scholarly journals PF555 PROGNOSTIC VALUE OF NEXT GENERATION FLOW MONITORING CIRCULATING TUMOR PLASMA CELLS IN PERIPHERAL BLOOD ON MYELOMA PATIENTS AFTER TREATMENT

HemaSphere ◽  
2019 ◽  
Vol 3 (S1) ◽  
pp. 230
Author(s):  
L. Sanoja Flores ◽  
J. Flores Montero ◽  
T. Contreras-Sanfeliciano ◽  
N. Puig ◽  
A. Corral Mateos ◽  
...  
Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4353-4353 ◽  
Author(s):  
Shayna Sarosiek ◽  
Vaishali Sanchorawala ◽  
Mariateresa Fulcinti ◽  
Allison P. Jacob ◽  
Nikhil C. Munshi ◽  
...  

Background: AL amyloidosis is a bone marrow disorder in which clonal plasma cells produce light chains that misfold and deposit in vital organs, such as the kidneys and heart, leading to organ failure and eventual death. Treatment is directed towards the clonal plasma cell population in an effort to halt the production of toxic light chains and recuperate organ function. Pallidini et al. demonstrated that almost 50% of patients with AL amyloidosis who achieved a complete hematologic response to prior therapy had minimal residual disease (MRD) detectable in their bone marrow by multiparametric flow cytometry (MPF).1. Next generation gene sequencing (NGS) has been a successful tool in measuring MRD among patients with multiple myeloma2 though the data regarding its use in AL amyloidosis are limited. AL amyloidosis is a disease with a much smaller plasma cell burden at baseline (typically 5-10%), making the task of isolating an initial clonal sequence even more challenging. We sought to evaluate NGS as a method of isolating a clonal population of plasma cells among patients with systemic AL amyloidosis in a first-ever feasibility study. Methods: Patients were eligible if they had systemic AL amyloidosis and no clinical evidence of concurrent active multiple myeloma. In this study, feasibility was deemed successful if discovery of a clone could be achieved in 3 out of 10 of patients. Approximately five cc's of peripheral blood and bone marrow aspirate were collected from each patient and processed for CD138 selection and DNA isolation/purification. De-identified samples were sent to Adaptive Biotech Inc. (Seattle, WA) for initial clonal identification using the ClonoSEQ immunoglobulin heavy chain (IGH) assay. Genomic DNA was amplified by implementing consensus primers targeting the IGH complete (IGH-VDJH) locus, IGH incomplete (IGH-DJH) locus, immunoglobulin κ locus (IGK) and immunoglobulin l locus (IGL). The amplified product was sequenced and a clone identified based on frequency. After proof of feasibility in the first 10 patients an additional 27 patients had initial clonal identification via the same process mentioned above. Results: In total, 37 patient samples underwent NGS via the ClonoSEQ IGH assay method. The median patient age was 66 years old (range: 44 to 83), 24% of which were female. All 37 patients had measurable disease based on serum electrophoresis and immunofixation and/or serum free light chain assay (Table 1). Four patients had no monoclonal protein detected on SIFE or UIFE and 13 patients had a normal sFLC ratio. Of the 33 patients with monoclonal disease on immunofixation, 12 patients had only a free lambda monoclonal protein and the remaining 21 patients had a clonal heavy chain with an associated light chain. Bone marrow biopsies demonstrated clonal plasmacytosis of 40% or lower. ClonoSEQ IGH assay identified trackable clones in 31 of 37 patients (84%) (see Table 1). Four patients had at least one trackable sequence (range: 1 to 5 sequences) in the peripheral blood and 29 patients had at least one trackable sequence in the bone marrow aspirate (range: 1 to 7 sequences). No correlation was seen between the detection of a clone and standard measures of plasma cell tumor burden (SIFE, SPEP, UIFE, UPEP, and sFLCs). Conclusion: NGS was successful in identifying an initial clone in 29 of 37 patients with systemic AL amyloidosis, four of which were detectable in the peripheral blood. Due to the low clonal burden in patients with AL amyloidosis, it is often difficult to assess disease status, especially post-treatment. These encouraging results may enhance disease monitoring and improve patient care in this rare disease. We are currently tracking MRD in the patients with identifiable clones as they receive systemic treatment, the results of which will be available for presentation in December 2019. REFERENCES 1. Palladini G, Massa M, Basset M, Russo F, Milani P, Foli A, et al. Persistence of Minimal Residual Disease By Multiparameter Flow Cytometry Can Hinder Recovery of Organ Damage in Patients with AL Amyloidosis Otherwise in Complete Response. Abstr 3261. 2016; 2. Ladetto M, Brüggemann M, Monitillo L, Ferrero S, Pepin F, Drandi D, et al. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders. Leukemia. 2014;28:1299-307. Table 1 Disclosures Sarosiek: Acrotech: Research Funding. Sanchorawala:Proclara: Consultancy, Honoraria; Takeda: Research Funding; Caelum: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Prothena: Research Funding; Celgene: Research Funding. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Munshi:Amgen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy.


Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 827-830 ◽  
Author(s):  
Animesh Pardanani ◽  
Thomas E. Witzig ◽  
Georgene Schroeder ◽  
Edwin A. McElroy ◽  
Rafael Fonseca ◽  
...  

Abstract This study examined the prognostic value of circulating peripheral blood plasma cells (PBPCs) in patients with primary systemic amyloidosis (AL). A sensitive slide-based immunofluorescence technique was used to assess 147 patients for circulating PBPCs. Circulating monoclonal plasma cells were quantified as a percentage of circulating cytoplasmic immunoglobulin-positive cells (PBPC%). The absolute circulating plasma cell count was also determined. When analyzed retrospectively, 24 (16%) of 147 patients were found to have detectable circulating PBPCs. Overall survival for patients with high PBPC%'s (> 1%) was poorer (median survival, 10 vs 29 months;P = .002). Similarly, overall survival for patients with high PBPC counts (> 0.5 × 106/L) was significantly poorer (median, 13 vs 31 months;P = .003). Increased percentages of bone marrow plasma cells (BMPC%; P = .0004), increased levels of serum β2-microglobulin (P = .04), and dominant cardiac amyloid involvement (P = .03) also predicted poorer survival. The combined consideration of circulating PBPCs and BMPC% identified low-, intermediate-, and high-risk groups with median survivals of 37.5, 15.5, and 10 months, respectively (P = .0003). Multivariate analysis revealed circulating PBPCs and BMPC% to be independent prognostic factors for survival. Patients with PBPC%'s of 2% or higher were significantly more likely to have a coexisting clinical diagnosis of multiple myeloma (50% vs 12%, P = .008). The prognostic value of circulating PBPCs may help select treatment for patients with AL.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 754-754
Author(s):  
Wilson I Gonsalves ◽  
Vinay Gupta ◽  
S. Vincent Rajkumar ◽  
William G Morice ◽  
Michael M Timm ◽  
...  

Abstract Background The presence of circulating plasma cells (PCs) in multiple myeloma (MM) is a known poor prognostic marker. Prior studies have utilized a technically challenging slide-based immunofluorescence technique to detect and quantify the presence of circulating PCs which was not widely adapted in clinical practice. More recently, the routine use of flow cytometry has provided the opportunity to quantitatively assess circulating PCs in MM patients with relative ease. We report the prognostic value of quantifying circulating clonal PCs using multiparametric flow cytometry in MM patients with relapsed disease. Methods We evaluated all MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 with previous or ongoing relapsed disease and who had their peripheral blood samples evaluated by flow cytometry. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. Plasma cells were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal plasma cells based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal plasma cells detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences in survival assessed using the log rank test. Results There were 647 consecutive patients with a history of treated MM who had a peripheral blood flow cytometry as part of their routine clinical evaluation. The median patient age was 62 years (29-88) and 55% were male. The median time since diagnosis was 12 months (range: 1-363) and the median number of lines of treatment received was 2 (1-11). There were 81 (13%) patients with clonal circulating PCs with a median of 368 cells (4 – 133,464). The 2-year overall survival (OS) for the 81 (13%) patients with any circulating PCs was 17% compared with 65% for those with none (P<0.001). The presence of circulating clonal PCs was associated with high-risk disease by FISH (P <0.001) as well as higher PCLI (P <0.001). We then correlated the presence of circulating clonal PCs with the disease status at flow cytometry assessment. Among the study patients, only 145 (22%) had actively relapsing disease with the remaining 502 (78%) in a plateau including patients in CR. Circulating clonal PCs were more likely to be detected in patients with actively relapsing disease compared with plateau phase (43% vs. 4%, P < 0.001); none of the CR patients and <5% of the remaining plateau phase patients had any detectable circulating clonal PCs. We then restricted the analysis to the actively relapsing patients; the best cutoff predicting 1-year mortality by ROC analysis was 100 events. Based on this, we defined >=100 events as a cutoff for defining the prognostic role of circulating clonal PCs in actively relapsing patients. The median OS for those with >=100 clonal PC events was 12 months compared with not reached for those with <100 events (p<0.001; Figure 1 ). Among patients with actively relapsing disease, >= 100 circulating PCs was associated with a higher ISS stage, plasma cell labeling index and bone marrow PC% compared with those with <100 circulating PCs. In a multivariable model, only LDH > 222 (HR: 2.08, P = 0.045) and >= 100 circulating PCs (HR: 2.48, P = 0.048) were found to adversely affect OS in actively relapsing patients. Conclusion The utilization of flow cytometry to quantify circulating clonal PCs in patients with relapsed MM appears to have significant prognostic relevance. In patients with actively relapsing disease, >= 100 circulating PCs predicted for worse OS with a median of 12 months. Future studies are needed to determine if this would allow an opportunity to develop a more risk adapted approach for patients with relapsed disease. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.


Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 258-258 ◽  
Author(s):  
Hiroyuki Takamatsu ◽  
Naoki Takezako ◽  
Rachel K Wee ◽  
Takeshi Yoroidaka ◽  
Takeshi Yamashita ◽  
...  

Abstract Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognosis of patients with multiple myeloma (MM). However, most patients with MM are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper responses are required. Next-generation sequencing (NGS) and multiparameter flow cytometry (MFC) methods have been used to assess MRD. However, the lack of standardization of conventional MFC approaches has had a negative impact on its reproducibility. Recently, a next-generation MFC method (EuroFlow, NGF) has been developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for a highly sensitive and standardized detection of MRD in MM. Aims: To compare the prognostic value of MRD detection in autografts in MM between NGS (Adaptive) and 8-color MFC method (EuroFlow, NGF), and also MRD levels between fresh and cryopreserved autografts. Methods: A total of 39 newly-diagnosed MM patients who underwent ASCT were enrolled in this study. Median age 60 at ASCT (range 41-69); males 22, females 17; ISS 1 (n=10), 2 (n=19), 3 (n=10). 10 patients showed high-risk chromosomal abnormalities (t(4;14) (n=9), del17p & t(4;14) (n=1)). The induction regimen was bortezomib-based chemotherapy. All patients received melphalan 200 mg/sqm as conditioning regimen before ASCT. 34 of 39 (87%) patients received maintenance therapy until progressive disease. The best response post-ASCT was as follows: 23sCR, 2CR, 12VGPR, 2PR. 39 autografts, one from each MM patient, were analyzed using NGF and NGS methods. The NGF method was based on a standardized lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of cells and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC) (J 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). To assess the correlation of MRD levels between fresh and cryopreserved autografts using NGF, 6 additional MM patients' autografts were used. Results: MRD levels in all 39 autografts were assessed using EuroFlow, while those in 32 of 39 (82%) were assessed with NGS due to limited availability of material for calibration. We identified abnormal plasma cells (aPC) in autografts based on multivariate analysis of individual cells from each patient (e.g. CD56+, CD19-, CyIgκ+, CD117+). Since there was a good correlation in MRD levels between fresh and thawed frozen autografts detected by EuroFlow (R=0.943, P=0.02), we assessed the MRD levels in thawed frozen autografts. For the MM MRD in autografts, the events from tube 1 and tube 2 were combined and a median of 7.3×106 (range: 2.2×106-37.6×106) events was acquired. The sensitivity of EuroFlow was 1×10-5-2×10-6 while that of NGS was 10-7 due to the high number of DNA derived from autografts (Takamatsu et al., Ann Oncol 2017). 21 of 39 (54%) cases were MRD positive by 8-color MFC while 22 of 32 (69%) cases were MRD positive by NGS. The correlation of MRD levels between 8-color MFC and NGS was relatively high (Fig. 1A). MRD negative by NGF (MRDMFC (-)) cases tended to show better PFS than MRDMFC (+) cases (P=0.145) (Fig. 1B) while MRD negative by NGS (MRDNGS (-)) cases showed significantly better PFS than MRDNGS (+) cases (P=0.03) (Fig. 1C). Furthermore, MRDMFC (-) MRDNGS (-) cases showed significantly better PFS than MRDMFC (-) MRDNGS (+) cases (P=0.01), but the PFS of MRDMFC (-) MRDNGS (+) cases was not different from that of MRDMFC (+) MRDNGS (+) cases (P=0.70). MRDMFC (-) and MRDNGS (-) cases showed better OS than MRDMFC (+) (P=0.14) and MRDNGS (+) (P=0.08) cases, respectively. Conclusions: Although EuroFlow is a fast and accurate method for detecting MRD of MM in autografts, in this study the NGS platform had a higher sensitivity and prognostic value than EuroFlow. The homogenous nature of the mobilized autograft relative to the focal nature of myeloma in bone marrow might provide a better sample to assess MRD. Figure 1. Figure 1. Disclosures Takamatsu: Celgene: Honoraria, Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4338-4338 ◽  
Author(s):  
Evangelos Terpos ◽  
Ioannis V. Kostopoulos ◽  
Aristea-Maria Papanota ◽  
Konstantinos Papadimitriou ◽  
Panagiotis Malandrakis ◽  
...  

Introduction: The apparent heterogeneity of multiple myeloma (MM) constitutes a key challenge in the clinical management and the design of effective therapeutic interventions, while it entails the identification of biomarkers with a strong prognostic value. In this context and taking into account patients' inconvenience to invasive bone marrow (BM) aspiration, the assessment of circulating plasma cells (CPCs) in liquid biopsies, at the time of diagnosis, has been proposed as a useful assay with prognostic value. Different methodologies have been applied for the detection of CPCs; the most common is the use of multicolor flow cytometry (MFC), mainly of 2-, 4-, or 6-color combination panels, which however yielded heterogeneous results due to variations in the detection efficacy of each approach. In the present study, we applied the standardized and highly sensitive Next Generation Flow Cytometry (NGF) approach, to detect CPCs in diagnostic MM peripheral blood (PB) samples, we compared their phenotypic characteristics with the aberrant clonal cells of BM matched samples and we correlated their presence with disease characteristics. Patients and Methods: PB and BM matched samples from 182 consecutive MM patients, at diagnosis, were evaluated for the presence of aberrant plasma cells (APCs) following the standard operating procedures (SOP) of NGF, according to EuroFlow guidelines. All these patients were diagnosed and treated in a single center (Department of Clinical Therapeutics, N.K. University of Athens, Greece). Samples were collected in EDTA-anticoagulated tubes and treated with the bulk-lysis procedure. Recovered cells were stained with antibodies against surface CD19-PEC7, CD27-BV510, CD38-FITC, CD45-PERCP, CD56-PE and CD138-BV421 and the intracellular CyIgκ-APC and CyIgλ-APCC750 to verify clonality. Six to ten million cells were acquired per sample, thus reaching a median Limit of Detection (LOD) of 3.5x10-6. Optimal PMT voltages were set according to the EuroFlow SOP for instrument set-up and daily performance status of FACSCANTOII was monitored with both CS&T (BD) and Rainbow beads (Spherotech Inc, Lake Forest, IL). Results: CPCs were detected in 158/182 (86.8%) MM diagnostic samples within a range of 0.0002% to 63.8% of total PB nucleated cells (PBNCs). The CPCs showed the same aberrant phenotype as the one detected in the BM for all cases, although with a significantly reduced intensity for the markers CD27, CD38, CD138 and CD56. When more than one phenotypically distinct subgroups were detectable in the BM, the same phenotypic subsets were present in the PB with the same relative frequency for >90% of bi/multi-phenotypic cases. The higher number of CPCs (>0.1% of all PBNCs) strongly correlated with an increased BM infiltration rate by myeloma cells (p<0.0001), with ISS-3 disease stage (p<0.0001) and with the presence of high-risk cytogenetics [t(4;14), t(14;16) and/or del(17p53); p<0.0001]. There was also weaker correlation between high number of CPCs and high serum creatinine levels (p=0.015). Inversely, the absence of CPCs or the presence of CPCs at numbers ≤0.001% correlated with lower serum β2-microglobulin (p<0.0001), with higher hemoglobin levels (p<0.0001) and with the presence of an elevated normal plasma cell compartment within the BM (i.e. ≥5% of all PCs; r2=0.84, p<0.0001). There was no association between the CPC number and the therapeutic response to induction treatment (IMWG criteria). Despite the short follow-up period (median of 16 months), there is a trend for inferior PFS in patients with high CPCs (p=0.16). Conclusions: The NGF approach using the EuroFlow protocol enables the detection of even rare CPCs in diagnostic MM PB samples, due to the high number of cells acquired and the elegantly elaborated 8-color marker combinations which allows for the detection of CPCs with even a non-typical phenotype. Our matched PB and BM analysis revealed that BM APCs and CPCs share very similar characteristics suggesting that liquid biopsy offers a representative alternative for the phenotypic characterization of BM APCs. The correlation of high CPCs with adverse disease characteristics suggests that the quantification of CPCs by standardized NGF may emerge as a valuable surrogate prognostic biomarker which could replace other invasive methods or other less informative assays. Disclosures Terpos: Janssen: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Amgen: Honoraria, Research Funding; Takeda: Honoraria, Other: Travel expenses, Research Funding; Genesis: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria. Gavriatopoulou:Amgen: Honoraria; Janssen: Honoraria, Other: Travel expenses; Genesis: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses. Kastritis:Genesis: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria. Dimopoulos:Sanofi Oncology: Research Funding.


2008 ◽  
Vol 87 (7) ◽  
pp. 587-589 ◽  
Author(s):  
Kazuhiko Natori ◽  
Haruka Izumi ◽  
Daisuke Nagase ◽  
Yoshinori Fujimoto ◽  
Susumu Ishihara ◽  
...  

2018 ◽  
Vol 46 (9) ◽  
pp. 3970-3978 ◽  
Author(s):  
Shujun Guo ◽  
Qingqing Chen ◽  
Xiaoli Liang ◽  
Mimi Mu ◽  
Jing He ◽  
...  

Objective To investigate levels of regulatory B (Breg) cells, plasma cells, and memory B cells in the peripheral blood, and interleukin (IL)-10 in the serum of multiple sclerosis (MS) patients, and to determine the correlation between Breg cell levels and the Expanded Disability Status Scale (EDSS) score. Methods Levels of Breg cells, plasma cells, and memory B cells in the peripheral blood of 12 MS patients were measured using flow cytometry. IL-10 serum levels were measured by enzyme-linked immunosorbent assay. The correlation between Breg cell levels and MS EDSS score was measured using Pearson’s correlation coefficient. Results Compared with healthy controls, MS patients had decreased levels of CD19+CD24hiCD38hi Breg cells in their peripheral blood and reduced serum levels of IL-10; however, the ratios of CD19+CD27hiCD38hi plasma cells and CD19+CD27+CD24hi memory B cells to total B cells did not differ significantly between healthy controls and MS patients. CD19+CD24hiCD38hi Breg cell levels in the peripheral blood of MS patients were not significantly correlated with MS EDSS score. Conclusion Peripheral blood CD19+CD24hiCD38hi Breg cell levels and serum IL-10 levels were reduced in MS patients compared with controls, but Breg cell levels were not correlated with MS EDSS score.


Sign in / Sign up

Export Citation Format

Share Document