Minimal Residual Disease (MRD) Detection By Deep Sequencing In Newly Diagnosed Multiple Myeloma Patients Treated With Carfilzomib, Lenalidomide and Dexamethasone

Abstract Background Multiple myeloma (MM) therapies are becoming increasingly effective with deeper responses. Indeed, recent prospective clinical trials based on carfilzomib, lenalidomide and dexamethasone (CRd) show complete response (CR)/near (n)-CR rates of over 75%. Consequently, newer techniques are needed to detect minimal residual disease (MRD). We are conducting a prospective Phase II clinical trial studying response to CRd in newly diagnosed MM patients. We assessed treatment responses obtained by traditional IMWG response criteria, MRD measurement by 8-color multiparameter flow cytometry (MFC) based on a minimum of 3 million events (maximum detection rate: 0.0005%), and MRD measurement by high throughput sequencing in 14 newly diagnosed MM patients who achieved very good partial response (VGPR)/CR/nCR. Methods Bone marrow (BM) and/or plasma samples were obtained from 14 newly diagnosed MM patients receiving CRd therapy. Samples were subjected to deep sequencing using the LymphoSIGHT™ platform, which has a sensitivity to detect one cancer cell per million leukocytes in peripheral blood (Faham et al, Blood 2012). Briefly, using universal primer sets, we amplified immunoglobulin heavy and kappa chain (IGH and IGK) variable, diversity, and joining gene segments from genomic DNA obtained from CD138+ BM cell lysate at baseline, as well as post-treatment plasma samples. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency within the B-cell repertoire in the CD138+ cell lysate BM sample. The presence of the myeloma-specific clonotype was then quantified in plasma samples obtained post-treatment. A quantitative and standardized measure of clone level among all leukocytes in the sample was determined using internal reference DNA. For patients who achieved VGPR/CR/nCR, we compared the results from MFC of the BM with those obtained by sequencing the cell free DNA in plasma samples. Results We detected a high frequency myeloma-specific gene rearrangement in 13 of 14 (93%) CD138+ BM cell lysate samples obtained at diagnosis. We assessed for MRD by flow cytometry and deep sequencing for the seven patients who were in VGPR/CR/nCR based on traditional protein response criteria. One patient was MRD positive by flow cytometry of the marrow and negative by deep sequencing in blood. Another patient was MRD positive by deep sequencing but negative by flow cytometry. Additionally, in the diagnostic BM sample of one patient, we observed two distinct high-frequency IGH clones that were related through a process of somatic hypermutation. The specific mutation pattern observed is consistent with a branched model of evolution where the two observed clones did not evolve from each other but rather from a common ancestor. We are currently analyzing samples collected at baseline and post-treatment in an additional 23 MM patients enrolled on the CRd trial, and results will be presented. Conclusions The development of sensitive, non-invasive MRD assays is becoming increasingly important to assess the impact of modern anti-myeloma therapies. One novel approach for MRD detection, termed the LymphoSIGHT™ platform, relies on high-throughput sequencing of VDJ rearrangements at the immunoglobulin locus. Based on our CRd clinical trial for newly diagnosed MM patients, we found sequencing of cell free tumor DNA in bone marrow aspirates and peripheral blood (plasma) to be technically feasible. Among 6 patients who obtained VGPR/CR/nCR and were found to be MRD negative by MFC of the BM, we found one patient to be MRD positive by sequencing in the peripheral blood (plasma compartment). One patient was MRD positive by flow cytometry but negative by deep sequencing. These preliminary results suggest that a sequencing-based MRD blood test may be more sensitive than standard protein response criteria and complementary to MFC-based BM tests. Analysis of additional samples will be presented. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Weng:Sequenta, Inc.: Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Research Funding.

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Identification of molecular signatures of cystic fibrosis disease status with plasma-based functional genomics

Physiological Genomics ◽

10.1152/physiolgenomics.00109.2018 ◽

2019 ◽

Vol 51 (1) ◽

pp. 27-41 ◽

Cited By ~ 6

Author(s):

Hara Levy ◽

Shuang Jia ◽

Amy Pan ◽

Xi Zhang ◽

Mary Kaldunski ◽

...

Keyword(s):

Cystic Fibrosis ◽

Flow Cytometry ◽

Functional Genomics ◽

Treatment Response ◽

Peripheral Blood ◽

Disease Status ◽

Enzyme Linked Immunosorbent Assay ◽

Molecular Signatures ◽

Healthy Controls ◽

Plasma Samples

Although cystic fibrosis (CF) is attributed to dysfunction of a single gene, the relationships between the abnormal gene product and the development of inflammation and progression of lung disease are not fully understood, which limits our ability to predict an individual patient’s clinical course and treatment response. To better understand CF progression, we characterized the molecular signatures of CF disease status with plasma-based functional genomics. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured with plasma samples from CF patients ( n = 103) and unrelated, healthy controls ( n = 31). Gene expression levels were measured with an Affymetrix microarray (GeneChip Human Genome U133 Plus 2.0). Peripheral blood samples from a subset of the CF patients ( n = 40) were immunophenotyped by flow cytometry, and the data were compared with historical data for age-matched healthy controls ( n = 351). Plasma samples from another subset of CF patients ( n = 56) and healthy controls ( n = 16) were analyzed by multiplex enzyme-linked immunosorbent assay (ELISA) for numerous cytokines and chemokines. Principal component analysis and hierarchical clustering of induced transcriptional data revealed disease-specific plasma-induced PBMC profiles. Among 1,094 differentially expressed probe sets, 51 genes were associated with pancreatic sufficient status, and 224 genes were associated with infection with Pseudomonas aeruginosa. The flow cytometry and ELISA data confirmed that various immune modulators are relevant contributors to the CF molecular signature. This study provides strong evidence for distinct molecular signatures among CF patients. An understanding of these molecular signatures may lead to unique molecular markers that will enable more personalized prognoses, individualized treatment plans, and rapid monitoring of treatment response.

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

Blood ◽

10.1182/blood.v120.21.321.321 ◽

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.

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Overcoming Drug Resistance of Pre-B ALL Cells By Targeting Integrin alpha6 Associated Cell-Adhesion Mediated Drug Resistance Using a Novel Antibody, P5G10

Blood ◽

10.1182/blood.v126.23.2525.2525 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2525-2525

Author(s):

EunJi Gang ◽

Yao-Te Hsieh ◽

Hye Na Kim ◽

Yann Duchartre ◽

Shishido Stephanie ◽

...

Keyword(s):

Flow Cytometry ◽

Drug Resistance ◽

Cell Adhesion ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

Post Treatment ◽

Leukemia Cells ◽

Laminin 1

Abstract Background. The presence of chemotherapy-resistant cells can be detected in the bone marrow (BM) and peripheral blood (PB) and is called minimal residual disease (MRD). The exact mechanism for cell adhesion-mediated drug resistance leading to MRD and how to therapeutically target MRD is unsolved.Integrin alpha-6 (alpha6) is expressed on normal hematopoietic cells but has recently been described as a novel marker for MRD+ ALL cells. We previously described a role of alpha6 as a critical molecule in drug resistance of ALL. Here we extend our studies evaluating a novel non-humanized antibody targeting alpha6, P5G10, as a novel therapy against drug resistant ALL. Method. For in vitro studies patient-derived (primary) pre-B ALL cells co-cultured with murine calvaria-derived mesenchymal stromal (OP9) cells or counter-ligand laminin-1. Annexin V/7-AAD staining was used for viability determination by flow cytometry. A NOD/SCID IL2Rγ-/- (NSG) xenograft model of primary pre-B ALL was used for in vivo experiments. Results. We evaluated integrin alpha6 blockade in four primary ALL cells (LAX7R, PDX2, TXL3, SFO2) using an anti-functional alpha6 antibody, P5G10, with and without the counter ligand laminin-1 or OP9. Alpha6 blockade de-adhered all four cases from laminin-1 compared to control-treated cells and percentage of adherence was significantly different (3.3%±0.6% vs. 77.7%±3.3%, p= 0.0002 for LAX7R; 10.5%±4.9% vs. 72.5%±0.7%, p= 0.003 for PDX2; 2.0%±1.3% vs. 66.9.6%±2.6%, p=0.0002 for TXL3; 9.6%±2.8% vs. 68.0%±5.7%, p=0.0006 for SFO2).. P5G10 de-adhered leukemia cells to a lesser degree from OP9-coated plates indicating that other adhesion molecules also contribute to leukemia cell adhesion. To determine the effect of alpha6 modulation in chemoresistant ALL, primary BCR-ABL1- ALL cells (LAX7R, SFO2) were treated with Vincristine, Dexamethasone and L-Asparaginase (VDL) and BCR-ABL1+ ALL cells (TXL3, PDX2) were treated with a tyrosine kinase inhibitor (TKI), Nilotinib (NTB). Primary ALL cells showed decreased viability after monotreatment with P5G10 in a short-term assay of 2 days with laminin-1 and were sensitized when P5G10 was combined with VDL or TKI, compared to TKI monotreatment (Cell viabilities were as follows: LAX7R, 13.9%±0.6% vs. 28.8%±2.6%, p=0.009; PDX2, 12.7%±1.4% vs. 19.9%±1.5%, p= 0.037: TXL3, 32.9%±2.6% vs. 48.3%±2.5%, p=0.026; and SFO2, 34.4%±7.9% vs. 47.6%±0.1%, p=0.047). Critically, in a long-term co-culture assay of primary ALL cells with OP9 cells, alpha6 blockade in combination with VDL or NTB lead to marked decrease in viability of ALL cells compared to VDL or NTB treatment (26.5%±10.0% vs. 74.2%±2.7%, p=0.002 for LAX7R and 33.5%±11.4% vs. 84.9%±15.1%, p=0.031 for SFO2 on day 17 post treatment, respectively). To determine if P5G10 induces mobilization of leukemia cells to the peripheral blood, patient-derived ALL cells, 3 cases (TXL3, PDX2 and LAX7R) were injected into NSG mice. After determination of engraftment of leukemia by flow cytometry of human CD45 in the PB, recipient mice were treated with 30mg/kg P5G10 or PBS control by i.v. or i.p. injection. The % of human CD45+ and CD19+ in peripheral blood (PB) was analyzed by flow cytometry before (pre), 1 and 3 days after (post) treatment with P5G10. In all 3 cases, we did not observe an increase of leukemia cells in the PB compared to before P5G10 treatment (Day 0) or compared to the control recipient mice. Critically, we determined, if P5G10 can restore chemosensitivity of leukemia cells in vivo. For this purpose, we injected luciferase-labeled LAX7R cells into NOD/SCID mice. Three days after leukemia cell injection leukemia cell-bearing mice received four weekly injections of 30mg/kg P5G10 or saline ± VDL. Mice treated with P5G10 survived similarly as untreated mice (PBS: MST = 39 days vs. P5G10: MST = 31 days; p=0.05). In marked contrast, mice treated with VDL plus P5G10 survived disease-free compared to chemotherapy-only treated mice until the experiment was terminated Day 186 post-leukemia injection (MST= 185 days vs. MST=71 days; p=0.0012). Human CD19 or CD45 was undetectable in the peripheral blood by flow cytometry in surviving P5G10+VDL-treated animals before they were sacrificed. Conclusion. Taken together, we demonstrate that alpha6 may be a novel therapeutic target in ALL and modulating the function of integrin alpha6 using P5G10 can overcome drug resistance in ALL. Disclosures No relevant conflicts of interest to declare.

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Increased Circulating Plasma Cells Predicts Poor Overall Survival in Symptomatic Plasma Cell Myeloma Patients

Blood ◽

10.1182/blood.v126.23.4205.4205 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4205-4205

Author(s):

Mi Hyun Bae ◽

Sang Hyuk Park ◽

Chan-Jeoung Park ◽

Bo Hyun Kim ◽

Young-Uk Cho ◽

...

Keyword(s):

Flow Cytometry ◽

Overall Survival ◽

Plasma Cell ◽

Peripheral Blood ◽

Plasma Cells ◽

Survival Curve ◽

Plasma Cell Myeloma ◽

Newly Diagnosed ◽

Kaplan Meier ◽

Meier Survival Curve

Abstract Backgrounds Flow cytometry can rapidly determine immunophenotypes of neoplastic plasma cells (PCs) and quantify PCs in patients with plasma cell myeloma. Flow cytometric immunophenotyping and quantification of neoplastic plasma cells is sensitive and reliable tool for diagnosis and disease monitoring in patients with monoclonal gammopathy. Circulating PCs (cPCs) in peripheral blood (PB) after autologous hematopoietic stem cell transplantation is a marker of high-risk disease in patients with plasma cell myeloma. We assessed the utility of quantification of cPCs using flow cytometry for risk stratification in newly diagnosed plasma cell myeloma patients in the era of novel agents. Methods PB and bone marrow (BM) aspirates of 85 newly diagnosed patients with symptomatic plasma cell myeloma from August 2013 to July 2014 were analyzed by five-color flow cytometry using monoclonal antibodies against CD45, CD19, CD56, CD38, and CD138. The gating strategy employed first used the expression of CD38 and CD138 to identify plasma cells among 100,000 to 200,000 events. cPCs in PB was determined according to the patient's specific immunophenotype of neoplastic PCs in BM. Results The median age of the patient population was 68 years (45~87) and 58% were female. Median follow-up duration was 19.2 months. Six out of 85 patients (7%) did not show cPCs. Among 79 patient (93%) who had detectable cPCs, the median cPCs was 0.09% (0.006~3.612%). Patients without cPCs or cPCs under 0.05% were assigned to low cPCs group (n=32, 38%) and others to high cPCs group (n=53, 62%) according to receiver operating characteristics analysis. High cPCs group showed higher level of BM neoplastic PCs detected by both methodologys of morphology and flow cytometry (P=0.002, 0.033, respectively), higher BM cellularity (P=0.011), higher serum M protein level (P=0.013), lower hemoglobin (P=0.008), and lower platelet level (P=0.034) than low cPCs group. High cPCs group was associated with adverse cytogenetics such as t(4;14) and monosomy 13 (P=0.008), and CD45 negative immunophenotype (P=0.007). In survival analysis, high cPCs presented shorter overall survival (OS) than low cPCs group (P=0.013) (Fig. 1). It was independent with patient age and cytogenetic risks (P =0.011). Conclusion By flow cytometry cPCs was detected in most symptomatic plasma cell myeloma patients. Increased cPCs ≥0.05% among PB leukocytes could be an independent prognostic factor showing adverse effect in overall survival in symptomatic plasma cell myeloma patients. Figure 1. Kaplan-Meier survival curve of patients with plasma cell myeloma who showed 0.05% or more circulating plasma cells in peripheral blood and patients with circulating plasma cells less than 0.05%. Figure 1. Kaplan-Meier survival curve of patients with plasma cell myeloma who showed 0.05% or more circulating plasma cells in peripheral blood and patients with circulating plasma cells less than 0.05%. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v128.22.2076.2076 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2076-2076

Author(s):

Barbara Muz ◽

Pilar De La Puente ◽

Justin A King ◽

Daniel R Kohnen ◽

Ravi Vij ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Complete Remission ◽

Peripheral Blood ◽

Research Funding ◽

Progressive Disease ◽

New Method ◽

The Novel ◽

Newly Diagnosed ◽

Novel Strategy

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

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TIM3 Dynamically Expressed on Peripheral Blood NK Cells in Acute Myeloid Leukemia Patients: The Expression Decreased When Untreated, and Restored While Disease Improved

Blood ◽

10.1182/blood.v128.22.5243.5243 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5243-5243

Author(s):

Caixia Li ◽

Xiaochen Chen ◽

Caihong Gu ◽

Tong Wang ◽

Qiu Zou ◽

...

Keyword(s):

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Nk Cells ◽

Clinical Significance ◽

Normal Control ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Newly Diagnosed ◽

Immunofluorescence Labeling ◽

Acute Myeloid

Abstract Objective: Preliminary study the expression and clinical significance of TIM3 on peripheral blood NK cells in patients with acute myeloid leukemia (AML). Methods: We investigated the expression characteristics of TIM3 on the peripheral blood NK cells of newly diagnosed AML patients and its clinical significance. Peripheral blood was obtained from 50 patients with newly diagnosed AML before intervention, with peripheral blood from 30 cases of healthy volunteers collected as normal control. Expression levels of TIM3 on the peripheral blood NK cells and subsets were assayed with flow cytometry. Then the expression of gamma interferon in Tim3+CD56+NK cells and Tim3-CD56+NK cells were analyzed by intracellular immunofluorescence labeling and flow cytometry. Results: Compared with the normal control, in the newly diagnosed AML patients, the NK cells percentage and the proportion of CD56dimNK/CD56+NK reduced, while the proportion of CD56brightNK/CD56+NK increased significantly, and the proportions of Tim3+CD56+NK/CD56+NK and Tim3+CD56dimNK/CD56dimNK decreased significantly. However, in AML patients with complete remission after chemotherapy, the proportion of peripheral blood NK cells and cell subsets recovered. Furthermore, compared with Tim3-CD56+NK cells, IFN-γsecretion level of Tim3+CD56+NK cells in the newly diagnosed AML patients is increased significantly. Conclusion: The dynamic expression of TIM3 on NK cell and its cell subsets before and after chemotherapy in AML patients suggest that Tim3 and NK cells may play an important role in the occurrence and development of AML. Disclosures No relevant conflicts of interest to declare.

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Robust Detection Of Minimal Residual Disease In Unselected Patients With B-Cell Precursor Acute Lymphoblastic Leukemia By High-Throughput Sequencing Of IGH

Blood ◽

10.1182/blood.v122.21.2550.2550 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2550-2550

Author(s):

David Wu ◽

Ryan O Emerson ◽

Anna Sherwood ◽

Mignon L. Loh ◽

Anne Angiolillo ◽

...

Keyword(s):

Flow Cytometry ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

High Throughput ◽

High Throughput Sequencing ◽

Lymphoblastic Leukemia ◽

Post Treatment ◽

Employment Equity ◽

Equity Ownership ◽

Pre Treatment

Abstract High-throughput sequencing (HTS) of immunoglobulin heavy chain genes (IGH) may be useful for detecting minimal residual disease (MRD) in acute lymphoblastic leukemia. We previously demonstrated the first application of high-throughput sequencing for the detection of minimal residual disease in T-cell precursor acute lymphoblastic leukemia (TPC-ALL) (Sci. Transl. Med. 4(134):134ra63. 2012). Recently, Faham and colleagues considered deep sequencing for MRD detection in B-cell precursor acute lymphoblastic leukemia (BPC-ALL) (Blood 120(26):5173-80, 2012). As this prior analysis in BPC-ALL apparently focused only on samples known to have a clonal rearrangement in IGH, the potential applicability and wide-spread utility of sequencing of IGH in unselected clinical samples for MRD has not been tested. Here, we consider an unselected cohort of patients enrolled in Children Oncology Group AALL0932 trial and use residual material from 99 patient samples submitted for routine multi-parametric flow cytometry (mpFC) at U. of Washington. One sample failed in the initial DNA extraction step and was not further considered. We show using high-throughput sequencing that clonal IGH rearrangements can be identified in 92 of the remaining 98 pre-treatment samples, using a definition of a V-D-J or D-J rearrangement comprising at least 10% of total nucleated cells (Fig. 1A). Similar to our prior findings in TPC-ALL, we find three subsets of patients—1) those for whom MRD is not detected by either flow cytometry or HTS; 2) those for whom MRD is detected both by flow cytometry and HTS; and 3) those for whom MRD is detected only by HTS, but not flow cytometry (Fig. 1B). There were no false negative results by HTS as compared to flow cytometry.Figure 1Measurement of clonal IGH rearrangement by high-throughput sequencing (HTS) or immunphenotypically abnormal B lymphoblast population by multi-parametric flow cytometry in pre-treatment (A) or day 29 post-treatment (B) residual samples. Results are reported for both HTS (red) and mpFC (blue) as clone frequency per total nucleated cells.Figure 1. Measurement of clonal IGH rearrangement by high-throughput sequencing (HTS) or immunphenotypically abnormal B lymphoblast population by multi-parametric flow cytometry in pre-treatment (A) or day 29 post-treatment (B) residual samples. Results are reported for both HTS (red) and mpFC (blue) as clone frequency per total nucleated cells. In the third group (HTS+positive, flow cytometry-negative), a subset of these patients, (5 of 28) had MRD detectable by HTS at a level within the expected sensitivity of flow cytometry. We hypothesized that in these cases that post-treatment MRD sequences may be present within the maturing B cell compartment that is not immunophenotypically aberrant by flow cytometry. To test this hypothesis, we analyzed eight additional post-treatment samples that were negative for MRD by flow cytometry. The mature B-cell fraction was collected by triple, flow cytometry-sorting and then sequenced by HTS for IGH rearrangements to search for the index clone defined in the corresponding, paired pre-treatment samples. Although a limited finding, diagnostic index IGH sequence was indeed identified in one of eight samples, in only the mature B-cell fraction, which is consistent with the proportion of cases with high-level MRD detected by HTS but which was missed by flow cytometry. Taken together, our results provide additional support for assessment of MRD in acute lymphoblastic leukemia by high-throughput sequencing. Our findings argue that precise quantification of the level of MRD by HTS will be important, and suggest that clonal IGH rearrangement sequences may be detected in an immunophenotypically normal population of mature B cells that may not be detected by flow cytometry. Disclosures: Emerson: Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Kirsch:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Wood:Becton Dickinson and Company, NJ, USA: Research Funding. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

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Circulating Plasma Cells in Newly Diagnosed Symptomatic Multiple Myeloma As a Prognostic Marker for Patients with Standard-Risk Cytogenetics

Blood ◽

10.1182/blood.v124.21.3353.3353 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3353-3353

Author(s):

Davide Vagnoni ◽

Fosco Travaglini ◽

Stefano Angelini ◽

Alessia Dalsass ◽

Francesca Mestichelli ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Peripheral Blood ◽

Poor Prognosis ◽

Plasma Cells ◽

Cox Model ◽

Significant Proportion ◽

Newly Diagnosed ◽

Operating Characteristics ◽

Standard Risk

Abstract Multiple Myeloma (MM) is a clonal B-cell disorder characterized by accumulation of malignant plasma cells (PCs) in the bone marrow (BM). Circulating PCs can be detected in the peripheral blood of a significant proportion of patients with MM and their presence is a well-known prognostic factor. Indeed, the appearance of circulating PCs in the blood could indicate relative indipendence from adhesion to the microenvironment, thus implying more aggressive disease. In this study, we examined the relationship between the number of PCs and citogenetic risk in patients with newly diagnosed MM. We analyzed peripheral blood from patients with Monoclonal Gammopathy of Undetermined Significance (MGUS; n=15), Smoldering Myeloma (SM; n=28), Solitary Plasmacytomas (SP; n=3) and active Multiple Myeloma (MM; n=105). These patients were followed by the U.O.C. Ematologia at the "Mazzoni" Hospital from January 2006 to December 2013, with a median follow-up of 25 months. We analyzed clinical, laboratory and cytogenetic data of patients with active MM. However, cytogenetic analysis was not evaluable for 15 patients. The number of circulating PCs was detected by flow cytometry using a simple two-colours approach. Cells were stained with fluorescence-labeled CD38 and CD45 antibodies and 50,000 events were acquired and analyzed for each patient. PCs were identified by gating on CD38bright+/CD45- cells. Using a receiver operating characteristics (ROC) analysis, we assessed that ³41circulating PCs is the optimal cut-off for defining poor prognosis. The 8-years probability of Overall Survival (OS) and Progression-Free Survival (PFS) in patients with <41 and ³41circulating PCs, was 32% vs 8% (p=0.017) and 29% vs 0% (p=0.0008), respectively. Patients with high-risk cytogenetics (n=24) had poor prognosis, independently of circulating PCs (PC<41 vs PC³41: OS=0% vs OS=16%, p=n.s.; PFS=0% vs 17%, p=n.s.). Patients with standard-risk cytogenetics (n=66) showed a better prognosis associated to a lower number of circulating PCs (PC<41 vs PC³41: OS=36% vs 10%, p=0.026; PFS=37% vs 0%, p=0.0001). These data were confirmed by multivariate analysis (Cox model) for the subgroup with standard-risk cytogenetics, in which the presence of ³41 circulating PCs, older age, DS stage >I and lack of maintenance therapy, adversely affected OS and PFS. All patients with SP showed no circulating PCs. In all cases of MGUS or SM, circulating PCs, when detected, were <20. In summary, our results suggest that the quantification of circulating PCs by flow cytometry could provide useful prognostic information in newly diagnosed MM patients with standard-risk cytogenetics. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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High-Throughput Sequencing of T-Cell Receptor Gene Loci for Minimal Residual Disease Monitoring in T Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.2545.2545 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2545-2545

Author(s):

David Wu ◽

Anna Sherwood ◽

Stuart S. Winter ◽

Kimberly Dunsmore ◽

Mignon L Loh ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

High Throughput ◽

High Throughput Sequencing ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

Post Treatment ◽

Pre Treatment ◽

Higher Sensitivity

Abstract Abstract 2545 There is increasing evidence for the utility of minimal residual disease (MRD) assessment in predicting clinical outcomes of patients with T cell lymphoblastic leukemia (T-ALL). Evaluation of MRD by PCR-based analysis of T-cell receptor (TCR) genes has a sensitivity of 10−5, but requires the use of individualized patient-specific primers, which is laborious, expensive and difficult to implement for real-time, clinical decision-making. Multi-parametric flow cytometry is currently limited to a sensitivity of 10−4, requires viable cells, and is poorly standardized. High-throughput DNA sequencing offers the potential to equal or surpass the higher sensitivity of PCR-based MRD testing with reduced cost, improved turn-around time, and better standardization. Paired samples of pediatric T-ALL from 14 patients enrolled on Children's Oncology Group AALL0434 were obtained at diagnosis and at day 29 post-induction therapy. The complementarity determining regions (CDR3) regions of TCRB and TCRG were sequenced for all 28 specimens using an Illumina GA2 platform as previously described (see Blood, 114(19):4099–4107, 2009 and Sci Transl Med. 3(90):90ra61, 2011). Pre-treatment samples were used to obtain unique TCR sequences for the leukemic clone, and post-treatment samples were assessed for the frequency of each TCR sequence as a percentage of the total. The frequency of each sequence was also enumerated in post-treatment samples from all other patients to evaluate specificity. These results were compared to MRD results obtained by 9-color flow cytometry per trial protocol. Eleven of 14 pre-treatment samples (78.6%) had a detectable clonal population based on TCRG sequence analysis, and 10 of these also had a clonal TCRB sequence. Five samples exhibited an additional unique TCRG sequence, consistent either with rearrangement of both TCRG loci or the presence of two clonal subpopulations. Two of 3 cases without a detectable clonal TCR gene sequence had the immunophenotype of early thymic precursor (ETP) T-ALL and would be expected to have germline TCRB and TCRG genes. No other cases were ETP. Clones were found in all 5 informative post-treatment samples positive for MRD by flow cytometry, as well as at a low level in 3 additional patients without MRD by flow cytometry, suggesting superior sensitivity for sequencing. The background sequence frequencies were very low (0–10−5) in other patient post-treatment samples, being slightly higher for TCRG than for TCRB, consistent with germline sequence diversity. We demonstrate the potential of high-throughput sequencing for analysis of MRD in pediatric T-ALL. The number of cases in which the assay is informative (78.6%) is similar to that seen with standard PCR MRD methods, but evaluation of more cases is needed. MRD by sequencing appears to have a higher sensitivity than current flow cytometric methods, although direct comparison of MRD frequencies from the two techniques is problematic and will require normalization. The strong association of ETP status and lack of clonal TCR sequence identification at diagnosis suggests utility in identifying this poor outcome subset of T-ALL. Disclosures: Sherwood: Adaptive TCR, Seattle, WA: Employment, Equity Ownership. Wood:Becton, Dickinson and Company, NJ, USA: Research Funding. Robins:Adaptive TCR, Seattle, WA: Consultancy, Equity Ownership, Patents & Royalties.

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Pilot Study Of Erlotinib In Patients With Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.5037.5037 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5037-5037

Author(s):

Hamid Sayar ◽

Magdalena Czader ◽

Chirag Amin ◽

Mary Cangany ◽

Larry D Cripe

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Pilot Study ◽

Disease Progression ◽

Peripheral Blood ◽

Bone Marrow Aspirate ◽

Molecular Profile ◽

Refractory Disease ◽

Newly Diagnosed ◽

Blast Count

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

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