Evaluation of a 5-Color Flow Cytometric Technique for Immunophenotyping and Quantitation of Plasma Cell Myeloma in Post-Therapy Bone Marrows.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5036-5036
Author(s):  
Tove Isaacson ◽  
Andrzej Jakubowiak ◽  
Lloyd Stoolman ◽  
Usha Kota ◽  
William Finn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Plasma Cell Myeloma ◽  
Color Flow ◽  
Flow Cytometric ◽  
Minimal Residual

Abstract Multiparameter flow cytometry is a useful tool for comprehensive immunophenotyping of plasma cell myeloma, and has been proposed as a sensitive method for the evaluation of minimal residual disease in patients following treatment. This study aimed to assess the value of flow cytometry in quantitation of residual disease, in comparison to routine morphologic examination of first-pull bone marrow aspirate smears, in myeloma patients post-therapy. Heparinized bone marrow aspirates were obtained from 27 treated patients with plasma cell myeloma. Cells were prepared for 5-color flow cytometric analysis within 24-hours of specimen draw. Surface membrane staining with anti-CD19, CD20, CD38, CD45, CD56, and CD138 was followed by ammonium chloride lysis of red cells. Fixed and permeabilized cells were analyzed for cytoplasmic light chains to confirm clonality. Data were acquired using an FC500 flow cytometer (Beckman-Coulter), analyzed with CXP software with plasma cells isolated based on bright CD38+ or CD138+ expression. A median of 97,639 cellular events (range 14,279 to 262,508) were collected per analysis. Flow cytometric enumeration of plasma cells was compared to 500-cell differential counts of Wright-Giemsa-stained first-pull aspirate smears from the same cases. The median plasma cell count as determined by flow cytometry was 0.5% (range 0–7.9%). The median plasma cell count estimated by morphologic review was 8.0% (range 0–84.4%). Flow cytometry underestimated the plasma cell content in all but one case. Clonal plasma cells expressed CD38 and CD138 in all cases; 87.5% (21/24) coexpressed CD56, 25% (6/24) coexpressed CD45, and 4.2% (1/24) coexpressed CD19. None was positive for CD20. Although detection of minimal residual disease after therapy for acute leukemia is routinely achieved by flow cytometric analysis, successful quantitation of minimal residual disease in treated myeloma patients using flow cytometry remains limited as it usually underestimates the plasma cell content of bone marrow samples compared to routine morphology of first-pull aspirates. We have observed that this holds true for both pre-treatment and post-treatment specimens. Causes for the discrepancy may include hemodilution of second-pull aspirates used for flow cytometry, fragility and loss of plasma cells during preparation for flow cytometry, and incomplete disaggregation of plasma cells from bone marrow spicules. With improved outcome of treatments, better and more reliable methods of detection of minimal residual disease are needed for optimal prognostic stratification. We are currently validating alternative methods, which may offer more sensitivity while at the same time allow more objectivity, for assessing the amount of minimal residual disease in myeloma patients.

Detection of Minimal Residual Disease of Plasma Cell Myeloma by Five Color Flow Cytometric Immunophenotype Analysis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1903-1903
Author(s):  
Linsheng Zhang ◽  
Sally E. Self ◽  
John Lazarchick
Keyword(s):  
Plasma Cell ◽  
Light Chain ◽  
Plasma Cells ◽  
Residual Disease ◽  
Plasma Cell Myeloma ◽  
Immunoglobulin Light Chain ◽  
Color Flow ◽  
Flow Cytometric ◽  
Chain Analysis ◽  
Minimal Residual

Abstract Abstract 1903 Flow cytometry is widely used to identify a monoclonal proliferation of plasma cells through cytoplasmic immunoglobulin light chain analysis, with a better sensitivity than immunocytochemical staining. Recent studies have demonstrated that neoplastic plasma cells express aberrant surface antigens and immunophenotyping of plasma cells by multiparameter flow cytometry is able to reveal neoplastic plasma cells by their surface antigen profile to a level that is meaningful for the detection of prognostically relevant minimal residual disease. In this study, we compare the sensitivity of detecting abnormal plasma cells by five color flow cytometric immunophenotyping and concurrent cytoplasmic immunoglobulin light chain analysis. Multiparameter analysis was performed with cell surface markers CD45, CD38, CD138, CD19, CD20, CD56, CD117, CD27 and CD28. Plasma cells were identified by bright CD38 and CD138 expression. The bone marrow plasma cells from 8 newly diagnosed or recurrent plasma cell myelomas with 10% or more morphologically identifiable plasma cells were initially analyzed. At least one antigen was found to be abnormally expressed in all 8 cases. CD45, CD19, CD56 and CD117 were most useful in recognizing abnormal plasma cells. Both CD45 and CD19 were negative in all 8 cases. CD56 and CD117 were each positive in 6 cases; at least one of them was positive in all 8 cases; and 4 cases co-expressed both antigens. Thirteen additional cases of plasma cell myeloma in clinical remission with less than 10% plasma cells by bone marrow morphology were studied with antibodies to CD38, CD138, CD45, CD56 and CD117 in a single tube. Eleven cases revealed an abnormal immunophenotype, however, immunoglobulin light chain restriction was detected only in 6 cases. Two cases demonstrated normal phenotype and did not show immunoglobulin light chain restriction. Immunoglobulin light chain restriction was not demonstrated in any cases with less than 0.5% bright CD38 plasma cells. In one case with 9% plasma cells by morphologic examination, the immunoglobulin light chain analysis failed to reveal monoclonal proliferation whereas abnormal expression of both CD56 and CD117 was identified in 50% of the bright CD38 and CD138 positive plasma cells, although flow cytometry only detected a total of 0.5% plasma cells. Abnormal phenotype was detected at a level as low as 0.05% plasma cells by flowcytometry, in cases that less than 1% plasma cells were identified by morphologic examination. Our result suggests that 5 color flow cytometric immunophenotyping is a sensitive and practical way to detect minimal residual disease of plasma cell myeloma in patients under clinical remission. Because rare neoplastic plasma cells may not have abnormal surface antigen profile, and the abnormal phenotype may change after chemotherapy, combination with cytoplasmic immunoglobulin light chain analysis may be necessary to increase the sensitivity and specificity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Marked Variability in Reported Minimal Residual Disease Lower Level of Detection of 4 Hematolymphoid Neoplasms: A Survey of Participants in the College of American Pathologists Flow Cytometry Proficiency Testing Program

2015 ◽  
Vol 139 (10) ◽  
pp. 1276-1280 ◽  
Author(s):  
Michael Keeney ◽  
Jaimie G. Halley ◽  
Daniel D. Rhoads ◽  
M. Qasim Ansari ◽  
Steven J. Kussick ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Plasma Cell Myeloma ◽  
Minimal Residual

Context Flow cytometry is often applied to minimal residual disease (MRD) testing in hematolymphoid neoplasia. Because flow-based MRD tests are developed in the laboratory, testing methodologies and lower levels of detection (LODs) are laboratory dependent. Objectives To broadly survey flow cytometry laboratories about MRD testing in laboratories, if performed, including indications and reported LODs. Design Voluntary supplemental questions were sent to the 549 laboratories participating in the College of American Pathologists (CAP) FL3-A Survey (Flow Cytometry—Immunophenotypic Characterization of Leukemia/Lymphoma) in the spring of 2014. Results A total of 500 laboratories (91%) responded to the supplemental questions as part of the FL3-A Survey by April 2014; of those 500 laboratories, 167 (33%) currently perform MRD for lymphoblastic leukemia, 118 (24%) for myeloid leukemia, 99 (20%) for chronic lymphocytic leukemia, and 91 (18%) for plasma cell myeloma. Other indications include non-Hodgkin lymphoma, hairy cell leukemia, neuroblastoma, and myelodysplastic syndrome. Most responding laboratories that perform MRD for lymphoblastic leukemia reported an LOD of 0.01%. For myeloid leukemia, chronic lymphocytic leukemia, and plasma cell myeloma, most laboratories indicated an LOD of 0.1%. Less than 3% (15 of 500) of laboratories reported LODs of 0.001% for one or more MRD assays performed. Conclusions There is major heterogeneity in the reported LODs of MRD testing performed by laboratories subscribing to the CAP FL3-A Survey. To address that heterogeneity, changes to the Flow Cytometry Checklist for the CAP Laboratory Accreditation Program are suggested that will include new requirements that each laboratory (1) document how an MRD assay's LOD is measured, and (2) include the LOD or lower limit of enumeration for flow-based MRD assays in the final diagnostic report.

Novel Leukemia Associated Immunophenotype (LAIP) Absent from Normal and Regenerating Bone Marrow Identified by Multiparameter 6-Color Flow Cytometry.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2362-2362
Author(s):  
Denis Guyotat ◽  
Daniela Olaru ◽  
Pascale Flandrin ◽  
Nathalie Nadal ◽  
Lydia Campos
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Color Flow ◽  
Significant Difference ◽  
Blast Cells ◽  
Disease Study ◽  
New Generation ◽  
Minimal Residual

Abstract Flow cytometry analysis of minimal residual disease (MRD) in acute myeloid leukemia (AML) is based on the detection of aberrant phenotypes responsible for the relapse. Until now, all studies were performed by 3 or 4 color immunostaining, allowing the identification of LAIP in 80% of cases. Moreover, no data is available regarding the existence of such phenotypes in regenerating bone marrow. The new generation of cytometers allows the study of 8 parameters that permit a better distinction of malignant from normal phenotypes. In our study we analyzed 20 bone marrow samples from allogeneic donors, 20 ALL regenerating bone marrows after chemotherapy and 53 AML samples at diagnosis. Multiparameter 4 colour and 6 colour flow cytometry was used in order to define antigen combinations which are totally absent or present at very minimal levels in normal and regenerating hematopoiesis. “Blast cells” were gated according to CD45/SSC properties.For the first time we describe by 6 color flow cytometry 47 phenotypes totally absent from “blasts” gate in all normal bone marrow (ex: CD34+DR−117+33−15+, CD34+38+33−56+19−, CD14−DR+4+11B+64+). Another 41 phenotypes were identified as presents at a frequency < 0,05% of total cells (ex: CD34+DR+117−33+15+, CD14−DR+4+11B+64−, CD34+65−56+4−16−). There was no significant difference between normal and regenerating marrows. The 4 color panel of moAbs allowed us to identify only 30 phenotypes presents at a frequency < 0,05% of total cells (ex: CD34+33−13+, CD34+117+11b+, CD34+DR−13+). 53 AML at diagnosis were studied using 6 color immunophenotyping and 58 % of phenotypes described as aberrant or infrequent in normal myeloid hematopoiesis were found in at least one AML at diagnosis in more than 1% of total cells. All AML cases show at least one LAIP but frequently we observed more than one LAIP blast subpopulation in the same sample. Some examples of LAIP observed are CD34+ 38+ 33+ 56+ 19−, CD34+ 38+ 33+ 56− 19+, CD34− DR− 117+ 33+ 15−. In conclusion our results shows that (1) the ability to clearly distinguish leukemic from the healthy cells is considerably increased by 6 color approach (8 parameters analyzed) than 4 color. (2) Furthermore that these aberrant or infrequent phenotypes in normal or regenerating bone marrow samples are identified in AML cases and can be utilized in AML minimal residual disease study. (3) Knowledge of the expression of different markers in normal hematopoietic development provides a frame of reference for identification of abnormal differentiation patterns.

Augmentation of Sensitivity of FLT3/ITD Assay Allows Detection of Minimal Residual Disease In Stem Cell Transplant Recipients-Correlation with Flow Cytometric MRD Assessment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1717-1717
Author(s):  
Maya Danielle Hughes ◽  
Rong Zeng ◽  
Kristen L. Miller ◽  
Soheil Meshinchi
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Residual Disease ◽  
Flow Cytometric ◽  
Detection Assay ◽  
Minimal Residual

Abstract Abstract 1717 FLT3 internal tandem duplication (FLT3/ITD) is a somatic mutation that is associated with therapy resistance in acute myeloid leukemia (AML). Early data demonstrated low sensitivity for this assay, thus limiting its utility to the evaluation of diagnostic specimens, and precluding its utility in remission samples. We inquired whether the standard FLT3/ITD assay can be modified to enable its utility to detect presence of residual disease in remission specimens. Enhanced FLT3/ITD assay sensitivity was accomplished by altering annealing temperature, increasing the number of cycles as well as amount and concentration of the product that was subjected to capillary electropheresis. To assess the sensitivity of the enhanced assay, FLT3/ITD positive cells M4V11 were serially diluted in a population of ITD negative cells (HL60). The concentration of M4V11 cells in each sample ranged from 10% to 0.0001%. PCR product was subjected to capillary electropheresis and the appropriate region of the electropherogram was examined for the presence of the appropriate mutant product length. Appropriate FLT3/ITD signal was detected in dilutions down to 0.01%, validating our ability to detect extremely low levels of FLT3/ITD. We subsequently examined the remission marrows from patients with a history of FLT3/ITD who had undergone stem cell transplantation. Available bone marrow specimens (N = 51) from patients who underwent stem cell transplantation for FLT3/ITD-positive AML were analyzed and the result was correlated with the available standard PCR as well as the available MRD assessment by muti-dimensional flow cytometry; samples negative for FLT3/ITD by standard assay (N=11) were then subjected to the enhanced PCR methodology. Available ITD length for each patient was used for examination of the appropriate region of the electropherogram in each case. Of the available 51 bone marrow specimens analyzed, 23 specimens had FLT3/ITD detectable by standard PCR protocol. Using our modified PCR method and capillary electrophoresis, an additional 13 specimens had identifiable FLT3/ITD. In 6/11 patients, where initial FLT3/ITD was negative by standard methodology, enhanced assay identified FLT3/ITD signal. In each case, detection of FLT3/ITD by the enhanced assay was followed by morphologic or immunophenotypic emergence of disease, prompting therapeutic intervention. We further evaluated the ability to detect FLT3/ITD in patients with minimal residual disease by flow cytometry. 33 of the bone marrow specimens analyzed had a less than 5% abnormal blast population as detectable via flow cytometry. Among these samples, 7 had FLT3/ITD detectable using standard detection techniques. An additional 11 samples had detectable FLT3/ITD when our modified protocol was employed. Of the specimens that had less than 1% abnormal blast population as detectable via flow cytometry (N = 27), 4 had FLT3/ITD detectable using the standard detection assay; when our modified protocol was employed, an additional 6 samples had detectable FLT3/ITD. 17 bone marrow specimens had no abnormal blast cells detectable via flow cytometry; of these samples 1 had detectable FLT3/ITD using the standard detection assay, while an additional 3 had detectable FLT3/ITD using our modified assay. In four patients, FLT3/ITD was detected in bone marrow specimens found to have flow cytometric MRD of 0% (N=2), 0.1% (N=1) and 0.4% (N=1). In two patients with no detectable disease by MDF, both had emergence of morphologic (60% blast) or immunophenotypic disease by MDF (1.1%) within 4–6 weeks of detection of FLT3/ITD by enhanced assay. In this study, we demonstrate that simple modifications to the FLT3/ITD genotyping assay significantly increases its sensitivity and provides a highly sensitive and very specific assay for identifying this disease associated mutation in remission specimens. The enhanced assay can be incorporated into the standard evaluation of remission status for patients with FLT3/ITD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Novel Single Panel Method for Minimal Residual Disease Detection for Plasma Cells By Flow Cytometry

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
David Kimmel ◽  
Mohammed A Aljama ◽  
Stephen Ronan Foley ◽  
Hira S Mian ◽  
Catherine A Ross
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Large Data ◽  
Final Analysis ◽  
Distinct Advantage ◽  
Limit Of Quantitation ◽  
Minimal Residual

Introduction: Advancement in myeloma therapy has significantly improved outcomes including minimal residual disease (MRD) negativity that may be a surrogate for overall survival. We describe an assay using a single 10-colour panel to detect minimal residual disease (MRD) in plasma cell neoplasms to a level &lt; 10-5. Methodology: Bone marrow aspirate specimens for this MRD assay must be from the first pull of 1.0 to 1.5 mL of bone marrow. A 1 in 10 dilution of the bone marrow is prepared and run on the instrument using a WBC count from the following calculation to determine the volume of specimen to process: For the MRD method, a corrective factor of 4 has been empirically determined to provide a sufficient number of cells to achieve the goal of 10-13 million viable cells during analysis. The required volume of bone marrow is added directly to Versalyse (Beckman Coulter) with 10% bovine serum albumin (BSA) in a bulk RBC lysis step. The cells are incubated for 15 minutes at room temperature while rocking. The cells are centrifuged and the following antibodies are added (Table 1): The cells and surface antibodies are incubated for a total of 20 minutes, specimen is gently vortexed at 10 minutes. Intracellular staining is achieved using the IntraPrep Kit (Beckman Coulter) using our standardized laboratory process. Cells are suspended in approximately 2mL of RPMI 1640 with 10% FCS. The specimen is loaded on to the Navios EX flow cytometer (Beckman Coulter)and data is acquired at approximately 5000 to 10000 events per second. The Navios EX is not capable of collecting more 1 700 000 events at acquisition when all 10 fluorescent detectors are in use plus light scatter detectors, so the specimen is repeatedly reloaded a total of 7 or 8 times. All data files are opened in Kaluza (Beckman Coulter) and merged in to a single file. This large data file is then imported in to the analysis template and analyzed for plasma cells. Analysis: A pilot of 20 specimens from patients with varying plasma cell disorders have been analyzed. Half of these specimens contained populations of monoclonal plasma cells. Ranked in order of smallest to largest (Figure 1): The smallest clone detected at 10x10E-4 is comprised of 1311 events. If a theoretical lower limit of quantitation of 50 events or 5x10E-6 in 10 000 000 total cells analyzed is required, this method will meet this criteria .Notably, all bone marrow specimens of adequate quality (not clotted, non-hemodilute) required less than 1.5mL of bone marrow to achieve &gt; 10 000 000 nucleated cells in the final analysis. Analysis is complex using several dozen plots. Plasma cells are identified using CD38 and CD138. Gated plasma cells are analyzed for the immunophenotype of CD56, CD117, CD27, CD45, CD81 and cytoplasmic light chains simultaneously using n-dimensional radar plots (Figures 2, 3a, 3b): Qualitative results can be calculated from adjusted gates (Table 2): Conclusion: This rapid, high-sensitivity assay for immunophenotypically abnormal and clonal plasma cells requires low volumes of bone marrow. Results are ready in approximately 4 hours which is a distinct advantage and sensitivity can be shown to reach 5 X10-6. Disclosures Foley: Amgen,CelgeneJanssen: Honoraria. Mian:Takeda: Consultancy, Honoraria; Sanofi: Consultancy; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy.

Pretreatment Patient Characteristics Associated with Achieving Bone Marrow Minimal Residual Disease-Free Status with Frontline Fludarabine, Cyclosphosphamide, Rituximab (FCR) Chemoimmunotherapy for CLL,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3902-3902
Author(s):  
William G. Wierda ◽  
Susan O'Brien ◽  
Alessandra Ferrajoli ◽  
Charles Asa Koller ◽  
Jan A. Burger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Odds Ratio ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Color Flow ◽  
End Of Treatment ◽  
Free Status ◽  
Minimal Residual

Abstract Abstract 3902 Chemoimmunotherapy (CIT) is highly effective treatment and standard of care for patients (pts) with CLL. Response to treatment by NCI-WG/IWCLL criteria correlates with outcome; pts who achieve complete remission (CR) have superior progression-free and overall survival compared to pts who achieve partial remission (PR); and pts who fail therapy have the poorest outcome. Emerging data indicate improved outcomes for pts who achieve minimal residual disease (MRD)-free status in blood or bone marrow (BM) by end of treatment. We are conducting a clinical trial to prospectively evaluate pretreatment pt characteristics and prognostic factors and correlations with NCI-WG response, MRD-free status, and time to event outcomes with standard frontline fludarabine, cyclophosphamide, and rituximab (FCR) CIT. A total of 197 pts have been registered, 160 have completed treatment and are evaluable for response by NCI-WG criteria, and 127 have BM MRD status evaluated by standard 4-color flow cytometry at Course 3 and/or end of treatment. We report on pretreatment characteristics associated with MRD-free status at end of treatment. For the 160 pts evaluable for response by NCI-WG criteria, 63% were male; the median (range) age, β2M, and absolute lymphocyte count (ALC) were 58 yrs (38–84), 3.6 mg/l (1.3–14.1), and 78.7 K/μl (.8–394), respectively. The percent pts with Rai high-risk disease, unmutated IGHV status, ZAP70+ by immunohistochemistry (IHC) and CD38+ (30% cutoff) was 35%, 60%, 63%, and 37%, respectively. According to the hierarchical categorization, FISH demonstrated 17p del in 9%, 11q del in 18%, +12 in 17%, 13q del in 36%, and no abnormality in 20% of pts. The median number of FCR courses given was 6; 57% received all intended 6, 21% received 4–5, and 23% received ≤3. Of the 160 pts, 63% achieved CR, 12% nodular PR (nPR), 23% PR and 3% did not respond. Of 127 pts with BM evaluated by 4-color flow cytometry at end of treatment, 56% were MRD-free. Of 71 MRD-free pts, 27 were negative at end of course 3, 33 converted to negative after course 3, and 11 were negative at end of treatment but did not have a course 3 evaluation. Univariable Chi-square analyses demonstrated pretreatment β2M, IGHV mutation status, 17p del, and +12 correlated with MRD-free status at end of treatment (Table). The following did not correlate: age, Rai stage, WBC, ALC, HGB, PLT, ZAP70, CD38, or number of FCR courses received. Multivariable logistic regression model identified β2M≥4 mg/l (odds ratio=.78; p=.007) and unmutated IGHV (odds ratio=.77; p=.006) as independently associated with lower likelihood to achieve MRD-free status. In conclusion, mutated IGHV and β2M <4 mg/l are independently associated with increased likelihood of achieving MRD-free status with frontline FCR CIT; further follow up is needed to correlate MRD-free status with improved survival outcomes for patients treated on this trial.TableNCI-WG Responsen% MRD-NegativeCR8071nPR150PR3047*NR20Pretreatment Characteristicn% MRD-Negativep-valueAge (yrs) <65100600.07≥652741Rai Stage Low & Int-risk82610.12High-risk4347b2M (mg/l) <473640.02≥44942ALC (K/ml) <5040560.86≥508755IGHV Mutated47700.006Unmutated6244ZAP70 IHC Negative41610.28Positive7351CD38+ ≤7%4863Ref**8–29%27480.23≥30%42550.46FISH 13q del4556RefNone22680.32+1220800.0611q del20400.2417p del10200.04*All MRD-free are PR due to cytopenia, with no evidence of CLL**Used as reference or comparison group Disclosures: No relevant conflicts of interest to declare.

Carfilzomib, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma: Final Results from the NCI Phase 2 Pilot Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4746-4746 ◽  
Author(s):  
Ola Landgren ◽  
Mark Roschewski ◽  
Sham Mailankody ◽  
Mary Kwok ◽  
Elisabet E. Manasanch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
High Risk ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Color Flow ◽  
Smoldering Multiple Myeloma ◽  
Generation Sequencing ◽  
Minimal Residual

Abstract BACKGROUND: Early treatment with lenalidomide and dexamethasone delays progression and increases overall survival in patients with high-risk smoldering multiple myeloma. The addition of the selective proteasome inhibitor carfilzomib to a lenalidomide and dexamethasone backbone has proven effective in patients with newly-diagnosed multiple myeloma; this combination may allow patients with high-risk smoldering multiple myeloma to obtain deep and durable responses. METHODS: In this phase 2 pilot study, patients with high-risk smoldering multiple myeloma received eight 28-day cycles of induction therapy with carfilzomib (at a dose of 20/36 mg per square meter on days 1, 2, 8, 9, 15, and 16), lenalidomide (at a dose of 25 mg on days 1–21), and dexamethasone (at a dose of 10 or 20 mg on days 1, 2, 8, 9, 15, 16, 22, and 23). Patients achieving stable disease or better after combination therapy received 2 years of maintenance therapy with lenalidomide. Minimal residual disease was assessed with multi-color flow cytometry, next-generation sequencing by the LymphoSIGHT method, and fluorodeoxyglucose-positron emission tomography-computed tomography (FDG-PET/CT). Myeloma clonotypes were identified in genomic DNA obtained from CD138+ bone marrow cell lysate or cell-free bone marrow aspirate at baseline for each patient based on their high frequency within the B-cell repertoire. Per study protocol, minimal residual disease assessment by next-generation sequencing, multi-color flow cytometry and FDG-PET/CT was repeated when patients achieved a complete response or completed 8 cycles of induction treatment. A sample size of 12 evaluable patients was calculated as being minimally necessary based on the following probability calculations: If the true probability of a very good partial response was 20% or 50%, we calculated that there would be a 7.3% or 80.6% probability, respectively, if 5 or more patients exhibiting a very good partial response (VGPR). Thus, if 5 or more patients out of 12 achieved a very good partial response, there would be strong evidence that the true probability of a VGPR was 50% or more. RESULTS: Twelve patients were enrolled. All 11 patients (100%) who completed 8 cycles of combination therapy obtained VGPR or better (primary end point). Minimal residual disease assessment by next-generation sequencing was performed on bone marrow supernatant to detect cell-free myeloma clonotypes, while flow cytometry analysis utilized bone marrow cells. Overall (N=12), 100% of patients achieved a complete response or better over the study period, including 11 patients (92%) negative for minimal residual disease based on multi-color flow cytometry. Based on next-generation sequencing, two of the 12 patients were positive for minimal residual disease in the bone marrow supernatant; one of these two patients was also positive for minimal residual disease based on multi-color flow cytometry in the bone marrow cells. Information regarding longitudinal minimal residual disease status will be available and presented at the meeting. Adverse events were manageable. CONCLUSIONS: Early treatment with carfilzomib, lenalidomide, and dexamethasone was associated with high rates of complete response and minimal residual disease negativity by multi-color flow cytometry, next-generation sequencing, and FDG-PET/CT in patients with high-risk smoldering multiple myeloma. Disclosures Landgren: Onyx Pharmaceuticals: Consultancy; Medscape: Consultancy; Millennium Pharmaceuticals: Independent Data Monitoring Committee (IDMC), Independent Data Monitoring Committee (IDMC) Other. Off Label Use: Carfilzomib and lenalidomide for high-risk smoldering multiple myeloma.

scholarly journals The Use of Next Generation Gene Sequencing to Measure Minimal Residual Disease in Patients with AL Amyloidosis and Low Plasma Cell Burden: A Feasibility Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4353-4353 ◽  
Author(s):  
Shayna Sarosiek ◽  
Vaishali Sanchorawala ◽  
Mariateresa Fulcinti ◽  
Allison P. Jacob ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Plasma Cells ◽  
Residual Disease ◽  
Al Amyloidosis ◽  
Next Generation ◽  
Minimal Residual

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.

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