Bone Marrow Aspirate Specimens Research Articles

Del20q and Gain/Loss of Sex Chromosomes in Patients with Plasma Cell Neoplasm

Abstract Aim Plasma cell neoplasm (PCN) is a clonal proliferation of plasma cells involving bone marrow and extramedullary anatomic sites. The purpose of this study is to investigate the prevalence and clinical significance of del20q and gain/loss of sex chromosomes in patients with PCN. Materials and Methods Routine karyotype analysis was performed on bone marrow aspirate specimens from patient with plasma cell neoplasm in the cytogenetics laboratory using the G-banding technology. Results from January 1, 2012 to June 21, 2019 were retrieved for analysis. Results Among the 8,665 karyotype results, 1,116 were abnormal and 593 demonstrated del20q and/or gain/loss of sex chromosomes. 33% (113/347) have bone marrow PCN when del20q and gain/loss of sex chromosomes occur in isolation, while 94% (256/250) have bone marrow PCN when 1 or more other autosomal abnormalities are also present. In those with isolated del20q or gain/loss of sex chromosomes, the frequency of bone marrow PCN ranges from 29–50%. Del20 occurs 1–6 years after therapy and in some patients is transient. Gain/loss of sex chromosome is generally present during the follow-up period. Conclusion Del20q and gain/loss of sex chromosomes do not appear to correlate with bone marrow PCN. Isolated Del20q likely represents transient treatment-related abnormality, while isolated gain/loss of sex chromosomes is probably age-related.

IgG4 plasma cell myeloma: Clinicopathological characteristics and diagnosis.

Plasma cell myeloma (PCM) is usually associated with the presence of M-protein in the serum and urine of patients, and about half of the PCM cases exhibit the IgG M-protein and increased gamma-globulin fraction on membrane electrophoresis. The IgG4 subclass is located in the beta-2 fraction on membrane electrophoresis. The aim of this study was to develop a method to evaluate IgG4-producing PCM (IgG4-PCM) and its clinicopathological characteristics. We found three cases of IgG4-PCM among 80 cases of IgG-producing PCM by membrane electrophoresis, which were confirmed by IgG4 immunostaining. None of the cases had a clinical history of IgG4-related disease, although they exhibited high levels of serum IgG4. A bone marrow aspiration specimen had an increased number of plasma cells with a relatively mature morphology. No cases exhibited lymphoplasmacytic inflammation, obliterative phlebitis or fibrosis. Immunohistochemistry revealed that tumor cells expressed CD138 and IgG4 and showed monoclonal expression of kappa. We revealed that IgG4-PCM might not be associated with IgG4-related disease and that the detection of M-protein with beta-globulin fraction by electrophoresis may be useful for screening IgG4-PCM.

Intramarrow injection needle: a new technique to treat patients with haematological and non-haematological malignancies by directly injecting therapeutic agents into the bone marrow cavity

AimsThe purpose of this report is to describe a new instrument designed for the injection of therapeutic agents directly into the bone marrow cavity as well as to aspirate bone...

Blastic plasmacytoid dendritic cell neoplasm of the uterus

Introduction. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and very aggressive hematological malignancy derived from precursor of the plasmacytoid dendritic cell. We present a case with cervix uteri involvement without skin lesions, which is, to the best of our knowledge, the first case of BPDCN localized in the cervix. Case outline. A 66-year-old previously healthy women initially presented with a four-week history of vaginal bleeding. Gynecologic examination revealed a tumorous bleeding formation on cervix uteri. Except paleness of the skin, physical examination results were normal. Complete blood counts showed anemia and thrombocytopenia. Computed tomography scans showed an expansive tumorous formation at the level of the isthmus and cervix uteri, 60 ? 42 mm in size. Cervical biopsy was done and final pathohistological diagnosis was BPDCN. Karyotype analysis results from the bone marrow aspiration specimen demonstrated tetrasomy of chromosome 2 and monosomy of chromosome 16. The patient did not accept treatment and died two months after the initial diagnosis was established. Conclusion. Attributes such as aggressive clinical course of BPDCN, demonstrated unusual localization, infrequency, and the absence of consensus about standard treatment options, demand constructive clinical reasoning and tight cooperation between medical professionals of various fields.

PB1906 FROM ACADEMY TO REAL WORLD: THE GRAY AREA OF THE LARGE GRANULAR LYMPHOCYTES LEUKEMIA

Background:Large granular lymphocytes (LGLs) are defined as large white blood cells (WBC) that are morphologically characterized by an abundant cytoplasm with azurophilic granulation. LGLs belong to CD3+CD56− cytotoxic T cells (CT) or natural killer (NK) cells. The NK cells are roughly defined by a lack of the T cell marker CD3 and a presence of CD56 (or NCAM). LGL leukaemia represents a chronic lymphoproliferative disorder of CTLs, a malignancy that involves lymphocyte infiltration of multiple organs, including the BM, liver, and spleen.Aims:The aim of this retrospective study was to achieve information about LGL and the association between LGL and clinical characteristics.Methods:A retrospective study analyzing the global diagnosis of LGL leukaemia and factors influencing the disease evolution at the district hospital and included cases with the previous or recent diagnosis of LGL leukaemia between January 2015 and December 2018. Special emphasis was taken on the results of bone marrow examination, flow cytometry, immunophenotype, and results of molecular studies to assess for T‐cell receptor (TCR) gene rearrangements. A total of 26 patients were confirmed as having LGL leukaemia, and they fulfilled the WHO classification criteria. May‐Grünwald‐Giemsa–stained peripheral blood smears were examined for the presence of large granular lymphocytes. Bone marrow trephine biopsy and aspirate specimens (when was applicable) were obtained. Bone marrow cellularity and the pattern of lymphocyte infiltration were determined on core biopsy or aspirate clot specimens stained with H&E. Statistic The Fisher exact test was used to analyze differences between the T‐LGL and T‐LGL/other LPD groups. The median time from completion of last treatment with rituximab to the diagnosis of LGL disorder was 30 months.Results:LGL leukaemia or T‐LGL expansion were identified in the global group. Near total of the patient 21 (80.7%) had a direct history of a clonal B‐cell lymphocytosis, monoclonal B‐cell lymphocytosis (MBL), or clonal B‐cell disorder concomitantly with or shortly after the LGL diagnosis. The other predominant diagnosis was rheumatoid conditions in 9 patients (34.6%) (including Felty syndrome) and monoclonal gammopathy of unknown significance (MGUS) 7 patients (26.9%). The median age at the time of diagnosis was 68.5 years (range, 20‐86 years). B‐symptoms were reported by 4 (15.38%) patients, as well as Lymphadenopathy/splenomegaly was identified in 5 (19.23%). 4 individuals received treatment prior to the diagnosis of LGL, and the predominant treatment was methotrexate/rituximab. The favourite regimens in our daily practice were cyclosporin, rituximab or intermittent G‐CSF. The overall response rate at 3 months and complete response rate for the various treatments were as follows: steroids (11% and 3%), methotrexate (55% and 21%), rituximab (64% and 47%), cyclosporine (19% and 3%), respectively. All patients responded to rituximab after methotrexate treatment had failed. The mean number of treatments was 3.4 (range, 1‐5).Summary/Conclusion:Whether or not the T‐LGL and T‐LGL/other LPD groups are correlated with specific clinical‐biological features remains uncertain and is the subject of ongoing research. We don’t perform routinely in our clinical practice an investigation for the Constitutive activation of STAT3. In our series, we have not reported direct LGL‐related deaths.

Clinical validation of long-range PCR assay for MLL partial tandem duplications for acute myeloid leukemia.

7033 Background: Partial tandem duplication of the mixed lineage leukemia gene ( MLL-PTD) occurs in about 3% to 5% of adult acute myeloid leukemia (AML), mostly in patients with normal karyotypes, and is associated with poor prognosis. Real-time PCR on cDNA or RT-PCR followed by gel electrophoresis are the most commonly used methods for the detection of the mutation. However, those methods require RNA extraction, which is not often performed for molecular cytogenetic testing or next generation sequencing (NGS) for AML. In this study, we developed an MLL-PTD detection assay that uses long-range PCR on extracted DNA, a commonly processed sample type for AML molecular testing, to reduce sample processing requirements. Methods: DNA was extracted from de-identified 72 whole blood and 30 bone marrow aspirate specimens. Two PCR reactions were performed for each DNA sample: one to detect MLL-PTD and another to detect CHEK2 to monitor DNA integrity. The 3 most frequent forms of MLL-PTD (exons 2~8, 2~9, and 2~10) were detected using a forward DNA primer on exon 8 and a reverse primer on exon 2. MLL-PTD positivity was determined by the intensity of amplified DNA fragment at or above the lower detection limit control of this assay. Intra-assay precision, inter-assay precision, and limit of detection (LOD) were performed according to standard laboratory protocols. Results of this method were compared to those of NGS. Results: Intra-assay precision studies on 5 specimens yielded 100% genotype concordance. Inter-assay precision studies on 20 specimens yielded 100% concordance between replicates. LOD studies using a human eosinophilic leukemia cell line (EoL-1) with 12.5-kb PTD in size showed assay sensitivity of 5%; for smaller MLL-PTD positives (PTD ~3 kb and ~5 kb), LOD was 1%. For method comparison studies, 71 specimens (52 whole blood and 19 bone marrow) were also analyzed by NGS; concordance between the methods was 97.2% (69/71). Conclusions: We developed and validated long-range MLL-PTD detection assay for AML. The developed method uses DNA, which is commonly used for molecular testing for AML, and thus may reduce sample processing time by consolidating sample type across tests.

Development of an unbiased, semi-automated approach for classifying plasma cell immunophenotype following multicolor flow cytometry of bone marrow aspirates.

Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes. In this retrospective study, flow cytometry results from bone marrow aspirate specimens from 570 patients referred to the Myeloma Institute at UAMS were evaluated. Mean and median FI data were obtained from 8-color MFC of non-neoplastic, malignant, and mixed PC populations using antibodies to CD38, CD138, CD19, CD20, CD27, CD45, CD56, and CD81. Of 570 cases, 252 cases showed only non-neoplastic PCs, 168 showed only malignant PCs, and 150 showed mixed PC populations. Statistical analysis of median FI data for each CD marker showed no difference in expression intensity on non-neoplastic and malignant PCs, between pure and mixed PC populations. ROC analysis of the median FI of CD expression in non-neoplastic and malignant PCs was used to develop an algorithm to convert quantitative FI values to qualitative assessments including "negative," "positive," "dim," and "heterogeneous" expression. FI data derived from 8-color MFC can be used to define marker expression on PCs. Translation of FI data from Infinicyt software to an Excel worksheet streamlines workflow and eliminates transcriptional errors when generating flow reports. © 2018 International Clinical Cytometry Society.

62 - SNP/CGH Microarray Analysis in MDS: Correlation With Conventional Cytogenetic, FISH and Flow-Cytometric Findings

To assess the correlation between microarray results and findings from conventional cytogenetic, FISH and flow-cytometric studies on myelodysplastic bone marrow specimens, we analyzed the testing results for 185 bone marrow aspirate specimens submitted for myelodysplastic syndromes work-up. Thirty-six (19.5%) of these were positive by SNP/CGH microarray analysis with clinically significant LOH regions detected in 28 (78%) cases and clonal heterogeneity/evolution detected in 11 (31%) cases. Compared to cytogenetic analysis, microarray detected abnormalities in 11 normal cytogenetic cases, revealed additional chromosomal gains/losses in 18 cases, and defined the origin of marker chromosomes in 10 cases. However, cytogenetic analysis revealed balanced translocations or low level abnormalities not identified by microarray in 10 cases. In contrast, all the abnormalities found by FISH were detected by microarray. Flow cytometry was used to assign FCSS (flow cytometric scoring system) scores for myeloid abnormalities in 31 of these 36 array-positive specimens. Acute myeloid leukemia was classified in 6/31 cases by flow cytometry. For the remaining 25 cases, complex (≥5) abnormalities were detected by microarray in 1/14 (7.1%) specimens with a FCSS of 0~3, in 1/4 (25.0%) specimens with a FCSS of 4, and in 4/7 (57.1%) specimens with a FCSS of 5~6, respectively. These results emphasize the additional value that CGH/SNP microarray analysis adds to conventional cytogenetic analysis and suggest a high correlation between our phenotypic FCSS and microarray findings for myeloid abnormalities. Higher FCSS scores correlate with increasing complexity of genomic abnormalities. Analyses of recently processed cases will be included in the final presentation.

Lysosomal storage disorders: Morphologic appraisal in Indian population.

Lysosomal storage disorders (LSDs) comprise a group of at least 50 distinct genetic diseases, each one resulting from the deficiency of a particular lysosomal enzyme involved in metabolism. We attempt to study and further subclassify pediatric LSDs into Gaucher's and non-Gaucher's category based on the morphologic variables seen in the bone marrow aspiration smears and trephine biopsy sections. Pediatric (<12 years age) cases of LSDs diagnosed by bone marrow aspiration and trephine biopsy specimens, in the last 12 years period, were retrieved. The archival material and the relevant clinical as well as hematologic parameters were reviewed. From January 1997 to December 2008, 55 cases were diagnosed as LSDs. Based on bone marrow morphology, 56% (n = 31) cases were diagnosed as non-Gaucher's and the remaining 44% (n = 24) cases as Gaucher's disease, the ratio being 1.29:1. Anemia and thrombocytopenia were more commonly observed in Gaucher's disease (91.67 and 62.5%) as compared to non-Gaucher's group (74.19 and 19.35%). Neurologic symptoms and signs were more frequently present in non-Gaucher's cases (45.16%) as compared to Gaucher's group (29.17%). LSDs can be classified into Gaucher's and non-Gaucher's subtypes based on the characteristic cytomorphology of the storage cells in Giemsa-stained bone marrow aspiration smears and on hematoxylin and eosin-stained trephine biopsy sections. This approach would be fairly adequate for therapeutic and prognostic purposes in resource.constrained settings, where enzyme studies and mutational analysis may not be easily available.

Chromosome Preparation for Acute Lymphoblastic Leukemia.

The chromosome abnormalities observed in acute lymphoblastic leukemia have been demonstrated to contribute to patient management and treatment stratification. This chapter provides a basic protocol for the procurement, culture, harvest, and slide preparation of bone marrow aspirate and unstimulated peripheral blood specimens.

Minimal Residual Disease (MRD) Assessment By Multiparametric Flow Cytometry Is Prognostic for Progression-Free Survival in Phase 1/1b Relapsed/Refractory Acute Myeloid Leukemia (AML) Patients Treated with Idasanutlin MDM2 Antagonist

Introduction. Despite considerable efforts towards novel therapeutics research and discovery, outcomes in AML remain poor. Although composite Complete Remission (cCR; CR, CRp, CRi) is routinely used as a measure of therapeutic clinical activity, cCR frequently does not translate to survival benefit. On this basis, the characterization and tracking of minimal residual disease (MRD) has emerged as a tool to help better define the depth of such cCR to offer prognostic utility on AML patients likely to experience survival benefit from a given experimental therapeutic.The MDM2 antagonist idasanutlin has shown promising clinical activity in AML. Idasanutlin enhances p53 activity through antagonism of the MDM2:p53 interaction. Disruption of this protein:protein interaction inhibits MDM2 targeting of p53 for ubiquitination and degradation, thus stabilizing p53 protein to exert tumor suppressor transcriptional regulation and induction of apoptotic pathways.Patients and Methods. Trial NP28679 (NCT01773408) is a Phase 1/1b study evaluating idasanutlin as monotherapy or in combination with cytarabine in relapsed or refractory AML patients with safety as primary and complete remission as secondary endpoints, respectively (Martinelli, EHA, 2016.) Duration of response was available as exploratory clinical data for a subset of patient. Patients' pre-treatment bone marrow aspirate specimens were evaluated by multiparametric flow cytometry using an 18 marker surface antigen-based panel. MRD assessment occurred per protocol recommendation at the time of hematological malignancy response assessment (HMRA) at Day 28 and for those patients initially experiencing cCR at each subsequent HMRA. Further flow cytometry analyses were conducted for expression changes of known p53 regulated proteins in CD45+(dim) blasts correlating with drug exposure, consistent with mechanistic engagement.Results. PFS analyses for cCR patients versus non-CR Kaplan-Meier plots indicate the median for responders is 315d (95%CI: 282, NA) versus non-responders is 43.5d (95%CI: 30, NA). When MRD<1% versus >1% is applied as a cut-point, analytics show a statistical association with median PFS (log-rank p-value < .001) at 367d (95%CI: 219, NA) for <1%MRD versus >1% is 84d (95%CI: 30, NA.) Median MRD values for CR/CRp/CRi vs PR/HI vs PD are 0.42%, 1.79%, and 19.16%, respectively, again consistent with MRD serving as a surrogate for clinical activity (log rank test for trend p-value = .001) Additionally, multiparametric flow cytometry analysis of idasanutlin pharmacodynamic (PD) protein expression changes comparing pretreatment patient blood specimens to 24 hours following first dose administration indicates that increases in both p53 and MDM2 in CD45+(dim) blasts is associated with steady state drug exposure levels ([Spearman’s Correlation =0.27; p=.013] and [Spearman’s Correlation=0.24; p=.026,] respectively.) Interestingly, these changes in protein expression for p53 and MDM2 are associated with orthologous PD changes in serum protein macrophage inhibitory cytokine 1 (MIC-1) ([Spearman’s Correlation=0.23; p=.035] and [Spearman’s Correlation=0.22; p=.042,] respectively.) These PD changes are mechanistically consistent with enhanced p53 resulting from diminished MDM2 ubiquitination and degradation of p53.Conclusions. In summary, the results presented here are consistent with multiparametric flow cytometry MRD assessment as an early indication aligning with cCR in relapsed/refractory AML patients treated with idasanutlin. Further, assessment for association with progression-free survival indicates that the lowest quartile patients by MRD flow cytometry measurement (<1%) are likely to exhibit a pronounced survival benefit over those patients with higher detectable levels of MRD (>1%). As such, the data presented here support inclusion of MRD assessment by flow cytometry as a prognostic indicator to provide guiding information for assignment of depth of AML patient response. We will continue to monitor this biomarker for diagnostic potential as a prognostic indicator of survival-based outcomes in future randomized clinical studies of idasanutlin. DisclosuresLanza:Roche-Genentech: Employment. Martinelli:Pfizer: Consultancy, Speakers Bureau; MSD: Consultancy; Novartis: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; BMS: Speakers Bureau. Yee:Novartis Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jukofsky:Roche Pharma: Employment. Reis:Roche Pharma: Employment. Blotner:Roche Pharma: Employment. Drummond:Pfizer: Honoraria, Speakers Bureau; celgene: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Roche: Honoraria, Speakers Bureau. Vey:Sunesis: Honoraria. Dickinson:GlaxoSmithKline: Consultancy, Research Funding. Kelly:Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Speakers Bureau. Theron:Roche Pharma: Employment. Venstrom:Roche-Genentech: Employment. Middleton:Roche Pharma: Employment. Chen:Roche Pharma: Employment. Kinnersley:Roche-Genentech: Employment, Equity Ownership. Nichols:Roche Pharma: Employment. Pierceall:Roche Pharma: Employment.

Treatment and Clinical Outcomes of High Grade B-Cell Lymphomas with MYC and BCL2 and/or BCL6 Rearrangements (Double Hit/Triple Hit Lymphomas)

Background: B-cell lymphomas with a MYC/8q24 rearrangement coupled with translocations involving BCL2 and/or BCL6, often referred to as double hit or triple hit (DH/TH) lymphomas, typically have an aggressive clinical course. A new WHO subtype, "high grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements", has been established for this entity (Swerdlow et al. Blood; 127: 2375-2390, 2016). Herein we report the treatment and outcomes of DH/TH patients at our institution.Materials and Methods: Cases were identified through the Mayo Clinic Lymphoma Database, University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER), and the Mayo Clinic Cytogenetics Laboratory. Interphase FISH was performed clinically on either paraffin sections of the tissue specimens or on smears of the bone marrow aspirate specimens using break-apart probes for MYC and BCL6, dual fusion probes for IGH/MYC, IGL/MYC, and IGK/MYC, and either a BCL2 break-apart probe or an IGH/BCL2 dual fusion probe. Consensus pathology re-review was performed to define morphology as diffuse large B-cell lymphoma (DLBCL) vs. high-grade B-cell lymphoma (HGBCL). Patients with de-novo DH/TH or diagnosis of DH/TH at the time of transformation of a previously diagnosed low-grade lymphoma and who were treated with anthracycline-based chemotherapy were included in this analysis. Patients with DH/TH first identified at the time of recurrent aggressive B-cell lymphoma were excluded. Overall survival (OS) was defined as time from DH/TH diagnosis to death from any cause. EFS12 was defined as event-free status 12 months from diagnosis of DH/TH.Results: Clinical outcome analysis was performed on 71 patients (Table 1). The median age was 61 years (range 29-82). 60 patients had de-novo DH/TH and 11 patients had transformation of a previously diagnosed low-grade lymphoma. In 65 cases in which there was consensus morphology on pathology re-review, there were 39 (60%) with HGBCL morphology and 26 (40%) with DLBCL morphology. At a median follow-up of 21 months (range 1-87), the event free survival at 12 months was 43%, and the median overall survival (OS) was 22 months (95% CI: 13-NA). The OS at 5 years was 48% (95% CI: 36%-63%). Patients with DH/TH at transformation of previously diagnosed low-grade lymphoma had a very poor outcome (median OS=10.8 months, EFS12 = 10%), which was inferior to patients with de-novo DH/TH (P=0.0069). When compared to all other therapies, patients treated with R-CODOX-M/IVAC had better EFS12 (p=0.03) with a trend in improved OS (p=0.08) though this may be confounded by patient selection bias as these patients were significantly younger (p=0.0001) than patients treated with other anthracycline based regimens (table 1).Conclusion: In "high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements", induction failure occurs early. Although transformation to DH/TH from previously diagnosed low-grade lymphoma is associated with a particularly poor outcome, the overall survival for the entire cohort at 5 years was 48%, suggesting long-term cures in a subset of patients. Treatment with R-CODOX-M/IVAC may result in superior outcomes in patients less than 60 years of age. [Display omitted] [Display omitted] DisclosuresAnsell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Nowakowski:Celgene: Research Funding; Morphosys: Research Funding; Bayer: Consultancy, Research Funding.

Human myeloid inhibitory C-lectin: a highly specific and stable acute myeloid leukemia marker.

The prognosis of acute myeloid leukemia (AML) is poor because of relapses occurring on conventional chemotherapy. The distinction between leukemic and normal stem cells relies on the expression of antigen combinations defining leukemia-associated immunophenotypes (LAIPs), which are absent or extremely infrequent in normal bone marrow. However, LAIPs are very different from patient to patient and are not necessarily stable over the course of the disease. Accordingly, we addressed the applicability of human myeloid inhibitory C-lectin (hMICL) by flow cytometry as a specific leukemic myeloid stem cell marker for the diagnosis of AML in CD34+ and CD34- cases and evaluated the stability of hMICL during the course of the disease. hMICL expression was assessed in 78 bone marrow aspirate specimens obtained from AML patients at diagnosis (n=40), complete remission (CR) (n=28), and relapse (n=10). AML patients at diagnosis were compared to 20 newly diagnosed acute lymphoblastic leukemia (ALL) patients and 20 healthy controls. hMICL was reevaluated in CR and relapse specimens. hMICL was expressed in 100% AML patients at diagnosis (mean±standard deviation [SD], 60.3±19.9%), both CD34+ and CD34- , but not in ALL (mean±SD, 3.3±1.9%) or healthy controls (mean±SD, 3.4±2.6%) (P<.001). hMICL median fluorescence intensity ratio was higher in AML (mean±SD, 15.9±11.7) compared to ALL (mean±SD, 4.5±1.4) and healthy controls (mean±SD, 4.4±1.6) (P<.001). hMICL was expressed in all studied AML morphologic subtypes. Preserved stable expression of hMICL was found in CR and relapse specimens with no antigen loss. hMICL is a robust pan-AML-associated antigen with excellent diagnostic impact, extreme specificity to AML blasts, and stability throughout the course of the disease. hMICL could be incorporated into the routine flow cytometry setting within the initial diagnostic work-up and follow-up of AML.

Hemophagocytic Lymphohistiocytosis and Bone Marrow Hemophagocytosis: A 5-Year Institutional Experience at a Tertiary Care Hospital.

The purpose of this study was to correlate the significance of bone marrow hemophagocytosis and analyze outcome data in patients with suspected hemophagocytic lymphohistiocytosis (HLH) at a tertiary care hospital during the course of 5 years. The pathology database of State University of New York Upstate Medical University, Syracuse, was searched for the terms "hemophagocytosis," "hemophagocytic syndrome," and "hemophagocytic lymphohistiocytosis" encompassing the period January 2009-December 2014. Bone marrow aspirate and biopsy specimens, along with ancillary laboratory studies, clinical course, and outcome data, were reviewed for each case. Of the 23 patients included in our study, HLH was diagnosed in 14 (60.8%). Bone marrow hemophagocytosis (HPC) was seen in a higher proportion of patients (78.5%) who were diagnosed as having HLH; however, 55.5% of the patients who were not diagnosed as having HLH also showed evidence of bone marrow HPC. Patients with malignancy-associated HLH had a markedly worse outcome compared with patients with nonmalignancy-associated HLH. Although bone marrow HPC is fairly sensitive, it is not specific to establish a diagnosis of HLH. A high index of clinical suspicion together with early diagnosis and treatment is imperative to improve outcomes in patients suspected of having HLH.

Abstract 5225: CDK11B, PTPRN2 and WDPCP were frequently duplicated genes in refractory/relapsed normal karyotype AML patients: Identifying structural variations using whole genome sequencing

Abstract Background According to the NCCN guideline, the prognosis of acute myeloid leukemia with normal karyotype (NK-AML) is classified based on the mutational status of NPM1, FLT3-ITD or CEBPA. However, patients with NK-AML without the above mutations, who are in intermediate risk, still show various clinical outcomes. Unfortunately, refractory/relapsed patients with intermediate risk NK-AML cannot be predicted before cytotoxic chemotherapy, yet. With the advent of next generation sequencing technology, we tried to reveal prognostic molecular marker in intermediate risk NK-AML, especially focusing on large structural variations (SVs). Methods A total of nine patients with intermediate risk NK-AML patients were included. Whole genome sequencing (WGS) was performed using a pair of tumor and germline DNA of the patients. Leukemic blasts were obtained from bone marrow aspiration specimens of each patient at the time of diagnosis. As a germline control, epithelial cells were collected from saliva of each patient at the time of complete remission. Then, genomic DNA was massively sequenced using HiSeq X10 system (Illumina Inc., San Diego, CA, USA). Possible SVs including inversion, duplication and translocation were identified with BreakDancer (Washington University School of Medicine, St. Louis, MO, USA), Pindel (The Wellcome Trust Sanger Institute, Cambridge, UK) and Delly (European Molecular Biology Laboratory, Heidelberg, Germany). Possible SVs were considered when they were recurrently identified from the three pipelines. Results This cohort consisted of 7 males and 2 females (median age = 55.6 years old). None of them had secondary AML. Eight patients were relapsed after complete remission and the other patient was refractory to 1st line induction chemotherapy. Through WGS, 25 inversions, 67 duplications and 10 translocations were identified in nine patients. Among them, 4 inversions, 13 duplications and 2 translocations were recurrently mutated. Especially, CDK11B, PTPRN2 and WDPCP were frequently duplicated more than 40% of the patients. Conclusions In this study, we performed WGS to investigate SVs of refractory/relapsed NK-AML patients with intermediate risk. Among various mutations, duplications of CDK11B, PTPRN2 and WDPCP were highly prevalent, so they could be a possible prognostic biomarker in NK-AML with intermediate risk. Further functional validation is required in order to clarify their roles. Citation Format: Jeonghwan Youk, Sunghoon Cho, Daeyoon Kim, Youngil Koh, Inho Kim, Murim Choi, Sung-Soo Yoon. CDK11B, PTPRN2 and WDPCP were frequently duplicated genes in refractory/relapsed normal karyotype AML patients: Identifying structural variations using whole genome sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5225.

T cell receptor rearrangements in a patient with γ-heavy chain disease: A case report.

Heavy chain diseases (HCDs) are rare B cell lymphoplasma cell proliferative disorders that are characterized by the production of incomplete monoclonal immunoglobulin (Ig) heavy chains without the associated light chains. γ-HCD (IgG subtype) is a rare subtype, with ~150 cases reported in the literature to date; however, to the best of our knowledge, no reports of T cell receptor (TCR) gene rearrangement in γ-HCD exist in the literature. The present study reports the case of an 81-year-old man with γ-heavy chain disease associated with TCR gene rearrangement, identified in lymph node biopsy and bone marrow aspirate specimens. The present case revealed an alternative manifestation of γ-HCD, which may provide additional biological insights into this rare B cell disorder.

Mutation Analysis in 276 Cases of Newly Diagnosed Acute Myeloid Leukemia By Next Generation Sequencing: Association with Established Prognostic Variables and MRC Risk Groups

INTRODUCTIONAcute myeloid leukemia (AML) is known to have numerous genomic aberrations that predict response to treatment and overall survival. We aimed to assess various mutations in newly diagnosed AML cases by next generation sequencing (NGS) and their association with various well-established clinicopathologic parameters and Medical Research Council (MRC) risk groups.MATERIALS AND METHODSWe performed molecular studies on DNA extracted from bone marrow aspirate specimens in 276 newly diagnosed treatment na•ve AML patients presenting at a single referral institution from 08/2013 to 03/2015 as part of routine clinical work up in a CLIA certified molecular diagnostics laboratory. Cases met criteria for AML per WHO 2008 criteria. The entire coding sequences of 28 genes (ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1) were sequenced using a NGS-based custom-designed assay using TruSeq chemistry on Illumina MiSeq platform. FLT3 internal tandem duplications (ITD) and codon 835/836 point mutation were detected by PCR followed by capillary electrophoresis. CEBPA mutation analysis was performed on 262 patients by PCR followed by Sanger sequencing. Cases were categorized as favorable, intermediate and adverse groups as per revised MRC cytogenetic risk group classification.RESULTSMedian age was 67 years. Patients included 167 (60.5%) males and 109 (39.5%) females. 38 (14%) and 6 (2%) patients had prior diagnosis of myelodysplastic syndrome and myeloproliferative neoplasms respectively. Hematologic parameters are as follows [median (range)]: Hb 8.7 g/dL (2.8-13.9), platelets 50.5 K/μ L (1-1109), WBC 5.4 K/μ L (0.4-620.4), ANC 0.9 K/μ L (0-145.7), AMC 0.3 K/μ L (0-98.1). Bone marrow (BM) blast % [median (range)] was 45.5% (5-96). LDH was 733 IU/dL (225-13156). Of 275 patients with cytogenetic analysis performed, 98 (35.64%) had diploid karyotype, 75 (27.27%) had one, 38 (13.82%) had two, 8 (2.91%) had three, 56 (20.36%) had > three abnormalities, 75 (27.27%) had monosomies and 62 (22.55%) had trisomies. Of 34 cases classified as AML with recurrent cytogenetic abnormalities per WHO 2008, 10 (3.64%) had t(8;21), 13 (4.73%) had inv(16), 1 (0.36%) had t(15;17), 3 (1.09%) had inv (3), 4 (1.45%) had t(9;11)(p22;q23) and 3 (1.09%) had t(6;9)(p23;q34). MRC risk categorization of the cases was as follows: favorable 24 (8.72%), intermediate 161 (58.55%) and adverse 90 (32.73%). Mutations identified by NGS are as detailed in Table 1. Of 56 patients with FLT3 mutations detected by PCR, the breakdown is as follows: FLT3 ITD (39, 14.13%), FLT3 D835 (16, 5.80%), FLT3, ITD + D835 (1, 0.36%). Of 262 patients assessed, CEBPA mutation was detected in 26 (9.92%). Thirty one (11.23%) cases had no mutations detected in the genes analyzed by NGS or PCR, 93 (33.70%) had mutations in one, 80 (28.98%) in two, 42 (15.22%) in three and 30 (10.87%) in > three genes. We found positive associations between mutated genes and various parameters as detailed in Table 2. CONCLUSIONSAML is a heterogeneous group of myeloid neoplasms at the genetic level. Multiple genetic mutations in a large subset of cases likely indicate clonal evolution. A subset of mutations has significant association with well-established clinico-pathologic parameters like WBC. With longer follow-up, we could use this data to refine prognostic models for AML.Table 1GenesNumber of CasesPercentage of CasesFLT36122.10NPM14817.39NRAS4817.39DNMT3A4717.03TP534516.30IDH24014.49IDH13311.96TET23211.59ASXL13010.87RUNX13010.87PTPN11134.71KRAS113.99KIT82.90WT182.90GATA272.54EZH262.17JAK241.45MPL20.72ABL110.36EGFR10.36GATA110.36IKZF210.36MDM210.36MLL10.36MYD8810.36NOTCH110.36Table 1Mutated genesp valueHbNRAS, NPM1<0.05, <0.04PlateletsTP53, IDH2<0.03, <0.02WBCFLT3, NRAS, TP53<0.05, <0.05, <0.05AMCNRAS, NPM1, TP53<0.001, <0.02, <0.02ABCFLT3 NPM1<0.049, <0.02PB blast %FLT3, NPM1, TP53, CEBPA<0.000, <0.002, <0.005, <0.000BM blast %FLT3, NRAS, NPM1, TP53, IDH1, CEBPA>0.000, <0.0000, <0.014, <0.004, <0.002, <0.012 AMCabsolute monocyte count, ABC: absolute basophil count, PB: peripheral blood, BM: bone marrow DisclosuresNo relevant conflicts of interest to declare.

Mutation Analysis in 200 Cases of Newly Diagnosed Myelodysplastic Syndrome By Next Generation Sequencing: Association with Established Prognostic Variables and IPSS-R

INTRODUCTIONMyelodysplastic syndrome (MDS) is known to have numerous genomic aberrations that predict response to treatment and overall survival. We aimed to assess various mutations in newly diagnosed MDS cases by next generation sequencing (NGS) and their association with various well-established clinicopathologic parameters and the Revised International Prognostic Scoring System (IPSS-R).MATERIALS AND METHODSWe performed molecular studies on DNA extracted from bone marrow aspirate specimens in 200 newly diagnosed treatment naïve MDS patients presenting at a single institution from 08/2013 to 03/2015 as part of routine clinical work up in a CLIA certified molecular diagnostics laboratory. Cases met criteria for MDS per WHO 2008 criteria. The entire coding sequences of 28 genes (ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1) were sequenced using a NGS-based custom-designed assay using TruSeq chemistry on Illumina MiSeq platform. FLT3 internal tandem duplications (ITD) and codon 835/836 point mutation were detected by PCR followed by capillary electrophoresis. CEBPA mutation analysis was performed by PCR followed by Sanger sequencing on 186 patients.RESULTSMedian age was 67 years. Patients included 139 males (69.5%) and 61 females (30.5%). Hematologic parameters are as follows [median (range)]: Hb 9.6 g/dL (5-16.7), platelets 75 K/μ L (5-652), WBC: 2.8 K/μ L (0.4-20.8), ANC 1.3 K/μ L (0.0 -12.0), AMC 0.2 K/μ L (0.0-3). Bone marrow (BM) blasts [median (range)] were 4% (0-19). Of 192 patients with cytogenetic analysis performed, 65 (33.85%) had diploid karyotype, 53 (27.6%) had one, 21 (10.93%) had two, 13 (6.77%) had three, 40 (20.83%) had > three abnormalities. IPSS-R risk categorization of the 200 cases is as follows: very low (17 cases, 8.5%), low (46, 23%) intermediate (42, 21%), high (47, 23.5%), very high (48, 24%). Mutations identified by NGS are as detailed in Table 1. Of the 4 patients with FLT mutations detected, the breakdown is as follows: FLT3 ITD (3, 75%), FLT3 D835 (1, 25%), FLT3, ITD + D835 (0, 0%). CEBPA mutation was detected in 12 of 186 (6.45%) cases assessed. CEBPA was detected in 12 (6.45%). Sixty three (31.5%) cases had no mutations detected in the genes analyzed by NGS or PCR, 80 (40%) had mutations in one, 42 (21%) had mutations in two, 8 (4%) in three and 7 (3.5%) in > three genes. We found positive associations between mutated genes and various parameters as detailed in Table 2. No association was found between frequency of any particular mutation and the IPSS-R score. CONCLUSIONSMDS is a heterogeneous group of myeloid neoplasms at the genetic level. Multiple genetic mutations in a large subset of cases likely indicate clonal evolution. A subset of mutations has significant association with well-established clinico-pathologic parameters like WBC and BM blast percentage. With longer follow-up, we could use this data to refine IPSS-R.Table 1Number of cases% casesTP534623TET23316.5RUNX12713.5ASXL12512.5DNMT3A178.5EZH2126IDH284IDH173.5NRAS73.5JAK252.5FLT342PTPN1131.5EGFR21MPL21WT121GATA210.5KIT10.5KRAS10.5MYD8810.5NPM110.5BRAF10.5Table 2Mutated genesp valueWBCASXL1<0.042AECTET2<0.016BM blast %RUNX1, CEBPA<0.008, p<0.02BM myelocyte %TP53, TET2, RUNX1, DNMT3A<0.014, <0.014, <0.015, <0.038AEC: absolute eosinophil count, BM: bone marrow DisclosuresNo relevant conflicts of interest to declare.

Transfer RNA detection by small RNA deep sequencing and disease association with myelodysplastic syndromes.

BackgroundAlthough advances in sequencing technologies have popularized the use of microRNA (miRNA) sequencing (miRNA-seq) for the quantification of miRNA expression, questions remain concerning the optimal methodologies for analysis and utilization of the data. The construction of a miRNA sequencing library selects RNA by length rather than type. However, as we have previously described, miRNAs represent only a subset of the species obtained by size selection. Consequently, the libraries obtained for miRNA sequencing also contain a variety of additional species of small RNAs. This study looks at the prevalence of these other species obtained from bone marrow aspirate specimens and explores the predictive value of these small RNAs in the determination of response to therapy in myelodysplastic syndromes (MDS).MethodsPaired pre and post treatment bone marrow aspirate specimens were obtained from patients with MDS who were treated with either azacytidine or decitabine (24 pre-treatment specimens, 23 post-treatment specimens) with 22 additional non-MDS control specimens. Total RNA was extracted from these specimens and submitted for next generation sequencing after an additional size exclusion step to enrich for small RNAs. The species of small RNAs were enumerated, single nucleotide variants (SNVs) identified, and finally the differential expression of tRNA-derived species (tDRs) in the specimens correlated with diseasestatus and response to therapy.ResultsUsing miRNA sequencing data generated from bone marrow aspirate samples of patients with known MDS (N = 47) and controls (N = 23), we demonstrated that transfer RNA (tRNA) fragments (specifically tRNA halves, tRHs) are one of the most common species of small RNA isolated from size selection. Using tRNA expression values extracted from miRNA sequencing data, we identified six tRNA fragments that are differentially expressed between MDS and normal samples. Using the elastic net method, we identified four tRNAs-derived small RNAs (tDRs) that together can explain 67 % of the variation in treatment response for MDS patients. Similar analysis of specifically mitochondrial tDRs (mt-tDRs) identified 13 mt-tDRs which distinguished disease status in the samples and a single mt-tDR which predited response. Finally, 14 SNVs within the tDRs were found in at least 20 % of the MDS samples and were not observed in any of the control specimens.DiscussionThis study highlights the prevalence of tDRs in RNA-seq studies focused on small RNAs. The potential etiologies of these species, both technical and biologic, are discussed as well as important challenges in the interpretation of tDR data.ConclusionsOur analysis results suggest that tRNA fragments can be accurately detected through miRNA sequencing data and that the expression of these species may be useful in the diagnosis of MDS and the prediction of response to therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1929-y) contains supplementary material, which is available to authorized users.

Pathological diagnosis of pediatric Burkitt lymphoma involving bone marrow

To investigate pathologic and differential diagnostic features of pediatric Burkitt lymphoma (BL). A total of 20 cases of pediatric BL were retrospectively reviewed for their clinical and pathologic profiles. Bone marrow aspiration specimens were available in all cases and bone marrow biopsies were available for immunohistochemical study in 18 cases. Flow cytometry study was available in 16 cases. MYC translocation by FISH method was performed in 11 cases. Atypical lymphocytes with cytoplasmic vacuoles were found in bone marrow smears in all 20 cases and peripheral blood films in all 19 available cases. The bone marrow biopsies showed infiltration by uniform medium-sized atypical lymphocytes with multiple small nucleoli but without the starry-sky pattern in all 18 cases. Immunohistochemistry showed the following results in all 18 cases: positive for CD20, PAX-5, CD10, CD34 and TdT, but negative for bcl-2 and CD3 with Ki-67 > 95%.Flow cytometry showed CD19+CD20+CD10+FMC7+CD22+TdT-CD3- in 16 cases, including κ+ in 8 cases, λ+ in 7 cases, and κ-λ- in 1 case. MYC gene rearrangement by FISH was observed in 10 of the 11 cases. The histopathology of BL is distinct, including atypical lymphocytes with cytoplasmic vacuoles in bone marrow aspirate, lack of starry-sky patternin bone marrow biopsy. Generally, the diagnosis should be made with a combined immunophenotype and FISH approach. Pediatric BL must be distinguished from DLBCL and B-cell lymphoma, unclassifiable, which has intermediate features between DLBCL and Burkitt lymphoma.