Detection of Multiple Myeloma Cells in Peripheral Blood Using High-Throughput Sequencing Assay

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 321-321
Author(s):  
Amitabha Mazumder ◽  
Malek Faham ◽  
Mark Fiala ◽  
Mark Klinger ◽  
Thomas Martin ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
High Frequency ◽  
Genomic Dna ◽  
Research Funding ◽  
High Throughput Sequencing ◽  
Plasma Dna ◽  
Employment Equity ◽  
Clonal Rearrangement ◽  
Dna And Rna ◽  
Equity Ownership

Abstract Abstract 321 Background: Multiple myeloma (MM) is characterized by the presence of monoclonal protein (M-protein) in serum and/or urine, clonal plasma cell accumulation in bone marrow (BM), and related organ or tissue impairment. MM patients are monitored during therapy and posttherapy using immunoglobulin, M-protein and free light chain assays. Assessing pathological myeloma cells using flow cytometry and RT-PCR has been shown to have superior prognostic value. However, the sensitivity of these techniques has generally limited their use to assessment of BM. In order to determine whether myeloma minimal residual disease could be detected in peripheral blood (PB), we assessed a cohort of MM patients using a sequencing based platform, LymphoSIGHT, with a sensitivity of 1 cancer cell per million leukocytes. Methods: We obtained from 4 sources (UCSF, NYU, Washington Univ, commercial source) pairs of BM and PB samples from 60 MM patients at different points of disease. BM samples were used to identify the clonal MM sequence and detection of that sequence in the PB was assessed. For some patients BM/PB sample pairs were obtained from >1 time point resulting in a total of 78 pairs. In addition blood and bone marrow plasma samples were available for 44 and 6 patients, respectively, to assess presence of the myeloma clonotype in cell free DNA. Altogether there were 206 samples. BM samples were either available as BM mononuclear cells (BMMC) or bead enriched CD138+ cells. Using universal primer sets, we amplified IgH@ variable (V), diversity (D), and joining (J) gene segments from genomic DNA and/or RNA samples, the incomplete IgH rearrangement (DJ), and IgK from genomic DNA. Amplified products were sequenced to obtain >1 million reads and analyzed using standardized algorithms for clonotype determination. Myeloma-specific IgH, IgK, and DJ clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in all PBMC (DNA and RNA), BM plasma (DNA), and PB plasma (DNA) samples using the same IgH and in some samples using the IgK sequencing assays. A quantitative and standardized measure of clone level among all leukocytes in each PB or BM sample was determined using internal reference DNA. Here we describe data on 46/60 patients; data from all 60 patients will be presented. Results: In BM samples, we detected the myeloma clonal rearrangement of at least one receptor (“calibrating receptor”) in 34/46 (74%) of MM patients (Table 1). The calibration rate varied by receptor, with 30/46 (65%) patients calibrating with IgH, 14/43 (33%) with IgH DJ, and 22/43 (51%) with IgK (Table 1). Identification of myeloma-specific clonal rearrangement is based on presence at high frequency and may not occur in samples from patients with low disease load (e.g., post-treatment). Of the 12 non-calibrating patients, only 3 had high disease load. The myeloma clonotype that was identified in the BM was also detected in PBMC in 22/30 (73%) and 28/30 (93%) patients with the DNA and RNA IgH analysis, respectively (Table 2). IgK DNA analysis showed the presence of the myeloma clonotype in 9/10 PBMC samples, all of which were concordant with IgH results. The myeloma clonotype that was identified in the BM was also detected in the cell-free BM and PB samples in 5/5 and 7/11 patients, respectively, using the IgH DNA assay. The evaluation of blood plasma and PBMC were at times complementary in detecting the myeloma. Conclusions: Results from the application of a high-throughput sequencing method for detection of myeloma-specific clonotypes in 46 MM patients are shown. A clonal rearrangement was detected in 74% of MM BM samples. Importantly, 93% of peripheral blood samples from 30 patients showed evidence of circulating myeloma in PBMC. Analysis of BM and PB samples from 14 additional MM patients as well as association of the level of myeloma in PBMC and BM with clinical measures is ongoing. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Klinger:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Comparison of High-Throughput Sequencing and Flow Cytometry for Measuring Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia: A Children's Oncology Group Cohort

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1440-1440
Author(s):  
Charles Gawad ◽  
Michael J. Borowitz ◽  
Gary V. Dahl ◽  
Meenakshi Devidas ◽  
Malek Faham ◽  
...  
Keyword(s):  
High Throughput ◽  
Research Funding ◽  
High Throughput Sequencing ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Cell Repertoire ◽  
Employment Equity ◽  
Clonal Rearrangement ◽  
Post Induction ◽  
Equity Ownership

Abstract Abstract 1440 Background: Measurement of minimal residual disease (MRD) during and after induction therapy has emerged as the most important predictor of outcome in pediatric acute lymphoblastic leukemia (ALL). Despite this, over 1/3 of relapses occur in patients who are MRD negative. In addition, ∼50% of children that have detectable MRD do not relapse. The Children Oncology Group (COG) trials use flow cytometry (FC) with a sensitivity of 10−4 for MRD detection and subsequent intensification of therapy in MRD+ patients. A more sensitive tool for monitoring MRD could lead to the identification of more patients who are likely to relapse, while a more specific assay could prevent unwarranted therapy intensification. To this end, we are employing the LymphoSIGHT platform developed by Sequenta Inc., which utilizes high-throughput sequencing for identification of clonal gene rearrangements in the B-cell repertoire and subsequent MRD measurement. In this blinded pilot study (COG AALL12B1), we compared the ability of the sequencing assay to measure MRD to that of FC in diagnostic and post-induction samples from 6 ALL patients. Methods: Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity (D), and joining (J) gene segments from genomic DNA in diagnostic and follow-up bone marrow samples from 6 ALL patients. Amplified products were sequenced to obtain >1 million reads per sample and were analyzed using algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the diagnostic sample. The presence of the tumor-specific clonotype was then monitored in post-induction samples. Absolute quantification was performed by normalizing the patient's reads to internal reference DNA. We then analyzed concordance between MRD results obtained by sequencing and FC. Results: We detected a high-frequency IgH clonal rearrangement in 5/6 diagnostic ALL samples. MRD was assessed in the 5 post-induction samples from these patients (Table 1). Deep coverage of all MRD samples was obtained, with each original IgH molecule generating ∼20 sequencing reads, ensuring the detection of a single leukemic cell if present in the sample. Leukemic clones were detected in 4/5 follow-up samples (Table 1). In the positive samples, the number of detected leukemic molecules ranged from 12 to over 6,000 and the MRD level ranged from 0.008% to 0.3%. MRD results were concordant with FC in 3 of 5 patients and were consistent with the patient's clinical courses. In one patient we detected MRD at 0.008%, a level below the sensitivity of FC, which was negative. In another sample, FC detected MRD of 0.01–0.1%, but no leukemic clones were detected by the sequencing assay despite the fact that the sample contained sufficient cell input (almost 2 million cells). The patient remained in continuous remission. Evaluation of additional paired diagnostic and post-induction samples and their association with clinical outcomes is ongoing. Conclusions: We show the application of a high-throughput sequencing method for MRD detection in childhood ALL. IgH clonal rearrangements were detected in 5/6 (83%) of samples using the sequencing assay. The absence of a clonal rearrangement in 1/6 of patients was anticipated and is likely to be mitigated by the presence of a clonal rearrangement in another immunoglobulin or T cell receptor gene. Experiments are ongoing to assess the presence of clonal rearrangements in these receptors (i.e., IgH D-J, IgK, TRB@, TRD@ or TRG@) in the diagnostic samples. In 3/5 patients there was concordance between FC and sequencing-based MRD detection. In one patient, sequencing detected MRD at a level below the threshold of FC. The last patient was negative by sequencing but positive by FC and has not relapsed. Further analysis of the sensitivity and specificity of the sequencing platform compared to FC using additional paired diagnostic and post-induction samples is ongoing. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Comparison of Minimal Residual Disease Detection Using High-Throughput Sequencing and Allele-Specific Oligonucleotide PCR Methods in Pediatric B-Lineage Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2532-2532 ◽  
Author(s):  
Marian Harris ◽  
Malek Faham ◽  
Lei Fang ◽  
Martin Moorhead ◽  
Jianbiao Zheng ◽  
...  
Keyword(s):  
High Frequency ◽  
Research Funding ◽  
High Throughput Sequencing ◽  
Lymphoblastic Leukemia ◽  
Patient Specific ◽  
Employment Equity ◽  
Allele Specific ◽  
Equity Ownership ◽  
B Lineage ◽  
Minimal Residual

Abstract Abstract 2532 Background: The clinical management of patients with pediatric B-lineage acute lymphoblastic leukemia (B-ALL) relies on combinations of multiagent anticancer drugs and risk-stratified treatment. The prognostic significance of minimal residual detection (MRD) in pediatric B-ALL has been demonstrated in multiple cohorts. Allele-specific oligonucleotide PCR (ASO-PCR) amplification of immunoglobulin or T-cell receptor rearrangements, a method for MRD detection, requires the development of patient-specific reagents and cannot detect clonal evolution. ASO-PCR also has limited coverage, with clonal rearrangements being detected in only 90% of patients. We developed the sequencing-based LymphoSIGHT platform to address these limitations. Here we report the results of a pilot study of MRD detection using both the sequencing assay and ASO-PCR in paired diagnostic and end-of-induction samples from 7 B-ALL patients. Analysis of 82 additional patients is ongoing. Methods: Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity, and joining gene segments from genomic DNA in diagnostic and follow-up bone marrow samples. Amplified products were sequenced to obtain >1 million reads (20× coverage per B-cell), and were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then assessed in the end-of-induction sample. A quantitative and standardized measure of MRD level among all leukocytes in the sample was determined using internal reference DNA. Following identification of IgH clonal rearrangements and MRD assessment using the sequencing assay, we examined the MRD results obtained at Boston Children's Hospital using ASO-PCR. Among the 7 patients analyzed to date, 6 patients were in complete remission at the time of the second sample; 1 patient had persistent evidence of disease. Sequencing was performed blinded to all clinical and ASO-PCR information on these patients. Results: With the sequencing platform, we detected a high-frequency IgH clonal rearrangement in all 7 diagnostic ALL samples. The leukemic clonotype that was identified at diagnosis was detected in the end-of-induction sample in each of the 7 patients. The quantitative range of the leukemic sequence in MRD samples ranged over 5 orders of magnitude. MRD results were concordant between sequencing and ASO-PCR in 5 of 7 patients. The detected MRD level differed by > 10 fold in 2 patients. In patient 1, sequencing detected high MRD, while low MRD was detected by ASO-PCR; this patient relapsed 1 year later while still on therapy. In patient 6, sequencing detected low MRD, while ASO-PCR detected high MRD. This patient remains in complete remission after 7 years. In patient 7, sequencing and ASO-PCR concordantly detected low MRD; this patient relapsed after completion of therapy. Conclusions: Results from the application of a high-throughput sequencing method for MRD detection in childhood B-ALL are shown. The sequencing assay does not require development of patient-specific reagents, which will reduce cost and laboratory turnaround time. This data, along with the laboratory workflow improvements, support the use of the sequencing assay as a next-generation MRD test for B-ALL. Analysis of samples from 82 patients is ongoing. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Fang:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Next-Generation Sequencing Based Minimal Residual Disease Assessment in Peripheral Blood RNA from Multiple Myeloma Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3286-3286
Author(s):  
Jeffrey L. Wolf ◽  
Katherine A. Kong ◽  
Jenai Wilmoth ◽  
Joshua Bayes ◽  
Amy Marsala ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
High Frequency ◽  
Dna Analysis ◽  
Residual Disease ◽  
Employment Equity ◽  
Dna And Rna ◽  
Equity Ownership ◽  
Generation Sequencing ◽  
Minimal Residual

Abstract Introduction: Multiple myeloma (MM) is characterized by the presence of monoclonal protein (M-protein) in serum and/or urine, clonal plasma cell accumulation in bone marrow, and related organ or tissue impairment. MM patients are monitored during and after therapy using immunoglobulin, M-protein and free light chain assays. Minimal residual disease (MRD) assessment of bone marrow samples from MM patients in complete remission (CR) using next-generation sequencing (NGS)-based technology has been shown to have prognostic value, and previous studies have demonstrated evidence of circulating myeloma cells in peripheral blood. In this prospective study, we compared the sensitivity of RNA- and DNA-based MRD assays in peripheral blood (PB) and bone marrow (BM) samples collected from MM patients. Methods: Matched BM and PB samples were obtained from 50 MM patients in various stages of their disease. Diagnostic BM samples were used to identify clonal immunoglobulin sequences (i.e. clonotypes) unique to each MM patient. Matched BM and PB samples were then assessed for the presence of the MM clonotype. Using universal primer sets, we amplified immunoglobulin (IgH) variable (V), diversity (D), and joining (J) gene segments, the incomplete IgH-DJ rearrangement, and IgK receptors. Genomic DNA samples were assessed for all 3 receptors, while RNA samples were only assessed for the functional IgH-VDJ and IgK receptors. Amplified products were sequenced to obtain >1 million reads and analyzed using standardized algorithms for clonotype quantification. Myeloma-specific IgH, IgK, and IgH-DJ clonotypes were identified for each patient based on their high frequency at diagnosis. The presence of the myeloma clonotype was then assessed in BM cells (DNA), PB cells (DNA and RNA), and PB plasma (DNA) samples. Myeloma clonotype levels were calculated as previously described (Martinez-Lopez et al. 2014). Results: High-frequency myeloma clonotypes were identified in the BM of all 50 patients. We performed a sequence-level assessment of each clonotype to determine whether it was also evaluable in RNA. Non-functional clonotypes (e.g. IgH-DJ and kappa deleting element clones, frameshift/nonsense mutations, nonfunctional V or J segments, loss of cysteine in CDR3) were excluded from the analysis. 37 evaluable RNA clonotypes were identified in 28 of the 50 patients (56%). We compared the sensitivity of MRD assessment using DNA and RNA extracted from PB mononuclear cells. 20 clonotypes were qualitatively concordant in both DNA and RNA. 17 clonotypes were discordant, with all 17 clonotypes being detectable in RNA but not in DNA (Figure 1A). Only 4 of 21 (19%) clonotypes detectable in RNA were also detectable in DNA in the PB. Thus, RNA provides a clear sensitivity advantage over DNA analysis in PB cellular samples. We then investigated whether the sensitivity advantage conferred by RNA in PB cells was equivalent or superior to the sensitivity of DNA analysis in BM cells. On a patient level, 23 patients were qualitatively concordant in the PB and BM assays. 5 patients were discordant, with all patients being positive in BM and negative in PB. 4 of the discordant patients were positive at low levels in the BM (1-10 MM clonotype molecules per 1 million cells). On a clonotype level, 28 clonotypes were qualitatively concordant in BM DNA and PB RNA. 9 clonotypes were discordant, with all 9 clonotypes being positive in BM DNA and negative in PB RNA (Figure 1B). Nevertheless, PB RNA detected MRD in 21 of 30 with measurable disease in the BM DNA (ie, 70% sensitivity). These results demonstrate that MRD assessment using PB RNA is more informative than PB DNA in MM; however, despite the sensitivity improvement provided by RNA, MRD assessment in the BM DNA remains the superior sample source for MRD assessment, particularly when achievement of MRD negativity is the goal of therapy. Conclusions: NGS-based MRD assessment of BM in myeloma patients has been shown to have prognostic value. PB-based MRD assessment would improve clinical MRD assessment and monitoring paradigms. Our results demonstrate that RNA provides increased sensitivity for blood-based MRD assessment. Additional assay optimization to improve the fraction of clonotypes evaluable in RNA and to enhance sensitivity compared with BM is necessary before PB MRD monitoring in MM patients can be incorporated into routine clinical practice. Figure Figure. Disclosures Wolf: Telomere Diagnostics: Consultancy; Pharmacyclics: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria. Kong:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Bayes:Adaptive Biotechnologies Corp: Equity Ownership. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Martin:Sanofi: Research Funding; Amgen: Research Funding.

scholarly journals Itolizumab As a Potential Therapeutic for the Prevention and Treatment of Graft Vs Host Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5603-5603 ◽  
Author(s):  
Cherie Tracy Ng ◽  
Jeanette Ampudia ◽  
Robert J. Soiffer ◽  
Jerome Ritz ◽  
Stephen Connelly
Keyword(s):  
Weight Loss ◽  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Chronic Gvhd ◽  
Vehicle Control ◽  
Control Group ◽  
Employment Equity ◽  
Equity Ownership

Background: CD6 is a co-stimulatory receptor, predominantly expressed on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation, differentiation and trafficking and is central to inflammation. While effector T cell (Teff) are CD6hi and upregulate expression upon activation, regulatory T cells (Treg) remain CD6lo/-, making this an attractive target to modulate Teff activity while preserving Treg activity. Early studies by Soiffer and colleagues demonstrated using T12, an anti-CD6 monoclonal antibody (mAb) that ex-vivo depletion of CD6+ donor cells prior to transplantation decreased the incidence of both acute and chronic GVHD, highlighting the importance of CD6+ cells in GVHD pathogenesis and validating it as a therapeutic target. However, it remains to be shown whether modulating the CD6-ALCAM pathway in vivo can attenuate GVHD. We investigated the use of itolizumab, a humanized anti-CD6 mAb that has demonstrated clinical efficacy in other autoimmune diseases, as both a preventive and therapeutic treatment for GVHD, using a humanized xenograft mouse model. Methods: Humanized xenograft mice were generated by intravenous transfer of 2x10^7 human PBMCs into 6-8 weeks old NOD/SCID IL2rγ-null (NSG). To investigate the ability of itolizumab to prevent GVHD, mice were dosed with either 60μg or 300μg of itolizumab, 150μg of abatacept (CTLA4-Ig), or vehicle, starting one day prior to PBMC transplantation. To investigate the therapeutic effect of itolizumab, mice were dosed with either 150μg of itolizumab or vehicle, starting at Day 5 post-PBMC transfer, when transplanted T cells are already activated. All treatments were administered IP every other day. Weight and disease scores were monitored throughout the study. At Days 18 and 35, peripheral blood was evaluated by flow cytometry to examine T cell prevalence, and tissues were collected for histological examination of pathology and T cell infiltration. Results: When administered as prevention (Day -1), treatment with either 60μg or 300μg of itolizumab significantly decreased mortality compared to the vehicle control (100% vs. 10%); this decrease was similar to the positive control group treated with abatacept (Figure 1). At 60μg, itolizumab-treated mice demonstrated significant reductions in the prevalence of human T cells in peripheral blood vs. vehicle-treated mice at Day 18 (<0.2% vs. 74.5%; p < 0.001). The reduction in peripheral T cells was accompanied by reductions in tissue-infiltrating T cells in lung (85-fold) and gut (9.5-fold), as well as reductions in disease scores and weight loss. When administered therapeutically, treatment with itolizumab was associated with a survival rate of 50% compared to 10% in the control group (Figure 2). Similarly, peripheral T cell prevalence (34.3% vs. 65.1%; p < 0.001), weight loss, and disease scores were inhibited by itolizumab compared to vehicle control mice. Conclusions: These data suggest that systemic treatment with itolizumab can modulate pathogenic Teff cell activity, establishing this antibody as a potential therapeutic for patents with GvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Ng: Equillium: Employment, Equity Ownership. Ampudia:Equillium: Employment. Soiffer:Mana therapeutic: Consultancy; Kiadis: Other: supervisory board; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Jazz: Consultancy. Ritz:Equillium: Research Funding; Merck: Research Funding; Avrobio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy; LifeVault Bio: Consultancy; Celgene: Consultancy; Aleta Biotherapeutics: Consultancy; Kite Pharma: Research Funding. Connelly:Equillium: Employment, Equity Ownership.

Detection of Diffuse Large B-Cell Lymphoma in Peripheral Blood Using High-Throughput Sequencing Assay.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2666-2666
Author(s):  
Yasuhiro Oki ◽  
Malek Faham ◽  
Victoria Carlton ◽  
Sattva S. Neelapu ◽  
Anas Younes
Keyword(s):  
B Cell ◽  
Peripheral Blood ◽  
High Throughput Sequencing ◽  
B Cell Lymphoma ◽  
Lymph Node Biopsy ◽  
Employment Equity ◽  
Tumor Biopsy ◽  
Blood Samples ◽  
Large B Cell Lymphoma ◽  
Equity Ownership

Abstract Abstract 2666 Background: In patients with diffuse large B-cell lymphoma (DLBCL), circulating lymphoma cells in the bloodstream are rarely detected by conventional morphology or flow cytometry evaluation. We developed a high-throughput sequencing based platform, LymphoSIGHT, to detect evidence of lymphoid malignancies in peripheral blood samples, as this could potentially be used for detection of minimal residual disease after treatment. This sequencing method has a sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood. We herein report the results of our pilot study assessing the ability of this method to detect the lymphoma clone in peripheral blood samples from 5 DLBCL patients at the time of diagnosis. Methods: This study has been approved by IRB and consent has been obtained from patients. Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and peripheral blood samples (plasma and peripheral blood mononuclear cell (PBMC) compartments) collected at initial diagnosis. Amplified products were sequenced to obtain >1 million reads (>10× sequencing coverage per IgH molecule), and were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the lymph node biopsy sample. The presence of the tumor-specific clonotype was then quantitated in cell-free and PBMC compartments from the diagnostic blood sample. A quantitative and standardized measure of clone level among all leukocytes in the diagnostic sample was determined using internal reference DNA. Results: We detected a high-frequency IgH clonal rearrangement in all 5 lymph node biopsy samples. The lymphoma clonotype that was identified in the tumor biopsy was also detected in the plasma and/or PBMC compartment in all 5 patients at diagnosis. Specifically, the lymphoma clonotype was detected in the plasma compartment in 4 patients, while 3 patients demonstrated the presence of the lymphoma clonotype in the PBMC compartment (Table 1). We hypothesize that the positive lymphoma clone in the plasma is due to rapid proliferation and necrosis of the primary tumor, releasing the degraded component of lymphoma into the blood stream. However, in this small sample size, we did not observe an obvious correlation between the level of detection (PBMC or plasma) and clinical parameters (LDH, stage, size of tumor, tumor Ki67, cell-of-origin). All patients achieved complete response after initial treatment and four are being followed. We plan to analyze blood specimens while they are in remission. Conclusions: IgH clonal rearrangements were detected by sequencing in all tumor biopsy samples. Importantly, all peripheral blood samples showed signs of circulating lymphoma material in either the plasma or PBMC compartment at diagnosis. Analysis of diagnostic and post-therapy samples from additional DLBCL patients is ongoing. These data will determine whether the sequencing assay is a strong indicator for response to therapy and relapse monitoring. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Carlton:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Deep Sequencing Reveals Oligoclonality At The Immunoglobulin Locus In Multiple Myeloma Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 401-401 ◽  
Author(s):  
Joaquin Martinez-Lopez ◽  
Mariateresa Fulciniti ◽  
Santiago Barrio ◽  
Victoria Carlton ◽  
Martin Moorhead ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
High Frequency ◽  
Genomic Dna ◽  
Gene Rearrangement ◽  
Common Ancestor ◽  
B Cell Development ◽  
Employment Equity ◽  
A Cell ◽  
Equity Ownership

Abstract Introduction Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. Multiple myeloma (MM) is a malignancy of the plasma cells, which are at the terminal stage of B cell development. MM is a clonal disease originating from the transformation process of a single plasma cell and, thus, myeloma cells are traditionally thought to have one clonal Ig gene sequence that remains stable throughout the course of the disease. Based on preliminary evidence of oligoclonality, we utilized the LymphoSIGHT™ platform, a high-throughput sequencing method, to detect evidence of oligoclonality at the Ig heavy and kappa chain (IGH and IGK) loci. The sequencing approach can be used to examine two general models for oligoclonality. In the first model, two unrelated clonal Ig sequences are observed indicating the presence of two independent myelomas. Alternatively, in the second model, two related myeloma clonal Ig sequences are observed indicating that both myeloma clones are derived from a common ancestor that arose after the pro B cell stage when VDJ recombination is completed. The common ancestor can be a cell with premalignant lesion or after the MM has developed. Using the sequencing platform, we looked for evidence of these two models of oligoclonality in 193 MM patients. Methods Two cohorts of newly diagnosed MM patients were included in this analysis (N=125, N=68). Using universal primer sets, we amplified IGH and IGK variable, diversity, and joining gene segments from genomic DNA or RNA from bone marrow collected at initial diagnosis. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination (Faham et al, Blood 2012). In the first cohort (N=125), we assessed gene rearrangement at the IGH-VDJ and IGK loci in 120 patients using RNA only and in 5 patients, we used both DNA and RNA to assess the IGH-VDJ, IGH-DJ and IGK loci. In the second cohort (N=68), we analyzed gene rearrangement at the IGH-VDJ, IGH-DJ and IGK loci using genomic DNA. Myeloma-specific clonotypes were identified for each patient based on their high frequency (5%) within the B-cell repertoire in the diagnostic sample. To identify clonotypes that are present in more than one cell we looked for patterns that are not consistent with having a maximum of one functional and one non-functional clonotype in a cell. Results We observed oligoclonality in 23 of 193 (12%) MM patients. Unrelated Ig sequences, which are consistent with the first model of oligoclonality, were present in 8 of the 193 (4%) patients. Fifteen of 193 (8%) patients exhibited related Ig clones, which is consistent with the second model of oligoclonality. In 4 of the 15 patients clones were related to each other via a somatic hypermutation process and differed by only a few bases (Figure 1), while in other 11 patients, the same VDJ sequence was associated with two distinct isotypes (IgA and IgG). Interestingly, in cases with both RNA and DNA sequencing and oligoclonality, we observed differential expression levels compared to clonal content at the DNA level, suggesting that a low frequency clone could be contributing as a predominant secretory clone. Conclusions This study demonstrates frequent oligoclonality in MM patients and suggests that this phenomenon does occur due to two distinct processes, either as unrelated sequences consistent with independent clones or as related sequences consistent with evolution after the MM malignant lesions occur. These findings shed light on the biology and pathogenesis of MM and may provide prognostic information. Currently, this analysis was limited to high frequency clones, using a threshold of 5% for identification of the myeloma-specific clones. Additional analysis is being performed to assess the presence of lower frequency clones, and data will be presented. Disclosures: Carlton: Sequenta, Inc. : Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc. : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Defining the Incidence and Clinical Impact of Genomic Alterations Across Different Histologic Types of Lymphoma Using a Clinically Validated Comprehensive Targeted Sequencing Assay

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2668-2668
Author(s):  
Connie Batlevi ◽  
Franck Rapaport ◽  
Andrew M. Intlekofer ◽  
Anne Reiner ◽  
Craig H Moskowitz ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Peripheral Blood ◽  
Clinical Relevance ◽  
Research Funding ◽  
Advisory Board ◽  
Genomic Sequencing ◽  
Genomic Alterations ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Background: Lymphoma is a clinically and molecularly heterogenous disease. Next generation sequencing of primary lymphoma samples has identified common recurring genomic alterations (GAs). The distribution and frequency of recurring GAs across lymphoma subtypes remains unknown because prior studies vary in sequencing methods, depth of coverage, and specimen source. In this study, we benchmark the distribution of GAs across different lymphoma subtypes by prospectively analyzing lymphoma cases and performing comprehensive DNA/RNA targeted sequencing of genes commonly found in hematologic malignancies using the Foundation One Heme (F1H) clinical assay. Methods: After obtaining proper consent, archived specimens from 183 samples [formalin fixed paraffin embedded (FFPE) N=141, peripheral blood N=28, BM aspirate N=14] distributed across lymphoma subtypes (including 62 DLBCL, 38 T cell lymphoma, 32 FL, 17 CLL, 13 MCL) were sequenced to high, uniform coverage averaging >600x for DNA, >20 million pairs for RNA. GAs were determined, including base substitutions, small insertions and deletions, rearrangements, and copy number alterations. Significant non-synonymous variants were identified as mutations from the COSMIC database, amplifications of established oncogenes, or homozygous deletions and/or clear loss-of-function mutations of known tumor suppressors. Fisher's exact test with Monte Carlo estimation corrected by false discovery rate was used for associations. Results: Samples from prospectively consented patients were banked for a median of 30 days prior to genomic analysis, range 1 day to 6.5 yr. Sequencing data was reported a median of 16 days from sample date receipt. GAs were identified in 95% of samples, with a median of 4 GAs/sample. The most common GAs were TP53 (29%), MLL2 (27%), BCL2 (25%), CDKN2A/B (17%) and CREBBP (14%). Alterations of chromatic modifiers (80%), BCR/NFkB components (51%), and cell cycle pathway (44%) were most common. In our group of unpaired follicular lymphoma samples (N=7 treatment naïve, N=25 treatment exposed), the number of GAs increased with treatment exposure. We found similar gene and biological signatures regardless of prior therapy; however differences emerge in genes of potential clinical relevance. Sequencing profiles augmented or altered the pathologic diagnosis in 11 of 183 (6%) of the cases. Importantly we were able to classify the GAs as actionable, potentially actionable and variants of unclear significance to better define the clinical relevance of targeted genomic sequencing. Conclusions: Integration of comprehensive next generation targeted genomic sequencing and clinical analysis in lymphoma provides an opportunity to describe the spectrum and incidence of GAs across different lymphoma subtypes and provide guidance on application of genomic profiling. This work serves to benchmark GAs across all lymphoma subtypes in a clinically relevant population and enables design of basket trials selecting patients based on shared genomic and biologic similarity instead of lymphoma subtype. To our knowledge, this is the largest repository of clinically annotated genomic sequencing in lymphoma. Table 1. Total Specimens N = 183 Median Age at Diagnosis 57 Range 21 - 84 Median Age at Biopsy 61 Range 21 - 91 Sex • Male • Female 113 70 62% 38% Biospecimen source • Paraffin embedded • Peripheral blood • Marrow aspirate 141 28 14 77% 15% 8% Patient consent • Prospective consent • Retrospective consent 145 38 79% 21% Prospectively consented patients (N=145) Median Days to Result Median Age of Sample 16 30 days 8 - 81 1 day - 6.5 yr Disclosures Palomba: Janssen: Consultancy. Gerecitano:Genentech: Consultancy, Other: Advisory Board; AbbVie: Consultancy, Other: Advisory Board. Matasar:Spectrum: Consultancy; Genentech: Consultancy. Straus:Millenium Pharmaceuticals: Research Funding. He:Foundation Medicine, Inc.: Employment, Equity Ownership. Balasubramanian:Foundation Medicine: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine: Employment. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy. Younes:Celgene: Honoraria; Johnson and Johnson: Research Funding; Novartis: Research Funding; Bayer: Honoraria; Bristol Meyer Squibb: Honoraria; Sanofi-Aventis: Honoraria; Seattle Genetics: Honoraria, Research Funding; Curis: Research Funding; Janssen: Honoraria; Takeda Millenium: Honoraria; Incyte: Honoraria.

scholarly journals An All Antibody Approach for Conditioning Bone Marrow for Hematopoietic Stem Cell Transplantation with Anti-cKIT and Anti-CD47 in Non-Human Primates

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4428-4428
Author(s):  
Kristopher D Marjon ◽  
James Y Chen ◽  
Jiaqi Duan ◽  
Timothy S Choi ◽  
Kavitha Sompalli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Mast Cell Degranulation ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership

Background Hematopoietic stem cell (HSC) transplantation (HSCT) is a well-established procedure that, with or without gene therapy, is curative for numerous severe life-threatening diseases including genetic blood disorders and blood cancers. While advances have been made, there are still substantial concerns since these chemo- and radiation therapy based procedures cause long-term toxicities such as infertility and secondary malignancies or even result in high mortality. We have previously established in a series of preclinical studies a novel chemo- and radiation-free non-toxic monoclonal antibody (Ab) -based conditioning regimen for autologous and allogeneic HSCT (Czechowicz et al., Akanksha et al. and George et al.). This cKIT-CD47 Ab-based regimen selectively depletes host HSCs for HSCT while sparing off-target toxicities caused by chemotherapy/radiation. By significantly decreasing morbidity/mortality associated with traditional conditioning regimens, antibody-mediated conditioning could expand the patient population eligible to receive HSCT for a variety of disorders. We developed a novel cKIT Ab (FSI-174), with an active Fc, and in combination with our CD47 magrolimab (previously 5F9, blocks the don't eat me pathway) could be utilized to translate the promising preclinical findings into clinical studies for safe and less toxic bone marrow conditioning for HSCT. Here we present the functional characterization of FSI-174 as single Ab and in combination with magrolimab in vitro and in non-human primate (NHP) studies. Methods We tested if FSI-174 could block stem cell factor signaling and we explored if FSI-174 alone or in combination with magrolimab could promote phagocytosis of cKIT positive cells (Kasumi-1). In addition, we determined if FSI-174 could cause mast cell degranulation. Subsequently, we explored the potential of FSI-174 alone (Phase A) or in combination with magrolimab (Phase B) to deplete HSCs in NHPs (rhesus macaques)in vivo. In Phase A, single doses of FSI-174 (0.3, 1, or 3 mg/kg) were administered alone. In Phase B, FSI-174 (0.3 or 3 mg/kg) was administered in combination with magrolimab (5mg/kg priming and 20 mg/kg maintenance dose). Bone marrow aspirates and core biopsies and peripheral blood were sampled before the study start and throughout the study. Frequency of bone marrow HSCs and cKIT receptor occupancy (RO) was determined by flow cytometry. In addition, the PK profile of FSI-174 was determined. Results In-vitro analysis demonstrated that FSI-174 decreases proliferation of HSPCs and enhances phagocytosis of cKIT positive cells, and the addition of magrolimab synergistically enhances the phagocytosis. Strikingly, FSI-174 did not cause mast cell degranulation in vitro. In the NHPs, complete (100%) cKIT receptor occupancy was achieved at all FSI-174 dose levels and was maintained for 1 to 9 days correlating with increasing doses and pharmacokinetics. The FSI-174 Cmax was found to be proportional to dose and mean Cmax increased from 6.25 ug/mL to 49.2 ug/mL. In Phase A, FSI-174 alone did not decrease the frequency of bone marrow HSCs compared to PBS control and had no effect on the peripheral blood cell counts. However, in Phase B, when FSI-174 was combined with magrolimab it significantly decreased the frequency of bone marrow HSCs with the nadir at day 9 and no recovery over 85 days compared to PBS control. Notably, there were no changes in peripheral blood cell counts over the course of the studies with no cytopenias in combination treatment. Conclusions We have developed a novel cKIT Ab (FSI-174) that meets the desired profile of stem cell factor block, promotion of phagocytosis, but without promoting mast cell degranulation. Furthermore, in the NHPs studies we have confirmed our chemo- and radiation-free cKIT-CD47 Ab -based conditioning approach with FSI-174 and magrolimab. As anticipated by our previous preclinical studies, monotherapy with FSI-174 does not deplete bone marrow HSCs in NHPs. Notably, no cytopenias are observed with either monotherapy or combination therapy. These data demonstrate the specificity, efficacy and safety of FSI-174/ magrolimab combination have great potential for conditioning regimen for HSCT in a chemotherapy and radiation free manner. Given the favorable safety profile of magrolimab across several clinical studies, these results are paving the way to the first-in-human trials for this novel conditioning for HSCT. Disclosures Marjon: Forty Seven Inc: Employment, Equity Ownership. Chen:Forty Seven Inc.: Consultancy, Equity Ownership. Duan:Forty Seven Inc.: Employment, Equity Ownership. Choi:Forty Seven inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Feng:Forty Seven Inc: Employment, Equity Ownership. Mata:Forty Seven inc: Employment, Equity Ownership. Chen:Forty Seven Inc: Employment, Equity Ownership. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding; Kymab: Consultancy; Jazz: Research Funding. Chao:Forty Seven Inc: Employment, Equity Ownership. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Agoram:Forty Seven Inc.: Employment, Equity Ownership. Majeti:FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties.

Results from Phase 1/2 Trial of Tagraxofusp in Combination with Pomalidomide and Dexamethasone in Relapsed or Refractory Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3145-3145 ◽  
Author(s):  
Paul G. Richardson ◽  
Myo Htut ◽  
Cristina Gasparetto ◽  
Jeffrey A. Zonder ◽  
Thomas G. Martin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Phase 1 ◽  
Single Agent ◽  
Advisory Board ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Background: The bone marrow microenvironment of many multiple myeloma (MM) patients contains high levels of CD123-expressing plasmacytoid dendritic cells (pDCs). These pDCs have been shown to augment MM growth and contribute to drug resistance (Chauhan, et al., Cancer Cell, 2009). Tagraxofusp, a novel CD123 targeted therapy, has demonstrated high levels of anti-tumor activity in patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive CD123+ malignancy of pDC origin. Tagraxofusp demonstrated potent in vitro and in vivo activity against MM cell lines and primary tumor samples via both a direct anti-MM effect and indirect pDC-targeting effect (Ray, et al., Leukemia, 2017), as well as demonstrating synergy in these systems when used in combination with traditional MM therapies including pomalidomide (POM). As such, targeting pDCs with tagraxofusp may offer a novel therapeutic approach in MM. Methods: This multicenter, single arm Phase 1/2 trial enrolled patients with relapsed or refractory (r/r) MM and tested two different doses of tagraxofusp (7 or 9 mcg/kg). Patients received tagraxofusp as a daily IV infusion for days 1-5 of a 28-day cycle as a single agent for the initial run-in cycle (cycle 0) and in combination with standard doses/administration of POM and dexamethasone (DEX) in cycles 1 and beyond. Objectives included evaluation of safety and tolerability, identification of the maximum tolerated or tested dose, and efficacy. Results: 9 patients with r/r MM received tagraxofusp (7 mcg/kg, n=7; 9 mcg/kg, n=2). 5 males, median age 65 years (range: 57-70), median 3 prior therapies (range 2-6). Median follow-up was 12 months (range: 7 - 19). The most common treatment-emergent AEs (TEAEs) were hypoalbuminemia 67% (6/9); chills, fatigue, insomnia, nausea and pyrexia each 56% (5/9); and dizziness, headache, hypophosphatemia, and thrombocytopenia each 44% (4/9). The most common grade 3 and 4 TEAEs were thrombocytopenia 44% (4/9) and neutropenia 33% (3/9). No grade 5 events reported. 5 patients treated with tagraxofusp and POM+DEX had a partial response (PR) after tumor evaluation. These patients demonstrated a rapid decrease in a set of myeloma-related laboratory values from pre-tagraxofusp treatment levels after the first combination cycle of tagraxofusp and POM+DEX. Additionally, these 5 patients demonstrated >50% decreases in peripheral blood pDC levels after both tagraxofusp monotherapy and combination therapy. Conclusions: Tagraxofusp was well-tolerated, with a predictable and manageable safety profile, when dosed in combination with POM+DEX in patients with r/r MM. Evidence of pDC suppression in peripheral blood and BM was observed in this patient population. 5 patients that received tagraxofusp and POM+DEX combination had PRs and decreases in pDC levels while on treatment with tagraxofusp. Given CD123 expression on pDCs in the tumor microenvironment and the potential synergy of tagraxofusp with certain MM agents including POM, tagraxofusp may offer a novel mechanism of action in MM. NCT02661022. Disclosures Richardson: Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding. Gasparetto:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Janssen: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; BMS: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed . Zonder:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees. Martin:Roche and Juno: Consultancy; Amgen, Sanofi, Seattle Genetics: Research Funding. Chen:Stemline Therapeutics: Employment, Equity Ownership. Brooks:Stemline Therapeutics: Employment, Equity Ownership, Patents & Royalties. McDonald:Stemline Therapeutics: Employment, Equity Ownership. Rupprecht:Stemline Therapeutics: Employment, Equity Ownership. Wysowskyj:Stemline Therapeutics: Employment, Equity Ownership. Chauhan:C4 Therapeutics.: Equity Ownership; Stemline Therapeutics: Consultancy. Anderson:Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder .

Detection Of Classical Hodgkin Lymphoma In Peripheral Blood Using High-Throughput Sequencing Assay

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 627-627 ◽  
Author(s):  
Yasuhiro Oki ◽  
Sattva S. Neelapu ◽  
Michelle A. Fanale ◽  
Larry W. Kwak ◽  
Luis E Fayad ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
High Throughput Sequencing ◽  
Residual Disease ◽  
Early Stage ◽  
Lymph Node Biopsy ◽  
Employment Equity ◽  
Blood Samples ◽  
Node Biopsy ◽  
Stage Disease ◽  
Equity Ownership

Abstract Background It was only a little more than a decade ago that the origin of the pathognomonic Reed Sternberg cells of Classical Hodgkin lymphoma (CHL) was established as B-cell malignancy characterized by a clonal expansion (Marafioti et al. Blood 2000). A previous report suggested that clonotypic B cells may be present in the blood of newly diagnosed CHL patients by virtue of identifying some light chain restricted populations by flow cytometry (Jones et al. Blood 2009). However, definitive investigation to assess whether CHL clones are detectable in the blood has been lacking. We developed the LymphoSIGHT™ platform, a high-throughput sequencing-based method, to detect evidence of lymphoid malignancies in peripheral blood samples, as this could potentially be used for detection of minimal residual disease (MRD) after treatment (Faham et al. Blood 2012). This sequencing platform has a sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood. We conducted a pilot study to assess the ability of this platform to detect the lymphoma clone in peripheral blood samples from 13 CHL patients at the time of diagnosis or disease recurrence. Methods This study was IRB-approved and consent was obtained from all patients. Using universal primer sets, we amplified immunoglobulin heavy chain (IGH) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and peripheral blood samples (plasma and peripheral blood mononuclear cell [PBMC] compartments) collected at initial diagnosis or at the time of recurrence. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the lymph node biopsy sample. The presence of the tumor-specific clonotype was then quantitated in plasma and PBMC compartments from a blood sample obtained around the time of primary tissue biopsy. A quantitative and standardized measure of clone level among all leukocytes in the sample was determined using internal reference DNA. Results Clinical and histopathological characteristics are summarized in Table 1. We detected a high-frequency IGH clonal rearrangement in 8 of 13 (62%) lymph node biopsy samples. We observed a trend for a low rate of lymphoma clonotype identification in untreated patients with early stage disease (50%, 4 of 8 patients), and a higher rate of identification in patients with relapsed disease (80%, 4 of 5). In the 8 patients with an identified lymphoma specific clonotype in the biopsy sample, the clonotype was also detected in the plasma and/or PBMC compartment in 7 (88%) patients (Table 1). We detected a lymphoma clonotype more frequently in plasma (88%, 7 of 8) than in PBMC (33%, 2 of 6). Conclusions Our data is the first to show that circulating clonal tumor cells can be detected in the blood of patients with CHL, providing new opportunities to explore novel methods to detect minimal residual disease. Additional cases are currently being analyzed to determine the sensitivity and specificity in early stage versus advanced stage disease, and to determine whether the detection of MRD post therapy would correlate with clinical relapse. Disclosures: Carlton: Sequenta, Inc: Employment, Equity Ownership. Kong:sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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