Multiparameter Flow Cytometry For Detection Of Minimal Residual Disease In Multiple Myeloma After T-Cell Depleted Allogeneic Stem Cell Transplant

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4647-4647
Author(s):  
Satyajit Kosuri ◽  
Katherine M Smith ◽  
Deborah Kuk ◽  
Sean M. Devlin ◽  
Peter G. Maslak ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Cell Population ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry

Introduction Multiparameter flow cytometry (MFC) has been shown to be a sensitive, reproducible and broadly applicable method for the early detection of minimal residual disease (MRD) in the bone marrow (BM) of pts with multiple myeloma (MM) following induction chemotherapy and/or autologous stem cell transplantation. In this study, we were interested in assessing the potential of MFC as a reliable and potentially predictive marker in pts with multiple myeloma who have undergone T-cell depleted allogeneic hematopoietic stem cell transplantation (TCD HSCT). Methods We analyzed the results of MFC obtained in 35pts with multiply relapsed MM, who also have high-risk cytogenetics undergoing allo TCD-HSCT from HLA compatible related (n= 15) and unrelated (matched (n=8), mismatched (n=12) donors. We compared these results to standard myeloma markers obtained from the blood and marrow of these pts at days 30, 60-90, 120-180, 12 and 24 months routinely and as clinically indicated thereafter post TCD HSCT. Disease evaluation included serologic immunoglobulin levels, serum protein electrophoresis/immunofixation, and serum analysis of free light chains, bone marrow biopsy and aspirate. Bone marrow specimens from each time point were also analyzed by MFC with a panel including CD38, CD56, CD45, CD19, CD138, cyKAPPA, and cyLAMBDA by gating on distinct populations of bright CD38+/CD45- plasma cells at 200,000 acquired events total or at least 100 gated plasma cell events. Malignant plasma cells (MPC) were defined as CD38+/CD138+/CD56+/CD45- and/or positive for light chain clonal excess. MPC were detected in the BM sample at the MFC sensitivity of 10-4(>1 MPC in 104normal cells). Results Thirty-five pts with multiply relapsed MM undergoing allo TCD HSCT were analyzed over median follow up of 27 months (range 6.2 – 53.3). Eighteen/35 pts did not relapse during the follow up period and none of these pts had a detectable CD38+/CD138+/CD56+/CD45- cell population by MFC. Seventeen/35 pts developed relapsed disease at a median of 12.5 months (range 3.2 – 52.5) post allo TCD-HSCT by standard serologic markers and all pts were found to be positive by MFC. The percentages of bright CD38+/CD45- cells in these pts ranged from 0.01% to 16.05% at time of first detection. In 14/17 pts, MFC became positive concurrently with standard serologic myeloma markers at relapse. In 3/17 pts, MFC detected a malignant plasma cell population with aberrant phenotype of 0.068%, 0.043% and 0.012% at 48.2, 24 and 25.4 months, respectively, post TCD HSCT in the absence of other positive markers in blood and bone marrow. These pts were also immunofixation (IF) negative at conversion to MFC positivity. Subsequent follow up of studies of these 3 pts lead to detection of recurrence by IF and/or M-spike/ aspirate at 3.8, 1.8 and 8.7 months with median follow up of 150 days after first MFC detection. The populations of MPC initially detected by MFC had increased upon relapse to higher levels. Interestingly, in 2 pts we detected 6 and 8% plasma cells by bone marrow aspirate at 90 days and 180 days, respectively, post TCD HSCT, while flow cytometry detected only CD138+/CD56-/CD45+ cells. These 2 pts never relapsed and continued to remain in CR without further intervention. Conclusions These analyses demonstrate that MFC performed on marrow specimen of pts with relapsed MM who underwent a TCD HSCT provides additional important results to assess the overall disease status. A negative MFC indicated non relapse 100% of the time attesting to its negative predictive value. In all of our patients diagnosed with relapsed MM by traditional parameters, MFC was concurrently positive. Importantly, in 3/17 pts (18%) MRD detected MPC prior to overt relapse. Interestingly, MFC was able to detect false positive marrow relapses as well. Therefore, MFC permits the detection of MRD preceding frank relapse and can distinguish a malignant plasma cell population from proliferating recovering marrow post transplant. In the post allo TCD-HSCT setting MFC may serve as an early marker which can help formulate the timing of therapeutic interventions, such as adoptive immunotherapeutic approaches, as MFC detection provides a window of several weeks to initiate treatment before disease recurrence by serology. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Daratumumab Interferes with Flow Cytometric Evaluation of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5630-5630 ◽  
Author(s):  
Sudhir Perincheri ◽  
Richard Torres ◽  
Christopher A Tormey ◽  
Brian R Smith ◽  
Henry M Rinder ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Population ◽  
Plasma Cell ◽  
Light Chain ◽  
Plasma Cells ◽  
Kappa Light Chain ◽  
Positive Events ◽  
History Of

Abstract The diagnosis of multiple myeloma (MM) requires the demonstration of clonal plasma cells at ≥10% marrow cellularity or a biopsy-proven bony or extra-medullary plasmacytoma, plus one or more myeloma-defining events. Clinical laboratories use multi-parameter flow cytometry (MFC) evaluation of cytoplasmic light chain expression in CD38-bright, CD45-dim or CD138-positive, CD45dim cells to establish plasma cell clonality with a high-degree of sensitivity and specificity. Daratumumab, a humanized IgG1 kappa monoclonal antibody targeting CD38, has been shown to significantly improve outcomes in refractory MM, and daratumumab was granted breakthrough status in 2013. Daratumumab is currently approved for treatment of MM patients who have failed first-line therapies. It has been noted that daratumumab can interfere in blood bank assays for antibody screening, as well as serum protein electrophoresis (SPEP). We describe for the first time daratumumab interference in the assessment of plasma cell neoplasms by MFC; daratumumab interfered with both CD38- and CD138-based gating strategies in three MM patients. Patient A is a 68 year old man with a 10 year history of MM who had failed multiple therapies. He had then been treated with daratumumab for two months, stopping therapy 25 days prior to bone marrow assessment. Patient B is a 53 year old man with a 3 year history MM who had failed numerous treatments. He had been receiving daratumumab monotherapy for two months at the time of his bone marrow studies. On multiple marrow aspirates at times of relapse prior to receiving daratumumab, both patients had demonstrated CD38-bright positive CD45dim/negative plasma cells expressing aberrant CD56, as well as kappa light chain restriction; mature B cells were polyclonal in both. Patient C is a 65 year old man with a four-year history of MM status post autologous stem cell transplantation, who had been receiving carfilzomib and pomalidomide following relapse and continues to have rising lambda light chains and rib pain. He now has abnormal plasma cells in blood worrisome for plasma cell leukemia. Bone marrow aspirates from patients A and B, and blood from patient C demonstrated near absence of CD38-bright events as detected by MFC (Figure 1). Hypothesizing that these results were due to blocking of the CD38 antigen by daratumumab, gating on CD138-positive events was assessed; surprisingly, virtually no CD138-positive events were detected by MFC. All 3 samples demonstrated a CD56-positive CD45dim population; when light chain studies were employed using specific gating on the CD56-positive population, light chain restriction was demonstrated in all patients (Figure 1). Aspirate morphology confirmed numerous abnormal, nucleolated plasma cells (Figure 2A), thus excluding a sampling error. CD138 and CD38 expression was also tested on the marrow biopsy cores from both patients. In contrast to MFC, immunohistochemistry (IHC) showed positive labeling of plasma cells with both CD138 (Figure 2B) and CD38 (Figure 2C). The reason for the labeling discrepancy between MFC and IHC is unknown. The different antibodies in the assays may target different epitopes; alternatively, tissue fixation/decalcification may dissociate the anti-CD38 therapeutic monoclonal from its target. Detection of clonal plasma cell populations is important for assessing response to therapy. Laboratories relying primarily on MFC to assess marrow aspirates without a concomitant biopsy may falsely diagnose remission or significant disease amelioration in daratumumab-treated patients. MFC is generally highly sensitive for monitoring minimal residual disease (MRD) in MM, but daratumumab-treated patients should have their biopsy evaluated to confirm the MRD assessment by MFC. We were able to detect large numbers of plasma cells and also demonstrate clonality in our patients based on an alternative MFC marker, aberrant CD56 expression, an approach that may not be possible in all cases. Figure 1 Flow cytometry showing near-absence of CD38-bright elements in the marrow of patient A (top panels). Gating on CD56-positive cells in the same sample reveals a kappa light chain-restricted plasma cell population (bottom panels). Figure 1. Flow cytometry showing near-absence of CD38-bright elements in the marrow of patient A (top panels). Gating on CD56-positive cells in the same sample reveals a kappa light chain-restricted plasma cell population (bottom panels). Figure 1 The marrow aspirate from Fig. 1 shows abnormal plasma cells (A). Immunohistochemistry on the concomitant biopsy shows the presence of numerous CD138-positive (B) and CD38-positive (C) plasma cells. Figure 1. The marrow aspirate from Fig. 1 shows abnormal plasma cells (A). Immunohistochemistry on the concomitant biopsy shows the presence of numerous CD138-positive (B) and CD38-positive (C) plasma cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals The importance of flowcytometry study with the first aspirate taken during bone marrow aspiration in the diagnosis of multiple myeloma and follow-up of minimal residual disease

2021 ◽  
Vol 8 (4) ◽  
pp. 219-224
Author(s):  
Ali Eser
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Plasma Cells ◽  
Residual Disease ◽  
Bone Marrow Aspiration ◽  
Morphological Examination ◽  
Flow Cytometric ◽  
Flow Cytometric Study

Objective: Flow cytometry (FC) is a diagnostic method supporting traditional morphological examination in disease follow-up and the diagnosis of Multiple myeloma (MM). Normal and atypical plasma cells (PCs) can be told apart from each other by means of FC method. The plasma cell rate is the highest in the blood obtained in the first aspirate during bone marrow aspiration in MM. Material and methods: A total of 60 patients that have been diagnosed with MM between 2018 and 2020, including 30 patients whom flow cytometry was studied with the first aspirate during bone marrow aspiration, and 30 patients whom FC was studied with the second aspirate were included in our study. The characteristics of the patients were analyzed retrospectively from their files. Results: The median ratio of plasma cells (PCs) detected by FC and bone marrow biopsy  was 17,5% and 44%, respectively. While this rate was median 37,5% in patients that flow cytometric study was performed with the first aspirate, the rate was found to be median 7% in patients that FC was performed with the second sample. The PCs rates were statistically significantly higher with the flow cytometric study with the first aspirate than the second one (p=0.000). Conclusion: Flow cytometric study with the first aspirate during bone marrow aspiration in patients with MM is diagnostically important.  

Multiparameter Flow Cytometry Remission Is the Most Relevant Prognostic Factor for Multiple Myeloma Patients Who Undergo Autologous Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 737-737
Author(s):  
Bruno Paiva ◽  
Maria-Belén Vidriales ◽  
Jorge Cerveró ◽  
Gema Mateo ◽  
Jose J. Pérez ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Progression Free Survival ◽  
Patient Specific ◽  
Multiparameter Flow Cytometry ◽  
Cell Transplant ◽  
Post Transplant

Abstract Minimal residual disease (MRD) assessment is standard in many hematologic malignancies but is considered investigational in multiple myeloma (MM). We report a prospective analysis of the prognostic importance of MRD detection by multiparameter flow cytometry (MFC) in 295 newly diagnosed MM patients uniformly treated in the GEM2000 protocol (VBMCP/VBAD induction plus autologous stem cell transplant [ASCT]). MRD status by MFC was determined at day 100 post-ASCT. Persistent myelomatous plasma cells (MM-PCs) were detected by MFC in 170 patients (58%), who were considered MRD-positive. Progression-free survival (PFS; median 71 vs 37 months, P < .0001) and overall survival (OS; median not reached vs 89 months, P = .002) were longer in patients who were MRD-negative versus MRD-positive at day 100 post-ASCT, with a 5-year PFS rate of 60% and 22% (P < .0001), respectively. Similar prognostic differentiation was seen in 147 patients who achieved immunofixation (IFx) negative complete response post-ASCT. The 5-year PFS rate was 62% in MRD-negative patients (n=94) versus 30% in MRD-positive patients (n=53; P < .0001), and the respective 5-year OS rates were 87% versus 59% (P = .009). Moreover, MRD− IFx− and MRD− IFx+ patients had significantly longer PFS than MRD+ IFx− patients (median 71, 65, and 37 months, respectively, P = .0002). By multivariate analysis, only MRD status by MFC at day 100 post-ASCT and FISH cytogenetics were identified as independent prognostic factors for PFS, and only MRD status by MFC and age were identified for OS. The relative risks of progression and death among MRD-positive versus MRD-negative patients were 3.64 (P = .002) and 2.02 (P = .02), respectively. Finally, a subgroup of 157 patients in which MRD information was available both pre- and post-ASCT were analyzed. Patients who were MRD-positive both pre- and post-transplant (n=93) had the worst prognosis; patients who were MRD-positive pre-ASCT but improved to MRD-negative post-ASCT (n=48) had an intermediate prognosis, and patients who were MRD-negative both pre- and post-transplant (n=16) had the best prognosis. The 5-year PFS and OS rates in these three prognostic subgroups were 25%, 57%, and 80%, respectively (P = .0001), and 59%, 78%, and 100%, respectively (P = .06). In summary, our results show that MRD evaluation by MFC is a very useful technique to identify patients at different risk of progression. This type of analysis, particularly when performed post-ASCT, may contribute to the design of patient-specific maintenance treatment approaches, as well as the evaluation of the potential benefits of consolidation therapies.

scholarly journals Ten Color Multiparameter Flow Cytometry in Bone Marrow and Apheresis Products for Assessment and Outcome Prediction in Multiple Myeloma Patients

2021 ◽  
Vol 11 ◽  
Author(s):  
Veronika Riebl ◽  
Sandra Maria Dold ◽  
Dagmar Wider ◽  
Marie Follo ◽  
Gabriele Ihorst ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Disease Burden ◽  
Outcome Prediction ◽  
Plasma Cells ◽  
Residual Disease ◽  
Progression Free Survival ◽  
Multiparameter Flow Cytometry ◽  
Highly Sensitive

ObjectiveIn clinical trials (CTs), the assessment of minimal residual disease (MRD) has proven to have prognostic value for multiple myeloma (MM) patients. Multiparameter flow cytometry (MFC) and next-generation sequencing are currently used in CTs as effective tools for outcome prediction. We have previously described 6- and 8-color MFC panels with and without kappa/lambda, which were equally reliable in detecting aberrant plasma cells (aPC) in myeloma bone marrow (BM) specimens. This follow-up study a) established a highly sensitive single-tube 10-color MFC panel for MRD detection in myeloma samples carrying different disease burden (monoclonal gammopathy of unknown significance (MGUS), smoldering multiple myeloma (SMM), MM), b) evaluated additional, rarely used markers included in this panel, and c) assessed MRD levels and the predictive value in apheresis vs. BM samples of MM patients undergoing autologous stem cell transplantation (ASCT).Methods + ResultsThe 10-color MFC was performed in BM and apheresis samples of 128 MM and pre-MM (MGUS/SMM) patients. The markers CD28, CD200, CD19, and CD117 underwent closer examination. The analysis revealed distinct differences in these antigens between MM, MGUS/SMM, and patients under treatment. In apheresis samples, the 10-color panel determined MRD negativity in 44% of patients. Absence of aPC in apheresis corresponded with disease burden, cytogenetics, and response to induction. It also determined MRD negativity in BM samples after ASCT and was associated with improved progression-free survival.ConclusionThese results highlight the significance of the evaluation of both BM and apheresis samples with a novel highly sensitive 10-color MFC panel.

scholarly journals The Prognostic Significance of Minimal Residual Disease Detection after Induction and ASCT to the Patients with Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4956-4956
Author(s):  
Weiqin Yao ◽  
Zhu Mingqing ◽  
Yao Feirong ◽  
Lingzhi Yan ◽  
Song Jin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Induction Therapy ◽  
Plasma Cells ◽  
Residual Disease ◽  
Prognostic Significance ◽  
Depth Of Response ◽  
Minimal Residual

Abstract Objective: In the last decade the outcome in multiple myeloma in CHINA has greatly improved due to the new, effective therapies including PIs and Imids. But responses to treatment and survival remains heterogeneous because of patient characteristic, disease biology and mechanisms of drug resistance. More and more studies have established the link between depth of response and improved PFS and OS. multiparameter-flow cytometry (MFC) is a main method to detect minimal residual disease(MRD) in myeloma. Sensitivity will be at least at 10-4 to 10-5 by 10-color MFC. Imaging techniques such as PET-CT are important for EMD and bone MRD detection. whole body DWI-MRI is a new imaging technique by mean of the apparent diffusion coefficient(ADC) which can qualify the depth of response to antineoplastic treatment. This study was designed to evaluate the prognostic significance of MRD by 10-color MFC and imaging to the MM patients after induction.Methods: 102 patients with newly diagnosed MM were enrolled at the First Affiliated Hospital of Soochow University from July 2015 to July 2017. All patients were diagnosed and the response were assessed by IMWG criteria. The median of age was 58 (31-75).There were 46 patients with IgG type , 24 IgA , 14 light chain, 18 others. 34 Patients in ISS stageⅠ,34 in stage Ⅱ, 30 in stage Ⅲ. All patients received 4-6 cycles of triplet bortezomib based or lenalidomide based induction therapy. Transplantation available patients received APBSCT with BUCY condition followed by 4-6 cycles of bortezomib based or lenalidomide based consolidation which were given to transplantation unavailable patients too. Lenalidomide and thalidomide were used for over 2y of maintenance therapy. Bone marrow aspirates for MRD imaging MRD assessment were obtained at the end of induction and 1year after ASCT.The median of follow-up was 13 (2-29) months.Results: According to MRD by MFC and imaging after induction therapy and 1 year after ASCT, the patients were divided into different groups. MFC negativity was 33%(29/88) after induction therapy compared with 63%(32/51) after ASCT (X2=11.636,P=0.001). After induction therapy, the median PFS was 22 months for MRD positive group compared with not reached with MRD negative group by MFC (P=0.042) in patients with very good partial remission(VGPR) and above. The 2 years PFS was 100% for those with MRD negative compared with 60% for MRD positive by imaging. The 2 years PFS was 80% for those have multiclonal normal plasma cells compared with 52.6% for those without. The median PFS was not reached for MFC MRD negative patients 1 year after ASCT compared with 20 months for positive patients. (P=0.002). Multivariate analysis including high risk cytogenetics(17p-, t(4;14), t(14;16)), sex, age, ISS, chemotherapy, ASCT, CR/VGPR, normal PCs showed that the MFC MRD and ASCT were independent prognostic factor.Conclusions: Patients with MFC MRD negative after induction therapy or ASCT is a better prognostic marker than CR or even the best marker. Imaging MRD negativity and the appearance of normal plasma cells in the bone marrow suggests a better prognosis.We will have a try to do more research on overall survival(OS),include longer follow-up and a larger number of patients enrolled. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Minimal Residual Disease in Autografts and Bone Marrow of Patients with Multiple Myeloma: 8-Color Multiparameter Flow Cytometry (EuroFlow-NGF) Vs. Next-Generation Sequencing

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Hiroyuki Takamatsu ◽  
Naoki Takezako ◽  
Takeshi Yoroidaka ◽  
Takeshi Yamashita ◽  
Ryoichi Murata ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Research Funding ◽  
Prognostic Value ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry ◽  
Next Generation ◽  
Generation Sequencing ◽  
Minimal Residual

Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognoses of patients with multiple myeloma (MM). However, most patients with MM ultimately relapse due to minimal residual disease (MRD). Next-generation multiparameter flow cytometry (MFC) (EuroFlow-NGF) and next-generation sequencing (NGS) are currently the standard methods to assess MRD. Aims: To compare the prognostic value of MRD detection in autografts and bone marrow (BM) cells using 8-color MFC (EuroFlow-NGF) and NGS (Adaptive Biotechnologies), and also MRD levels between fresh and cryopreserved autografts using NGF. Methods: The study enrolled 52 newly-diagnosed MM patients who underwent ASCT. The median age ASCT was 61 (range 41-69) years and included 29 males and 23 females at ISS I (n = 17), II (n = 23), and III (n = 12). Of these, 18 patients harbored high-risk chromosomal abnormalities including t(4;14) (n = 15), del17p and t(4;14) (n = 2), and complex (n = 1). Bortezomib-based chemotherapy was used for induction together with melphalan at 140 mg/m2 (n = 1) and 200 mg/m2 (n = 51) for conditioning before ASCT. 39 of 52 (75%) patients received maintenance therapy until progressive disease. The best responses achieved post-ASCT included 30 sCR, 4 CR, 15 VGPR, and 3 PR. Forty autografts, one from each MM patient, were analyzed using NGF and NGS protocols, and BM cells at pre/post-ASCT and autografts derived from 16 patients were analyzed using NGS. The EuroFlow-NGF method uses standard sample preparation; large numbers of cells are evaluated using an optimized 8-color antibody panel that facilitates accurate identification of discrimination between phenotypically aberrant plasma cells (aPCs) and their normal counterparts (Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Martinez-Lopez et al., Blood 2014). Eight additional autografts were used to assess MRD in both fresh and cryopreserved samples by NGF. Results: MRD was evaluated in 48 of 52 autografts (92%) using NGF and in 44 of 52 autografts (85%) using NGS. We identified aPCs in autografts based on multivariate analysis of individual cell populations (e.g., CD56+, CD19−, CyIgκ+, and CD117+). As the results of NGF revealed a strong correlation with respect to MRD in fresh vs. thawed autografts (r = 0.999, P &lt; 0.0001), MRD was subsequently evaluated in thawed autografts. The sensitivity of NGF was 1 × 10−5-2 × 10−6; the sensitivity of NGS was 1 × 10−6. 28 of 48 (58%) of the autografts were MRD-positive by NGF; 30 of 44 (68%) of the autografts were MRD-positive by NGS. MRD levels in autografts using NGF and NGS correlated with one another (r = 0.69, P &lt; 0.0001; Fig. 1A). MRD negative in autografts by NGF cases (MRDNGF (-)) and MRDNGS (-) tended to show better progression-free survival (PFS) than MRDNGF (+) (P = 0.195) and MRDNGS (+) (P = 0.156), respectively. Furthermore, MRDNGS (-) showed significantly better overall survival (OS) than MRDNGS (+) (P = 0.03) (Fig. 1C) while MRDNGF (-) showed better OS than MRDNGF (+) (P = 0.09) (Fig. 1B). Our data revealed only a minimal correlation between MRD in the autografts (median 1.1 × 10−5,range 0-7.29 × 10−4) and in the BM cells at pre-ASCT (median 5.05 × 10−3,range 6 × 10−6-2.64 × 10−1; r = 0.09, P = 0.7) or at post-ASCT (median 2.11 × 10−4,range 0-9.09 × 10−3; r = 0.14, P = 0.6); MRD detected in the autografts was &gt; 27 times lower than that detected in pre-ASCT BM cells, and MRD detected in the post-ASCT BM cells was &gt; 3 times lower than that detected in pre-ASCT BM cells except for one case in which the ratio was increased by two times. Interestingly, while MRD was detected in all BM cells at pre-ASCT (n = 16), 4 of 16 (25%) of these autografts were MRDNGS-negative. The median of MRD levels of the 4 cases in pre-ASCT and post-ASCT BM cells were 4.14 × 10−4 (range 6-583 × 10−6)and 1.8 × 10−5 (range 0-27 × 10−6), respectively. Conclusion: Although EuroFlow-NGF is a rapid and accurate method for detecting MRD, NGS was more sensitive and provided greater prognostic value than EuroFlow-NGF. Disclosures Takamatsu: Adaptive Biotechnologies: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria, Research Funding; Ono pharmaceutical: Honoraria, Research Funding; SRL: Consultancy, Research Funding. Takezako:Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Research Funding; Abbvie: Research Funding. Nakao:Symbio: Consultancy; Kyowa Kirin: Honoraria; Alexion: Research Funding; Novartis: Honoraria.

scholarly journals Patient-Tailored Analysis of Minimal Residual Disease in Acute Myeloid Leukemia Using Next Generation Sequencing

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3841-3841
Author(s):  
Erik Malmberg ◽  
Sara Ståhlman ◽  
Anna Rehammar ◽  
Tore Samuelsson ◽  
Sofie J Alm ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Exome Sequencing ◽  
Deep Sequencing ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Multiparameter Flow Cytometry ◽  
Targeted Deep Sequencing

Abstract Background and aim: The importance of sensitive minimal residual disease (MRD) analysis for determination of response to treatment in acute myeloid leukemia (AML) is becoming increasingly evident. Routinely, this analysis is performed using multiparameter flow cytometry, and in select cases with fusion transcripts using reverse transcription polymerase chain reaction. The drawback with flow cytometry is that it is associated with false negativity due to immunophenotypic shifts during treatment and in pending relapse. In addition, leukemia immunophenotypes often overlap with the normal regenerating bone marrow cell populations. Therefore, other means of identifying remaining leukemic cells are warranted. Leukemic cells in AML are characterized by somatic mutations in recurrently mutated genes as well as in random genes, in most cases as single nucleotide variations (SNVs). We have previously reported that leukemia-specific mutations can be readily identified at the time of diagnosis of AML using exome sequencing of high purity sorted leukemic cells and lymphocytes. The aim here was to show that leukemia-specific mutations identified with exome sequencing at diagnosis can serve as markers for MRD, quantified with targeted deep sequencing, during follow-up. Method: Seventeen cases of AML, age 2-71 years old, were included in the study. Leukemic cells and lymphocytes were sorted using fluorescence activated cell sorting (FACS), from blood or bone marrow at diagnosis of AML. Exome sequencing of sorted cell populations was performed on the Illumina platform. Variant calling was performed with Mutect for SNVs and with Strelka and Varscan for short insertions/deletions. The data was subjected to an in-house statistical algorithm to identify variants present in all leukemic cells and thus suitable for MRD analysis. For targeted deep sequencing, the Truseq-library system was used for in-house PCR and sequencing on the Illumina Miseq platform (2x150 bp). The acquired reads were stitched using PEAR, aligned to the human reference genome and the resulting alignments were analyzed with in-house scripts with respect to specific SNVs and NPM1 insertion. Results: Exome sequencing of the paired leukemia/lymphocyte samples identified 240 leukemia-specific SNVs (14 (0-29) per case (median, range) and 22 small insertions and deletions (1 (0-5) per case). The most common type of mutation was, as expected, substitution of cytosine to thymine (CàT). The number of leukemia specific SNVs correlated with age (r=0.76, p<0.001). Mutations suitable for MRD analysis were identified in all but one of the investigated AML cases. Targeted deep sequencing of leukemic cells in serial dilutions established linearity down to a determined variant allele frequency (VAF) of 0.025% for SNVs and of 0.016% for insertion in NPM1. The level of detection (mean+3SD of normal samples) was VAF 0.025% for SNVs and VAF 0.007% for insertion in NPM1. Targeted deep sequencing was then performed on DNA prepared from follow-up bone marrow slides from a patient with AML with mutations suitable for MRD analysis according to our algorithm. Targeted deep sequencing of three SNVs (in the genes CPS1, ITGB7 and FAM193A) and NPM1 type A mutation could detect mutations at all eight time points tested. There were strong correlations between the detected mutation load of the SNVs and the NPM1 type A mutation and all four mutations were present at relapse 10 months after diagnosis. Targeted deep sequencing of SNVs was in this case more sensitive and robust than multiparameter flow cytometry, which could not detect leukemic cells (<0.1% of all cells) at two of the tested time points (5 and 8 months after diagnosis) and showed a completely switched immunophenotype of leukemic cells at relapse. Conclusions: Exome sequencing of high purity sorted leukemic cells and lymphocytes at the time of diagnosis of AML can identify leukemia-specific mutations suitable for MRD analysis. With targeted deep sequencing of leukemia-specific SNVs identified in this manner, leukemic cell burden can be estimated with high sensitivity during follow-up. The method could be used for patient-tailored MRD analysis in AML. Disclosures No relevant conflicts of interest to declare.

The Natural History of Smoldering (Asymptomatic) Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3396-3396 ◽  
Author(s):  
Robert Kyle ◽  
Ellen Remstein ◽  
Terry Therneau ◽  
Angela Dispenzieri ◽  
Paul Kurtin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Light Chain ◽  
Plasma Cells ◽  
M Protein ◽  
Cell Content ◽  
Bone Marrow Plasma ◽  
M Spike

Abstract Smoldering multiple myeloma (SMM) is characterized by a serum M protein ≥ 3g/dL and/or 10% or more of plasma cells in the bone marrow. However, the definition is not standardized, and it is not known whether both serum M protein levels and bone marrow plasma cell counts are necessary for diagnosis or if one parameter is sufficient. We reviewed the medical records and bone marrows of all patients from Mayo Clinic seen within 30 days of recognition of an IgG or IgA M protein ≥ 3g/dL or a bone marrow containing ≥ 10% plasma cells from 1970 to 1995. This allows for a minimum potential follow-up of 10 years. Patients with end-organ damage at baseline from plasma cell proliferation, including active multiple myeloma (MM) and primary amyloidosis (AL) and those who had received chemotherapy were excluded. A differential of the bone marrow aspirate coupled with the bone marrow biopsy morphology and immunohistochemistry using antibodies directed against CD138, MUM-1 and Cyclin D1 were evaluated in every case in order to estimate the plasma cell content. In all, 301 patients fulfilled either of the criteria for SMM. Their median age was 64 years and only 3% were less than 40 years of age; 60% were male. The median hemoglobin value was 12.9 g/dL; 7% were less than 10 g/dL, but the anemia was unrelated to plasma cell proliferation. IgG accounted for 75%, IgA 22%, and biclonal proteins were found in 3%. The serum light-chain was κ in 67% and λ in 33%. The median serum M spike was 2.9 g/dL; 11% were at least 4.0 g/dL. Uninvolved serum immunoglobulins were reduced in 81%; only 1 immunoglobulin was reduced in 31% and both were decreased in 50%. The urine contained a monoclonal κ protein in 36% and λ in 18% and 46% were negative. The median size of the urine M spike was 0.04 g/24h; only 5 (3%) were &gt; 1 g/24h. The median bone marrow plasma cell content was 15 – 19%; 10% had less than 10% plasma cells, while 10% had at least 50% plasma cells in the bone marrow. Cyclin D-1 was expressed in 17%. Patients were categorized into 3 groups: Group 1, serum M protein ≥ 3g/dL and bone marrow containing ≥ 10% plasma cells (n= 113, 38%); Group 2, bone marrow plasma cells ≥ 10% but serum M protein &lt; 3g/dL (n= 158, 52%); Group 3, serum M protein ≥ 3g/dL but bone marrow plasma cells &lt; 10% (n= 30, 10%). During 2,204 cumulative years of follow-up 85% died (median follow-up of those still living 10.8 years), 155 (51%) developed MM, while 7 (2%) developed AL. The overall rate of progression at 10 years was 62%; median time to progression was 5.5 yrs. The median time to progression was 2.4, 9.2, and 19 years in groups 1, 2, and 3 respectively; correspondingly at 10 years, progression occurred in 76%, 59%, and 32% respectively. Significant risk factors for progression with univariate analysis were serum M spike ≥ 4g/dL (p &lt; 0.001), presence of IgA (p = 0.003), presence of urine light chain (p = 0.006), presence of λ urinary light chain (p = 0.002), bone marrow plasma cells ≥ 20% (p &lt; 0.001) and reduction of uninvolved immunoglobulins (p &lt; 0.001). The hemoglobin value, gender, serum albumin, and expression of cyclin D-1 were not of prognostic importance. On multivariate analysis, the percentage of bone marrow plasma cells was the only significant factor predicting progression to MM or AL.

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