Comparison of MALDI‐TOF mass spectrometry analysis of peripheral blood and bone marrow‐based flow cytometry for tracking measurable residual disease in patients with multiple myeloma

e19525 Background: Minimal residual disease (MRD) negativity after completed therapy is associated with longer progression-free survival (PFS) in patients with multiple myeloma (MM). Current standard of care for MRD testing use flow cytometry and/or next generation sequencing (NGS)-based assays applied on bone marrow (BM) aspirate samples. To develop a strategy for MRD tracking in peripheral blood (PB), we were motivated to evaluate MALDI-TOF head-to-head with established bone marrow-based MRD assays. Methods: We used MALDI-TOF mass spectrometry to detect M-proteins in PB. Our cohort included patients who had serum samples available at 2 timepoints including during active disease and within 60 days of MRD results as determined by flow cytometry of BM aspirates. The cohort enrolled 71 patients (26 females, 45 males) with a median age of 61 years (37-78 years). Twenty-seven patients had high-risk cytogenetics at baseline. Patients were classified at diagnosis as ISS1 (n = 38), ISS2 (n = 18) or ISS3 (n = 6). The flow cytometry based MRD assay was performed using MSKCCs 10-color, single-tube method. MALDI-TOF analysis was performed as described by Mills et al. Samples taken during active disease were used to identify the mass/charge ratio of the M-protein at baseline and in follow-up samples. MALDI-TOF results were compared to flow cytometry bone marrow-based MRD results. Results: The median time between diagnosis and the MRD timepoint was 13.4 months (3.4-91 months). MALDI-TOF in PB and flow cytometry BM-based MRD results were concordant for 44/71 (62%) patients (8+/+, 36 -/- respectively) while 27 were discordant (10 +/-, 17-/+). Fifty-four of 71 patients were in complete response (CR) (45/54 in sCR) at the time of MRD. MALDI-TOF was still positive in 13 of these 54 CR patients. In this cohort, the median PFS since MRD assessment was not reached in the 2 subgroups of double negative patients (n = 31) or in patients with a positive result in at least one technique (n = 23) with a median follow-up of 11.2 months (0-34.6 months). Conclusions: In 44/71 (62%) samples, MALDI-TOF of PB results and flow cytometry BM-based MRD results were concordant. MALDI-TOF of PB may be useful for detecting measurable residual disease and for the monitoring of MM patients during maintenance therapy with the future goal to rule out early recurrent disease.

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Analysis of minimal residual disease in bone marrow by NGF and in peripheral blood by mass spectrometry in newly diagnosed multiple myeloma patients enrolled in the GEM2012MENOS65 clinical trial.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.8010 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 8010-8010

Author(s):

Noemi Puig ◽

Bruno Paiva ◽

Teresa Contreras ◽

M. Teresa Cedena ◽

Laura Rosiñol ◽

...

Keyword(s):

Mass Spectrometry ◽

Multiple Myeloma ◽

Bone Marrow ◽

Minimal Residual Disease ◽

Peripheral Blood ◽

Prognostic Value ◽

Residual Disease ◽

Newly Diagnosed ◽

Time Points ◽

Minimal Residual

8010 Background: Analysis of minimal residual disease (MRD) in the bone marrow (BM) of patients with multiple myeloma (MM) is accepted by the IMWG to evaluate treatment efficacy and is a well-established prognostic factor. However, there is an unmet need to explore the clinical value of MRD in peripheral blood (PB). Methods: Newly diagnosed MM patients enrolled in the GEM2012MENOS65 trial received six induction (Ind) cycles of bortezomib, lenalidomide, and dexamethasone (VRD) followed by autologous stem cell transplantation (ASCT) and 2 further cycles of consolidation (Cons) with VRD. MRD was analyzed in BM using Next Generation Flow (NGF) and in serum by Mass Spectrometry (MS) using IgG/A/M, κ, λ, free κ and free λ specific beads, both after Ind, at day 100 after ASCT, and after Cons. Sequential samples from the first 184 patients were analyzed. Results: Results of both methods were in agreement (NGF+/MS+ and NGF-/MS-) in 83% of cases post-Ind (152/184), 80% post-ASCT (139/174) and 76% post-Cons (128/169). Stratifying by the log range of MRD by NGF, discordances (NGF+/MS- and NGF-/MS+) seemed to increase at the lower MRD ranges, being 22%, 21% and 19% from ≥10−5 to <10−4 and 21%, 21%, 23% at ≥x10−6(post-Ind, ASCT and Cons, respectively). Analysis of discordances showed that they could be partly explained by the higher percentages of cases found to be positive by MS as compared by NGF at part of the time-points analyzed and at each log range of MRD. From ≥10−5 to <10−4, MRD was detected by NGF in 36%, 28%, 20% of cases post-Ind, ASCT and Cons, respectively vs MS in 37%, 29%, 21% of them; at ≥x10−6, NGF was positive in 11%, 14%, 19% of cases vs MS in 23%, 19% and 16% of them. Considering NGF as a reference, the negative predictive value (NPV) of MS per MRD range (≥10−5 to <10−4 and ≥x10−6, respectively) was: post-Ind: 83% (p<0,0001), 94% (p=0,034); post-ASCT 86% (p<0,0001), 90% (p=0,022); post-Cons 89% (p<0,0001), 85% (p=0,0469). Despite these discordances, the prognostic value of each technique in terms of undetectable MRD and progression-free survival (PFS) was consistent at all time-points (Table) and further, discordant cases (NGF+/MS- and NGF-/MS+) did not display a significantly different PFS as compared to NGF-/MS- cases. Conclusions: The results of MRD assessed by NGF in BM and by MS in PB show a significant concordance and are associated with a similar prognostic value analyzed in terms of PFS. Given its high NPV, MRD in peripheral blood by MS provides a gateway for BM aspiration/biopsy and MRD assessment by NGF.[Table: see text]

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Measurable Residual Disease in Extracellular Vesicles from Bone Marrow and Peripheral Blood of Patients with Multiple Myeloma: A Proof-of-Concept Study

Blood ◽

10.1182/blood-2021-149398 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4712-4712

Author(s):

Rui Bergantim ◽

Mélanie A.G. Barbosa ◽

Sara Peixoto da Silva ◽

Bárbara Polónia ◽

Hugo R. Caires ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Complete Remission ◽

Research Funding ◽

Residual Disease ◽

Disease Diagnosis ◽

Invasive Monitoring ◽

Protein Markers ◽

Measurable Residual Disease

Abstract BACKGROUND: Multiple myeloma (MM) treatment improved substantially in the last years, with unprecedented survival outcomes. However, even when achieving complete remission, patients ultimately relapse. Therefore, monitoring measurable residual disease (MRD) is crucial to assess treatment response and define the depth of patients' remission status. However, this currently still requires invasive bone marrow (BM) aspirates, which severely hinders real-time monitoring of the disease. Therefore, the identification of biomarkers of MRD in the peripheral blood (PB) of patients would allow a more frequent and minimally invasive monitoring of MRD. Extracellular Vesicles (EVs) are small particles (30-1000nm) shed by all cells, which are found in all biofluids including the BM and PB. These particles carry a specific cargo from their cell of origin, including proteins, enclosed by a lipidic layer. Therefore, they have been described as a possible source of cancer biomarkers, with potential to monitor MRD. AIMS: This study aimed to implement a protocol for the isolation of EVs from the BM and PB of MM patients at distinct stages of the disease (diagnosis and remission), in order to detect and compare the levels of known MRD biomarkers in their cargo. METHODS: The study was previously approved by the Ethical Committee of CHSJ and patient's consent was obtained. EVs from BM and PB Platelet-Poor Plasma (PPP) were isolated by size-exclusion chromatography (SEC), and further concentrated by ultrafiltration (UF). Then, the EVs were characterized according to their size and concentration (by Nanoparticle Tracking Analysis), morphology (by Transmission Electron Microscopy), protein concentration (Lowry protein assay) and presence of EV-associated protein markers (Western Blot - WB). In addition, 16 known MRD and MM biomarkers were analyzed by WB in the isolated EVs from PB and BM of seven patients, at two main stages of the disease - diagnosis versus response after autologous stem cell transplant (ASCT). Clinical features regarding cytogenetics and immunophenotypic markers using multi-parameter flow cytometry (MFC) were analyzed and compared. RESULTS: The two-step protocol described allowed the isolation of size-resolved EVs from both PB and BM of MM patients. The EVs isolated (both from PB and BM) presented a size-range from 50 to 500nm and presented EV-associated protein markers, such as CD81 and CD63. Moreover, several MM MRD biomarkers (e.g. CD56, CD45, CD38 and light chain) were detected in the cargo of the EVs from BM and PB at diagnosis and complete remission. The biomarkers of MM and MRD detected in the cargo of PB EVs were mainly the same as the ones detected in the cargo of BM EVs. The complete remission after ASCT was mostly associated with a decrease in the expression of EV-associated MM markers in both the BM and the PB; however, in some patients a few of the markers persisted at this stage when compared to diagnosis. In fact, the expression of CD45 and HLA-DR persisted at the remission stage in 3 and 2, respectively, out of 5 patients presenting these markers at diagnosis. Moreover, an increased expression of CD56 was also detected at remission in 3 out of 7 patients. By correlating these data with patient's routine work-up it was found that patients with persistent CD45 didn't reach 10^-5 MRD negative by flow cytometry. CONCLUSIONS: Taken together, this work suggests that it is possible to detect MM markers in EVs from either BM or PB of MM patients and compare their expression at different stages of the disease (diagnosis and remission after ASCT). Importantly, our results demonstrate the importance and potential of analyzing EVs cargo from PB, suggesting the possibility of using them for minimally invasive monitoring of MRD in MM patients. ACKNOWLEDGEMENTS: The authors acknowledge Celgene/BMS for providing funding to this work (Project Looker - Grant_138800). The authors acknowledge Cytogenetics Laboratory, Department of Clinical Hematology, Centro Hospitalar e Universitário São João and Flow Cytometry Laboratory, Department of Clinical Pathology, Centro Hospitalar e Universitário São João. Disclosures Bergantim: Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Barbosa: BMS: Research Funding. Silva: BMS: Research Funding. Polónia: BMS: Research Funding. Caires: BMS: Research Funding. Guimarães: BMS: Research Funding; Amgen: Research Funding. Vasconcelos: BMS: Research Funding; Amgen: Research Funding.

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Acute Myeloid Leukemia Measurable Residual Disease Detection by Flow Cytometry in Peripheral Blood Versus Bone Marrow

Blood ◽

10.1182/blood.2020006219 ◽

2020 ◽

Author(s):

Colin Douglas Godwin ◽

Yi Zhou ◽

Megan Othus ◽

Mallette M. Asmuth ◽

Carole Shaw ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Residual Disease ◽

Disease Detection ◽

Measurable Residual Disease ◽

Acute Myeloid

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Measurable residual disease assessed by mass spectrometry in peripheral blood in multiple myeloma in a phase II trial of carfilzomib, lenalidomide, dexamethasone and autologous stem cell transplantation

Blood Cancer Journal ◽

10.1038/s41408-021-00418-2 ◽

2021 ◽

Vol 11 (2) ◽

Author(s):

Benjamin A. Derman ◽

Andrew T. Stefka ◽

Ken Jiang ◽

Amanda McIver ◽

Tadeusz Kubicki ◽

...

Keyword(s):

Mass Spectrometry ◽

Multiple Myeloma ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Peripheral Blood ◽

Phase Ii Trial ◽

Residual Disease ◽

Measurable Residual Disease

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Detection of Circulating Plasma Cells in Multiple Myeloma with Extramedullary Plasmacytoma

Blood ◽

10.1182/blood.v126.23.5328.5328 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5328-5328 ◽

Cited By ~ 1

Author(s):

Jing Wang ◽

Shuang Geng ◽

Yuping Zhong ◽

Mingyi Chen ◽

Wenming Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Peripheral Blood ◽

Disease Surveillance ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Residual Disease ◽

Bone Marrow Aspiration ◽

Extramedullary Plasmacytoma

Abstract Objective: To detect the circulating plasma cells (cPCs) in patients of multiple myeloma (MM) with or withoutextramedullary plasmacytoma (EMP). Methods: The 21 patients of MM samples were collected from April 2014 to April 2015. There were 12 males, 9 females, with a median age of 60 (49 to 76 years old). Peripheral blood and bone marrow were examined before treatment or after EMP. Multi parameter flow cytometry (MFC) was used to analyze abnormal plasma cells (tumor cells) in samples of bone marrow and CD138 MACS positive sorting peripheral blood. The antibodies used in the flow cytometry were CD38-APC, CD138-PE, CD81-PE-Cy7, CD45-PacBlue, CD19-Percp-Cy5.5, CD56-mCherry-PE-ef610, CD117-AmCyan, CD16, Zombie-APC-Cy7. Results: In these 21 patients, he ratio of sex is 1.33:1, the median age is 60 (49-76). The immunoglobin type is as follows: IgG κ 7 cases, IgG λ 5 cases, IgG 2 cases, IgA κ 3 cases, IgA λ 2 cases, λ light chain 1 cases. The morphology of bone marrow aspiration showed more than 15% plasma cells and abnormal plasma cells can be seen in bone marrow in cytometry. 9 of 21 patients diagnosed MM with EMP, 2 of them find EMP when initial diagnosis and 7 of them find EMP in the course of disease (6 months to 8 years). The sites of the EMP included head, jaw, chest wall, side of the rib, the soft tissue of the sacral region and the vertebral body and all patients had bone involvement. In 17 patients with complete clinical data, bone marrow and peripheral blood specimens, the cPCs negative rate was 87.5%(7/8) in EMP negative patients, while the cPCs positive rate was 66.7% (6/9) in EMP positive patients, the difference among groups was statistically significant (chi square values: 5.13, p = 0.024). Conclusion: MFC has been widely used in diagnosis and minimal residual disease surveillance of MM, here we established the detection method of cPCs in MM patients, and it is valuable for clinical diagnosis and prognosis judgment. Disclosures No relevant conflicts of interest to declare.

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MASS-FIX versus standard methods to predict for PFS and OS among multiple myeloma patients participating on the STAMINA trial.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.8009 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 8009-8009

Author(s):

Angela Dispenzieri ◽

Amrita Y. Krishnan ◽

Bonnie Arendt ◽

Surendra Dasari ◽

Yvonne Adeduni Efebera ◽

...

Keyword(s):

Mass Spectrometry ◽

Multiple Myeloma ◽

Flow Cytometry ◽

Multiple Testing ◽

Residual Disease ◽

Progression Free Survival ◽

Complete Response ◽

Independent Effect ◽

Next Generation ◽

Different Response

8009 Background: Measuring response among patients with multiple myeloma is essential for the care of patients. Deeper responses have been associated with better progression free survival (PFS) and overall survival (OS). Serum (SIFE) and urine immunofixation are the currently used markers for biochemical documentation of CR after which marrow is tested for plasma cell clearance. Next generation flow cytometry and sequencing are used to document the presence of minimal residual disease (MRD). Mass spectrometry of blood by MALDI (Mass-Fix) is a new simple, inexpensive, sensitive, and specific means of detecting monoclonal immunoglobulins. To better test the hypothesis that Mass-Fix is superior to existing methodologies to predict for survival outcomes—especially SIFE-- samples from the STAMINA trial (NCT01109004), a trial comparing 3 transplant approaches among patients who have already received induction, were employed. Methods: Five-hundred and seventy-five patients were included. Samples from enrollment post-induction (post-I) and 1-year post enrollment (1YR) were tested when available. Four response parameters were assessed univariately: Mass-Fix, SIFE, complete response, and MRD by next generation flow cytometry. Mass spectrometry spectra were evaluated in a blinded fashion. Complete response was according to the 2006 International Myeloma Working Group criteria. Multivariate Cox proportional hazard models using stepwise regression were developed to explore the independent effect of the different response parameters on PFS and OS and interactions with other risk factors. Results: Of the 4 response measures, only MRD and Mass-Fix predicted for PFS and OS at multiple testing points on multivariate analyses (Table). Of the 4 post-I measurements, only MRD predicted for PFS; however, Mass-Fix was the only post-I measurement to predict for OS. Of all the 1-year measures, both 1YR Mass-Fix and 1YR MRD positivity predicted for inferior PFS and OS. In models including MRD and Mass-Fix, SIFE and CR were not prognostic for PFS or OS. Conclusions: Mass-Fix is a powerful means to track monoclonal proteins. The full utility of Mass-Fix was not exploited given the absence of a diagnostic sample and the fact that only serum (and not urine) was tested. Despite these limitations, it performed well at pre-induction and at 1 year. Mass-Fix provides a convenient and non-invasive means of predicting for myeloma outcomes. Clinical trial information: NCT01109004. [Table: see text]

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