The Frequency of Genetic Anomalies According to Clonal Plasma Cells' Phenotype in Patients with Newly Diagnosed (ND) Multiple Myeloma (MM)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5316-5316
Author(s):  
Andrei Garifullin ◽  
Irina Martynkevich ◽  
Sergei Voloshin ◽  
Alexei Kuvshinov ◽  
Ludmila Martynenko ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Risk Group ◽  
Risk Groups ◽  
Fish Analysis ◽  
Newly Diagnosed ◽  
Definition Of ◽  
Standard Risk

Abstract Background. Genetic anomalies (GA) are primary link of pathogenesis in MM. GA lead to formation of clonal plasma cells, which has different phenotype. Aim. To estimate the incidence of GA and their correlation with clonal plasma cells' phenotype in patients with ND MM. Methods. We analysed 22 patients with ND MM (median age 57 years, range 38-80; male/female - 1:1.75). Cytogenetic analysis was performed on bone marrow samples using standard GTG-method. Metaphase FISH analysis was performed according to the manufacturer's protocol using DNA probes: LSI 13(RB1)13q14, IGH/CCND1, IGH/FGFR3, LSI TP53 (17q13.1). 8-color immunophenotypic by flow cytometry using antibody to CD45, CD38, CD138, CD56, CD19, CD20, CD27 and CD117 antigenes. Results. Translocation t(11;14) was detected in 3/14 (21.4%) patients, del(13q) - 2/14 (14.3%), t(11;14) - 3/14 (21.4%), hypodyploidy - 1/20 (5%), del(17р) - 0% patients. Clonal plasma cells' phenotype CD38+CD138+CD45- was detected in 100%. Expression CD56+ was revealed in 11/22 (50%) patients, CD19+ in 9/22 (40.9%), CD117+ in 5/22 (22.7%), CD20+ in 1/22 (4.5%), CD27+ in 1/22 (4.5%). The frequency of GA didn't depend on clonal plasma cells' phenotype and was 27.3%(3/11) in CD56+ phenotype, 23.8%(5/21) - CD20-, 23.8%(5/21) - CD27-, 23.5%(4/17) - CD117-, 23%(3/13) - CD19-, 22.2%(2/9) - CD19+, 20%(1/5) - CD117+, 18.2%(2/11) - CD56-, 0%(0/1) - CD20+, 0%(0/1) - in CD27+ phenotype. Patients of standard risk group according to mSMART 2.0 with GA had CD19-negative plasma cells' phenotype vs. CD19-positive phenotype in patients of intermediate and high-risk groups (p<0.05). 3-years overall survival in standard risk group with CD19- phenotype was 92,3%, CD19+ - 77,7% (p>0.05). Conclusion . Identification of GA, which has adverse forecast, correlates with CD19+ plasma cells phenotype. The combined definition of plasma cells phenotype and GA can improve the system of risk stratification in MM. Disclosures No relevant conflicts of interest to declare.

Cohort Analysis of FISH Testing of CD138+ Cells in Relapsed Multiple Myeloma: Implications for Prognosis and Choice of Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3399-3399
Author(s):  
Dean Smith ◽  
Clemency Stephenson ◽  
Anna Lach ◽  
Steve Chatters ◽  
Helena Kempski ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Conflicts Of Interest ◽  
Risk Group ◽  
Cohort Analysis ◽  
Fish Analysis ◽  
Newly Diagnosed ◽  
Interphase Fish ◽  
Line Of Therapy ◽  
Standard Risk

Abstract Introduction: Interphase FISH on CD138-selected bone marrow cells enables genetic risk stratification in newly diagnosed multiple myeloma (MM), however as MM remains incurable, most centres still treat newly diagnosed MM uniformly, utilising the most active regimens available. At relapse an increasing choice of regimens, coupled with co-morbidities and treatment-emergent toxicities, means no uniform approach is possible. Instead, therapy is tailored to disease and patient related risk factors. In this setting, FISH testing may be particularly useful if not done at diagnosis and to identify progression events that may alter prognosis. Aim: To evaluate the outcome of FISH analysis in consecutive patients with relapsed MM undertaken at our centre: success rate, frequency of abnormalities, incidence of progression events and correlation of FISH abnormalities with treatment outcomes. Methods: FISH analysis was performed on 192 samples from 154 relapsed patients (2012-13). Plasma cells were selected using magnetic CD138 MicroBeads and interphase FISH carried out using probes as recommended by the EMN (Ross et al, 2012). If patients had no prior results, a full FISH MM panel was performed, using probes for t(4;14), t(14;16), t(11;14), deletion 17p (17p-), Chr 1 abnormalities (1p-/1q+) and deletion 13q (13q-). If patients had been previously tested for an IgH translocation (Tx), a progression event panel was used: 1p-/1q+, 17p- and 13q-. Patients underwent FISH testing prior to starting the next line of therapy. Results: 79% of samples were successfully analysed, with analysis limited in 16% and failed in 5%. Common reasons for failure were poor quality/aged slides, insufficient material and poor hybridisation. 17% of patients had no cytogenetic abnormality. The most common abnormality was 13q- (43.1%), followed by 1q+ (41.4%), t(11;14) (18.3%), t(4;14) (12.4%), 17p- (12.0%) 1p- (8.9%), and t(14;16) (5.6%) Progression events were more common in t(14;16) and t(4;14) groups. All patients with t(14;16) and 82% with t(4;14) had an additional genetic lesion. Only 21% of patients with t(11;14) and 54% with no IgH Tx had an additional event. 80 patients (51.3%) had prior FISH results and 13 (16.3%) had developed a new abnormality on the later test. In 9 cases the progression event was 17p-, in 2 it was 1q+ and 2 cases developed 17p- and 1q+. The patients developing 1q+ were previously standard risk, so repeat testing altered risk group. Acquisition of 17p- indicates especially poor outcome, thus in all 13 cases repeat FISH analysis altered risk. Among patients with progression events none harboured t(11;14), 8 (64%) had no IgH Tx, 3 had t(14;16) and 2 had t(4;14). FISH results were correlated with clinical outcome. Patients were stratified as having high risk genetics [t(4;14), t(14,16), 17p- in ≥50% cells, 1p-/1q+] or standard risk [t(11;14), normal cytogenetics]. 63 (41%) patients were high risk, 83 (54%) standard risk, with no information available for 8 (5%). Both groups had received a median of 2 prior lines of therapy. Response rates (≥PR) to the next line of therapy were similar (60.4% standard risk vs 56.0% high risk). PFS from time of FISH was significantly longer in the standard risk group (9.8 months vs 5.9, p<0.01) as was OS (not reached vs 17.1 months, p<0.01, Fig. 1). In the high risk group, PFS was significantly longer in patients receiving a proteasome inhibitor (PI) as the next line of treatment versus those receiving other therapies (9.6 months vs 4.6, p=0.01) as was OS (not reached vs 9.7 months, p<0.01, Fig. 2). In the standard risk group, PFS was similar if patients received PI or not (9.5 months PI vs 9.8 other) as was OS (not reached both groups, Fig. 2). Conclusions: FISH analysis on MM patients at relapse was achievable. 74/154 patients had no prior results and a further 13 developed new poor prognostic markers, thus FISH at relapse provided new information in 56% of patients. Progression events were more common in patients harbouring t(4;14) or t(14;16). FISH at relapse was prognostic with high risk abnormalities associated with significantly shorter PFS and OS. The use of PI appeared to abrogate this poor prognosis, suggesting FISH at relapse could be a predictive and prognostic marker. Given the availability of second generation PI and the option of bortezomib re-treatment, results of FISH testing at relapse could directly influence clinical practice. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Bortezomib Plus Melphalan and Prednisone (VMP) Followed By Lenalidomide and Dexamethasone (Rd) in Newly Diagnosed Elderly Myeloma Patients Overcome the Poor Prognosis of High-Risk Cytogenetic Abnormalities (CA) Detected By Fluorescence in Situ Hibridization (FISH)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4243-4243 ◽  
Author(s):  
Maria-Victoria Mateos ◽  
Norma C Gutierrez ◽  
María-Luisa Martín ◽  
Joaquín Martínez-López ◽  
Miguel T Hernandez ◽  
...  
Keyword(s):  
High Risk ◽  
Elderly Patients ◽  
Poor Prognosis ◽  
Plasma Cells ◽  
Risk Group ◽  
Fish Analysis ◽  
Newly Diagnosed ◽  
The Poor ◽  
Risk Patients ◽  
Standard Risk

Abstract Background: Novel insights into the biology of myeloma cells have led to the identification of relevant prognosis factors. CA has become one of the most important prognostic factors, and the presence of t(4;14), t(14;16) or del(17p) are associated with poor prognosis. Although there are some reports indicating that 1q gains may be considered as a poor-risk feature, the information is not uniform. Furthermore, there are important controversies about whether or not novel agents-based combinations are able to overcome the poor prognosis of CA. Bortezomib-based combinations have shown to improve the outcome of patients with high-risk CA but they do not completely overcome their adverse prognosis. Here we report a preplanned analysis, in a series of elderly newly diagnosed myeloma patients included in the Spanish GEM2010 trial and receiving VMP and Rd, in a sequential or alternating approach, in order to evaluate the influence of CA by FISH on the response rate and outcome. Patients and methods: 242 pts were randomized to receive a sequential scheme consisting on 9 cycles of VMP followed by 9 cycles of Rd or the same regimens in an alternating approach (one cycle of VMP alternating with one Rd, up to 18 cycles. VMP included the iv administration of weekly bortezomib (except in the first cycle that was given twice weekly) at 1.3 mg/m2 in combination with oral melphalan 9 mg/m2 and prednisone 60 mg/m2 once daily on days 1-4. Rd treatment consisted on lenalidomide 25 mg daily on days 1-21 plus dexamethasone 40 mg weekly. FISH analysis for t(4;14), t(14;16), del(17p) and 1q gains was performed at diagnosis according to standard procedures using purified plasma cells. Results: In 174 out of the 233 patients evaluable for efficacy and safety, FISH analysis at diagnosis were available and two groups were identified: high-risk group (n= 32 patients with t(4;14) and/or t(14;16) and/or del(17p)) and standard-risk group (n=142 patients without high-risk CA). There weren't differences in the rates of CA according to the treatment arm. Response Rates (RR) were no different in the high-risk vs standard-risk groups, both in the sequential (74% vs 79% RR and 42% vs 39% CR) and alternating arms (69% vs 86% RR and 39% vs 38% CR). After a median follow-up of 37 months, high-risk patients showed shorter PFS as compared to standard risk in the alternating arm (24 versus 36 months, p=0.01, HR 2.2, 95% IC 1.1-4.2) and this also translated into a significantly shorter 4-years OS (27% vs 72%, p=0.006, HR 3.3, 95% IC 1.4-7.7). However, in the sequential arm, high-risk and standard-risk patients showed similar PFS (32 months vs 30 months) and 4-years OS (64% vs 60%). This effect was observed only in the sequential arm applied to either t(4;14) or del(17p). As far as 1q gains is concerned, 151 patients had 1q information and 76 of them had 1q gains (50.3%), defined as the presence of more than 3 copies in at least 10% of plasma cells. The rate of 1q gains was well balanced in both sequential and alternating arms. The ORR was similar in patients with or without 1q gains (83% vs 80%) as well as the CR rate (45% vs 31%), and no differences were observed between sequential and alternating arms. Patients with or without 1q gains had a similar PFS (33 months vs 30 months) and 4-years OS (58% vs 65%) in the whole series and no differences were observed in the sequential and alternating arms. This effect has been observed in patients with 1q gains as isolated CA and the outcome was slightly but not significantly worse when 1q gains were present plus either t(4;14) and/or del17p. Conclusions: The total therapy approach including VMP and Rd administered in a sequential approach is able to overcome the poor prognosis of the presence of high-risk CA in elderly patients with newly diagnosed MM. The presence of 1q gains has no impact in the PFS and OS of elderly patients treated with VMP and Rd. Disclosures Mateos: Celgene: Consultancy, Honoraria; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Takeda: Consultancy. Gironella:Celgene Corporation: Consultancy, Honoraria. Paiva:BD Bioscience: Consultancy; Binding Site: Consultancy; Sanofi: Consultancy; EngMab AG: Research Funding; Onyx: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Celgene: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. San Miguel:Millennium: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria; Sanofi-Aventis: Honoraria.

Chromosomal Translocation (14;16)-Positive Multiple Myeloma Shows Negativity for CD56 Expression and Unfavorable Outcome Even in the Era of Novel Drugs

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3349-3349
Author(s):  
Tomoko Narita ◽  
Atsushi Inagaki ◽  
Tsutomu Kobayashi ◽  
Yoshiaki Kuroda ◽  
Toshihiro Fukushima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Features ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Unfavorable Outcome ◽  
University Hospital ◽  
Fish Analysis ◽  
Newly Diagnosed ◽  
Novel Drugs

Abstract Introduction Multiple myeloma (MM) is an incurable plasma cell neoplasm developing through long-term multistep genetic events. Biological and clinical features of the MM are known to be associated in part with relatively early genetic aberrations such as chromosomal translocations involving IGH. The t(14;16)(q32;q23) involving c-MAF oncogene locus is an important chromosomal aberration observed in approximately 5 percent of newly diagnosed MM. Various studies have suggested that MM carrying t(14;16) is associated with specific clinical characteristics. However, these studies were not definitive, since the number of the patients analyzed was relatively small. The aim of this study is to clarify the clinical features of patients with newly diagnosed MM harboring t(14;16) detected by double-color fluorescence in situ hybridization (FISH) in Japan. Methods Clinical and laboratory features of t(14;16)-positive MM diagnosed between 2002 and 2013 were collected retrospectively as a nationwide study in Japan after approval by each institutional ethical committee. The t(14;16) translocation was detected by FISH analysis using bone marrow or peripheral blood samples from all patients. Expression of surface antigens such as CD56 and CD20 was detected by flow cytometric analysis (FCM) and defined as positive when more than 20% of the CD38-positive plasma cells were positive. To compare t(14;16)-positive and t(14;16)-negative MM, we also assessed 132 patients with newly diagnosed symptomatic MM and without c-MAF mRNA expression, as confirmed by global RQ/RT-PCR using purified plasma cells (Tajima E, et al.:Haematologica 2005; 90: 559, Inagaki A et al.: Leuk Res 2013; 37: 1648) at Nagoya City University Hospital. Results In total, 37 patients carrying t(14;16)-positive MM were enrolled from 19 institutions. Median ages of the MM patients with or without t(14;16) at diagnosis were 62 and 68, respectively. Regarding the cell surface phenotype, none of the t(14;16)-positive MM cells was positive for CD56 (Fig. 1), whereas 82 of 118 (69%) t(14;16)-negative ones were positive. Positivity for CD20 antigen was more common in t(14;16)-positive MM cells (11/23, 48%) than in t(14;16)-negative ones (16/115, 14%)(p= 0.001). The proportion of patients with additional chromosome aberrations other than t(14;16), determined by G-banded karyotyping, was higher in patients with t(14;16) (16/30, 53%) than in those without (19/131 cases, 15%) (p< 0.001). Moreover, MM patients with t(14;16) showed higher frequencies of IgG subtype M protein, leukocytosis (p= 0.001), thrombocytopenia (p< 0.001) and hyperproteinemia (p= 0.001), and a lower frequency of hypercalcemia (p= 0.001), compared to those without t(14;16). Overall survival (OS) of the patients with t(14;16) was significantly shorter than that of those without t(14;16) even though the patients received one or more lines of treatment containing novel drugs such as bortezomib, thalidomide and lenalidomide (p= 0.014) (Fig. 2). Poor PS (PS ≥ 2-4), low PLT count (<100x103/ƒÊL), or high LDH levels (>1.0N) were significantly unfavorable prognostic factors for OS in patients with t(14;16)-positive MM, whereas they were not in those without t(14;16). Progression-free survival (PFS) of the patients with t(14;16) was also significantly shorter than those without t(14;16) (p= 0.002) Conclusion The t(14;16)-positive MM comprises a specific category in MM, which is featured by negativity for CD56, higher positivity for CD20 and unfavorable outcome, even in the novel drug era. Unraveling biological characteristics of this specific disease category will lead us to establish novel treatment strategies. Disclosures No relevant conflicts of interest to declare.

A Fully Automated Method for Isolating Highly Enriched Population of Multiple Myeloma Plasma Cell From Whole Bone Marrow for Enhanced Sensitivity in Downstream Assays

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5064-5064
Author(s):  
Hossein Mossafa ◽  
Sabine Defasque ◽  
Hamid Belaouni ◽  
Adrian Arechiga
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Dna Extraction ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Snp Array ◽  
Fish Analysis ◽  
Automated Method

Abstract Abstract 5064 Introduction, Multiple myeloma (MM) is characterized by a huge clinical heterogeneity despite the homogenous morphologic appearance of malignant plasma cells (PCs). The advent of interphase fluorescence in situ hybridization (FISH) or MicroArrays (MA) allows an increased rate of aberration detection and identification of some recurrent cryptic changes, which have been increasingly implemented as additional diagnostic and prognostic factors. To heighten sensitivity of Single Nucleotide Polymorphism (SNP) arrays, or FISH it is necessary to have a purified population of cells as starting material. Screening must be performed systematically on the purified CD138+ PCs. After testing different systems for cell purification, we encountered some challenges. We didn't obtain enough PCs for FISH and SNP array studies. This was due to excess M-protein accumulating in the blood stream, increasing hyper viscosity and also due to the morphology and size variations of PCs at various stages of differentiation. Additionally, downstream DNA extraction can be a challenge since EDTA found in most buffers is an inhibitor for chemical PCR reaction for some MA chips. Given the challenges, CERBA laboratory and Miltenyi Biotec GmbH have developed a fully automated process (FAP) for purification for CD138+ PCs. In a study of 100 BM patient samples, we compared the specificity, efficiency, performance, purity, ease of use, technologists' time and the quality of DNA after CD138+ PCs purification. Two methods were compared. In the first method, cells were directly purified from bone marrow samples by FAP using Automated Magnetic Cell Sorter (AMCS). In the second method, mononuclear cells from fresh whole bone marrow (WBM) were enriched by Ficoll, followed by cell selection procedure with anti-CD138+ MicroBeads using the AutoMACS®. Before separation and following the separation, the percentage of PCs was determined by Flow cytometry (FC) on WBM by multiparameter FC (MFC) for CD138/CD38 expression. Additionally, DNA quality on separated cells was assessed by Nanodrop. A fraction of the CD138+ PCs were used after hypotonic shock and Carnoy fixation, applied to glass slides for FISH application and another fraction for DNA extraction for MA (SNP.6 Affymetrix®) FISH was performed with the recommended unbalanced alterations & reciprocal rearrangements: del(13) (q14)(D13S25), del(17)(p13)(TP53),+3(D3Z), +9(D9Z1), +15(D15Z14), t(4;14)(p16;q32)/IGH-FGFR3. Results, the specificity and purity were the same for both process but the efficiency and performance were considerably better for FAP than mononuclear cells enriched by Ficoll (MCEFicoll) process. With FAP, in 95% of the MM cases we obtained enough PCs for performance of the recommended panel of FISH and for 50% of them we could extract DNA for SNP array. For the MCEFicoll, we observed inferior performance, with very few plasma cells after isolation. Having enough PSc for only 65% of the cases and we could only extract DNA for 28% of them. The quality of DNA was the same for both process and the technologists' time was longer by 30' /patient for MCEFicoll process than for FAP. Currently in CERBA lab, we realize more than 20 plasma cells isolation per week for patients with MM and from October 2007 to July 2011 we have separated more than 5.000 specimens using CD138 Whole Blood MicroBeads (CD 138 WBMB) from Miltenyi Biotec, in combination with the AMCS. This has allowed isolation directly from WBM without any sample preparation required, such as density gradient centrifugation (ficoll) or erythrocyte lysis. The detection rate of chromosomal abnormalities and the number of abnormalities per case in MM and PCs dyscrasia significantly improves when there are enough CD138+PCs for analysis. Conclusion, in this report we describe the benefits of fully automated isolations of CD138+ cells from WBM. We have developed an SOP for an automated reliable and standardized method which allows the processing of multiple samples in a single day, while maintaining sample integrity and increasing sensitivity of FISH analysis and WG arrays for a diagnosis lab. Disclosures: No relevant conflicts of interest to declare.

Extramedullary Relapse of Multiple Myeloma - Plasma Cells Characteristics.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2935-2935
Author(s):  
Ludek Pour ◽  
Sabina Sevcikova ◽  
Lucie Rihova ◽  
Lenka Kubiczkova ◽  
Henrietta Greslikova ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Soft Tissues ◽  
Plasma Cells ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Fish Analysis ◽  
Extramedullary Relapse ◽  
Igh Rearrangement ◽  
Lower Expression

Abstract Abstract 2935 Background: Multiple myeloma (MM) is the second most common hematological malignancy in the world. The introduction of new drugs (thalidomide, bortezomib, revlimid) has dramatically improved survival of MM patients, but MM still remains an incurable disease. Unfortunately, an increase in the incidence of extramedullary relapse of MM (EM), an aggressive mostly resistant entity with abysmal prognosis for patients has been reported. EM can affect any area of tissue - soft tissue involvement can be with or without relationship to bone. A recent study of 936 MM patients by Usmani et al (2012) reported presence of EM in the skin and soft tissues at the time of diagnosis while liver involvement was common at relapse or progression. Aims: The objective of this study was to evaluate cytogenetic and flowcytometric data of available set of EM patients, and also to compare characteristics of plasma cells isolated from bone marrow and the extramedullary tumor. Material and methods: In total, we evaluated 29 EM patients. Patients' characteristics were as follows: males/females 18/11, median age was 61.2 years, ISS stage I/II/III 1/5/23, IgG/IgA/ LC only 20/6/3. I-FISH analysis was performed on bone marrow (BM) samples obtained at the time of diagnosis of EM. Flowcytometric analysis was performed on plasma cells (PC) isolated from BM as well as the EM tumor. Results: Using flowcytometry, PC were identified as CD138+CD38+ leukocytes and surface expression of CD20, CD27, CD28, CD33, CD40, CD54, CD117, CD19 and CD56 were analysed on PC in whole BM and the tumor. We found statistically significant decrease of CD27 (60.0 vs. 9.1% positivity in BM vs. tumor, resp.; p&lt;0.02) and CD19 (35.0 vs. 8.3%; p=0.001). Other markers were non-significantly decreased: CD33 (27.3 vs. 12.5%), CD40 (84.6 vs. 75.0%), CD54 (84.6 vs. 50.0%), CD117 (26.7 vs. 16.7%), CD56 (70.0 vs. 58.3%) while expression of CD28 was increased (13.3 vs. 33.3%) on tumor PC compared to BM PC. In the BM PC, we found del(13)(q14) in 67% (18/27), del(17)(p13) in 22% (6/27), IGH rearrangement in 58% (11/19), t(4;14) in 33% (6/18), 1q21 gain in 58% (15/26), hyperdiploidy in 43% (10/23) of EM patients. The total number of aberrations per patient was: 0–1 aberration in 31%, 2–3 aberrations in 62%, 4 aberrations in 7% of MM patients BM. For 4 patients, we were able to analyze both BM and the EM tumor. We found that in 2/4 patients, there was no agreement in chromosomal abnormalities found in the BM and EM tumor. The differences were in del(13)(q14) and IGH rearrangement. del(13)(q14) was present in all 100% (4/4) samples of BM but only 75% (3/4) of EM tumors. del(17)(p13) was present in 25% (1/4) of patients in the BM as well as EM. IGH rearrangement was present in 75% (3/4) of BM but only 25% (1/4) of EM. 1q21 gain was present in 50% (1/2) of patients in the BM and EM and hyperdiploidy was not present in the BM or EM tumor (0/2). Conclusion: Chromosomal abnormalities connected to worse prognosis are more common in EM patients. PC phenotype seems to be different in cells obtained from BM and EM tumor. PC from EM tumor had significantly lower expression of CD27 and CD19. CD27 is a tumor necrosis factor receptor and plays a key role in regulating B-cell activation and immunoglobulin synthesis. Its low expression could be one of the main reasons for resistance in MM while loss of CD19 can create a proliferative advantage for the malignant plasma cell clone. Other interesting markers are CD54 and CD56 which were non-significantly decreased. CD54 also known as ICAM-1 plays a key role in stabilizing cell-cell interactions and migration, and CD56 (NCAM) is important for adhesion of PC to the bone marrow microenvironment. CD54 and CD56 lower expression may be the reason for EM development in MM but their role needs to be further elucidated. Acknowledgment: This study was supported by grants NT12130, MSM0021622434, NS10207, NT11154. Disclosures: No relevant conflicts of interest to declare.

Expression and Clinical Significance of Notch1 On the Membrane of Bone Marrow CD38+CD138+Plasma Cells in the Patients with Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4981-4981
Author(s):  
Rong Fu ◽  
Yiran Zhao ◽  
Zonghong Shao ◽  
Honglei Wang ◽  
Tian Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Lactate Dehydrogenase ◽  
Serum Level ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Newly Diagnosed ◽  
Term Outcome ◽  
Long Term Outcome

Abstract Abstract 4981 Objective: To investigate the expression of bone marrow CD38+CD138+, CD38+CD138-plasma cells and the expression of Notch1 on the membrane of them in the patients with multiple myeloma(MM), and explore the importance of Notch signaling pathway in the formation and progression of MM further. Methods: Thirty-three MM patients and 15 healthy controls were enrolled in this study. The expression of bone marrow CD38+CD138+, CD38+CD138-plasma cells and the expression of Notch1 on the membrane of them were analyzed by flow cytometry. The expression of Notch1 mRNA of bone marrow mononuclear cells were analyzed by RT-PCR. Results: The ratio of CD38+CD138+ plasma cells from 24 newly diagnosed MM patients was (51. 50%±12. 48%) which was significantly higher than CD38+CD138- plasma cells of MM patients (42. 88%±11. 41%)(P=0. 016)and controls 20. 13%±5. 8(P=0. 000). The expression of CD38+CD138+ plasma cells from 24 newly diagnosed MM patients was correlated to the level of malignant plasma cells in there bone marrow(r=0. 546, p=0. 006), serum level of lactate dehydrogenase(LDH)(r=0. 567, p=0. 004), and β2-MG(r=0. 431, p=0. 035). The ratio of Notch1 on the membrane of CD38+CD138+ plasma cells of MM patients was (60. 21%±25. 06%) which was significantly higher than those of CD38+CD138- plasma cells of MM patients 39. 84%±18. 94%(P=0. 000)and controls (38. 34%±19. 39%)(P=0. 004). There was no statistical difference between the two latter groups(P>0. 05). The expression of Notch1 on CD38+CD138+ plasma cells from 24 newly diagnosed MM patients was correlated to the level of malignant plasma cells in there brone marrow(r=0. 914, p=0. 000), serum level of lactate dehydrogenase(LDH) (r=0. 604, p=0. 002), and β2-MG(r=0. 455, p=0. 026). The ratio of Notch1 on the membrane of CD38+CD138+ plasma cells of MM patients who had renal dysfunction was correlated to their abnormal serum creatinine levels. The expression of Notch1 on CD38+CD138+ plasma cells from 17 MM patients who received VD chemotherapy was correlated to the ratio of plasma cell reduction after the first VD chemotherapy(r=0. 842, p=0. 000). The expression of Notch1 mRNA of bone marrow mononuclear from 10 MM patients was (0. 8252±0. 4079) which was significantly higher than those of controls (0. 3759±0. 0813)(p=0. 032). Conclusion: Notch1 over expressed on CD38+CD138+ plasma cells with relation to the effects of early VD therapy and long term outcome of MM. Disclosures: No relevant conflicts of interest to declare.

Validation of Immuno-Magnetic Chromatography As a Novel Method for the in Situ enrichment of Plasma Cells in Bone Marrow.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2930-2930
Author(s):  
Eric Crawford ◽  
Victoria Golembiewski-Ruiz ◽  
Mingya Liu ◽  
Robert D MacPhee
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Fish Analysis ◽  
Microscope Slide ◽  
Novel Method ◽  
Standard Risk ◽  
Supernatant Solution

Abstract Abstract 2930 Background: Multiple myeloma (MM) is characterized by the clonal proliferation of terminally differentiated plasma cells. These dyscrasias range from the phenotypically benign monoclonal gammopathy of unknown significance (MGUS), to the clinically significant form, multiple myeloma (MM). Molecular cytogenetic abnormalities are one of the measures that has been used to stratify patients. As the clinical cut-off values for individual fluorescence in situ hybridization (FISH) probes can exceed the total percentage of abnormal plasma cells within a specimen, the International Myeloma Workshop Consensus Panel recommends analysis for genetic abnormalities by FISH, preferably after plasma cell sorting, as one means to stratify patients into standard risk or high risk categories. Purpose: We present a novel method for plasma cell isolation in which antibody coated para-magnetic immunobeads are used in a two-step separation process that deposits an enriched population of CD138+ plasma cells directly onto a predefined area of a microscope slide in a manner suitable for subsequent staining and FISH analyses. Materials and Methods: The clinical materials utilized were appropriately anonymized extraneous volumes of bone marrow aspirates obtained after all routine clinical testing requests had been fulfilled. Briefly, 100ml of bone marrow is incubated with a blocking buffer and paramagnetic immunobeads covalently conjugated to anti-CD138 antibody (WaveSense, Irvine, CA) for 45 minutes at room temperature with gentle agitation. Primary enrichment is achieved when the tube is placed in a magnetic dock for 15 minutes allowing the paramagnetic immunobead-plasma cell-complexes to sediment on the side of the reaction tube. Unbound cells remain in the supernatant solution which is gently removed. The cells from the supernatant are still suitable for additional analysis for hematologic disorders not involving a CD138+ lineage. The residual CD138+ cell population is resuspended in blocking buffer. This enriched cell suspension is pipetted into an EpiSep® (WaveSense, Irvine, CA) chambered slide. As the solution diffuses through the unit, the suspended plasma cell-paramagnetic bead complexes are immobilized at the slide's surface as they pass close to a fixed magnet in the slide dock while the residual supernatant solution is eluted into adjacent chambers containing compact absorbent pads. A small volume of Carnoy's fixative is introduced through the EpiSep port to affect an initial fixation of the captured plasma cells deposited on the microscope slide's surface. The unit is then manually separated from the microscope slide. Samples were analyzed with a panel of FISH probes to assess copy number changes for chromosomes 5, 7, 13, 1p/1q, 17p and t(4;14), t(11;14) and t(14;16). Results: Bone marrow from 32 patients with indications of possible or known plasma cell disorders were enriched for CD138+ plasma cells. Conventional non-enriched FISH analysis detected reportable abnormalities in 11 (34%) of patients. With enrichment, abnormalities were detected in 21 (66%) of patients. When compared to abnormalities detected by FISH on non-enriched marrow, new abnormalities were detected in 14 (44%) of cases. Of the14 cases, 5 (36%) cases had abnormalities that shifted the prognosis from standard risk to high. An additional 3 cases (21%) had patterns consistent with an uncharacterized IGH rearrangement which may also hold a higher risk. The increased rate of abnormal cells ranged from 1.2x to 44.0x with an average increase of 9.7x. Cut-off values for the FISH analysis with this enrichment method were comparable to other methods of plasma cell enrichment. Discussion and Conclusions: We evaluated the use of a novel method to isolate and present only the target cell lineage. Deposition of a sorted plasma cell suspension via a slide chamber limits loss of plasma cells and confines these cells to a defined area for analysis. This method preserves cell morphology and avoids loss of cells that can occur with manual slide dropping methods. We found this strategy significantly increased both the sensitivity of detection levels and accuracy for determinations of these low-level, clinically prognostic genetic alterations while reducing the amount of bone marrow required for complete FISH analysis. Disclosures: MacPhee: WaveSense: Consultancy.

scholarly journals Novel Insight into Multi-Hit Multiple Myeloma Based on "Two-Hit" Theory of Cancer Causation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 47-48
Author(s):  
Chenxing Du ◽  
Xue-Han Mao ◽  
Jiahui Liu ◽  
Huishou Fan ◽  
Weiwei Sui ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Induction Therapy ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Staging System ◽  
Hazard Ratio ◽  
Drug Induction ◽  
Double Hit ◽  
Definition Of ◽  
Standard Risk

Background: Current definition of multi-hit multiple myeloma (MM) is based upon the number of high-risk cytogenetic abnormalities (CA). But we may overlook the influence of standard-risk CA and different concurrent patterns. In fact, standard-risk t(11;14) and del(13q) may bring extra danger when concurrent with other CA. And the concurrency of two secondary CA may do more harm to patients than that of one secondary CA with one primary CA. This study is to answer whether CA number or pattern exert an impact on outcomes of MM patients. Methods: This study was carried out based on the prospective, non-randomized clinical trial BDH 2008/02. 537 MM patients with complete cytogenetic data were enrolled, of whom 64% (341/537) patients were treated with bortezomib-based three-drug induction therapy, and the remaining patients with thalidomide-based therapy. Autologous stem cell transplantation (ASCT) was recommended post induction therapy in transplant-eligible patients, and all patients received maintenance therapy for two years. CA were divided into primary CA [pCA: any type of IgH breakage], and secondary CA [sCA: del(17p), del(13q), gain(1q) (≥3 copies)] Results: In the era of novel agents, patients with pCA only did not have outcomes different from those patients without any FISH abnormality. Patients with s1 CA or s1+p CA had hazard ratio for PFS or OS of 1.5-2.0. Patients with s2 CA or s2+p CA had hazard ratio for PFS or OS of 2.0-3.0. Patients with concurrent del(13q), del(17p) and gain(1q) (s3 CA) had hazard ratio for PFS of 3.11 and for OS of 3.00. Patients with s3+p CA had hazard ratio for PFS of 4.65 and for OS of 6.16. Based on these results, we divided patients into four subgroups: no CA or only pCA, s1 CA in the presence or absence of pCA, s2 CA in the presence or absence of pCA, and s3 CA in the presence or absence of pCA. Both the PFS and OS decreased in a stepwise fashion with the accumulation of CA (Figure 1). Therefore, we defined double-hit MM as any one sCA in the presence or absence of pCA. Triple-hit MM referred to two sCA plus pCA or not, and quadra-hit MM referred to three sCA plus pCA or not. Furthermore, we confirmed the prognostic independence of CA pattern in the multivariant analysis with International Staging System (ISS) and LDH (Table 1). Conclusion: In this study, we found that the primary CA as a whole lost its adverse effect when treated by bortezomib-based regimens. CA subtype conferred a prognostic value. In details, secondary CA imposed an accumulative risk to patients. For the first time, we indicated that double-hit or triple-hit MM should be defined upon the number of secondary CA. This definition coincides with the "Two-Hit" theory of cancer causation and fits well with MM evolution model. The prognostic significance of CA pattern needs validation in further prospective trials. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circulating plasma cells in newly diagnosed symptomatic multiple myeloma as a possible prognostic marker for patients with standard-risk cytogenetics

2015 ◽  
Vol 170 (4) ◽  
pp. 523-531 ◽  
Author(s):  
Davide Vagnoni ◽  
Fosco Travaglini ◽  
Valerio Pezzoni ◽  
Miriana Ruggieri ◽  
Catia Bigazzi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Prognostic Marker ◽  
Plasma Cells ◽  
Newly Diagnosed ◽  
Standard Risk

TIMP-1 and TIMP-2 Levels Are Directly Correlated with Neoangiogenesis and Are Prognostic Factors in Multiple Myeloma Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4866-4866
Author(s):  
Luciana Correa Oliveira de Oliveira ◽  
Juliana Alves Uzuelli ◽  
Ana Paula Alencar de Lima Lange ◽  
Barbara Amelia Aparecida Santana-Lemos ◽  
Marcia Sueli Baggio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Overall Survival ◽  
Prognostic Factors ◽  
Bone Disease ◽  
Cox Regression ◽  
Conflicts Of Interest ◽  
Newly Diagnosed ◽  
Significant Difference ◽  
The Impact

Abstract Abstract 4866 Background Multiple myeloma (MM) is an incurable malignant disease, characterized by increased angiogenesis in the bone marrow (BM) microenvironment and aberrant BM metabolism. Matrix metalloproteinases (MMP) are a family of zinc-dependent endopeptidases implicated in tumour progression, invasion, metastasis and angiogenesis, via proteolytic degradation of extracellular matrix. MMPs are inhibited by tissue inhibitors of metalloproteinase (TIMP). Although recent studies have implicated MMP 9 in MM bone disease, little is known about the role of the TIMPs. Objectives a) to compare levels of sRANKL, OPG, MMP-2, MMP-9, TIMP-1, TIMP-2, VEGF, bFGF, microvessel density (MVD) between newly diagnosed MM patients and healthy controls; b) to determine the association of these molecules with disease progression, bone disease and neoangiogenesis and c) to evaluate the impact of these variables on survival. Patients and Methods As of July 2009 38 newly diagnosed and untreated multiple myeloma patients were enrolled in the study. The median age was 61years-old (range 39-91) with 24 (63%) males. Patients were diagnosed and categorized according The International Myeloma Working Group criteria and ISS, respectively. Bone involvement was graded according to standard X-ray: patients with no lesions, or with one/ two bones involved or diffuse osteoporosis were classified as low score, whereas patients with lesions in more than two bones or presence of bone fracture were classified as high score. MMP-2 and MMP-9 were determined by PAGE gelatin zymography from plasma as previously described. MMP-9, TIMP-1 and TIMP-2, OPG and sRANKL concentrations were measured by ELISA. The levels of VEGF, bFGF were obtained using cytometric bead array. Ten healthy volunteers were used as controls. Bone marrow MVD measured in hotspots was evaluated in 26 out of 38 patients at diagnosis and 15 patients with Hodgkin Lymphoma stage IA and IIA (used as controls) by staining immunohistochemically for CD34. Comparisons among groups were analyzed by ANOVA and the correlation by the Spearman's correlation coefficient. Cox regression were performed for overall survival (OS) analysis. Results Patients with MM had elevated TIMP-1, TIMP-2 and OPG values compared with controls. No significant difference was found between plasma sRANKL, pro-MMP2, pro-MMP9 and MMP-9 levels. We found that plasma TIMP-1 levels correlated positively with bFGF, VEGF, MVD, beta-2 microglobulin (B2M) and OPG (r: 0.514, p=0,001, r: 0.350, p=0,031; r: 0.610, p<0.0001; r: 0.760, p<0.0001 and r: 0.701, p<0.0001, respectively) and TIMP-2 levels with bFGF, DMV, B2M and OPG (r: 0.512, p=0.002; r: 0.595, p<0.0001; r: 0.587, p<0.0001 and r: 0.552, p<0.0001, respectively). TIMP-1 and TIMP-2 levels correlated with the ISS stage (p<0.0001, p=0.006, respectively). The only variables that correlated with clinical bone disease staging were hemoglobin, B2M and albumin levels, whereas TIMP-1, TIMP-2, bFGF, VEGF and OPG correlated with DMV. On the univariate analyses, age, gender, proMMP2, TIMP-1, TIMP-2, creatinine, B2M and MVD were significantly associated with overall survival. In Cox regression model, TIMP-1, TIMP-2 and B2M levels remained to be significantly associated with OS. In conclusion, our results suggest that TIMP-1 and TIMP-2 levels are strongly associated with neoangiogenesis and are independent prognostic factors in MM. Disclosures No relevant conflicts of interest to declare.

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