relapsing MM who had BM biopsies analyzed via flow cytometry as part of their routine clinical evaluation. The median age of this group was 65 years (range: 40 - 87); 52% were male. At the time of this analysis, 104 patients had died, and the 2-year overall survival (OS) rate for the cohort was 58%. The median number of therapies received was 4 (range: 1 - 15). Of these patients, 61% received a prior ASCT, and almost all (99%) received prior regimens containing either immunomodulators or proteasome inhibitors. There were 55 (30%) patients with >5% pPCs among the total PCs in their BM. The median percentage of pPCs among total PCs in these 55 patients was 33% (range: 5 - 99). The median OS for those with >5% pPCs was not reached compared with 22 months for those with <5% pPCs (P = 0.028; Figure 1). Patients with <5% pPCs PCs had a higher likelihood of high-risk FISH cytogenetics compared with the rest of the patients. In a univariate analysis, increasing number of pPCs was associated with an improved OS, while higher labeling index, number of prior therapies, and the presence of high-risk FISH cytogenetics were associated with a worse OS. In a multivariate analysis, only the increasing number of pPCs (P = 0.006), higher labeling index (P = 0.0002) and number of prior therapies (P = 0.003) retained statistical significance. Conclusion: Quantitative estimation of the percentage of pPCs among the total PCs in the BM of patients with actively relapsing MM was determined to be a predictor of worse OS. As such, this parameter is able to identify a group of patients with MM with actively relapsing disease who have a particularly poor outcome. Further studies evaluating its biological significance are warranted. Figure 1 Kaplan-Meier curve comparing OS between patients with ≥5% pPCs and <5% pPCs among the total PCs in their BM. Figure 1. Kaplan-Meier curve comparing OS between patients with ≥5% pPCs and <5% pPCs among the total PCs in their BM. Disclosures Kapoor: Celgene: Research Funding; Amgen: Research Funding; Takeda: Research Funding. Gertz:Prothena Therapeutics: Research Funding; Novartis: Research Funding; Alnylam Pharmaceuticals: Research Funding; Research to Practice: Honoraria, Speakers Bureau; Med Learning Group: Honoraria, Speakers Bureau; Celgene: Honoraria; NCI Frederick: Honoraria; Sandoz Inc: Honoraria; GSK: Honoraria; Ionis: Research Funding; Annexon Biosciences: Research Funding. Kumar:AbbVie: Research Funding; Noxxon Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Research Funding; Kesios: Consultancy; Glycomimetics: Consultancy; BMS: Consultancy.

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Bone disease as the first manifestation of systemic AL-amyloidosis

Terapevticheskii arkhiv ◽

10.26442/00403660.2020.07.000775 ◽

2020 ◽

Vol 92 (7) ◽

pp. 85-89

Author(s):

L. P. Mendeleeva ◽

I. G. Rekhtina ◽

A. M. Kovrigina ◽

I. E. Kostina ◽

V. A. Khyshova ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Disease ◽

Plasma Cells ◽

Interventricular Septum ◽

Bone Destruction ◽

Amyloid Deposition ◽

Bone Lesions ◽

Al Amyloidosis ◽

Linear Type

Our case demonstrates severe bone disease in primary AL-amyloidosis without concomitant multiple myeloma. A 30-year-old man had spontaneous vertebral fracture Th8. A computed tomography scan suggested multiple foci of lesions in all the bones. In bone marrow and resected rib werent detected any tumor cells. After 15 years from the beginning of the disease, nephrotic syndrome developed. Based on the kidney biopsy, AL-amyloidosis was confirmed. Amyloid was also detected in the bowel and bone marrow. On the indirect signs (thickening of the interventricular septum 16 mm and increased NT-proBNP 2200 pg/ml), a cardial involvement was confirmed. In the bone marrow (from three sites) was found 2.85% clonal plasma cells with immunophenotype СD138+, СD38dim, СD19-, СD117+, СD81-, СD27-, СD56-. FISH method revealed polysomy 5,9,15 in 3% of the nuclei. Serum free light chain Kappa 575 mg/l (/44.9) was detected. Multiple foci of destruction with increased metabolic activity (SUVmax 3.6) were visualized on PET-CT, and an surgical intervention biopsy was performed from two foci. The number of plasma cells from the destruction foci was 2.5%, and massive amyloid deposition was detected. On CT scan foci of lesions differed from bone lesions at multiple myeloma. Bone fragments of point and linear type (button sequestration) were visualized in most of the destruction foci. The content of the lesion was low density. There was no extraossal spread from large zones of destruction. There was also spontaneous scarring of the some lesions (without therapy). Thus, the diagnosis of multiple myeloma was excluded on the basis based on x-ray signs, of the duration of osteodestructive syndrome (15 years), the absence of plasma infiltration in the bone marrow, including from foci of bone destruction by open biopsy. This observation proves the possibility of damage to the skeleton due to amyloid deposition and justifies the need to include AL-amyloidosis in the spectrum of differential diagnosis of diseases that occur with osteodestructive syndrome.

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An Unprecedented Case of p190 BCR-ABL Chronic Myeloid Leukemia Diagnosed during Treatment for Multiple Myeloma: A Case Report and Review of the Literature

Case Reports in Hematology ◽

10.1155/2018/7863943 ◽

2018 ◽

Vol 2018 ◽

pp. 1-5

Author(s):

Kosuke Miki ◽

Naoshi Obara ◽

Kenichi Makishima ◽

Tatsuhiro Sakamoto ◽

Manabu Kusakabe ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Plasma Cells ◽

Bone Marrow Examination ◽

Bone Lesions ◽

Dexamethasone Treatment ◽

Lytic Bone Lesions

We report the case of a 76-year-old man who was diagnosed as having chronic myeloid leukemia (CML) with p190 BCR-ABL while receiving treatment for symptomatic multiple myeloma (MM). The diagnosis of MM was based on the presence of serum M-protein, abnormal plasma cells in the bone marrow, and lytic bone lesions. The patient achieved a partial response to lenalidomide and dexamethasone treatment. However, 2 years after the diagnosis of MM, the patient developed leukocytosis with granulocytosis, anemia, and thrombocytopenia. Bone marrow examination revealed Philadelphia chromosomes and chimeric p190 BCR-ABL mRNA. Fluorescence in situ hybridization also revealed BCR-ABL-positive neutrophils in the peripheral blood, which suggested the emergence of CML with p190 BCR-ABL. The codevelopment of MM and CML is very rare, and this is the first report describing p190 BCR-ABL-type CML coexisting with MM. Moreover, we have reviewed the literature regarding the coexistence of these diseases.

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The Evolution in The Treatment of Multiple Myeloma Towards Targeted Magnetic Molecularly Imprinted Nanomedicines

Edelweiss: Cancer Open Access ◽

10.33805/2689-6737.101 ◽

2015 ◽

pp. 1-2

Author(s):

Edgar Pérez-Herrero

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bacterial Infections ◽

Plasma Cells ◽

The Body ◽

Western World ◽

Bone Lesions ◽

Non Hodgkin Lymphoma ◽

Clonal Proliferation ◽

Tract Infections

Multiple myeloma is the second more frequently haematological cancer in the western world, after non-Hodgkin lymphoma, being about the 1-2 % of all the cancers cases and the 10-13% of hematologic diseases. The disease is caused by an uncontrolled clonal proliferation of plasma cells in the bone marrow that accumulate in different parts of the body, usually in the bone marrow, around some bones, and rarely in other tissues, forming tumor deposits, called plasmocytomas. This uncontrolled clonal proliferation of plasma cells produces the secretion of an abnormal monoclonal immunoglobulin (paraprotein or M-protein) and prevents the formation of the other antibodies produced by the normal plasma cells that are destroyed. The anormal secretion of paraproteins unbalance the osteoblastosis and osteoclastosis processes, leading to bone lesions that cause lytic bone deposits and the release of calcium from bones (hypercalcemia) that may produce renal failure. Regions affected by bone lesions are the skull, spine, ribs, sternum, pelvis and bones that form part of the shoulders and hips. The substitution of the healthy bone marrow by infiltrating malignant cells and the inhibition of the normal production of red blood cells produce anaemia, thrombocytopenia and leukopenia. Multiple myeloma patients are immunosuppressed because of leukopenia and the abnormal immunoglobulin production caused by the uncontrolled clonal proliferation of plasma cells, being susceptible to bacterial infections, like pneumonias and urinary tract infections. The interaction of immunoglobulin with hemostatic mechanisms may lead to haemorrhagic diathesis or thrombosis. Also, disorders of the central and peripheral nervous system are part of the disease, being the more common neurological manifestations the spinal cord compressions and the peripheral neuropathies.

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CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.3457.3457 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3457-3457 ◽

Cited By ~ 3

Author(s):

Eric D. Hsi ◽

Roxanne Steinle ◽

Balaji Balasa ◽

Aparna Draksharapu ◽

Benny Shum ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Mononuclear Cells ◽

Plasma Cells ◽

Target Cells ◽

Dependent Manner ◽

Myeloma Cells ◽

Effector Cells

Abstract Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM). Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays. Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner. Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

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Multiple Myeloma Associated GI Polyposis

Blood ◽

10.1182/blood.v120.21.5009.5009 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5009-5009

Author(s):

Nassim Nabbout ◽

Mohamad El Hawari ◽

Thomas K. Schulz

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Case Reports ◽

Bone Marrow Failure ◽

Bone Lesions ◽

Rare Manifestation ◽

History Of ◽

Central Ulceration

Abstract Abstract 5009 Multiple myeloma is a neoplastic proliferation of monoclonal plasma cells that can result in osteolytic bone lesions, hypercalcemia, renal impairment, bone marrow failure, and the production of monoclonal gammopathy. The gastrointestinal tract is rarely involved in myeloma. GI polyposis is a rare manifestation of extra-medullary disease in multiple myeloma. Such cases usually present as gastrointestinal hemorrhage or intestinal obstruction. A 53-year-old African American male recently diagnosed with multiple myeloma presented with three-day history of rectal bleed and fatigue. EGD showed multiple raised, polypoid, rounded lesions with a superficial central ulceration in the stomach. Colonoscopy showed similar lesions in the ascending and transverse areas of the colon that ranged in size from 5 to 16 mm in diameter. Biopsies showed that these polyps were made of plasma cells. A bone marrow biopsy showed diffuse involvement (greater than 90%) of bone marrow with multiple myeloma with anaplastic features. The patient was started on bortezomib at diagnosis, however, he passed away a few weeks later. This type of metastatic disease has been described in isolated case reports in the literature, while solitary GI plasmacytoma has been reported more frequently. In rare cases, multiple myeloma can involve the GI tract which may lead to bleed or obstruction. This involvement is likely a marker of aggressivity. This example of extra-medullary disease in myeloma is an uncommon variant with features of poor prognosis and dedifferentiation. Disclosures: No relevant conflicts of interest to declare.

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All-Trans Retinoic Acid (ATRA) up-Regulates Cell Surface CD38 Expression Which Promotes Pro-Tumoral Mitochondrial Trafficking from Stromal Cells to Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-110323 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3153-3153

Author(s):

Christopher Richard Marlein ◽

Rebecca H Horton ◽

Rachel E Piddock ◽

Jayna J Mistry ◽

Charlotte Hellmich ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Stromal Cells ◽

Plasma Cells ◽

Atra Treatment ◽

Cd38 Expression ◽

Mitochondrial Transfer ◽

Mitotracker Green

Abstract Background Multiple myeloma (MM) is malignancy highly reliant on its microenvironment. In this study, we investigated whether mitochondrial transfer occurred between bone marrow stromal cells (BMSC) and malignant plasma cells. We then used our observations as a platform to investigate the mechanisms controlling pro-tumoral mitochondrial transfer with a view to identifying druggable targets. Methods Primary MM cells were obtained from patients' bone marrow after informed consent and under approval from the United Kingdom Health Research Authority. Animal experiments were conducted under approvals from the UK Home Office and the University of East Anglia Animal Welfare and Ethics Review Board. Primary BMSC were also obtained from patient bone marrow, using adherence and characterised using flow cytometry. Mitochondrial transfer was assessed using two methods; a MitoTracker Green based staining of the BMSC (in-vitro), rLV.EF1.AcGFP-Mem9 labelling of the MM plasma membrane with MitoTracker CMXRos staining of the BMSC (in-vitro) and an in vivo MM NSG xenograft model. CD38 expression on MM cells was tested after ATRA treatment, using RT-qPCR and flow cytometry. Mitochondrial transfer levels were assessed when CD38 was over expressed using ATRA or inhibited using lentivirus targeted shRNA. Results We report that mitochondria are transferred from BMSC to MM cells. First, we cultured MM cells on MitoTracker Green labelled BMSC and found increased MitoTracker Green fluorescence in the MM cells. We then transduced MM with rLV.EF1.AcGFP-Mem9 lentivirus and stained BMSC with MitoTracker CMXRos and used wide field microscopy to show MM derived tunnelling nanotubles (TNT) formed between MM cells and BMSC, with red mitochondria located within the GFP-tagged TNT. Next, we engrafted the MM cell lines MM1S and U266 into NSG mouse, after isolation we detected the presence of mouse mitochondrial DNA in the purified MM population. Together, these data show that mitochondria are transferred from BMSC to MM cells. We next analysed OXPHOS levels in MM cells grown on BMSC, using the seahorse extracellular flux assay. We found that the MM cells had increased levels of OXPHOS after culture with BMSC, which was also the case for MM cell lines analysed after isolation from NSG mice, showing the micro-environment of MM can alter the metabolism of the malignant cell. To examine whether the mitochondrial transfer process was controlled by CD38, we knocked down CD38 in MM cells using lentiviral targeted shRNA. We found reduced levels of mitochondrial transfer in CD38KD MM cells, with a consequent reduction of OXPHOS in the malignant cells. Finally, as ATRA has previously been shown to increase CD38 expression in AML, we next quantified CD38 mRNA and surface glycoprotein level on malignant plasma cells with and without ATRA treatment. We found ATRA increased CD38 expression at the mRNA and protein levels and this resulted in an increase in mitochondrial transfer from BMSC to MM cells. Conclusion Here we show that CD38 mediated mitochondrial transfer in the MM micro-environment forms part of the malignant phenotype of multiple myeloma. This finding develops our understanding of the mechanisms which underpin the efficacy of CD38 directed therapy in MM. Disclosures No relevant conflicts of interest to declare.

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Monitoring of Circulating Tumor Plasma Cells in Patients with Precursor Conditions of Multiple Myeloma: Data from the Prospective Iceland Screens, Treats, or Prevents Multiple Myeloma (iStopMM) Study

Blood ◽

10.1182/blood-2021-146363 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2645-2645

Author(s):

Jón Þórir Óskarsson ◽

Iris Petursdottir ◽

Sæmundur Rögnvaldsson ◽

Sigrun Thorsteinsdottir ◽

Gudrun Asta Sigurdardottir ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Research Funding ◽

Plasma Cells ◽

Advanced Disease ◽

Limit Of Detection ◽

Screening Tests ◽

Disease Monitoring ◽

Risk Of Progression

Abstract Background: A proportion of patient with monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) will progress to active multiple myeloma (MM). Optimization of follow-up strategies and diagnostic testing is needed to detect those who are at risk of imminent progression since they may benefit from early treatment. There is a considerable need for biomarkers that can accurately reflect disease status and risk of progression to MM. In recent years, circulating tumor plasma cells (CTPC) have gained interest in disease monitoring for their promising prognostic significance and the minimally-invasive nature of blood sampling. Aim: To evaluate the feasibility of using CTPC analysis by next-generation flow cytometry (NGF) for disease monitoring in precursor conditions of MM and early detection of progression to active MM. Methods: Participants were enrolled from the Iceland Screens, Treats, or Prevents Multiple Myeloma study (iStopMM). The study is a population-based screening study for MM precursors and randomized trial of follow-up strategies that enrolled 80,579 Icelanders and screened 75,422 of participants by serum protein electrophoresis (SPEP) and free light chain (FLC) assay. A total of 2/3 of participants who had abnormal screening tests were invited to the study clinic to undergo assessment and testing to detect SMM and MM. All cases of SMM and MM and a random conveniency sample of participants with MGUS were eligible for a flow cytometry sub study. The Euroflow NGF MM-MRD method was used for quantitation of tumor PC in the bone marrow (BM) and CTPC in peripheral blood (PB). Paired BM and PB samples were collected at baseline or after SMM/MM diagnosis at the next scheduled BM sampling during follow-up. The Infinicyt software (Cytognos SL, Salamanca, Spain) was used for flow cytometry data analysis. The limit of detection (LOD) was set at ≥ 20 tumor PC in both BM and PB. The Mann-Whitney U test or the Kruskal-Wallis tests were used to assess statistical significance of differences observed between two or more than two groups, respectively. Results: A total of 189 individuals have been included in the study at this point (90 MGUS, 73 SMM, and 26 MM). The frequency of cases in which CTPC were detected in PB increased (p <0.001) from MGUS (17.8%) to SMM (74.0%) and MM (96.2%) at a median LOD of 2.3x10 -6 (Fig. A). Similarly, progressively higher (p <0.01) numbers of CTPC were found from MGUS (median: 0 CTPC/µL), to SMM (0.028 CTPC/µL), and MM (0.16 CTPC/µL) (Fig. B). Analysis of PC in BM by NGF showed that patients with a detectable CTPC population in PB had a significantly (p <0.001) higher percentage of tumor PC within the BMPC compartment compared to patients with undetected CTPC, in both the MGUS and SMM groups (median: 31.5% vs 86.0% and 86.4% vs 94.9%, respectively) (Table). In the SMM group, 96% (27/28) of cases with over 95% tumor PC within the BMPC compartment had a detectable CTPC population in PB (Table). No difference was observed in the frequency of CTPC detection nor the number of CTPC (data not shown) between levels in the MGUS and the 2/20/20 SMM risk stratification models (Table). Conclusion: This is the first study evaluating CTPC in a screened cohort of patients with precursor conditions of MM. We found the frequency of CTPC detection to be lower than has been previously reported in a study by Sanoja-Flores et al. in 2018 using the same NGF method, particularly for MGUS (18% vs 59% [n=150]) and SMM (74% vs 100% [n=26]). This difference can likely be attributed to a higher frequency of patients with less advanced disease in the screened cohort of the iStopMM study, suggested by markedly lower median M-component levels in this study (3.3 vs 6, 7.8 vs 21, and 16.2 vs 27 g/L for MGUS, SMM, and MM, respectively). We found that the number of CTPC progressively increased from MGUS to SMM and MM. Furthermore, a detectable CTPC population by NGF was associated with a higher percentage tumor PC in the BMPC compartment in both MGUS and SMM. A BMPC compartment that is highly dominated by tumor PC (>95%) has been reported to be associated with a higher risk of progression in both MGUS and SMM and in our study a CTPC population was detected in a vast majority of SMM patients with over 95% tumor PC. Taken together, these results confirm that the detection and number of CTPC by NGF is associated with a more advanced disease and that their detection by NGF may have a clinical utility in the follow-up of myeloma precursor disease. Figure 1 Figure 1. Disclosures Durie: Amgen: Other: fees from non-CME/CE services ; Amgen, Celgene/Bristol-Myers Squibb, Janssen, and Takeda: Consultancy. Kristinsson: Amgen: Research Funding; Celgene: Research Funding.

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Flow Cytometric Disease Monitoring in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation: A Retrospective Study.

Blood ◽

10.1182/blood.v108.11.5014.5014 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5014-5014

Author(s):

Hong Liu ◽

Constance M. Yuan ◽

Raul C. Braylan ◽

Myron N. Chang ◽

John R. Wingard ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Plasma Cells ◽

Flow Cytometric ◽

Before And After

Abstract The persistence of abnormal neoplastic plasma cells (APC) detectable in the bone marrow by flow cytometry at more than 3 months after autografting for multiple myeloma (MM) has been reported to predict early disease progression. In this study, we retrospectively reviewed the flow cytometric data from bone marrow aspirates of MM patients before and after autologous stem cell transplantation (ASCT). Light scatter properties and CD38 expression were used to identify plasma cells, and CD19/CD45/CD56 further distinguished normal plasma cells (NPC) from APC. Conventional response criteria (Blade criteria) and survival data were also collected. Forty-seven (47) patients treated with the same conditioning regimen were screened. Median follow up from ASCT was 19 months. After ASCT, 66% (31/47) patients achieved complete remission (CR)/very good partial remission (VGPR), as compared to only 36% (17/47) prior to ASCT. In 39 patients with data before and after ASCT, all 39 (100%) had a detectable abnormal plasma cell population identified phenotypically by flow cytometry prior to ASCT. Of these patient, 18/39 (46%) had greater than 30 APC and these patients had significantly shorter PFS independent of other covariates (1-sided P=0.036, 2 sided P=0.072, logrank). Twenty-six out of 39 patients (67%) also had detectable NPC. Following ASCT, the number of patients with detectable NPC increased to 35/39 (89%), while 3/39 (8%) had no detectable NPC and 1/39 (3%) had neither NPC or APC. The proportion of APC decreased significantly after transplant (81% prior to transplant vs. 59% post-transplant, P=0.008, 2 tailed t-test). Patients with a APC to NPC ratio < 1 post transplant has higher PFS rate at 2 year (54%) when compared to patients with higher APC/NPC ratio (29% PFS at 2 year), however, the difference is not statistically significant. In addition to the presence of APC, the ratio of APC to NPC, age, beta-2 microglobulin levels, and the presence of normal immunoglobulin levels were analyzed. Patients who achieved CR/VGPR after transplant had significantly longer PFS (23 months vs. 11 months, P=0.03). All other covariates were not found to be significant. Because only 10 deaths were observed, covariate analysis for OS was not feasible. In conclusion, the recovery of NPC after ASCT is seen in a substantial propotion of patients with a trend towards better PFS in patients with low APC/NPC ratio. On the other hand, the presence of a significant population of APC (> 30) prior to transplant appears to correlate with poorer PFS in MM patients.

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Role of Selectins in the Pathogenesis of Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.951.951 ◽

2009 ◽

Vol 114 (22) ◽

pp. 951-951 ◽

Cited By ~ 1

Author(s):

Abdel Kareem Azab ◽

Phong Quang ◽

Feda Azab ◽

Costas M Pitsillides ◽

John T Patton ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Endothelial Cells ◽

Cell Adhesion ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Advisory Committees

Abstract Abstract 951 INTRODUCTION: Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role. METHODS AND RESULTS: We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p<0.05). Moreover, we found that blockade of P-selectin on endothelial cells reduced their interaction with MM cells (p<0.01), while blockade of E and L-selectin did not show any effect. Treating endothelial cells with GMI-1070 mimicked the effect of blocking P-selectin. Moreover, we found that treating endothelial cells with the chemokine stroma cell-derived factor-1-alpha (SDF1) increased their expression of P but not E or L-selectin detected by flow cytometry. Neither the blockade of each of the selectins and their ligands nor the GMI-1070 inhibited the trans-well chemotaxis of MM cells towards SDF1-alpha. However, blockade of P-selectin (p<0.001) on endothelial cells by GMI-1070 inhibited the trans-endothelial chemotaxis of MM cells towards SDF1-alpha. Both adhesion to endothelial cells and activation with recombinant P-selectin induced phosphorylation of cell adhesion related molecules including FAK, SRC, Cadherins, Cofilin, AKT and GSK3. GMI-1070 decreased the activation of cell adhesion molecules induced by both recombinant P-selectin and endothelial cells. Using in vivo flow cytometry we found that both anti P-selectin antibody and GMI-1070 prevented the extravasation of MM cells out of blood vessels into the bone marrow in mice. Moreover, we found that, in a co-culture system, endothelial cells protected MM cells from bortezomib induced apoptosis, an effect which was reversed by using GMI-1070, showing synergistic effect with bortezomib. CONCLUSION: In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma. Disclosures: Patton: GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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