Circulating Multiple Myeloma Cells Research Articles

Needle in a Haystack: A Pilot Study Combining Single-Cell Multiomics with Clinical NGS-MRD Sequencing to Search for Circulating Clonotypic Dedifferentiated Myeloma Cells

Multiple myeloma (MM) is a heterogenous disease in which clonal evolution or preexisting reservoirs of phenotypically divergent cells may be harbingers of therapeutic resistance. Prior research has suggested the presence of myeloma cells with immunophenotypic and expression profiles dedifferentiated from that of terminally differentiated plasma cells that may be found in circulation, marrow, or potentially other lymphoid compartments; however, this has not been conclusively demonstrated in patients. The identification of such clonotypic myeloma cells could lead to a better understanding of myeloma biology and resistance, and inform therapies targeted at these populations, and single-cell technologies provide the granularity to characterize them. Next-generation sequencing (NGS)-based measurable residual disease (MRD) clinical-grade assays (eg. clonoSEQ®, Adaptive Biosciences) identify individuals’ unique heavy and light chain sequences. Theoretically this sequencing data could be overlaid with research-grade single-cell multiomics to identify clusters of clonotypic cells with variable differentiation. In this pilot study using the Enhanced Single Cell Analysis with Protein Expression (ESCAPE) platform (Singleron Biotechnologies) we aimed to identify peripherally circulating clonotypic MM cells by their NGS-MRD sequence and simultaneously determine whether their multiomic profile reflected possible dedifferentiated clusters found in paired marrow. Using scRNA-Seq (10x Genomics) data previously generated at our site from bone marrow mononuclear cells we identified MM cell clusters enriched for differentially expressed genes known to be expressed in B-cells and plasmablasts. These genes included CD19, PAX5, BCL6, EZH2, STMN1, HMGB2, MKI67 and CD27. We next sought to assay the peripheral blood mononuclear cells (PBMC) of patients (n=2) with an overrepresentation of cells in these clusters. To evaluate both the transcriptomic and immunophenotypic profiles of the PBMCs at single-cell resolution we utilized Singleron's Immune Profiling panel on the ESCAPE platform. Using Singleron's MapSuite™ analysis pipeline, we annotated cell-type classifications, and highlighted cells within the mature B-cell/plasma cell differentiation spectrum (Figure A). Within this subset, we attempted to identify gene expression reflective of the previously identified "dedifferentiated” marrow clusters with specific genes of interest, but found that there was not significant overlap. Heatmaps comparing the 5 peripheral subclusters with marrow plasma cells demonstrate some concordance with the marrow subclusters, with examination of these overlaps ongoing, but these were not the "dedifferentiated” marrow clusters. The included patients also had clonoSEQ® identification sequences of heavy chain and/or light chain available. By cross-referencing the NGS-MRD sequence data (clonoSEQ®, Adaptive Biosciences), we were able to use ESCAPE-Seq to identify definitive clonotypic circulating MM cells within the PBMC samples at single cell granularity. Among PBMCs from sample 2 we were able to identify circulating clonotypic myeloma cells with 100% match to the IgH and IgK MRD sequences, which also represented the dominant V(D)J clonotype in peripheral circulating. Concomitant single-cell surface protein analysis of this clonotypic cell population demonstrated significant expression of CD38, without significant differential expression of CD138 nor surface markers of B-cells (Figure B). While these data are preliminary and studies are ongoing, we successfully leveraged clinical-level MRD sequencing data to inform single-cell multiomics, a potentially powerful translational research pathway for any hematologic malignancy in which NGS-MRD is clinically available. We definitively identified a small subset of circulating MM cells and used the ESCAPE platform to efficiently generate a multiomics profile. This profile suggested elevated CD38 expression without elevated CD138 expression, which previously has been associated with dedifferentiated plasma cells and plasmablasts, although further analyses are ongoing. In future studies, we will enrich for B-lymphoid spectrum cells prior to single-cell analytics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Free from Maintenance Drug Therapy in Multiple Myeloma (The FREEDMM Trial): A Pilot Study of Minimal Residual Disease (MRD)-Driven Discontinuation of Maintenance

Background Induction therapy followed by autologous stem cell transplant (ASCT) and maintenance is the current standard of care for the treatment of newly diagnosed multiple myeloma (NDMM). However, indefinite maintenance therapy after ASCT carries both significant clinical and financial toxicities. While minimal residual disease (MRD) testing has become part of routine standard of care, its use in guiding treatment decisions, in particular cessation of therapy, has not been established in the maintenance setting. We hypothesize that MRD monitoring could be used to guide clinical decision making, justifying cessation of maintenance therapy in a sub-group of patients with sustained MRD-negative remission. Study Design and Methods This is a phase 2 single-arm adaptive design study (NCT 05192122) for decision-making regarding maintenance therapy (Figure 1). Patients are eligible if they have a diagnosis of active MM, have completed at least 2 years of maintenance therapy post-ASCT, and meet International Myeloma Working Group (IMWG) criteria for very good partial response (VGPR) or complete response (CR). MRD testing is performed twice, at least one year apart, on bone marrow aspirate using next-generation sequencing (NGS) and MRD-negative status is defined at a threshold of 10-6. Patients with negative PET scan in VGPR or CR and sustained (i.e., twice, 1 year apart) MRD negative status discontinue maintenance therapy. MRD-positive patients continue continue maintenance and are taken off study. In patients who are MRD-negative and discontinue maintenance therapy, MRD testing will be re-assessed yearly for 3 years. At each time of MRD testing, MALDI-TOF spectrometry and measurement of circulating multiple myeloma cells (CMMCs) will be done in parallel to compare against MRD results. Health-related quality of life (HRQoL) will be assessed throughout and results on maintenance compared with those after discontinuation using the EORTC QLQ-MY20 questionnaire. This study will enroll 50 patients to address the primary objective of assessment for sustained MRD-negative status at 12 months after stopping maintenance therapy. For all patients, a 5-mL peripheral blood and bone marrow sample is collected and stored at the time of each standard of care bone marrow biopsy and will be used to characterize the immune microenvironment by Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) of paired peripheral blood and bone marrow in MRD-negative and MRD-positive patients as well as of patients progressing after stopping therapy. Preliminary Results Enrollment into FREEDMM trial began in December 2021, and to date 20 patients in CR have undergone a first bone marrow for MRD assessment, with results pending in 9 patients. The median age is 66.5 (range: 39-76) years, and the majority of patients are either Black (n=7; 35%) or Hispanic (n=7; 35%). One patient has high risk disease (17p deletion). The median number of years of maintenance therapy completed is 4.2 (range: 2.14-7.9) years. 17 (85%) patients had a negative MALDI-TOF test at the time of the first marrow. Seven (63.6%) of 11 patients were found to be MRD-negative. MALDI-TOF failed to accurately correlate with MRD results in 2 patients. 40 paired peripheral blood (n=20) and bone marrow aspirate (n=20) samples have been collected for CITE-seq analysis. Pilot CITE-seq experiments captured 6,700-11,000 cells with between 38,000-68,000 reads per cell on one MRD-negative and one MRD-positive bone marrow aspirate and paired peripheral blood sample. CITE-seq results for these 20 patients will be presented at the conference. Conclusion: This phase 2 trial applies a novel clinical trial design with an adaptive strategy to explore use of MRD testing as a guide to discontinuing maintenance therapy in myeloma and represents one of the first studies to explore in parallel various measures of disease monitoring (i.e., MRD, MALDI-TOF, CMMCs, CITE-seq). Ongoing results will be reported at the conference. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 640: Genome sequencing of circulating multiple myeloma cells for minimally invasive molecular characterization of precursor disease pathology

Abstract Introduction: Multiple myeloma (MM) develops from indolent stages monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Precursor conditions are incidentally diagnosed and require invasive bone marrow (BM) biopsies for complete characterization, highlighting the urgent need for improved early detection methods. Minimally invasive blood biopsies to measure circulating multiple myeloma cells (CMMCs) as markers of MM disease development are a promising solution to this unmet need. Here, we present our novel method CatchTheFISH, for whole genome sequencing (WGS) of CMMCs that enables genomic profiling and WGS based cytogenetic analyses from enriched liquid biopsy samples. Application of WGS in a cohort of 20 patients, revealed CMMCs were of tumor origin and able to faithfully match BM sequencing results and detect 100% of clinically reported events. Methods: Peripheral blood from 110 SMM patients from the PCROWD observational study (Dana-Farber Cancer Institute IRB #14-174) was collected and processed on CellSearch system (Menarini Silicon Biosystems), with enrichment and enumeration of CMMCs based on CD138+38+CD45-19- immunophenotype. In 20 patients, CMMCs and white blood cells were sorted for library construction, quantification and WGS on Illumina NovaSeq6000. Mutation analyses were performed with the cancer genome analysis pipelines of the Broad Institute. Results: CMMCs were detected in 84% of SMM patients enrolled in the study, with a median count of 13 CMMCs (range 0 to 43836). We first demonstrated the concordance of WGS results obtained from CMMCs with BM. In 100% of patients tested with paired BM and CMMCs (n = 8), we observed full agreement in structural event calling between our samples and clinical reports, including translocations and CNAs (trisomies, tetrasomy, monosomy 13 and 1q gain/amplification). Next, we showed WGS of CMMCs provided increased diagnostic yield compared to BM biopsy for the detection of structural events and MM-associated driver mutations. In 7 patients (88%) we detected additional aberrations not found by FISH. Unknown translocation events of IGH-MYC and t(14;20) were distinguished in two patients. Additionally, our method enabled detection of MM driver mutations. Three patients (38%) were found to harbor RAS mutations (KRAS and NRAS) in BM samples, which were also validated in matched CMMCs. Finally, we assessed a validation cohort of 12 SMM patients with CMMCs only. WGS detected comprehensive mutation data across all scales including clinically relevant translocations, trisomies, CNAs and mutations. Conclusion: Our findings provide proof of principle that capture and genomic profiling of CMMCs could be a robust surrogate to BM biopsy, allowing minimally invasive detection and monitoring of disease, unlocking the clinical potential of liquid biopsies for MM diagnostics. Citation Format: Ankit K. Dutta, Jean-Baptiste Alberge, Elizabeth D. Lightbody, Romanos Sklavenitis-Pistofidis, Cody J. Boehner, Tarek H. Mouhieddine, Anna Cowan, Nang Kham Su, Erica M. Horowitz, Andrew Dunford, Chip Stewart, Ziao Lin, Laura Hevenor, Hadley Barr, Amanda Cao, Ornkleaw Zepp, Thai Bui, Steve Gross, Daniel Auclair, Gad Getz, Irene M. Ghobrial. Genome sequencing of circulating multiple myeloma cells for minimally invasive molecular characterization of precursor disease pathology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 640.

Abstract 6087: Evaluation of intra-tumor heterogeneity by single-cell copy number profiling of circulating multiple myeloma cells using Shannon index

Abstract Tumors develop through an evolutionary process in which genomic lesions accumulate in different coexisting subclones leading to intra-tumor heterogeneity (ITH). ITH allows the tumor to adapt to microenvironments and to resist therapies and has been shown to be a prognostic marker. In this work we show the characterization of ITH in Multiple Myeloma (MM) at single cell resolution from a pool of Circulating Multiple Myeloma Cells (CMMCs) and we study the relationship between number of sampled cells and diversity detected. Peripheral blood was collected from 3 patients affected by MM. Using CellSearch® system, blood was enriched for target CMMCs, with immuno-magnetic selection of CD138+, and immunofluorescently stained cells, positive for CD38-PE, DAPI and negative for CD45/CD19-APC, were enumerated. The samples were then analyzed and CMMCs were isolated with DEPArray platform. The DNA of each single cell (n=261) was amplified with Ampli1™ WGA kit and the WGA output was used for genome-wide single cell copy number alteration analysis with Ampli1 LowPass kit. Copy number profiling revealed from 2 to 3 coexisting subclones per patient, with minor clones size going from 6.5% to 33%. ITH was assessed by determining the Shannon index, which showed a range of values from 0.44 (low diversity) to 1.01 (high diversity) depending on source sample. We evaluated the effect of number of cells/profiles on Shannon index by bootstrapping (200 times) a number of copy number profiles ranging from 2 to the total number of profiles available per patient. Results showed that the mean Shannon index almost reaches a plateau at 15-20 cells/profiles in the case of lower diversity samples and at about 30 cells/profiles for the highest diversity sample. Moreover, as few as 10 cells allowed to differentiate low vs. high diversity samples. Cell enrichment and sorting by CellSearch and DEPArray platforms allow to quantify ITH at diversity level with high precision even from a limited number of initial single-cells and provide a promising method to objectively measure heterogeneity, as required in order to study its clinical implications. Citation Format: Alberto Ferrarini, Claudio Forcato, Valentina del Monaco, Mario Terracciano, Marianna Garonzi, Massimiliano Pellicano, Genny Buson, Francesca Fontana, Paola Tononi, Nicolo Manaresi. Evaluation of intra-tumor heterogeneity by single-cell copy number profiling of circulating multiple myeloma cells using Shannon index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6087.

Non-Invasive Liquid Biopsy to Quantify and Molecularly Characterize Circulating Multiple Myeloma Cells in the Assessment of Precursor Disease Pathology

▪Introduction: Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by the abnormal growth of clonal plasma cells in the bone marrow (BM). In most cases MM develops from early, asymptomatic disease stages known as Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Despite effective new therapies, most MM patients inevitably relapse and require further treatment, highlighting the need for better early detection methods for precursor patients and targeted interventions to prevent early disease from progressing.The initial diagnosis of MGUS/SMM remains an incidental process following the identification of increased clonal immunoglobulin in the blood. BM biopsy is the gold standard for diagnosis and monitoring of MM progression, but is intrusive, painful, and comes with possible secondary complications for patients. Consequently, repeated assessment is not a feasible option for MGUS and SMM patients who are asymptomatic. Here we tested the utility of circulating multiple myeloma cells (CMMCs) from non-invasive blood biopsy to accompany BM as a method to monitor disease development, by enumerating CMMCs from MGUS/SMM patients.Methods: Peripheral blood from 185 precursor patients (75 MGUS and 110 SMM) from the Dana-Farber Cancer Institute observational PCROWD study (IRB #14-174) was collected in CellRescue TM Preservative Tubes and processed on the CellSearch CellTracks Autoprep system using the CMMC assay kit using 4mL of blood. This assay employs the enrichment of CMMCs through the immunophenotype of CD138 +CD45 -19 -, and leukocyte exclusion based on CD45 +CD19 +. Nucleated cells were identified using DAPI staining. The CellTracks Analyzer II fluorescence microscope system was subsequently used to scan captured CMMC cartridges, with software allowing the automated scoring and enumeration of CMMCs. Additional molecular analyses were carried out on SMM patients. Briefly, minipools of CMMCs were sorted by DEPArray and underwent whole genome amplification using Ampli1 kit, PCR-free library construction, quantification and low pass whole genome sequencing (~0.5x) on the Illumina HiSeq2500. To assess whether molecular analyses can be performed to detect hyperdiploidy as a genomic biomarker of MM disease, ichorCNA analyses was performed to determine copy number variant (CNV) events and infer tumour fraction.Results: CMMCs were detected in 27% of MGUS patients collected, with a median count of 2 CMMCs (range 0 to 1328). Comparably, CMMCs were detected in 57% of SMM patients, with a median enumeration of 13 CMMCs (range 0 to 43836). Enumeration of CMMCs illustrated a correlation with clinical measure of disease including the International Myeloma Working Group 2/20/20 risk stratification model. A higher CMMC count was associated with increasing risk group based on the 3-risk factor model, with a median of 5, 29 and 59 CMMCs detected at low, intermediate, and high-risk SMM groups, respectively. CMMC counts were significantly increased at intermediate (P = 5.0 x 10 -4) and high-risk stages (P = 3.7 x 10 -3) compared to low-risk.While enumeration provides a correlative measure of CMMCs that may be of tumor origin, downstream molecular characterization can confirm MM-associated genetic alterations. At the precursor stages, a low tumour burden is evident clinically, thus both normal and malignant plasma cells are present. Therefore, to determine the concordance between bone marrow and peripheral blood CMMCs, we performed genomic analyses to identify arm level gain or loss events. Molecular analyses of CMMCs was carried out in patients who had matched BM and clinical fluorescent in situ hybridization (FISH) results. We showed that CMMCs can capture 100% of clinically annotated BM FISH CNV events. Furthermore, CMMC samples identified additional yield, with further CNVs identified that were not observed by FISH. In cases that did not have BM biopsy results, sequencing of CMMCs revealed the existence of genetic aberrations.Conclusion:Our results demonstrate clinical correlation and molecular characterization of CMMCs from MGUS/SMM patients. This study provides a foundation for non-invasive detection, enumeration and genomic interrogation of rare CMMCs from the peripheral blood of MGUS/SMM, illustrating the clinical potential of using liquid biopsies for monitoring and managing disease in the precursor setting of MM. DisclosuresGetz: IBM, Pharmacyclics: Research Funding; Scorpion Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

A Next Generation Liquid Biopsy Approach for Multiple Myeloma

Direct-to-Patient (DTP) Multiple Myeloma (MM) research studies have been launched recently, including PCROWD (NCT02269592), PROMISE (NCT03689595) and the MMRF CureCloud Research Initiative (NCT03657251), aimed at enrolling thousands of individuals from whom comprehensive molecular and immune analyses will be generated from blood specimens and the resulting data aggregated with the correlating clinical information. To support the molecular characterization of liquid biopsies for such DTP efforts, a set of myeloma-specific liquid biopsy approaches were developed. First, a hybrid selection panel was developed that detects somatic variants present in a patient's circulating-free DNA (cfDNA) in 70 commonly altered MM and Clonal Hematopoiesis of Indeterminate Potential (CHIP) genes. For this MM 70-Gene cfDNA Assay, samples are received as blood in a StreckTM tube designed for stabilization of cfDNA and DNA is extracted from buffy coat using magnetic bead-based chemistry. Deep coverage sequencing (80,000x depth) is performed and duplex BAM files generated with UMI alignment and error correction allowing for sensitive detection of clinically relevant variants. Technical validation data on healthy donor cfDNA mixes was generated using samples with a range of cfDNA inputs. This data determined that the assay is capable of achieving >90% sensitivity for detecting somatic events present at 1% variant allele frequency with a specificity of <0.2 false positives per megabase. Using a clinical cohort, we observed a strong correlation between Bone Marrow Aspirate (BMA) and cfDNA samples with the vast majority of variants previously detected in BMA also identified via the MM-70 Gene panel analysis. Interestingly, we also saw evidence of additional somatic variants identified in cfDNA previously undetected in BMA analysis. Because one the aims of this effort is to return results to treating physicians, a clinical-grade (CLIA) pipeline was established. For that CLIA pipeline, the variants reported are a subset of all the events detected by the MM 70-Gene Assay. The events detected in the assay are reviewed by experienced molecular pathologists at the Dana Farber Cancer Institute (DFCI) who have developed a customized reporting process. These reports utilize an internally-developed knowledgebase of variant/gene annotations that leverages the DFCI expertise in hematologic malignancies and myeloma specifically. The reports are then provided back through providers to the patient via the CureCloud system for their use in clinical care and trial identification. In order to complement the MM-70 Gene panel with copy number and translocation information, we have been exploring Circulating Multiple Myeloma Cells (CMMCs). Our current approach involves automated capture of CMMCs using ferrofluids coated with MM-selective and discriminating antibodies to immunomagnetically enrich circulating plasma cells. The highly enriched CMMCs fractions generated in such a fashion are then submitted for molecular characterization. At the submission date of this abstract, 163 patients have been fully enrolled into CureCloud from which results will be presented. In summary, we have developed a robust and very sensitive clinical-grade next-gen liquid biopsy sequencing platform allowing for minimally invasive monitoring of MM disease genomics that can be used to complement other more classical approaches and to help support our Direct-To-Patient Initiatives. Especially in this post-COVID19 era, such liquid biopsy-based approaches that avoid clinic visits for the patients and can be performed through at-home mobile phlebotomy are emerging as important new options. Disclosures Kim: LabCorp: Consultancy; Quanterix, Inc: Consultancy; PapGene, Inc: Consultancy. Ghobrial:AbbVie: Consultancy; GNS Healthcare: Consultancy; GlaxoSmithKline: Consultancy; Genentech: Consultancy; Noxxon Pharma: Consultancy; Novartis: Consultancy; Adaptive Biotechnologies: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Cellectar: Honoraria; Karyopharm Therapeutics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Abstract A38: A novel clinical-grade liquid biopsy platform for multiple myeloma

Abstract Direct-to-patient (DTP) multiple myeloma (MM) research studies have been launched recently, including PCROWD (NCT02269592), PROMISE (NCT03689595), and the MMRF CureCloud Research Initiative (NCT03657251), aimed at enrolling thousands of individuals from whom comprehensive molecular and immune analyses will be generated from blood specimens and the resulting data aggregated with the correlating clinical information. To support the molecular characterization of liquid biopsies for such DTP efforts, a myeloma-specific hybrid selection panel was developed that captures 70 commonly altered genes. The assay detects somatic point mutations and indels present in a patient’s circulating-free DNA (cfDNA). For this MM 70-Gene cfDNA Assay, samples are received as blood in a Streck’s tube and DNA is extracted from buffy coat using magnetic bead-based chemistry. Coverage sequencing (80,000x depth) is performed and duplex BAM files are generated with UMI alignment and de-duplication. As will be presented, MM blood specimens present a unique challenge as circulating MM cells are often present at significant levels in the buffy coat blood fraction used as the source of normal genomic DNA. The performance of the 70-Gene cfDNA Assay was thoroughly validated in order to establish the sensitivity, specificity, and reproducibility of the technical approach. First, the reference genomic DNA from unrelated healthy individuals was sequenced in replicate at deep coverage. Next, two cohorts were used, one from Dana-Farber and one from the MMRF CureCloud pilot. For both cohorts, tumor DNA samples from bone marrow aspirates (BMAs) with matched normal DNA from blood were sequenced on an orthogonal platform and compared to results from the MM 70-Gene Assay on cfDNA extracted from the same individuals. The yield of extracted cfDNA ranged from 6 ng to 80 ng, and about two third of cases yielded enough material to attempt sequencing, with failures coming mostly from individuals in remission. As will be presented, there was a very strong correlation between BMA and cfDNA and additional events could actually be detected in the blood that were not seen in the BMAs. Because this MM 70-Gene cfDNA Assay may potentially be used by treating physicians for management of care, a clinical-grade (CLIA) pipeline was established. For that CLIA pipeline, the variants reported are a subset of all the events detected by the MM 70-Gene Assay. The events detected in the assay are reviewed by a Genomic Tumor Board within the appropriate subset of territory predefined for reporting. The territory limitations are defined by the Genomic Tumor Board knowledgebase of actionable genomic territory available. In summary, we have developed a robust and very sensitive clinical-grade next-gen liquid biopsy sequencing platform allowing for less invasive monitoring of MM disease genomics that can be used to complement other more classical approaches and to help support direct-to-patient Initiatives. Citation Format: Mark W. Bustoros, Carrie Cibulskis, Teni Dowdell, Svetlana Gavrilov, Cody Boehner, Jennifer Yesil, Steven E. Labkoff, Shaadi Mehr, Jihye Park, Romanos Sklavenitis Pistofidis, Salomon Manier, Annette S. Kim, Keith L. Ligon, Niall Lennon, Viktor Adalsteinsson, Jane Wilkinson, Irene M. Ghobrial, Daniel Auclair. A novel clinical-grade liquid biopsy platform for multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A38.

Abstract 2911: Genome-wide copy number profiling of single circulating multiple myeloma cells (CMMCs) reveals intra-patient convergent copy-number alterations (CNAs)

Abstract Introduction: Multiple Myeloma (MM) evolution and heterogeneity is interesting for its potential translational relevance. Recent studies using bulk sequencing of Smoldering MM cells obtained from Bone Marrow (BM) report recurring CNA patterns. By analyzing single-CMMCs isolated from enriched peripheral blood, we show here, for the first time in MM, evidence of frequent convergent lesions, i.e. alterations developed independently across different evolutionary branches, including CNAs often found in MM and previously reported as common truncal alterations. Methods: Peripheral blood samples (4.0 ml) were obtained from n=3 patients with MM. CMMCs were enriched with CellSearch® AutoPrep® using a custom kit with anti-CD138 or anti-CD138/CD38 antibody-conjugated ferrofluids for positive enrichment and CD38-PE, CD19/CD45-APC immunofluorescent staining for detection. Cell enumeration was based on the co-localization of nuclear DAPI staining and CD38-PE on CellSearch CTAII®. Single CMMCs (CD38+/CD19- and CD45-/DAPI+) and White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+) were then isolated with DEPArray NxT system. Single-cell genomic DNA was amplified using Ampli1™ Whole Genome Amplification (WGA) kit, Illumina®-compatible libraries were obtained using Ampli1™ LowPass kit and the process was automated on a Hamilton STARLet Liquid handler. Multiplexed, low-pass whole-genome sequencing was performed on HiSeq 2500 Illumina® platform. Genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using the cloud-based bioinformatic suite MSBio Suite (Menarini Silicon Biosystems). Results: 186/215 (86%) single CMMC in total passed QC criteria and were included in the analysis. Single WBCs were also included as normal controls. Cumulatively, CNA profiles of single CMMCs showed patterns typical of MM, including 1q gain, 13 monosomy, sub-chromosomal gain or trisomy 3, 5, 7, 9, 11, 15, 21. Of these, intra-patient single-cell profiling surprisingly revealed -in all three patients- convergent lesions, i.e. alterations developed independently across different evolutionary branches, along with conserved (common truncal), and divergent alterations (found only in specific sub-clusters). In addition, we found evidence that 1q gain, 13q deletion and 6p gain were actually subclonal, in contrast with recent publications reporting them as truncal early-onset lesions. Conclusion: Single CMMCs CNA profiling reveals patterns of frequent convergent alterations developed independently through branched evolution, undetectable through bulk sequencing. Citation Format: Claudio Forcato, Andrea Raspadori, Alberto Ferrarini, Mario Terracciano, Valentina del Monaco, Marianna Garonzi, Carrie Morano, Steven Gross, Genny Buson, Chiara Bolognesi, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolò Manaresi. Genome-wide copy number profiling of single circulating multiple myeloma cells (CMMCs) reveals intra-patient convergent copy-number alterations (CNAs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2911.

A Phase I Study of the Safety and Feasibility of Bortezomib in Combination With G-CSF for Stem Cell Mobilization in Patients With Multiple Myeloma

BackgroundWe previously reported that administration of bortezomib (BTZ) after 4 days of granulocyte colony-stimulating factor (G-CSF) significantly augments mobilization in mice. We hypothesized that administration of BTZ at peak G-CSF mobilization in patients with multiple myeloma (MM) would be safe, augment mobilization, and have an in vivo purging effect on circulating myeloma cells. Patients and MethodsThis was a phase I study using 3 dose levels of BTZ. G-CSF was administered for 5 days. On the evening of the fourth day, a single dose of BTZ was administered. Peripheral blood was drawn 1 to 2 hours before and 15 to 18 hours after BTZ administration (before day 5 G-CSF administration) to analyze the mobilization effect of BTZ. Standard apheresis was then performed starting on day 5. After mobilization, patients underwent autologous stem cell transplantation (ASCT) per institutional guidelines. ResultsTen patients were enrolled. There were no dose-limiting toxicities. Median peripheral blood CD34+ cells at day 4 before BTZ administration was 16 per microliter and 15 hours later was 32 per microliter suggesting that administration of BTZ at peak G-CSF mobilization augments the mobilization effect of G-CSF. The effect of BTZ on circulating MM cells was unclear. All patients had successful engraftment after ASCT. ConclusionAdministration of 1 dose of BTZ at peak G-CSF mobilization was safe and well tolerated, enhanced stem cell mobilization, and did not affect graft viability. The mobilization effect of BTZ at peak G-CSF mobilization shown in this phase I study needs to be confirmed in a larger randomized trial.

A High-Throughput Workflow for the Detection, Isolation and Genomic Analysis of Single Circulating Multiple Myeloma Cells

Introduction: Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells. The high heterogeneity of MM cells is one of the major cause of disease relapse. Detection of circulating MM cells (CMMC) from peripheral blood is a useful procedure to investigate tumor heterogeneity and provides a painless alternative to the classic bone marrow biopsy to monitor disease progression. Here we demonstrate that the synergy between CellSearch® (CS) and DEPArray™ (DA) technologies can be used to identify, isolate and characterize at the genetic level single and pure CMMCs .Methods: 4.0 ml of peripheral blood samples were obtained from 3 patients with MM. Putative CMMCs were enriched with CS using anti-CD138 or anti-CD138/CD38 as positive selection marker and subsequently stained with CD38-PE, CD19/CD45-APC immunofluorescent probes. Cells detection and enumeration was performed based on the co-localization of nuclei DAPI staining and CD38-PE. Single CMMCs (CD38+/CD19- and CD45-/DAPI+) and White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+) were then isolated using the DA NxT system. Single cells genomic DNA was amplified using Ampli1™ Whole Genome Amplification (WGA) kit and Illumina®-compatible libraries were obtained using Ampli1™ LowPass kit and a high-throughput, customized automated protocol using Hamilton STARLet Liquid handler. Highly-multiplexed, genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using HiSeq 2500 Illumina® platform.Results: CS and DA workflow* enabled the isolation of 215 single CMMC, selected for LPCNA analysis. 42 single WBCs were also included as normal controls. Copy-number profiles of single CMMCs showed relevant gains and losses of chromosomal segments, as result of a high-level genomic instability. Notably, intra-patient CMMCs revealed overall conserved CNA patterns with subclonal alterations, suggesting a certain level of branched tumor evolution. Conversely, a higher degree of heterogeneity in CMMCs CNA profiles was observed among different patients. Interestingly, CNAs detected in all patients are located in regions containing genes involved in cell cycle regulation (MAPK, NOTCH pathways) and cell signaling (IL6R), which might be involved in proliferative processes and immuno-surveillance escape.Conclusion: The combination of CS and DA workflow* with a streamlined automated protocol allowed to obtain hundreds of genomic libraries from pure single CMMCs. The presented workflow constitutes a non-invasive, rapid and high-throughput approach for characterizing MM tumor heterogeneity and progression, suggesting a possible future implementation in clinical applications.*For Research Use Only. Not for use in diagnostic procedures. DisclosuresRaspadori:Menarini Silicon Biosystems: Employment. Forcato:Menarini Silicon Biosystems: Employment. Edoardo:Menarini Silicon Biosystems: Employment. Papadopulos:Menarini Silicon Biosystems: Employment. Ferrarini:Menarini Silicon Biosystems: Employment. Del Monaco:Menarini Silicon Biosystems: Employment. Terracciano:Menarini Silicon Biosystems: Employment. Morano:Menarini Silicon Biosystems: Employment. Gross:Menarini Silicon Biosystems: Employment. Bolognesi:Menarini Silicon Biosystems: Employment. Buson:Menarini Silicon Biosystems: Employment. Fontana:Menarini Silicon Biosystems: Employment. Connelly:Menarini Silicon Biosystems, Inc.: Employment, Other: Chief R&D Officer, USA. Simonelli:Menarini Silicon Biosystems: Employment. Medoro:Menarini Silicon Biosystems: Employment. Manaresi:Menarini Silicon Biosystems: Employment.

Abstract 2198: An integrated workflow for liquid biopsy of circulating multiple myeloma cells (CMMCs) with single cell resolution reveals tumor heterogeneity

Abstract Background: Multiple myeloma (MM) is a bone marrow derived cancer of plasma cells, which remains an incurable disease. Because of the invasive and painful nature of bone biopsy, an alternate tumor monitoring strategy is needed. We have previously shown that Circulating Multiple Myeloma Cells (CMMCs) isolated by CellSearch® (CS) are prognostic and indicative of disease burden through remission and relapse. Here we report, for the first time, the molecular characterization of pure single CMMCs isolated from a multiple myeloma patient, by integrating CS and DEPArray™ (DA) NxT systems, providing access to copy-number alteration (CNA) profiling. Methods: 4.0ml of peripheral blood from a patient with multiple myeloma was tested. On CS, target CMMCs were enriched using anti-CD138 for cell capture, and stained immunofluorescently with CD38-PE, CD19 and CD45-APC. Nuclei were stained with DAPI and detected target cells counted. The enriched sample was then analyzed using the DA NxT system: single CMMCs (CD38+/CD19- and CD45-/DAPI+), along with some single White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+), were isolated. The DNA of each single cell was amplified using the Ampli1™ WGA kit and used for highly-multiplexed, genome-wide single-cell CNA analysis using a Ampli1™ LowPass kit (LPCNA) on Illumina® MiSeq. Results: CS identified 128 CMMCs. From DA NxT we harvested 20 individual CMMCs for LPCNA analysis. Copy-number profiles of CMMCs confirmed their tumor origin, showing a high-level of genome instability with several gains and few losses of chromosomal segments. Moreover, an unsupervised hierarchical cluster analysis highlighted a conserved pattern of alterations, enabling the separation between CMMCs and WBCs groups. A pattern of copy-number gains shared by all CMMCs, coupled with gains and losses shared by a subset of CMMCs, suggests a branched evolution of different tumor subclones. WBC profiles were flat as expected. Conclusions: Cell enrichment by CS followed by individual cell sorting using DA NxT, enabled the isolation of single CMMCs with 100% purity. Ampli1™ single-cell analysis demonstrated CMMC molecular heterogeneity suggestive of tumor subclones presence. This platform combination provides a reliable and non-invasive method for MM characterization enabling translational research and future clinical application. Citation Format: Mario Terracciano, Claudio Forcato, Edoardo Petrini, Alberto Ferrarini, Valentina del Monaco, Andrea Raspadori, Carrie Morano, Steven Gross, Chiara Bolognesi, Genny Buson, Thai Bui, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolò Manaresi. An integrated workflow for liquid biopsy of circulating multiple myeloma cells (CMMCs) with single cell resolution reveals tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2198.

Comprehensive Genetic Interrogation of Circulating Multiple Myeloma Cells at Single Cell Resolution

Continuous genomic evolution has been a major limitation to curative treatment of multiple myeloma (MM). Frequent monitoring of the genetic heterogeneity in MM from blood, rather than serial bone marrow (BM) biopsies, would therefore be desirable. We hypothesized that genomic characterization of circulating MM cells (CMMCs) recapitulates the genetics of MM in BM biopsies, enables MM classification, and is feasible in the majority of MM patients with active disease.Methods: To test these hypotheses, we developed a method to enrich, purify and isolate single CMMCs with a sensitivity of at least 1:10(5). We then performed DNA- and RNA-sequencing of single CMMCs and compared them to single BM-derived MM cells. We determined CMMC numbers in 24 randomly selected MM patient samples and compared them to numbers of circulating MM cells obtained by flow cytometry. We performed single-cell whole genome amplification of single cells from 10 MM patients, and targeted sequencing of the 35 most recurrently mutated loci in MM. A total of 568 single primary cells representing CMMCs, BM MM cells, CD19+ B lymphocytes, CD45+CD138- WBC from these patients were subjected to DNA-sequencing. By processing 80 single cells from four MM cell lines with known mutations we determined the mean sensitivity of mutation detection in single cells to be 93 ± 9%. In addition to DNA-sequencing we also isolated 57 single MM cells from the BM and peripheral blood of two MM patients and performed whole transcriptome single cell RNA-sequencing.Results: In 24 randomly selected MM patient samples we detected >12 CMMCs per 1ml of blood in all 24 patients. In comparison, by flow cytometry, we detected ≥10 CMMCs per 10(5) white blood cells in 10/24 cases (42%), ≥1 CMMC but ≤ 10 CMMCs in 13/24 cases (54%), and < 1 CTCs in 1/24 patients (4%). Mutational analysis of 35 recurrently mutated loci in 335 high quality single MM cells from the blood and BM of 10 patients, including one MGUS patient, revealed the presence of a total of 12 mutations (in KRAS, NRAS, BRAF, IRF4 and TP53). All targeted mutations that were detected by clinical-grade genotyping of bulk BM were also detected in single cell analysis of CMMCs. While in most patients, the fraction of mutated single cells was similar between blood and BM, in three patients, the proportion of MM cells harboring TP53 R273C, BRAF G469A and NRAS G13D mutations was significantly higher in the blood than in the BM, suggesting a different clonal composition. We developed an analytical model to predict whether a genetic locus underwent loss of heterozygosity, using the distribution of known allelic fractions of previously described mutations in MM cell lines as a benchmark. In two patients who simultaneously harbored two mutations, we predicted a BRAF G469E and a KRAS G12C mutation to be heterozygous, whereas the loci harboring a TP53 R273C and a TP53 R280T mutation were predicted to be associated with LOH with high statistical confidence. Whole transcriptome single cell RNA-sequencing of 57 MM cells from the BM and peripheral blood of two patients showed >3,700 transcripts per cell. Single-cell RNA-sequencing allowed for a clear distinction between normal plasma cells and MM cells, either based on analysis of CD45, CD27, and CD56 alone, or by unsupervised hierarchical clustering of detected transcripts in single cells. In addition, single cell CMMC expression analysis could be used to infer the existence of key MM chromosomal translocations. For example, CCND1 and CCND3 were highly upregulated in single MM cells from the blood and BM of two patients, whose MM was found by FISH analysis to harbor a t(11;14) and a t(6;14) translocation, respectively.Conclusion: We demonstrate that extensive genomic characterization of MM is feasible from very small numbers of CMMCs with single cell resolution. Interrogation of single CMMCs faithfully reproduces the pattern of somatic mutations present in MM in the BM, identifies actionable oncogenes, and reveals if somatic mutated loci underwent loss of heterozygosity. Single CMMCs also reveal mutations that are not detectable in the BM either by single cell sequencing or clinical grade bulk sequencing. Single cell RNA-sequencing of CMMCs provides robust transcriptomic profiling, allowing for class-differentiation and inference of translocations in MM patients. DisclosuresRaje:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding.

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

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. DisclosuresDe 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.

CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma

AbstractThe anti-CD38 monoclonal antibody daratumumab is well tolerated and has high single agent activity in heavily pretreated relapsed and refractory multiple myeloma (MM). However, not all patients respond, and many patients eventually develop progressive disease to daratumumab monotherapy. We therefore examined whether pretreatment expression levels of CD38 and complement-inhibitory proteins (CIPs) are associated with response and whether changes in expression of these proteins contribute to development of resistance. In a cohort of 102 patients treated with daratumumab monotherapy (16 mg/kg), we found that pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) compared with patients who achieved less than PR. However, cell surface expression of the CIPs, CD46, CD55, and CD59, was not associated with clinical response. In addition, CD38 expression was reduced in both bone marrow–localized and circulating MM cells, following the first daratumumab infusion. CD38 expression levels on MM cells increased again following daratumumab discontinuation. In contrast, CD55 and CD59 levels were significantly increased on MM cells only at the time of progression. All-trans retinoic acid increased CD38 levels and decreased CD55 and CD59 expression on MM cells from patients who developed daratumumab resistance, to approximately pretreatment values. This resulted in significant enhancement of daratumumab-mediated complement-dependent cytotoxicity. Together, these data demonstrate an important role for CD38 and CIP expression levels in daratumumab sensitivity and suggest that therapeutic combinations that alter CD38 and CIP expression levels should be investigated in the treatment of MM. These trials were registered at www.clinicaltrials.gov as #NCT00574288 (GEN501) and #NCT01985126 (SIRIUS).

Abstract 3163: Peripheral blood circulating multiple myeloma cells (CMMCs) correlate with disease burden and can be used to characterize high-risk cytogenetics in newly diagnosed and smoldering myeloma

Abstract There is an increasing interest in the ability to dynamically track disease burden and perform molecular subtyping of patients with plasma cell disorders without invasive bone marrow sampling. Circulating multiple myeloma cells (CMMC) have been detected in elevated numbers in the peripheral blood of patients with plasma cell disorders using flow cytometry or circulating cell enrichment platforms. We developed an automated CELLSEARCH® assay to enrich, enumerate, and perform a triplex FISH assay for t(4;14), t(14;16), and del 17p on CMMC (CD138+CD38+, CD45-CD19-) isolated from a 4 mL peripheral blood sample (Gross, et.al. Blood 2011; 118(21):1825). Here we present the enumeration and cytogenetic profiling of CMMC from separate cohorts of patients across the spectrum of plasma cell disorders. The first cohort consisted of newly diagnosed multiple myeloma patients enrolled in the CoMMpass study (ClinicalTrials.gov Identifier: NCT01454297). One or more CMMC per four ml blood were detected in 684/698 (98%) of newly diagnosed myeloma patients with median CMMC count of 413 per 4 mL of blood. CMMC counts decreased significantly from baseline when a remission was achieved due to treatment (p&amp;lt;0.001). CMMC counts &amp;lt;100 at remission were associated with improved PFS and OS compared to those patients whose CMMC counts were &amp;gt; 100 at remission. CMMC FISH results (n = 57) showed overall agreement of 85%, 91% and 80% with bone marrow FISH results and 81%, 91%, and 95% agreement with bone marrow CNV/RNAseq results for the t(4;14), t(14;16), and del 17p assays, respectively. The second cohort of patients consisted of intermediate/high risk smoldering myeloma patients enrolled in a Phase 2 study of Siltuximab (ClinicalTrials.gov Identifier: NCT01484275). One or more CMMC per 4 mL blood was detected at baseline in 74/79 (94%) of intermediate/high risk smoldering myeloma patients with median CMMC count of 100 per 4 mL of blood. Significantly higher CMMC counts were observed between patients in the placebo arm that progressed versus those without progression (n = 34, p = 0.031). This is in contrast to standard metrics of percentage of bone marrow plasma cells and serum M protein levels where statistically significant differences were not seen between progressors and non-progressors in the placebo arm (p = 0.068 and p = 0.070, respectively). CMMCs were collected from a third cohort of 35 patients across the plasma cell disease spectrum with an emphasis on MGUS and SMM. CMMC counts were associated with the disease burden of patients within this cohort. CMMC may be a useful non-invasive tool for disease monitoring and characterization across the plasma cell disorder spectrum. In myeloma, CMMC may be a useful prognostic marker at remission to delineate those patients at risk for relapse. In SMM, CMMC may be useful for predictive patients at risk of progression to MM. Citation Format: Brad Foulk, Mike Schaffer, Steve Gross, Chandra Rao, Denis Smirnov, Shalini Chaturvedi, Manjula Reddy, Madeline Repollet, Claudia Rojas, Daniel Auclair, Mary DeRome, The MMRF CoMMpass Network, Brendan Weiss, A. Kate Sasser. Peripheral blood circulating multiple myeloma cells (CMMCs) correlate with disease burden and can be used to characterize high-risk cytogenetics in newly diagnosed and smoldering myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3163.

A CD138-independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma.

CD138 (also termed SDC1) has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug-resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down-regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138-negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.

E-Selectin Ligand Expression Increases with Progression of Myeloma and Induces Drug Resistance in a Murine Transplant Model, Which Is Overcome By the Glycomimetic E-Selectin Antagonist, GMI-1271

There is increasing evidence that E-selectin and its ligands play an important role in the progression of multiple myeloma (MM) and drug resistance. We reported that the sialyltransferase ST3GAL6 influences homing and survival in MM, and postulated that it may function in the synthesis of E-selectin ligands (Glavey et al Blood, 2014). We also found that a small subpopulation of cells (~ 5%) from MM cell lines express functional E-selectin ligands, which could be expanded under hypoxic conditions typical of the bone marrow (BM) microenvironment. These cells were identified by reactivity with an antibody (HECA452), which binds the same carbohydrate epitope required for binding to E-selectin. Rolling of MM1S cells on E-selectin was blocked by a small molecule glycomimetic antagonist to E-selectin (GMI-1271). Moreover, GMI-1271 significantly enhanced the anti-myeloma activity of bortezomib (BTZ) in an in vivo murine transplant model (Natoni et al Blood, 2014). We now extend these observations to obtain a more complete understanding of the role E-selectin plays in MM biology and chemotherapy resistance for its potential clinical relevance.The parental, heterogeneous MM cell lines MM1S and RPMI8226 (MM1Spar, RPMI8226par, respectively) were sequentially sorted to obtain cell lines highly enriched (>85% and 80%) for the expression of cell surface carbohydrates bound by HECA452, and designated MM1SHECA452 and RPMI8226HECA452. The cell lines could be passaged in vitro and were stable for enriched E-selectin ligand expression. In contrast to parental cells, both MM1SHECA452 and RPMI8226HECA452 showed strong binding to E-selectin in static adhesion assays. Both MM1SHECA452 and RPMI8226HECA452 exhibited strong rolling on E-selectin under shear stress. MM1Spar or RPMI8226par failed to roll well on E-selectin. The addition of GMI-1271 during culture conditions led to a marked reduction in adhesion of MM1SHECA452 and profoundly inhibited rolling on E-selectin of both HECA452 enriched MM cell lines.The significance of these in vitro findings was studied in vivo. MM1Spar or MM1SHECA452 cells were injected i.v. into SCID beige mice. Beginning 5 days post tumor injection, the survival impact of treatment with saline control, GMI-1271, BTZ or a combination of both was determined. Mice transplanted with MM1SHECA452 had more aggressive disease with significantly shorter survival compared to those transplanted with MM1Spar. In contrast to MM1Spar cells, mice engrafted with MM1SHECA452 demonstrated a marked resistance to BTZ treatment. Whereas GMI-1271 treatment alone had no impact on survival, the combination of GMI-1271 and BTZ led to a highly significant improvement in survival of MM1Spar engrafted mice (P=0.0363), and more importantly broke the resistance and restored the anti-myeloma activity of BTZ in MM1SHECA452 engrafted mice (P=0.0123) (figure 1). The number of peripheral blood (PB) human CD138+ cells was increased in MM1SHECA452-engrafted mice within 60 min following a single injection of GMI-1271, and persisted for at least 24 hours (2.37% v. 0.03%, p <0.001). This effect was consistent with GMI-1271 disrupting the tumor microenvironment and mobilizing MM1SHECA-452 cells from the BM niche.Given these findings we wished to see if samples from patients with MM express E-selectin ligands and whether higher levels are seen with disease progression. BM and/or PB were obtained following informed consent from patients with MM and plasma cells (CD38+/CD138+) were analyzed for E-selectin ligand expression by flow cytometry using the HECA452 antibody. To date all primary MM samples (n=25) contained HECA452-reactive cell populations (median 22%). A consistently higher proportion of circulating MM cells express HECA452 when compared with paired BM samples (n=14), with a median difference of 33% (Wilcoxon signed rank test, p=0.02). HECA452 expression of MM in PB was significantly higher (on average 40% higher) in samples taken at relapse vs. diagnosis, (unpaired t test, p = 0.0008)These data provide compelling evidence that E-selectin ligand bearing cells play an important role in disease progression and drug resistance in MM, and a strong rationale for clinical strategies incorporating GMI-1271 to improve patient outcome. [Display omitted] DisclosuresSmith:GlycoMimetics, Inc.: Employment. Locatelli-Hoops:GlycoMimetics, Inc.: Employment. Oliva:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. O'Dwyer:Celgene: Honoraria, Research Funding.

Abstract 1597: A novel method for isolation and genetic analysis of pure populations of circulating plasma cells from multiple myeloma patients

Abstract Multiple Myeloma is bone marrow tumor that frequently results in the aberrant release of neoplastic plasma cells into circulation. The CELLSEARCH® system (Janssen Diagnostics, Raritan, NJ) has been used to capture, enumerate, and perform FISH analysis on circulating multiple myeloma cells (CMMC) from patients with active multiple myeloma, smoldering myeloma, and MGUS (Gross, et.al. Poster #1825, ASH 2011). This method utilizes a blood fixative and subsequently permeabilizes the cells, which confounds the ability to do mRNA analysis on these samples. Here we present a novel method to isolate pure circulating myeloma cells that is compatible with downstream genetic analysis. Blood was obtained through a commercial vendor (Conversant Bio) from multiple myeloma patients. Samples were processed in parallel using the existing method for enumeration and a novel method for genetic profiling. For CMMC enumeration, blood was collected in CellSave tubes (Janssen Diagnostics) and CMMC were enriched using paramagnetic ferrofluid recognizing CD138 and CS1 antigens. Cells were permeabilized and stained on the CELLSEARCH® AUTOPREP with CD38 (PE), CD45 (APC), CD19 (APC) and DAPI. CMMC were enumerated using the CELLTRACKS ANALYZER II® scanning platform. CMMC were defined as DAPI +, CD138+, CD38+, CD45-, CD19-. For genetic profiling, 4 ml EDTA blood was enriched using CD138 and CS1 ferrofluids on the AUTOPREP with no subsequent permeabilization. CMMC were stained offline using CD38 (Texas Red), CD45 (FITC), CD19 (FITC), CD3 (FITC) and NucBlue. Samples were transferred to glass a bottomed petri dish and observed through an inverted microscope using a Texas Red/FITC dual filter, individual FITC and Texas red filters, and a DAPI filter. CMMC were defined as NucBlue+, CD138+, CD38+, CD45-, CD19-, and CD3-. Pools of individual CMMC were picked from the sample using an Eppendorf Transferman micromanipulator. For comparison, contaminating leukocytes were also collected for analysis. RNA from picked cells was amplified using the SMARTer Ultra Low Input Kit (Clontech) and qPCR was performed for myeloma and leukocyte specific markers. Detection of CMMC using the novel method for genetic profiling was found to be concordant with the existing method for enumeration. We found this approach to provide a deep and unbiased characterization of gene pathways activated in CMMC by RT qPCR and RNASeq. This method will have applications in longitudinal studies of active myeloma patients as a way to gather information about relapse and minimal residual disease between bone marrow draws. Citation Format: Greg Brittingham, Chandra Rao, Peter Vulfson, Vipul Bhargava, Denis Smirnov, Brad Foulk. A novel method for isolation and genetic analysis of pure populations of circulating plasma cells from multiple myeloma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1597. doi:10.1158/1538-7445.AM2015-1597

Abstract 5305: Predicting relapse using CD138-independent strategy to detect residual myeloma plasma cells

Abstract Introduction: Despite the introduction of novel therapies, majority of multiple myeloma (MM) patients relapses. The major reason for the relapse in MM is the presence of a minimal residual disease (MRD) population that remains after treatment. MM cells are defined as CD138+; however, the presence of clonogenic CD138-negative MM cells and hypoxia-driven CD138 decrease in expression were previously shown. We propose a novel set of biomarkers (independent of CD138) to detect MRD in MM in order to predict patient relapse. Procedures: We tested the effect of hypoxia on CD markers expression in MM cell lines. We developed new method to detect MM cells by flow cytometry based on CD38(APC+) gating and negatively selecting other leukocytes including T cells, B cells, monocytes, NK cells, and dendritic cells using CD3, CD19, CD14, CD16, and CD123, respectively (FITC-conjugated), the MM cells were defined as APC+/FITC-. We compared traditional method (CD138-based) with the new set of biomarkers (1) to detect normoxic and hypoxic MM cells, (2) to detect MM cells in the peripheral blood (PB) and the bone marrow (BM) from MM patients, confirming cell clonality by internal staining with Kappa/Lambda light chain antibodies, and (3) to predict relapse in MM patients whose BM was defined a CD138-negative. Summary of data: We found that hypoxia decreased the expression of CD138 by ∼50%, while CD38 was not altered by hypoxia in MM cells. The use of CD138+ as a marker for MM detected only 60-75% of hypoxic MM cells, whereas APC+/FITC- strategy detected close to a 100% of the hypoxic MM cells in vitro. In patient samples, all patients had a higher number of BM-derived and circulating MM cells as detected by APC+/FITC- strategy compared to CD138+. In addition, the clonality of the APC+/FITC- population in BM negative fractions was the same as the original disease (CD138+ cells). Moreover, studies conducted on 16 patients with BM defined as a CD138-negative, showed that the novel APC+/FITC- strategy was more successful than CD138-based method or histology in predicting recurrence and time to relapse. The median time-to-progression with &amp;lt;2% or ≥2% of MM cells detected by novel strategy was 40 and 20 months, respectively. Moreover, patients who relapsed within 2 years had an average 4.4% of APC+/FITC- cells, while patients who relapse in more than 2 years had an average of 1.6% APC+/FITC- cells in their BM. Conclusion: CD138 cannot be used as a universal marker to detect MM cells, especially when they are hypoxic. A novel set of biomarkers CD38+/CD3-/CD19-/CD14-/CD16-/CD123- (APC+/FITC-) was designed to detect MM cells independent of CD138 expression and hypoxic status. This strategy was able to detect a clonal CD138-negative MM cell population in the BM and PB from MM patients. Moreover, this strategy predicted tumor recurrence and relapse in MM patients more accurately compared to traditional methods such as CD138-based or histology. Citation Format: Barbara Muz, Feda Azab, Pilar de la Puente, Justin King, Micah Luderer, Ravi Vij, Abdel Kareem Azab. Predicting relapse using CD138-independent strategy to detect residual myeloma plasma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5305. doi:10.1158/1538-7445.AM2015-5305

Circulating Multiple Myeloma Cells (CMMCs): A Novel Method for Detection and Molecular Characterization of Peripheral Blood Plasma Cells in Multiple Myeloma Precursor States

Background Circulating plasma cells (PCs) have been identified as a prognostic factor in patients with myeloma precursor states (MGUS and SMM) and active multiple myeloma (MM). Enumeration of circulating PCs by available methods is not suitable for widespread use and does not provide molecular characterization. We developed and evaluated a novel method for enumeration and molecular characterization of circulating PCs (circulating multiple myeloma cells, “CMMC”), based on the CELLSEARCH® System (Janssen Diagnostics LLC, Raritan, NJ), an automated technology for the capture, enumeration and characterization of rare cells in the peripheral blood.Methods We are performing a prospective study of patients with MGUS and SMM to evaluate CMMCs as biomarker for progression to active MM. Utilizing the CELLSEARCH® System CMMCs were captured by CD138 ferrofluid magnetic particles and identification was defined as CD38+ and CD19-, CD45-. Nonviable cells were excluded by DAPI. Isolated CMMCs were stored and FISH for t(4:14), t(14;16) and del17 was performed.Results We have enrolled 16 patients, MGUS = 3, SMM = 11, and newly diagnosed MM = 2. The Mayo Risk stratification for MGUS patients was: low risk = 2, low-intermediate = 1. All SMM patients were low risk by Mayo Model incorporating serum free light chains. The median number of bone marrow plasma cells for MGUS patients was 7 (range 7-9) and for SMM patients was 15 (range 10-40). The median CMMCs for MGUS = 6 (range 2-55), median CMMCs for SMM = 31 (5-1918). The two patients with NDMM had 5870 and 5 CMMCs, respectively. A single patient with SMM progressed with a symptomatic solitary lumbar plasmacytoma and had CMMCs of 5 and 3 at baseline and progression, respectively. Abnormalities by FISH were detected in both bone marrow and CMMCs. Accrual is ongoing and additional data will be presented at the meeting.Conclusions The CELLSEARCH® CMMC assay can detect, quantify and provide molecular characterization of circulating PCs in MGUS/SMM/MM; longer prospective follow-up is needed to test the prognostic value of CMMCs. DisclosuresWeiss:Janssen: Consultancy, Research Funding. Sasser:Janssen: Employment. Rao:Janssen: Employment, Equity Ownership. Foulk:Janssen: Employment. Gross:Johnson & Johnson: Employment, Equity Ownership. Cohen:Janssen: Membership on an entity’s Board of Directors or advisory committees. Vogl:Celgene Corporation: Consultancy; Amgen: Consultancy; Millennium/Takeda: Research Funding; GSK: Research Funding; Acetylon: Research Funding. Stadtmauer:Janssen: Consultancy.