Novel Flow Cytometry-Based Biomarkers Predict Recurrence in Myeloma Patients through Detection of MRD in the Bone Marrow and CTCs in Peripheral Blood

Abstract Introduction: The presence of a minimal residual disease (MRD) that remains after treatment is one of the main reasons for recurrence and relapse in multiple myeloma (MM) patients. Diagnosis of MRD is becoming vital to assess the effectiveness of the treatment as well as to predict survival among patients with complete remission. CD138 (syndecan-1) is the gold standard marker for detecting MM cells using multiparametric flow cytometry analysis or immunohistochemistry (IHC) staining of BM biopsies. However, the presence of highly clonogenic, drug resistant and stem-cell like CD138-negative MM cells have been previously demonstrated. Moreover, hypoxia which is known to drive MM progression, drug resistance and metastasis was shown to significantly decrease CD138 expression in MM cells. In this study, we utilized a novel set of biomarkers to detect MRD in the bone marrow as well as circulating tumor cells (CTCs) in the blood, in order to predict progression free survival (PFS) in MM patients defined as complete remission according to the detection of CD138+ cells in their bone marrow. Methods: To detect myeloma cells, we utilized a novel set of biomarkers, independent of CD138 expression and hypoxic state of MM cells (CD38+/CD3-/CD19-/CD14-/CD16-/CD123-) by two-color flow cytometry. We detected MM cells in the bone marrow of 25 patients with complete remission and very good partial response, whose bone marrow was defined as a CD138-negative (less than 0.5%). In order to retrospectively correlate the involvement of MM cells (%) with PFS, we used 24 months as a cut-off and compared the results acquired with the new method to traditional CD138-based flow or histology. In addition, based on the percentage of detected MM cells with the 2% cut-off, we analyzed the time to progression (months). Moreover, we detected MM cells in the peripheral blood from matching MM patients and performed analogous analysis. Furthermore, we also detected CTCs in 7 newly diagnosed patients and compared to 7 patients with progressive disease. Results: A study conducted on 25 patients with their bone marrow defined as a CD138-negative (less than 0.5%) showed that the novel strategy to detect MM cells was more precise than CD138-based flow or histology in predicting PFS. Patients who relapsed in less than 24 months had an average (±SEM) of 3.53±0.82%, while patients who relapsed later than 24 months had an average of 1.15±0.27% MM cells in the bone marrow when detected by the new method (p=0.004). In addition, the median PFS with <2% or ≥2% of MM cells detected by novel strategy was 31.6±2.8 and 18.5±2.2 months, respectively (p=0.004). Similarly, while CD138 detected very low amounts of CTCs and demonstrated no difference between fast and slow relapsing patients; we found that the new biomarkers detected significant differences in the number of circulating MM cells in patients who relapsed fast and patients who relapsed late or did not relapse. Patients who relapsed in less than 24 months had 1.74±0.42%, while patients who relapsed later than 24 months had 0.4±0.08% CTCs in the peripheral blood (p=0.01). In addition, we detected significantly more CTCs in patients with progressive disease (1.27±0.24%) compared to newly diagnosed MM patients (0.63±0.12%), respectively (p=0.03). Conclusions: The novel flow cytometry-based set of biomarkers provides an alternative strategy to detect MRD in the bone marrow and CTCs in the peripheral blood of MM patients, and both allowed prediction of PFS in MM patients. Disclosures De La Puente: Cellatrix LLC: Other: Co-founder. Vij:Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy; Jazz: Consultancy; Shire: Consultancy; Karyopharma: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy. Azab:Targeted Therapeutics LLC: Other: Founder and owner; Cleave Bioscience: Research Funding; Verastem: Research Funding; Cell Works: Research Funding; Karyopharm: Research Funding; Vasculox: Research Funding; Glycomimetics: Research Funding; Cellatrix LLC: Other: Founder and owner; Selexys: Research Funding.

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First-in-Human Phase I Trial of Adoptive Immunotherapy with Ex Vivo Expanded and Activated Γδ T Cells Following Haploidentical Bone Marrow Transplantation and Post-BMT Cyclophosphamide

Blood ◽

10.1182/blood-2020-142876 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-9

Author(s):

Lawrence S. Lamb ◽

Melissa Jo Beelen ◽

Samantha Langford Youngblood ◽

Rupal Soder ◽

Sunil Abhyankar ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Complete Remission ◽

Phase I ◽

Peripheral Blood ◽

Research Funding ◽

Ex Vivo ◽

Γδ T Cells ◽

Disease Recurrence ◽

Publicly Traded

INTRODUCTION: HAPLO BMT combined with cyclophosphamide infusion on days +3 and +4 following BMT (PTCy) for patients that lack an HLA-matched donor provides effective graft versus host disease (GVHD) prophylaxis but with a heightened risk of disease recurrence likely due to prolonged immunodeficiency. Multiple studies have shown that increases in circulating donor-derived gamma delta (γδ) T cells during the early post-transplant period is strongly associated with significant improvement in disease-free survival (DFS). We developed a clinical protocol to engineer this effect by infusing escalating doses of ex vivo expanded and activated donor γδ T cells (EAGD) during this period of immunodeficiency as prophylaxis against disease recurrence. As innate immune effectors γδ T cells immediately recognize and kill malignant cells in a broad-based non-MHC restricted manner and do not initiate GVHD. STUDY DESIGN AND METHODS: This single-center Phase I clinical trial of allogeneic haploidentical BMT + EAGD (NCT03533816), currently underway at the Kansas University Cancer Center, represents the first time BMT patients have received a large infusion of EAGD from a haploidentical donor during the early post-transplant time period. Subjects ≥18 years of age with AML, ALL, CML or MDS either in morphologic complete remission with high risk features, in first complete remission, or chronic phase (CML) eligible for BMT are enrolled in a 3 x 3 design with escalating EAGD doses from 1 x 106/kg to 3 x 106/kg to 1 x 107/kg. Subjects must have adequate organ function and a KPS ≥70. Any subjects with central nervous system neoplastic involvement, HIV infection, or a life expectancy <12 weeks are excluded. Haploidentical donors provide an initial non-mobilized leukapheresis product from which EAGD are manufactured and an unmanipulated bone marrow harvest as the source of hematopoietic stem cells. Closed system EAGD manufacturing is performed in the Miltenyi Prodigy® bioreactor by expansion of γδ T cells in conjunction with αβ T cell depletion. Specimens for composition, viability, sterility, identity, and purity are obtained prior to cryopreservation and results obtained prior to infusion. Subjects undergo standard of care conditioning therapy with fludarabine and cyclophosphamide followed by BMT and post-BMT cyclophosphamide (Cy). The EAGD product is thawed and infused directly within 5 days of neutrophil engraftment (ANC >500/mL for 3 consecutive days). Peripheral blood is collected at screening, pre and post EAGD infusion, then monthly thereafter through Day 100 with additional samples collected every 6 months through 1 year and annually thereafter. Biologic parameters include leukemia phenotype and genomics, multiparameter peripheral blood immunophenotyping, single-cell and serum Th1/Th2/Th17 cytokine analysis, and immunogenomics for both peripheral blood and the EAGD cell product. Primary endpoints include the following safety assessments; laboratory parameters, viral monitoring, physical exams, acute and chronic GVHD, as well as biopsy pathology. Relapse and overall survival will be measured as secondary endpoints. Descriptive statistics under each specific dose will be used to summarize baseline characteristics, safety variables and efficacy outcomes. Kaplan-Meier curves and summary statistics will be used to summarize time-to-event outcomes. STATUS: To date, 3 female subjects (age range, 44 - 54) with AML have been enrolled with 2 subjects receiving the EAGD infusion. To have no treatment-related adverse events have been recorded Disclosures Lamb: In8bio: Current Employment, Current equity holder in private company. Beelen:In8bio: Current Employment, Current equity holder in publicly-traded company. Youngblood:In8bio: Current Employment, Current equity holder in publicly-traded company. McGuirk:Pluristem Ltd: Research Funding; Allo Vir: Consultancy, Honoraria, Research Funding; Kite Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bellicum Pharmaceutical: Research Funding; Astellas: Research Funding; Fresenius Biotech: Research Funding; Novartis: Research Funding; Gamida Cell: Research Funding; Juno Therapeutics: Consultancy, Honoraria, Research Funding.

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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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Molecular Characterization of Bone Marrow and Peripheral Blood Compartments from Patients with Plasma Cell Leukemia in the Mmrf Commpass Study

Blood ◽

10.1182/blood-2019-130697 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1782-1782

Author(s):

Sheri Skerget ◽

Austin Christofferson ◽

Sara Nasser ◽

Christophe Legendre ◽

The MMRF CoMMpass Network ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Plasma Cell ◽

Peripheral Blood ◽

Research Funding ◽

Plasma Cells ◽

Cell Cycle Control ◽

Cell Leukemia

Plasma cell leukemia (PCL) is rare but represents an aggressive, advanced form of multiple myeloma (MM) where neoplastic plasma cells (PCs) escape the bone marrow (BM) and circulate in the peripheral blood (PB). Traditionally, PCL is defined by the presence of >20% circulating plasma cells (CPCs), however, recent studies have suggested that PCL be redefined as the presence of >5% CPCs. The Multiple Myeloma Research Foundation CoMMpass study (NCT01454297) is a longitudinal, observational clinical study with 1143 newly diagnosed MM patients. BM-derived MM samples were characterized using whole genome (WGS), exome (WES), and RNA (RNAseq) sequencing at diagnosis and each progression event. When >5% CPCs were detected by flow cytometry, PCs were enriched independently from both compartments, and T-cells were selected from the PB as a control for WGS and WES. This substudy within CoMMpass provides the largest, most comprehensively characterized dataset of matched MM and PCL samples to date, which can be leveraged to better understand the molecular drivers of PCL. At diagnosis, 813/1143 CoMMpass patients had flow cytometry data reporting the percent PCs in PB, of which 790 had <5%, 17 had 5-20%, and 6 had >20% CPCs. Survival analyses revealed that patients with 5-20% CPCs (median = 20 months) had poor overall survival (OS) outcomes compared to patients with <5% CPCs (median = 74 months, p < 0.001), and no significant difference in outcome was observed between patients with 5-20% and >20% (median = 38 months) CPCs. Patients with 1-5% CPCs (median = 50 months, HR = 2.45, 95% CI = 1.64 - 3.69, p < 0.001) also exhibited poor OS outcomes compared to patients with <1% CPCs (median = 74 months), suggesting that patients with >1% CPCs are a higher risk population, even if they do not meet the PCL threshold. Using a cutoff of >5% CPCs, 23/813 (2.8%) patients presented with primary PCL (pPCL) at diagnosis. Of these patients, 7 (30%) were hyperdiploid (HRD), of whom 1 had a CCND1 and 1 had a MYC translocation; while 16 (70%) were nonhyperdiploid (NHRD), all of whom had a canonical immunoglobulin translocation (6 CCND1, 5 WHSC1, 3 MAF, 1 MAFA, and 1 MAFB). Of 124 patients with serial sample collections, 5 (4%) patients without pPCL had >5% CPCs at progression, and thus relapsed with secondary PCL (sPCL). Of the 5 sPCL patients, 2 (40%) were NHRD with a CCND1 or MAF translocation; while 3 (60%) were HRD, 1 with a WHSC1 translocation. Median time to diagnosis of sPCL was 22 months (range = 2 - 31 months), and patients with sPCL (median = 22 months) and pPCL (median = 30 months) exhibited poor OS outcomes as compared to MM patients (74 months, p < 0.001). Sequencing data was available for 15 pPCL and 5 sPCL samples. For 12 patients with WES, WGS, and RNAseq performed on their PCL tumor sample, an integrated analysis identified recurrent, complete loss-of-function (LOF) events in only CDKN2C/FAF1, SETD2, and TRAF3. Five pPCL patients had complete LOF of a gene involved in G1/S cell cycle control, including CDKN2C, CDKN2A, CDKN1C, and ATM. These LOF events were not observed in NHRD t(11;14) PCL patients, suggesting that CCND1 overexpression and LOF of genes involved in G1/S cell cycle control may represent independent drivers of PCL. Comparing WES and WGS data between matched MM and PCL tumor samples revealed a high degree of similarity in mutation and copy number profile. However, differential expression analysis performed for 13 patients with RNAseq data comparing their MM and PCL tumors revealed 27 up- and 39 downregulated genes (padj < 0.01, FDR = 0.1) in PCL versus MM. Pathway analysis revealed an enrichment (p < 0.001) for genes involved in adhesion and diapedesis, including upregulation of ITGB2, PF4, and PPBP, and downregulation of CCL8, CXCL12, MMP19, and VCAM1. The most significantly downregulated gene in PCL (log2FC = -6.98) was VCAM1, which plays a role in cell adhesion, and where loss of expression (TPM < 0.01) was observed across all PCL samples. Upregulation of four S100 genes including S100A8, S100A9, S100A12, and S100P, which have been implicated in tumor growth, metastasis, and immune evasion, was also observed in PCL. Interestingly, a S100A9 inhibitor has been developed and may represent a novel treatment option for PCL patients. In summary, PCL was found to be associated with molecular events dysregulating G1/S cell cycle control coupled with subtle changes in transcription that likely occur in a subclonal population of the MM tumor. Disclosures Lonial: Genentech: Consultancy; GSK: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; Karyopharm: Consultancy; Takeda: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Amgen: Consultancy.

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Value of Immunohistochemistry-Based Direct Visualization for Localization, Lineage Determination and Monitoring of IDH1 p.R132H Mutant Clones in AML

Blood ◽

10.1182/blood.v128.22.1726.1726 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1726-1726

Author(s):

Habibe Kurt ◽

Carlos E. Bueso-Ramos ◽

Joseph D Khoury ◽

Mark Routbort ◽

Rashmi Kanagal-Shamanna ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Complete Remission ◽

Minimal Residual Disease ◽

Research Funding ◽

Residual Disease ◽

Advisory Board ◽

Post Treatment ◽

Immature Cells ◽

Minimal Residual

Abstract Background Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are important prognostic biomarkers in acute myeloid leukemia (AML). Although the clinicopathologic correlates of IDH mutations have been extensively studied, the distribution of abnormal myeloid cells carrying these mutations has not been studied. Specific localization of cells carrying IDH mutations will be useful in further understanding the pathophysiology and post-treatment biology of IDH mutant cases of AML. This characterization is becoming particularly relevant for identification of minimal residual disease, especially for patients treated with novel IDHinhibitors. In this study, we characterized IDH1 p.R132H clones in bone marrow specimens involved by AML using a mutation specific antibody. Materials and Methods Bone marrow tissue sections (biopsy or clot specimens) from 32 AML cases with IDH1 p.R132H mutation were stained with IDH1 p.R132H-mutation specific antibody. These cases include 20 de novoAML and 12 cases of AML with myelodysplasia-related changes (AML-MRC). We also included 10 AML cases with wild-type IDH1 as a control. After confirmation of the positive IDH1 immunohistochemical (IHC) signal in the primary specimens, follow up bone marrow specimens (n=67) including (a) persistent disease, (b) minimal residual disease by flow cytometry, (3) complete remission by morphology and flow cytometry, but, positive for mutation by PCR, as well as (4) relapsed cases after complete remission were included in the study (in progress). We also included pre- and post-treatment (unresponsive with increasing blast counts, stable disease, persistent disease with decreasing blast counts, complete remission, and relapse) bone marrow specimens (n=72) from 16 patients treated with IDH inhibitors (in progress). Results All the IDH1 wild type AML cases were negative for IDH1 IHC stain showing 100% specificity. Positive signal was detected in all de novo AML and AML-MRC (allelic frequency ranges from 1.8% to 47% by PCR) except one AML case with 8.9% allele burden which was a limited sample; overall sensitivity was 96%. The IHC signal was detected in the cytoplasm of myelomonocytic cells, their precursors, and megakaryocytes. Erythroid precursors, lymphoid cells, endothelial cells, and osteoblasts were consistently negative. The signal intensity ranged from weak (n=10) to moderate (n=9), to strong (n=13). The positive cells predominantly showed an interstitial distribution in the bone marrow. In the de novo AML group, only the immature cells were positive in 100% of pre-treatment AML cases. However, both mature and immature cells were positive in 7/13 (54%) post-treatment AML cases (6 cases treated with hypomethylating agents). One case was transformed from MPN which also showed positivity in mature and immature cells. In two cases with complete morphologic remission and one case with minimal residual disease detected by flow cytometry, IHC signal was detected in both mature and immature cells; both patients relapsed in 8 and 11 months. In the AML-MRC group, both immature and mature cells were positive in 11/12 (92%) cases of which 2 were not previously treated indicating the possibility that IDH1 mutation is an early event. Since the remaining 9 patients were treated with hypomethylating agents, the positivity of both mature and immature cells as a result of maturation effect versus an early event cannot be assessed. Additional studies for follow-up AML cases, including cases on an IDH inhibitor clinical trial are in progress. Conclusions Our preliminary data indicate that IDH1 IHC is a highly specific and sensitive tool to detect IDH1 R132H mutated cases and can be used as a primary method to localize the population of mutation-bearing cells in the bone marrow. IHC also allows determination of whether the IDH1 mutation in the post-treatment setting is arising from immature or mature cells. IHC provides an opportunity to understand the difference between these two populations and, based on characterization of cell type and distribution, may be helpful to predict whether the risk of relapse is high. Disclosures DiNardo: Agios: Other: advisory board, Research Funding; Novartis: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding; Celgene: Research Funding; Abbvie: Research Funding.

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Role Of Folate Receptor Targeted Therapy In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.4436.4436 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4436-4436

Author(s):

Sonali Panchabhai ◽

Katalin Kelemen ◽

Sinto Sebastian Chirackal ◽

Rafael Fonseca

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Targeted Therapy ◽

Cell Lines ◽

Research Funding ◽

Plasma Cells ◽

Newly Diagnosed ◽

Tumoricidal Activity ◽

Significant Difference

In multiple myeloma (MM) the interaction of plasma cells, bone marrow stromal cells and tumor associated macrophages (TAMs) plays a significant role in conferring resistance to therapy and in the maintenance of residual disease. Folate receptors (FR) are specifically expressed on metabolically active malignant cells and TAMs and their expression in normal tissues and resting macrophages is limited. FR mediates folate uptake by receptor-mediated endocytosis. This qualifies the receptor to be exploited for drug delivery of folate conjugated cancer therapeutics. Our primary goal in this study is to evaluate the expression of functional FR in MM cells and TAMs with a view to exploit this for folate conjugated targeted therapy for MM. First we evaluated the presence of TAMs in paraffin embedded bone marrow slides of newly diagnosed MM patients with CD68 (pan-Macrophage marker) and CD163 antibodies (specific marker of TAMs) and found extensive infiltration of macrophages in bone marrow from MM patients. Next to evaluate expression of FR in MM cells, we employed an FR antibody and evaluated MM cell lines with immunoblot, flow cytometry and confocal microscopy. In a panel of ten MM cell lines, we found that out of the three FR isoforms (α, β and γ), FR beta (FR-β) is expressed by all of them. Next to test whether this expressed receptor is indeed functional, we incubated the cells with folate deficient media, added different concentrations of EC17 (folate conjugated to FITC, Endocyte Inc.) to the medium and looked for the uptake by flow cytometry after washing off the drug at different time points (after 10, 20, 40 and 60 min).We observed that the uptake begins in 10 min and is saturated at 1 hour with 100nM Folate-FITC. With confocal imaging, Folate-FITC was found in the cytoplasm of MM cells suggesting internalization of Folate-FITC and localization in the cytoplasm. In addition to myeloma cell lines, we also confirmed the uptake of Folate-FITC in CD138+ plasma cells of a newly diagnosed myeloma patient by flow cytometry. This strongly suggests that MM cells express functional FR-β. To test the specificity of this FR mediated uptake, we pre-incubated cells with 0.1mM folic acid in medium for 30 min and then added EC17. This maneuver blocked the activity mediated by FR and no uptake was observed , which proves that the Folate-FITC is internalized only through the FR. To evaluate the expression of FR in-vivo samples, we stained paraffin embedded bone marrow slides of newly diagnosed MM patients with FR-β antibody and TAM specific markers. We observe that FR is expressed on both MM cells as well as TAMs. To assess the endurance of cytotoxic effect of folate conjugated chemotherapeutic agents, we treated MM cell lines with folate conjugated vinka alkaloids and compared them to unconjugated drug and found no significant difference in their action suggesting conjugation with folate does not alter its efficacy. To assess potential toxicity of folate conjugated therapeutics, we obtained CD34+ cells and looked for the uptake of Folate-FITC with flow cytometry. We found no uptake and this is in line with previous reports suggesting that CD34 positive cells express nonfunctional FR. So we propose that FR qualify as potential targets for cancer treatment. Folate targeted therapy using folate-conjugated drugs which can selectively act against both MM cells and supporting TAMs has the potential of specific anti-MM tumoricidal activity. This therapeutic approach would broaden the use of drugs that could be conjugated with folate for MM therapy. Additionally assessment of TAMs in bone marrow sections of MM patients would add another feature for grading, classifying and prognosticating MM. Disclosures: Fonseca: Cylene: Research Funding; AMGEN: Consultancy; Millennium: Consultancy; Binding Site: Consultancy; Onyx: Consultancy, Research Funding; Lilly: Consultancy; BMS: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Otsuka: Consultancy; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

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Pilot Study Of Erlotinib In Patients With Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.5037.5037 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5037-5037

Author(s):

Hamid Sayar ◽

Magdalena Czader ◽

Chirag Amin ◽

Mary Cangany ◽

Larry D Cripe

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Pilot Study ◽

Disease Progression ◽

Peripheral Blood ◽

Bone Marrow Aspirate ◽

Molecular Profile ◽

Refractory Disease ◽

Newly Diagnosed ◽

Blast Count

Abstract Backgound Preclinical data suggest “off-target” efficacy of EGFR tyrosine kinase inhibitor erlotinib in MDS and AML by inducing apoptosis and promoting differentiation in myeloblasts (Boehrer et al., Blood, 2008). The cytotoxic effect of erlotinib on leukemic myeloblasts ex vivo occurs at concentrations below the plasma levels achievable with regular dosing of this drug. Two case reports, in literature, of patients with concomitant AML and lung cancer, treated with erlotinib and demonstrated AML response, corroborate these findings. The exact targets of erlotinib in MDS/AML are unknown but inhibition of SRC family kinases, Syk, mTOR, and drug efflux system has been suggested. Since normal CD34+ bone marrow cells are not affected by this agent, and no myelosuppression is seen in patients taking erlotinib, clinical application of this drug for the treatment of MDS/AML seems attractive. Clinical studies testing efficacy of erlotinib in MDS are undergoing. We report a pilot study of administration of erlotinib in AML. Methods This was a single-institution pilot study. The study was approved by scientific review committee and IRB. All patients signed informed consent. Eligibility included a confirmed diagnosis of AML per WHO classification (>20% blasts), no pre-existing history of MDS, newly diagnosed disease in patients older than 70 or relapsed disease in younger patients unfit for chemotherapy or refractory disease at any age. All cytogenetics and molecular profile categories were eligible. An ECOG performance status of 0-3 was acceptable. WBC count had to be less than 20,000/cumm, and hydroxyurea or other therapies to be discontinued at least 14 days prior to treatment; therefore, patients with high proliferative rate disease were excluded. Smokers were excluded due to potential diminished erlotinib plasma concentration with cigarette smoking. Drugs with potential interaction with erlotinib metabolism were not allowed. Erlotinib was given orally at 150 mg per day continuously in 28-day cycles. Bone marrow at baseline was evaluated for morphology, cytogenetics, flow-cytometry, and molecular profile (FLT3, NPM1, CEBPA mutations). CD34+ cells were separated from bone marrow aspirates taken at baseline, day 3-4, and day 8+1 of treatment, and frozen/banked for future correlative studies. Response was evaluated by bone marrow examination after each cycle. A flow-cytometry was performed on the bone marrow aspirate after the first cycle to be compared with baseline, specifically for evidence of differentiation. Given possibility of “slow/delayed response” with erlotinib, patients were planned to continue at least 3 cycles of treatment in the absence of disease progression or toxicity. Assessment of overall response was the primary endpoint of the study. Results Between August 2010 and August 2012, a total of 11 patients were treated on this study at Indiana University Simon Cancer Center. One patient had relapsed AML, and 1 had relapsed/refractory disease; 9 patients were older than 70 with newly diagnosed AML. Two patients with newly diagnosed AML demonstrated modest response with the first cycle of treatment, with reduction of bone marrow blasts from 89% and 88% to 71% and 73%, respectively, but both experienced disease progression subsequently. Nine other patients had progression of the disease without any response. Of these, 6 completed at least one cycle of treatment, and 3 were taken off study earlier due to disease progression (per peripheral blood blast count) and clinical deterioration. None of the patients with circulating blasts showed meaningful response by total WBC or absolute peripheral blood blast count. Flow-cytometry of bone marrow aspirate following the first cycle compared to pre-treatment sample did not show evidence of differentiation in any of the 8 patients who completed at least one cycle of therapy. None of the patients experienced drug-related toxicity. Conclusion This pilot study, which enrolled mostly older AML patients with newly diagnosed disease, did not demonstrate clinical response to erlotinib monotherapy when administered continuously at 150 mg per day. No evidence of differentiation was observed in AML blasts after 4 weeks of therapy. The treatment was well tolerated without drug-related adverse events. Disclosures: No relevant conflicts of interest to declare.

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In Human Visualization of Ibrutinib-Induced CLL Compartment Shift

Blood ◽

10.1182/blood-2019-131469 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1750-1750

Author(s):

Marius E Mayerhoefer ◽

Alexander Haug ◽

Ulrich Jaeger ◽

Lukas Kazianka ◽

Verena Pichler ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Lymph Nodes ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Relative Increase ◽

Whole Body ◽

Advisory Committees ◽

Treatment Naïve

BACKGROUND. B-cell receptor (BCR) signaling is a central driver for chronic lymphocytic leukemia (CLL), and its targeting through irreversible inhibition of Bruton's tyrosine kinase (BTK) by ibrutinib has significantly improved the prognosis of CLL patients. Ibrutinib treatment has become standard of care, and, recently, has advanced to the first-line setting. A treatment-induced, sometimes dramatic increase of peripheral lymphocytosis, has emerged as a class effect of BTK inhibitors in CLL. Mechanistically, BTK blocking by ibrutinib might affect adhesion molecules and chemokine receptors, such as CXCR4 and CXCR5, thus interfering with the protective tissue microenvironment and mobilizing tissue-resident CLL cells from the lymph nodes and bone marrow into the peripheral blood. This hypostasized mechanism of a "compartment shift," however, has not yet been demonstrated experimentally or visually. Positron emission tomography (PET) with [68Ga]Pentixafor, a radiotracer that specifically targets the CXCR4 receptor, was recently established as a sensitive approach with which to detect CLL in vivo. As a proof-of-concept, we here present three CLL patients to demonstrate the potential of [68Ga]Pentixafor-PET/MR imaging to functionally track CLL cells along the redistribution induced by ibrutinib. METHODS. Three CLL patients were included: patient 1 was a 74-year-old treatment-naïve male, with unmutated IGHV status and high-risk cytogenetics, including del17p13 and TP-53 mutation; patient 2 was a 53-year-old female with late relapse (12 years after allogeneic stem cell transplantation), IGHV was unmutated, and cytogenetic abnormalities included del11q22 and del13q14; and patient 3 was a 59-year-old treatment-naïve male with unmutated IGHV and del11q22 and del13q14. Whole-body PET/MRI with injection of 150 MBq of [68Ga]Pentixafor was performed pre and on ibrutinib treatment (patient 1: one week; patient 2: two weeks; and patient 3: three weeks after start of the first therapy cycle, respectively). Treatment consisted of continuous oral administration of 420 mg of ibrutinib. Mean standardized [68Ga]Pentixafor uptake values (SUVmean) of involved lymph nodes, the bone marrow, and the spleen were measured. Isolated peripheral blood mononuclear cells (PBMCs) were stained with fluorescence-labeled antibodies, and measurements were performed on flow cytometer. RESULTS. In all three cases - at one week, two weeks, and three weeks on ibrutinib treatment - CXCR4 density, as measured on [68Ga]Pentixafor-PET, shifted from the bone marrow and lymph nodes toward the spleen (Figs. 1-3). In patient 1, the SUVmean decreased in the bone marrow (-9.8%) and lymph nodes (-12.0%), whereas it increased markedly in the spleen (+29.8%). At flow cytometry, this patient exhibited an increase of CXCR4-high (tissue-resident) CLL cells upon ibrutinib treatment (+56.7% relative to baseline). In patient 2, the SUVmean decreased in the bone marrow (-14.7%) and lymph nodes (-27.3%), whereas it more than doubled in the spleen (+133.0%). At flow cytometry, individual CLL cells demonstrated a relative increase (+39.1%) of CXCR4 positivity. In patients 3, the SUVmean decreased in the bone marrow (-27.6% %) and lymph nodes (-41.9%), whereas it increased markedly in the spleen (+26.1%). At flow cytometry, the relative increase of CXCR4-high CLL cells was +58.3%. CONCLUSIONS. We here provide the first pictures of the early functional treatment effects of ibrutinib. While our analyses confirmed a shift of CXCR4 positive CLL cells from lymph nodes to peripheral blood, they also revealed that ibrutinib rapidly released CLL cells from the bone marrow. Also, unexpectedly, CLL cells redistributed to the orthotopic splenic cavernous system. Visualization of CLL on ibrutinib supports the pre-existing clinical hypothesis of a "compartment shift", however it also modified and refined the mechanistic model by describing early clearing of the bone marrow and re-distribution to the peripheral blood and the spleen. Disclosures Jaeger: Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria. Wester:Scintomics: Other: Spouse CEO of Company; CXCR4-targeted radiopharmaceuticals: Other: Inventor; Scintomics GmbH, Germany: Other: Shareholder. Staber:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria, Speakers Bureau; Takeda-Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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Impact of FLT3 Mutation Clearance after Front-Line Treatment with Gilteritinib Plus Azacitidine, or Gilteritinib or Azacitidine Alone in Patients with Newly Diagnosed AML: Results from the Phase 2/3 Lacewing Trial

Blood ◽

10.1182/blood-2021-145362 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3445-3445

Author(s):

Eunice S. Wang ◽

Jessica K. Altman ◽

Mark D. Minden ◽

Ruishan Wu ◽

Elizabeth Shima Rich ◽

...

Keyword(s):

Bone Marrow ◽

Complete Remission ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Phase 2 ◽

Newly Diagnosed ◽

Front Line ◽

Advisory Committees ◽

Flt3 Mutation

Abstract Background: The presence of measurable residual disease (MRD) after achievement of remission with induction therapy is a prognostic marker of relapse risk in patients with acute myeloid leukemia (AML). Gilteritinib is an oral FLT3 inhibitor approved as a single agent for the treatment of patients with FLT3-mutated (FLT3mut+) relapsed or refractory AML. Evaluation of gilteritinib in the front-line setting is under way. We evaluated FLT3 internal tandem duplication (FLT3-ITD) mutation clearance using two different thresholds and correlated mutation clearance with survival outcomes in patients with newly diagnosed AML ineligible for intensive chemotherapy who were treated with front-line gilteritinib plus azacitidine (AZA) or either agent alone in the phase 2/3 LACEWING trial. Methods: Adult patients with newly diagnosed FLT3mut+ AML ineligible for intensive induction chemotherapy received 28-day cycles of once-daily gilteritinib plus AZA in the Safety Cohort (80 or 120 mg/day gilteritinib plus 75 mg/m 2 AZA, Days 1-7) and in Arm AC (120 mg/day gilteritinib plus 75 mg/m 2 AZA, Days 1-7), gilteritinib (120 mg/day) alone in Arm A, or AZA (75 mg/m 2, Days 1-7) alone in Arm C. A subset of patients who had a best overall response of composite complete remission (CRc; defined as the sum of patients who achieved complete remission with or without complete hematologic or platelet recovery) and who had bone marrow-derived DNA samples available at baseline and at least one additional post-baseline timepoint were assessed for FLT3-ITD mutation clearance using next-generation sequencing. An Illumina ® sequencing platform was used to quantify FLT3-ITD and total FLT3 alleles. The FLT3-ITD variant allelic frequency (VAF) was defined as the ratio of FLT3-ITD to total FLT3 frequency. Data were analyzed using two different mutation clearance thresholds, FLT3-ITD VAF <10 −4 or <10 −3, where 10 −4 was based on previously published findings in patients with relapsed or refractory FLT3mut+ AML who were treated with gilteritinib (Altman JK et al., Cancer Med. 2021;10[3]:797-805) and 10 -3 was an additional exploratory threshold used because it provided a more balanced distribution of patients, given the small number of patients achieving mutation clearance at the 10 -4 threshold. Results: The median age of patients enrolled in LACEWING was 77 years (range, 59-90), with 73% of patients aged >75 years. Although baseline characteristics of the overall LACEWING population were generally well balanced across treatment arms, higher proportions of patients treated with gilteritinib plus AZA (47%) or gilteritinib alone (59%) had an Eastern Cooperative Oncology Group (ECOG) performance status of ≥2 compared with patients treated with AZA alone (33%). Overall, 40 patients who achieved CRc and had sufficient DNA samples from bone marrow aspirates obtained at baseline and at least one additional post-baseline timepoint were included in the analysis (Safety Cohort, n=8; Arm A, n=7; Arm AC, n=17; and Arm C, n=8). Across both thresholds, the proportions of patients with FLT3 mutation clearance did not markedly differ between patients treated with gilteritinib or AZA (Table). In patients who received gilteritinib, FLT3-ITD mutation clearance using either threshold was associated with a similar increase in median overall survival (OS) compared to patients who did not achieve mutation clearance (Figure). Conclusions: Regardless of MRD threshold, rates of MRD negativity were not substantially different between newly diagnosed FLT3mut+ AML patients ineligible for intensive induction chemotherapy who received gilteritinib alone, gilteritinib plus AZA, or AZA alone. Advanced age coupled with a worse baseline ECOG performance score at baseline may have compromised treatment response and achievement of FLT3 mutation clearance in patients treated with gilteritinib. The mutation clearance thresholds used in this analysis showed similar median OS in patients who received gilteritinib. Figure 1 Figure 1. Disclosures Wang: Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; Genentech: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; Novartis: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Takeda: Consultancy, Honoraria, Other: Advisory board; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; Mana Therapeutics: Consultancy, Honoraria; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; DAVA Oncology: Consultancy, Speakers Bureau; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy. Altman: Kartos: Research Funding; Theradex: Consultancy, Other: Advisory boards; Biosight: Consultancy, Other: Travel fees, Research Funding; Daiichi Sankyo: Consultancy; AbbVie: Consultancy, Other: Advisory Board, Research Funding; BMS: Research Funding; Amgen: Research Funding; Astellas: Consultancy, Other: Advisory Board, Research Funding; Fujifilm: Research Funding; ALZ Oncology: Research Funding; Immunogen: Research Funding; GlycoMimetics: Other: Participation on an advisory board; Syros: Consultancy; Kura Oncology: Consultancy; Boehringer Ingelheim: Research Funding; Aprea: Research Funding; Kura: Research Funding. Minden: Astellas: Consultancy. Wu: Astellas: Current Employment. Rich: Astellas Pharma Global Development, Inc.: Current Employment. Hill: Ligacept, LLC: Current holder of individual stocks in a privately-held company, Other: Stockholder; Astellas Pharma Global Development: Current Employment.

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Bone Marrow-Based and Longitudinal Blood-Based MRD Tracking in Newly Diagnosed Multiple Myeloma Patients Treated with Daratumumab, Carfilzomib, Lenalidomide and Dexamethasone (DKRd): A Correlative and Clinical Phase II Study

Blood ◽

10.1182/blood-2018-99-114311 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3281-3281 ◽

Cited By ~ 3

Author(s):

Ola Landgren ◽

Katie Thoren ◽

Malin Hultcrantz ◽

Alexander M. Lesokhin ◽

Nikoletta Lendvai ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Newly Diagnosed ◽

Advisory Committees ◽

Single Tube ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Abstract INTRODUCTION Using Carfilzomib, Lenalidomide and Dexamethasone (KRd) combination therapy in newly diagnosed multiple myeloma patients lead to ~40% minimal residual disease (MRD) negativity rate. Here, we use KRd in combination with daratumumab (DKRd); and treatment response is assessed with extensive correlative science including parallel bone-marrow-based and blood-based MRD tracking, together with targeted DNA sequencing of baseline bone marrow samples. Primary end-point is to rule out 60% and to target up to 80% MRD negativity rate. METHODS This is a single-arm, Phase II clinical trial based on Simon's optimal two-stage design. The first cohort (twice-a-week carfilzomib) (N=41) has the following treatment schedule: 8 cycles of treatment; 28-day cycles with carfilzomib 20/36 mg/m2 days 1, 2, 8, 9, 15 and 16; lenalidomide 25 mg days 1-21; dexamethasone 40 mg weekly cycles 1-4, 20 mg after cycle 4; and daratumumab 16 mg/kg days 1, 8, 15, and 22 cycles 1-2, days 1 and 15 cycles 3-6, and day 1 cycles 7-8. The second cohort (once-a-week carfilzomib) (N=41): 8 cycles of treatment; 28-day cycles with carfilzomib 20/56 mg/m2 days 1, 8, and 15; lenalidomide, dexamethasone, and daratumumab are given at the same doses/schedules as the first cohort. For fit patients, stemcell collection is recommended after 4 to 6 cycles of therapy; DKRd therapy is resumed after collection to a total of 8 cycles DKRd. Treatment response is being assessed with parallel bone-marrow-based (10-color single tube flowcytometry, invivoscribe V(D)J sequencing) as well as blood-based (MALDI-TOF and QTOF-mass spectrometry [MS]) for MRD tracking. Baseline bone marrow samples are evaluated with targeted DNA sequencing for FISH-Seq and somatic mutational characteristics (myTYPE). Here, we present the first stage (N=28) of the first cohort (twice-a-week carfilzomib). We are waiting for the results to mature before the second stage (N=13) of the first cohort can open. The second cohort (once-a-week carfilzomib) is opening for enrollment in August 2018 (N=41). RESULTS The first stage of the first cohort is fully enrolled; 28 patients meeting eligibility criteria were enrolled onto study (14 males, 14 females) between October 2017 and July 2018. Baseline characteristics include; median age 60 years (range 32-80 years); 12(43%) patients had high-risk FISH/SNP signature defined as one or more of the following: 1q+, t(4,14), t(14,16), t(14,20), and 17p-. At the submission of this abstract, 20 patients have completed one or more cycles DKRd; among these, 3 patients have completed all 8 cycles. The median number of cycles delivered is currently 4 (range 1-8). Full assessments with MRD assays have been completed in 3 patients: -Pt #1 obtained complete response (CR) after 3 cycles, and workup after the last cycle of therapy showed MRD-negativity (by 10-color single tube flowcytometry and V(D)J sequencing) in the bone marrow; and peripheral blood (serum) was negative by MALDI-TOF MS after completion of cycle 2. -Pt#2 obtained CR after 4 cycles, however, workup after cycle 5 showed MRD-positivity (by 10-color single tube flowcytometry and V(D)J sequencing) in the bone marrow; and peripheral blood (serum) was positive by MALDI-TOF MS throughout the end of the last cycle. -Pt#3 obtained CR after 4 cycles and after 6 cycles both 10-color single tube flowcytometry and V(D)J sequencing showed MRD-negativity in the bone marrow. However, MALDI-TOF MS detected small abnormal serum proteins in peripheral blood and remained positive throughout the end of cycle 8. Overall, the DKRd therapy is well tolerated and it has similar toxicity profile as KRd. Grade >3 adverse events were hypotension, musculoskeletal deformity, back pain, dyspnea, lung-infection, and febrile neutropenia. So far, 5 patients underwent dose reductions of lenalidomide. CONCLUSIONS In this pre-planned interim analysis of our phase II study, we show that DKRd is a highly effective and well tolerated combination therapy for newly diagnosed multiple myeloma patients. Based on small numbers of patients who have completed the planned DKRd cycles and been evaluated by bone marrow-based MRD and peripheral-blood based assays, we show that highly sensitive protein assays may allow longitudinal MRD tracking in peripheral-blood. At the meeting, we will present updated results using longitudinal testing with MALDI TOF-MS and QTOF-MS on the entire cohort. Disclosures Landgren: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Pfizer: Consultancy; Karyopharm: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees. Lesokhin:Takeda: Consultancy, Honoraria; Janssen: Research Funding; Squibb: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; Serametrix, inc.: Patents & Royalties: Royalties. Mailankody:Juno: Research Funding; Physician Education Resource: Honoraria; Takeda: Research Funding; Janssen: Research Funding. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Hassoun:Oncopeptides AB: Research Funding. Shah:Amgen: Research Funding; Janssen: Research Funding. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Ho:Invivoscribe, Inc.: Honoraria. Korde:Amgen: Research Funding.

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Increased Circulating Plasma Cells On Multiparametric Flow Cytometry As An Independent Prognostic Biomarker In Newly Diagnosed Multiple Myeloma: Implications For Redefining High-Risk Myeloma

Blood ◽

10.1182/blood.v122.21.1842.1842 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1842-1842

Author(s):

Wilson I Gonsalves ◽

Vinay Gupta ◽

S. Vincent Rajkumar ◽

William G Morice ◽

Michael M Timm ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Overall Survival ◽

High Risk ◽

Peripheral Blood ◽

Research Funding ◽

Light Chains ◽

Newly Diagnosed ◽

Risk Status ◽

Multiparametric Flow Cytometry

Abstract Background Prior studies have shown that presence of increased circulating plasma cell (PCs) identified using a slide-based immunofluorescence method is an adverse prognostic marker for overall survival in multiple myeloma (MM), and increases the risk of progression in patients with MGUS and smoldering MM. However, utility in clinical settings has been limited by the cumbersome nature of the test and lack of widespread availability. We studied the prognostic value of circulating PCs using sensitive multiparametric flow cytometry that enable us to quantitatively assess circulating PCs in patients with MM. Methods We evaluated all newly diagnosed MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 who had their peripheral blood samples evaluated by flow cytometry prior to therapy. Patients with plasma cell leukemia were excluded. 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. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal PCs based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal PCs 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 assessed using the log rank test. Results There were 158 consecutive patients with newly diagnosed MM who had their peripheral blood evaluated via flow cytometry as part of their routine clinical evaluation. The median age of this group was 66 years (39-95) and 59% were male. At the time of this analysis, 25 patients had died and the 2-year OS rate for the cohort was 83%. The 2-year OS for the 89 (55%) patients with any circulating PCs was 76% compared with 91% for those with none (P=0.02). The median number of circulating clonal PCs was 33 (range, 0-46,413)/ 150,000 gated events. Using a ROC analysis the best cutoff predicting 1 and 2-year mortality were 435 and 376 events, respectively. Based on this, we defined >=400 events as a cutoff for defining patients with high-risk disease. The median time-to-next-treatment (TTNT) for patients with circulating clonal PCs >=400 (n=37, 23%) was 14 months compared with 26 months for those with <400 events (n=121, 77%) (p<0.001; Fig 1a). The median OS for those with >=400 clonal PC events was 32 months compared with not reached for those with <400 events (p<0.001; Fig 1b). Patients with >= 400 circulating PCs had higher ISS stage, creatinine, LDH, PC labeling index and bone marrow PC% compared with the rest. In a univariate analysis examining the presence of circulating PC >=400, LDH, labeling index, marrow PC% and FISH risk status, only circulating PCs were prognostic for overall survival (P = 0.0011). Among the 89 patients with any circulating PCs, 26 (30%) had CD45+ clonal PCs. The TTNT in patients with a clonal CD45+ population was inferior to those with no CD45+ clonal PCs in circulation (median 11 vs. 19 months, P = 0.034) as was the OS (P = 0.039). Conclusion Quantitative estimation of circulating clonal PCs in patients with newly diagnosed MM is a powerful predictor of early relapse from therapy and mortality. A cutoff of >=400 clonal events/150,000 gated mononuclear events predicts for a median TTNT of 14 months and OS of 32 months. This parameter is more powerful than the current high-risk parameters such as FISH risk status as well as traditional high-risk markers such as ISS stage and LDH levels, and is able to identify a group of patients with particularly poor outcome. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

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