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

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.

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The Prognostic Significance of Minimal Residual Disease and Possibility of Its Correction in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-118457 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5751-5751

Author(s):

Andrey Garifullin ◽

Sergei Voloshin ◽

Alexey Kuvshinov ◽

Anastasiya Kuzyaeva ◽

Alexander Sсhmidt ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

High Risk ◽

Minimal Residual Disease ◽

Induction Therapy ◽

Residual Disease ◽

Pet Ct ◽

Minimal Residual

Abstract Introduction. Most patients with multiple myeloma (MM) are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients, the optimal methodology to assess MRD is not clear. The results of previous studies demonstrated the potential of multiparameter flow cytometry (MFC) and (PET-CT) in evaluation of MRD in MM. MRD monitoring should be applied in prospective clinical trials to compare and evaluate the efficacy of different treatment strategies, particularly in the consolidation and maintenance settings. The impact of MRD negativity is important, but further studies are needed to quantify the pharmacoeconomic and quality-of-life differences between early and delayed transplant strategies. Therefore, with the currently available evidence, upfront autologous stem cell transplantation (ASCT) is standard of care regardless of MRD status. Aim. We are aiming to determine the role of MRD and role of autologous stem cells transplantation in MM. Materials and methods. We`ve recently started a prospective one-center pilot study in subjects with MM. We analyzed 18 transplant-eligible patients with MM (the median age is 57 years, a male/female ratio is 3.5:1).The induction therapy Bortezomib-based only regimens was used in 12/18 (67%) patients, combination of Bortezomib-Immunomodulator-based regimens - in 6/18 (33%). High dose therapy (Mel200) and ASCT is carried out on 100% patients. The standard risk was established on 15 patients, 1 patient has an intermediate risk and 2 patients have high risk according to mSMART 2.0 stratification. The MFC MRD status of bone marrow was evaluated after 4-6 cycles of induction therapy and after ASCT on 5-color flow cytometry with use anti- CD38, CD138, CD45, CD19, CD20, CD27, CD56 and CD117 antibodies. We were based on two levels: MFC MRD- (<10-4) and MFC MRD- (<10-5) for assessing the significance of factors that affect MRD and for identifying the prognostic potential of MRD-negative status. The evaluation of MRD was carried out by genetic (cytogenetic and FISH) analysis of bone marrow plasma cells and PET-CT with 18-FDG before ASCT and on 100 day post ASCT. The results. The MFC MRD- (<10-4) before carrying out an ASCT reached 22.2% (4/18), the MFC MRD- (<10-5) - 0% and was not depended on the variant of pre-transplantation regimen. After the ASCT had been carried out there was a tendency to decrease the tumor burden in bone marrow from 0.65% to 0.1% and to increase the frequency of MFC MRD- (<10-4) status to 44.4% (8/18), of which MFC MRD- (<10-5) was 16.7% (3/18). MRD status was determined before ASCT and after ASCT by MFC and FISH in patients with high risk. The use of maintenance therapy with bortezomib (n = 5) or lenalidomide (n = 13) did not increase the frequency of MRD status. The PFS median in MFC MRD+ (>10-4) group was 23 months, in the MFC MRD- (<10-4) was not achieved; 2-year PFS was 43% and 100%, respectively (p=.04) We compared PFC between MFC MRD+ (>10-4) before ASCT (n = 4) and MFC MRD- (<10-4) after ASCT (n = 6) to assess the effect of ASCT in MM. The median PFS was not reached in both groups; 2-year PFS was 67% and 100%, respectively. The reliable difference between PFS in MFC MRD- (10-4-10-5) group and MFC MRD- (<10-5) was absent: the median of PFS was not achieved in both groups. PET-CT has been tested on 15 patients, PET-CT- response was achieved in 53% (8/15) patients. The PFS median in PET-CT+ group and PET-CT- group was not achieved. The 2-year PFS was higher in PET-CT+ group then PET-CT- probably due to patients with MFC MRD-. The 2-year PFS in «MFC MRD-PET-CT-» group was 100% to 55% in other patients. Conclusion. Carrying out ASCT demonstrated a tendency to increase the percentage of MFC MRD negative responses and improvement of PFS. The use of MFC in evaluation of MRD should be complemented with PET-CT and genetic methods for further analysis of the MFC MRD role status on MM patients. Disclosures No relevant conflicts of interest to declare.

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Single Immunophenotypical Evaluation of Minimal Residual Disease before Autotransplantation of Non-Cryopreserved Bone Marrow Is Insufficient for Prediction of Outcome in High Risk Adult Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v104.11.4439.4439 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4439-4439

Author(s):

Beata M. Stella-Holowiecka ◽

Krystyna Jagoda ◽

Aleksandra M. Holowiecka-Goral ◽

Tomasz Czerw ◽

Sebastian Giebel ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Minimal Residual Disease ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Conditioning Regimen ◽

Long Term Results ◽

Color Flow ◽

Childhood All ◽

Minimal Residual

Abstract For high-risk adult ALL patients alloHCT is a preferable option. However, a significant proportion of those not having a suitable donor may be successfully treated with autotransplantation (autoHCT). Based on our experience this treatment ensures low transplant related mortality below 3% and a reasonable overall survival and disease free survival of 60% and 45% respectively. The status of the disease before transplantation is an important factor for long term results. In childhood ALL most studies suggest that the level of minimal residual disease (MRD) after induction evaluated immunophenotypically or with bio-molecular methods is predictive for outcome after different treatments including chemotherapy, alloHCT and autoHCT. The results in adult ALL are more controversial. Patients selection. Among 1205 haematopoetic cell transplantations performed in our institution 224 (147 autologous, 77 allogeneic) were performed in 205 adults with ALL. For this study we selected an uniform group of 81 patients fulfilling following criteria’s: Ph (-) ALL, status CR1, evaluable MRD, strictly defined autoBMT procedure performed until the end of 2003. Methods. MRD was tested before autoBMT (median interval 10 days) using 2 ore 3-color flow-cytometry, as appropriate. The atypical immunophenotypes were evaluated using the “quadrans” analysis in all cases and since 2002 also the “empty spaces” technique. The sensitivity equals at least 0.0001. For all autoHSCT bone marrow was used as a source of stem cells. The CAV conditioning regimen consisted of cyclophosphamide 60mg/kg on d. -3, -2, cytarabine 2 g/m2 d. -3, -2, -1, etoposide 800 mg/m2 d. -3, -2. Bone marrow was not cryo-preserved after collection but stored in 40 C and re-transplanted after 72h. Results. In 41 patients; age med. 26 y (15–53), F/M=12/29, the MRD level was <0,001: the MRD (−) group. In 40 patients; age med. 29 y (16–53), F/M=18/22, the MRD was detected at the level =/> 0,001; MRD+ group. The ALL-immunophenotypes of MRD−/MRD+ groups were as follows; proB 4/7, preB 2/6, Common 18/19, B 0/1, preT 5/2, T 12/1). The interval from DGN to BMT was similar in both groups. The probability of LFS and OS at 10y calculated with median follow up time of 5y equaled; in the MRD(−) group 47% and 62% and in the MRD+ one 48% and 57% respectively (p=ns). The main reason of failure in both groups was a relapse which occurred after a median time of 277 days in the MRD(−) group and 134 days in MRD+ one (p=0.19). Conclusion and comment. Based on this observation we conclude that a single evaluation stratifying patients before autoBMT according to MRD level below or above 0.001 is not predictive for DFS and OS, because it informs only about the current amount of the disease but not about its opportunistic nature. In this respect a repeatedly confirmed MRD positivity should be more significant. Taking into consideration that the main reason of failures were relapses, this finding suggests also that in patients with chemotherapy-responsive ALL confirmed by stabile CR, the myeloablative CAV regimen is sufficiently strong to eliminate the residual disease at the level ranging 0.01–0.001. It may be speculated only that the 72h lasting incubation of bone marrow product before re-transplantation has also some kind of purging effect for leukemic blasts.

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

Blood ◽

10.1182/blood.v108.11.2362.2362 ◽

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.

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Minimal residual disease in multiple myeloma: why, when, where

Hematology ◽

10.1182/hematology.2021000230 ◽

2021 ◽

Vol 2021 (1) ◽

pp. 37-45

Author(s):

Andrew J. Yee ◽

Noopur Raje

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Clinical Practice ◽

Minimal Residual Disease ◽

Residual Disease ◽

Limit Of Detection ◽

Improved Outcomes ◽

Future Clinical Practice ◽

Minimal Residual

Abstract Improvements in multiple myeloma therapy have led to deeper responses that are beyond the limit of detection by historical immunohistochemistry and conventional flow cytometry in bone marrow samples. In parallel, more sensitive techniques for assessing minimal residual disease (MRD) through next-generation flow cytometry and sequencing have been developed and are now routinely available. Deep responses when measured by these assays correspond with improved outcomes and survival. We review the data supporting MRD testing as well as its limitations and how it may fit in with current and future clinical practice.

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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 ◽

10.1182/blood-2020-137014 ◽

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 < 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 < 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 > 27 times lower than that detected in pre-ASCT BM cells, and MRD detected in the post-ASCT BM cells was > 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.

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

Blood ◽

10.1182/blood.v108.11.5036.5036 ◽

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.

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

Blood ◽

10.1182/blood.v118.21.3902.3902 ◽

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.

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Retrospective Analysis of Minimal Residual Disease Testing By High Throughput Immunosequencing Versus High Sensitivity Flow Cytometry in Multiple Myeloma

Blood ◽

10.1182/blood-2021-154418 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1625-1625

Author(s):

Anwar Khan ◽

Nagehan Pakasticali ◽

Omar Fathalla ◽

Taiga Nishihori ◽

Mohammad O Hussaini

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Minimal Residual Disease ◽

Correlation Coefficient ◽

Research Funding ◽

Residual Disease ◽

High Sensitivity ◽

Spearman Correlation ◽

Color Flow ◽

Minimal Residual

Abstract Introduction: Detection of minimal residual disease (MRD) is one of the strongest predictors of outcome in multiple myeloma (MM). Until recently, the most commonly available method to detect MRD in clinical practice has been high sensitivity flow cytometry (FC) which can detect MRD with at 10 -5 sensitivity. In recent years, next-generation sequencing (NGS) has become a viable method to assess the MRD in MM patients with a 10 -6 sensitivity. NGS appears to have some advantages over HC-FC by circumventing subjectivity of analysis. However, real-world comparison between these two methodologies in the literature is limited and is important to inform daily hematopathology and oncology ordering practices. Methods: We retrospectively identified all cases of MM with NGS MRD data from bone marrow specimens at the Moffitt Cancer Center and collated corresponding flow MRD data and clinical data (OS, patient demographics) electronically and via chart review. 10-color flow cytometry was performed on a Gallios System and analyzed on Kaluza (Beckman Coulter, IN). Two million events were collected on all cells. Validated lower limit of detection was at least 0.01%. Antibodies included CD28, CD81, CD56, CD138, CD319, CD20, CD19, CD117, CD38, CD45, CD27, CD200 (BD, Biolegend, Beckman Coulter). clonoSEQ ® (Adaptive Biotechnologies, Seattle, WA) testing was performed which uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor clonotypes of immunoglobulin (Ig) IgH (V-J), IgH (D-J), IgK, and IgL receptor gene sequences, and translocated BCL1/IgH (J) and BCL2/IgH (J) sequences Statistical analysis was performed by Spearman correlation coefficient and Kaplan-Meier analysis. Results: 192 samples from 122 unique patients were identified that had both NGS and FC data performed on the same sample. FC+ values ranged from 1x10 -7 to 0.39. NGS+ values ranged from 2.3 x 10 -7 to 0.15. Spearman correlation coefficient showed moderate concordance between NGS and FC at r=0.67 (p<0.001). Six samples were positive by FC (mean tumor burden (MTB)= 0.0007) but missed by NGS; whereas 59 samples were positive by NGS (MTB= 0.002) but missed by flow cytometry. Two cases by FC were equivocal and these were both definitively designated as MRD+ by NGS. Overall survival was worse for MRD+ (by NGS or FC) vs MRD(-) (Figure 1). Conclusion: Our study confirms the importance of MRD detection in MM and shows the robust utility of NGS for MRD detection in routine hematopathology practice. While both FC and NGS are complementary given that each can potentially detect MRD missed by another method, the data supports the increased sensitivity of NGS over FC. Figure 1 Figure 1. Disclosures Nishihori: Novartis: Research Funding; Karyopharm: Research Funding. Hussaini: Stemeline Therapeutics: Honoraria.

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Persistent bone marrow minimal residual disease as a “high‐risk” disease feature in multiple myeloma

American Journal of Hematology ◽

10.1002/ajh.26255 ◽

2021 ◽

Author(s):

Meera Mohan ◽

Aniko Szabo ◽

Naveen Yarlagadda ◽

Sravani Gundarlapalli ◽

Sharmilan Thanendrarajan ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

High Risk ◽

Minimal Residual Disease ◽

Residual Disease ◽

High Risk Disease ◽

Minimal Residual

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Validation of a Sensitive Flow Cytometry Assay to Assess Minimal Residual Disease of Multiple Myeloma Cells in Apheresis Product

Blood ◽

10.1182/blood-2018-99-119858 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5646-5646

Author(s):

Nicholas Jones ◽

Josette William Ragheb ◽

Brian Ngo ◽

David Uyeji ◽

Anselm L. Hii

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Stem Cell ◽

Minimal Residual Disease ◽

Patient Monitoring ◽

Residual Disease ◽

Cell Transplant ◽

Flow Cytometry Assay ◽

Color Flow ◽

Minimal Residual

Abstract For treatment of patients with multiple myeloma (MM), flow cytometry has become a widely used and valuable method for the evaluation of minimal residual disease (MRD) in bone marrow. Use of an optimized single-tube, 10-color flow cytometry panel for assessment of MM MRD has shown to be beneficial in patient monitoring and has shown correlation to the acknowledged EuroFlow 8-color, 2-tube method. In order to further correlate levels of MM and relapse in patients, validation of a sensitive MM MRD assay that can be applied to testing for residual disease in apheresis product prior to autologous stem cell transplant, a standard treatment for patients with multiple myeloma. This approach can exhibit great value in patient monitoring and treatment. As new combination therapies are developed for treatment of multiple myeloma in concert with autologous stem cell transplantation, evaluation of MRD in apheresis will likely continue to grow in importance. The purpose of this study is to show the validation and sensitivity of a flow cytometry assay designed to detect Multiple Myeloma (MM) cells in G-CSF and other mobilized apheresis samples from human Multiple Myeloma patients. Using GSM-mobilized apheresis product, spiked with different levels of patient de-identified MM plasma cells, validation of a 10-color MM MRD panel can be evaluated, intended to mimic patient apheresis at varying levels of residual disease post treatment. As with any minimal residual disease assessment, a high number of events are required to ensure sensitivity and precision. Typically, acquisition of 3-5 x 107 events is required to ensure precision of MM MRD levels to 0.001%. Disclosures No relevant conflicts of interest to declare.

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