Formalin-fixed Paraffin-embedded Bone Marrow Research Articles

Loss of EZH2 Protein Expression in Myelodysplastic Syndrome Correlates with EZH2 Mutation and Portends a Worse Outcome

INTRODUCTION Epigenetic alterations are frequent in MDS. EZH2 encodes a histone methyltransferase, which is a catalytic component of polycomb repressive complex 2 crucial for epigenetic silencing of genes involved in stem cell renewal. EZH2 mutation is an independent predictor of overall survival in MDS. Unlike EZH2 Y641 gain-of-function mutation in lymphomas, MDS shows a spectrum of EZH2 mutations without a hotspot that often leads to loss-of-EZH2 function. EZH2 function can also be altered by other mechanisms including mutations in non-coding region, copy number changes and other gene mutations. Assessment of EZH2 protein expression can identify alterations of downstream EZH2 function that may be missed by mutation assessment. In this study, we evaluated EZH2 mutations in MDS patients (pts) and correlated with clinicopathologic findings and outcomes. METHODS We searched our institutional database for newly diagnosed MDS pts with EZH2 mutation detected by targeted next-generation-sequencing panels. Consecutive MDS group with wild-type EZH2 served as matched control. EZH2 expression was evaluated by immunohistochemistry (IHC) on decalcified formalin-fixed paraffin embedded bone marrow (BM) biopsies. Nuclear EZH2 staining was independently scored by 3 hematopathologists semi-quantitatively (blinded to data). H-index was obtained by multiplying %positive cells [0, 0%; 1 (1-5%), 2 (6-20%), 3 (21-50%), and 4 (51-100%)] and staining intensity (0-3). H-index of 0-1 was considered negative (MDS-EZH2NONEXP) implying "loss of expression"; ≥2 was considered expressors (MDS-EZH2EXP). Overall survival (OS) was calculated from date of diagnosis to death. Statistical analysis was performed using R version 3.5.1. RESULTS We identified 40 (8.8%) MDS pts with 45 EZH2 somatic mutations (35 single; 5 double mutations). There were 33 men and 7 women with a median age of 74 years (range, 55-90). EZH2 mutations included missense (62%), frameshift (27%) and nonsense (11%). Mutations spanned the entire coding region; majority (36%) involved exons 18/19; ~50% were within catalytic SET domain. The median VAF was 31.5 (1.3-92%). EZH2 mutations were frequent within MDS with multilineage dysplasia (16, 40%) and excess blasts (15, 38%) WHO categories. Control group included 38 consecutive MDS pts with wild type EZH2 (MDSEZH2WT). Twenty-nine EZH2MUTand 37 EZH2WT cases were available for IHC. Assessment in 11 healthy controls (no mutations by targeted NGS panel) showed EZH2 expression in all cases [median score: 9 (range, 6 -12)]. EZH2 expression was lost more frequently in EZH2MUTMDS (69% vs. 26.5%, p=0.001) compared to EZH2WT, majority of which showed preserved staining. Of interest, 9 patients with EZH2WT MDS also showed loss of staining. Hence, we compared outcomes and clinicopathologic features between MDS-EZH2EXP/EZH2NONEXP as well as EZH2MUT/EZH2WT groups. Compared to EZH2WT, EZH2MUT MDS showed male predominance (54% vs. 83%; p=0.012), frequent chr(7) loss (2.9% vs. 39.3%; p=0.001) and less frequent mutations in splicing factor genes: SF3B1 (37.1% vs. 10%, p=0.006); SRSF2 (25.7% vs. 2.5%, p=0.005); U2AF1 (17.1% vs. 0%) and STAG2 (20% vs. 0%, p=0.003). Compared to MDS-EZH2EXP, MDS-EZH2NONEXP showed frequent chr(7) loss (5.9% vs. 35.7%, p=0.004), less frequent SF3B1 mutation (38.2% vs. 6.9%, p=0.006), lower median platelet count (112 vs. 68, p=0.041) and a trend for higher R-IPSS scores and BM blast%. Over the course of the study (median: 14 months), 34 (85%) received hypomethylating agent; 17 (42.5%) died and 12 (30%) patients transformed to acute myeloid leukemia (AML). By univariate analysis, EZH2WT MDS has a significantly better outcome compared to EZH2MUT [not reached (NR) vs. 15 mo, HR 3.39 (1.32-8.73), p=0.011]. MDS-EZH2NONEXP has a significantly better outcome compared to MDS-EZH2EXP (NR vs. 20 mo, HR 0.21 (0.08-0.56), p=0.0017). By multivariate analysis that also included age, gender and R-IPSS, both mutation and protein expression correlated with poor survival, independent of R-IPSS (p=0.027 vs. 0.0063). When mutation and protein expression were analyzed together, protein expression showed a stronger correlation with survival than mutation (p=0.061 vs. p=0.43). CONCLUSIONS Both EZH2 mutation and protein loss correlate with poor survival in MDS, independent of R-IPSS. EZH2 protein expression shows a stronger correlation with survival than mutation. Figure Disclosures Sasaki: Otsuka: Honoraria; Pfizer: Consultancy. Bueso-Ramos:Incyte: Consultancy. Khoury:Stemline Therapeutics: Research Funding; Angle: Research Funding; Kiromic: Research Funding. Kantarjian:Astex: Research Funding; Daiichi-Sankyo: Research Funding; BMS: Research Funding; Pfizer: Honoraria, Research Funding; Jazz Pharma: Research Funding; Ariad: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Cyclacel: Research Funding; Immunogen: Research Funding; Takeda: Honoraria; Novartis: Research Funding; Amgen: Honoraria, Research Funding. Garcia-Manero:Merck: Research Funding; Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding.

Interrogating the Spatial Architecture of Human Bone Marrow Via Imaging Mass Cytometry

Hematopoietic stem cell (HSC) proliferation, self-renewal, differentiation, and trafficking are dependent, in part, upon signals generated by stromal cells in the bone marrow. Stromal cells are organized into niches that support specific subsets of hematopoietic progenitors. Intimate interactions between HSCs and neighboring stromal cells coordinate hematopoietic responses during periods of physiologic stress, while also maintaining the lifelong integrity of the hematopoietic stem cell pool. Hematopoietic niches are comprised of a heterogeneous population of stromal and hematopoietic cells. The identity and function of the known stromal cell subsets have primarily been gleaned through genetic manipulation of mouse models. In humans, the spatial organization of these stromal cells in bone marrow and the signals they generate to regulate hematopoiesis are poorly understood. Current methods to characterize bone marrow mesenchymal stromal cells in humans include: 1) immunostaining of bone sections; 2) analysis of flow sorted stromal cells; and 3) analysis of ex vivoexpanded mesenchymal stem/progenitors. Major limitations to all of these approaches exist that relate to the heterogeneity of bone marrow stromal cells, the lack of markers that reliably distinguish different stromal cell populations, and inherent technical limitations of the assays, such as the number of markers that can be analyzed at one time. Here we report our efforts to use imaging mass cytometry imaging (IMC) to interrogate the complex cellular architecture of human bone marrow. IMC allows for the simultaneous detection of up to 40 markers through the use of antibodies conjugated to elemental metal tags acquired by time-of-flight mass spectrometry. We first used standard immunostaining techniques to develop a panel of antibodies compatible with archived formalin-fixed paraffin-embedded (FFPE) human bone marrow specimens using a heat-induced epitope retrieval method. Hematopoietic lineages have been successfully identified using CD11b, CD68, CD15, CD14, CD16, CD11c, CD20, CD3, CD4, CD8a, CD38, CD45RA, CD45RO, CD235a, CD71, CD34, CD31; stromal cells and structures with CXCL12, alpha-smooth muscle actin, collagen I, vimentin; and nuclear staining using Ki67, Histone H3, and DNA intercalator. We used panels consisting of up to 18 of these antibodies each to test IMC on FFPE human bone marrow specimens. Imaging was performed using the Hyperion Imaging System (Fluidigm), which consists of a UV laser scanning module to capture regions of more than 10,000 cells with 1-micron resolution coupled with a Helios Mass Cytometer. Image analysis was performed by creation of a cell segmentation mask using CellProfiler software and high dimensional analysis in histoCAT. Representative images and corresponding dimensional reduction with t-SNE are shown in Figure 1A-H, demonstrating successful discrimination of distinct hematopoietic lineages. Gating on subpopulations within the t-SNE clusters can be projected on to the original image to illustrate spatial distribution and marker co-expression, as an example CD3+ and CD8+ cells are shown in Figure 1I. These data indicate that IMC allows for highly multiplexed analysis of bone marrow cell populations. Developing imaging techniques for analysis of tissue-banked FFPE bone marrow samples would have broad applications for translational research on hematologic diseases. In particular, this technology has tremendous potential to advance understanding of the spatial architecture of human bone marrow and to investigate alterations in the bone marrow environment in malignant hematopoiesis. Disclosures Oh: Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Expression of Myeloma Cell and Soluble B-Cell Maturation Antigen (BCMA) in Relapsed and Refractory Multiple Myeloma Patients Treated with GSK2857916 in BMA117159

Introduction:B-cell maturation antigen (BCMA) is a cell surface receptor that is widely expressed on multiple myeloma (MM) cells. GSK2857916 is a humanized Fc enhanced IgG1 anti-BCMA antibody conjugated to the microtubule inhibitor monomethyl auristatin-F (MMAF). Safety and clinical efficacy of a Q3W GSK2857916 dosing schedule were assessed in relapsed/refractory MM subjects in the first time in human trial BMA117159. In the dose escalation phase, 38 subjects were treated with doses from 0.03 mg/kg up to 4.60 mg/kg, followed by an expansion cohort of 35 subjects at a dose of 3.40 mg/kg where an overall response rate (ORR) of 60% (21/35; 95% CI 42.1-76.1) by IMWG criteria was demonstrated (Trudel, et al. Blood 2017). Several novel biomarkers were followed during the BMA117159 study, including: the level of BCMA expression on MM cells and circulating soluble BCMA (sBCMA).Methods:The expression of BCMA on MM cells was measured in subjects enrolled in BMA117159 at baseline in formalin-fixed paraffin-embedded bone marrow aspirate samples using a BCMA Immunohistochemistry (IHC) assay and a dual color IHC assay with BCMA and CD138 (plasma cell marker). The antibody to detect BCMA (J6M0) uses the same CDR regions as GSK2857916. BCMA staining was quantified as a percentage positive of all cells, percentage of plasma cells as determined by morphology, or in CD138+ cells (for the dual color IHC assay). Soluble BCMA was measured in serum samples collected at pre- and post-infusion at cycle 1 using immunoassays to determine the levels of free and GSK2857916-bound sBCMA. All measurements were performed at central laboratories.Results:Among the subjects enrolled in BMA117159, the baseline BCMA expression was found specifically in plasma cells using BCMA IHC, with a median of 100% plasma cells expressing BCMA across all subjects, compared to a median of 5% of all bone marrow cells expressing BCMA. In the dose expansion cohort, no differences by response group were noted in BCMA staining with both responders (PR or better) and non-responders having 100% BCMA positivity in plasma cells. Comparable results were observed using a dual staining IHC assay for BCMA and CD138, where 95% of CD138+ cells expressed BCMA in non-responders compared with 88% in responders.Examination of circulating sBCMA in BMA117159 subjects revealed high sBCMA levels, with a baseline median concentration of free sBCMA of 58 ng/mL across all doses (n = 68; range 4 ng/mL to >1000 ng/mL). Among patients enrolled in the expansion phase, the median baseline sBCMA concentrations were higher in non-responders compared to responders (81 ng/mL, n = 12; compared to 43 ng/mL, n = 19). However, high baseline sBCMA levels were also found in responders with levels up to 262 ng/mL. To examine whether sBCMA may act as a surrogate for BCMA expression in tumor cells, the relationship between these measures was examined; however, no strong associations were observed between baseline sBCMA and BCMA IHC staining (Spearman rho = 0.27).The binding of GSK2857916 to sBCMA can be measured by comparing the post-infusion levels of free sBCMA measured 60 minutes after the start of infusion to those found at pre-infusion. It was found that the reduction in free sBCMA appeared to be related to the dose level administered, and doses above 1.92 mg/kg consistently achieved a greater than 90% reduction of free sBMCA.Conclusions:These preliminary data indicate that tumor cells from MM patients participating in BMA117159 consistently expressed the BCMA receptor at baseline, and the levels of expression did not appear to be associated with response to treatment. At higher dose levels, it was found that GSK2857916 bound a large fraction of sBCMA, and responses to GSK2857916 were observed in 60% of dose expansion subjects with either low or high baseline sBCMA. However, at present, the sample sizes are limited, and further investigations in future studies are necessary to understand the value of these biomarkers for treatment decisions.Study is funded by GlaxoSmithKline (NCT02064387); drug linker technology is licensed from Seattle Genetics; monoclonal antibody is produced using POTELLIGENT® Technology licensed from BioWa. DisclosuresDettman:GlaxoSmithKline: Employment, Equity Ownership. Rigat:GlaxoSmithKline: Employment, Equity Ownership. Albert:GlaxoSmithKline: Employment, Equity Ownership. Barnard:GlaxoSmithKline: Employment, Equity Ownership. Birchler:GlaxoSmithKline: Employment, Equity Ownership. Deghenhardt:GlaxoSmithKline: Employment, Equity Ownership. DeWall:GlaxoSmithKline: Employment, Equity Ownership. Gaye:GlaxoSmithKline: Employment, Equity Ownership. He:GlaxoSmithKline: Employment, Equity Ownership. Liu:GlaxoSmithKline: Employment, Equity Ownership. Opalinska:GlaxoSmithKline: Employment, Equity Ownership.

Molecular Quantification of Tissue Mast Cell Burden in Systemic Mastocytosis: A New Approach for Diagnostics and Prognostication

Background: The somatic KIT D816V mutation leads to an activation of the receptor tyrosine kinase KIT and represents a diagnostic criterion for systemic mastocytosis (SM). In the majority of patients, only a very few KIT D816V+ mast cells (MC) or MC precursors, if any, are found in peripheral blood (PB) and bone marrow (BM) aspirates. In contrast, the MC count in BM biopsies is typically much higher. Melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) is widely used for detecting KIT mutations in biopsies.1 We have previously proposed dPCR as a new standard method of KIT D816V testing in SM that can also reliably quantify the variant allele fraction (VAF) in formalin-fixed paraffin-embedded (FFPE) material.2 While multilineage involvement of KIT D816V indicated by a high VAF in PB was associated with an aggressive clinical course,3-6 the mutant allele burden in tissue sections has not been studied in a large series of SM patients.Methods: The VAF of KIT D816V in the tissue was assessed by dPCR (PrimePCR ddPCR, Biorad) on DNA isolated from 211 FFPE BM sections from 116 patients with SM (median age 53 years; 58 females, 58 males) and 57 controls (lymphoma patients undergoing staging biopsy). A total of 91 SM patients were diagnosed with indolent SM (ISM) and 25 with advanced SM (aggressive SM, SM with associated hematologic neoplasm, and mast cell leukemia according to WHO criteria). Results of diagnostic samples were compared to qualitative analysis of KIT D816V clamp-PCR. BM MC infiltration was quantified in a representative area of the tryptase stained section and expressed as % of nucleated cells. Total tryptase levels in serum were determined by a fluoroenzyme-immunoassay.Results: The KIT D816V mutation burden in FFPE BM sections of SM patients showed marked differences between patients ranging from 0.027% to 60% VAF (median: 1.9%). The VAF in BM sections was largely independent from that in liquid specimen (PB and BM aspirate). In particular, a number of ISM patients showed substantially higher VAF in FFPE tissue than in liquid specimen (Figure 1A). In line with this, a higher correlation of KIT D816V mutation burden to BM MC infiltration and serum tryptase levels was observed for FFPE tissue (Spearman's coefficient of correlation r = 0.68 and r = 0.68 respectively; Figure 1B-C) than for liquid specimen (r = 0.48 and r = 0.58 respectively). When analyzing subgroups of SM, FFPE BM sections of patients with advanced SM had a significantly higher KIT D816V allele burden (median 23.40%) compared to patients with ISM (median 1.65%; p<0.001 Figure 1D). Finally, we assessed the sensitivity and specificity of dPCR for KIT D816V analysis in FFPE BM section compared to clamp PCR. While a specificity of 100% was observed for both assays, dPCR showed a significantly higher sensitivity to detect the mutation in BM tissue of ISM patients compared to clamp PCR (97% vs. 89%; p<0.05).Conclusion: dPCR is a sensitive method to detect KIT D816V and reliably quantify the mutant allele burden in FFPE BM sections of SM patients. The mutational burden in the tissue represents a new molecular marker of the disease burden in SM since it reflects both the multilineage involvement of KIT D816V and the MC burden while the mutation burden in PB primarily reflects the KIT D816V multilineage involvement. Although the tissue mutational burden in patients with advanced SM is higher compared to ISM, the difference is not as substantial as described for PB. Thus, the prognostic value of KIT D816V allele burden measurement in SM needs to be redefined for BM sections. Potential future applications of KIT D816V quantification in FFPE BM sections include treatment response monitoring and definition of high disease burden in smoldering SM (SSM). We propose to include dPCR-based KIT D816V mutant allele burden measurement in BM tissue as a new biomarker for disease burden in SM in future clinical trials.

Transcriptional Characterization of Myelofibrotic Bone Marrow Microenvironment Reveals Distinct Tumor Microenvironment in JAK2+ and Calr+ PMF Marrows

Background: Primary myelofibrosis (PMF) is a clonal stem cell disorder associated with somatic mutations in three genes: Janus kinase 2 (JAK2), calreticulin (CALR) and thrombopoietin receptor (MPL). Although, our understanding of the microenvironment in PMF is limited, in PMF levels of Treg, cytotoxic T-cells, B-cells, macrophages and megakaryocyte cell populations have been reported to be elevated in either peripheral blood or bone marrow (BM) (Barosi Curr Hematol Malig Rep 2014). In addition, various cellular pathways including JAK/STAT, TGFβ1, and cytokine pathways (CXC family, hematopoietin family, PDGF family and TGF family), have been reported to play an important role in the dysregulation of hematopoietic cell proliferation and disease progression. Here-in we characterize the tumor microenvironment in formalin fixed paraffin embedded (FFPE) BM biopsies obtained from PMF patients and correlate these findings with mutational status.Methods: We applied the enzyme-free NanoString nCounter® PanCancer Immune Profiling Panel system (NanoString Technologies, Inc., Seattle, WA) to identify and assess immunological function in the microenvironement of archival FFPE bone marrow samples from patients with PMF. Twelve archival bone marrow FFPE biopsies from PMF patients along with clinical information and 5 normal controls were analyzed using upto 500ng of RNA (at 100ng/ul) for digital expression profiling. The panel included 109 genes that define 24 immune cell types and populations and forty housekeeping genes that facilitate sample-to-sample normalization. Data analysis was performed using nSolver software 3.0 and the Advanced Analysis Module (v.1.0.84).Results: Gene expression profiles for cellular immune pathways were analyzed for global changes based mutation. Globally, cellular functions involving immune cell development and cellular responses/functions were dramatically decreased in myelofibrotic marrow (chemokines, complement, cytokines, cytotoxicity) when compared to normal marrow. However, only in areas of adhesion, antigen processing, transporter function and senescence genes were transcription levels elevated over normal controls. Differential expression analysis of JAK2V617F+ marrow showed decreased expression of genes involved in cell regulation, NK cell function, T-cell functions and pathogen defense and increased expression of genes involved in inflammation, chemokines and transporter functions over normal marrow. Whereas CALR+ bone marrow biopsies showed fewer genes down regulated and an increased number of genes up regulated, particularly involved in fibrosis, inflammation, chemokines, adhesion, antigen processing and regulation. Pathway analysis suggested a particular role for FLT3 ligand in myeloid stem cell regulation, thrombospondin (THBS1) which has been reported to promote the activation of the latent forms of TGFβ1, and mitogen-activated protein kinases (JNK1, ERK) in PMF cell proliferation and differentiation.Conclusions: Digital immune expression profiling reveals a distinct PMF tumor microenvironment and illustrates potential transcriptional differences based on their mutational status. (JAK2+ or CALR+). These transcriptional changes in myelofibrotic marrow are reflected in global changes in immune cells and pathway activation These data provide for the first time in situ evidence of the importance of the immune system in PMF pathogenesis.Barosi G, 2014 An immune dysregulation in MPN. Curr Hematol Malig Rep 9:331-339. DisclosuresNo relevant conflicts of interest to declare.

Ex Vivo and In Vivo Evaluation of Overexpressed VLA-4 in Multiple Myeloma Using LLP2A Imaging Agents.

Very-late-antigen-4 (VLA-4, α4β1 integrin, CD49d/CD29) is a transmembrane adhesion receptor that plays an important role in cancer and immune responses. Enhanced VLA-4 expression has been observed in multiple myeloma (MM) cells and surrounding stroma. VLA-4 conformational activation has been associated with MM pathogenesis. VLA-4 is a promising MM imaging and therapeutic biomarker. Specificity of (64)Cu-LLP2A ((64)Cu-CB-TE1A1P-PEG4-LLP2A), a high-affinity VLA-4 peptidomimetic-based radiopharmaceutical, was evaluated in α4 knock-out mice and by competitive blocking in wild-type tumor-bearing mice. (64)Cu-LLP2A PET/CT (static and dynamic) imaging was conducted in C57BL6/KaLwRij mice bearing murine 5TGM1-GFP syngeneic tumors generated after intravenous injection via the tail. Blood samples were collected for serum protein electrophoresis. Bone marrow and splenic cells extracted from tumor-bearing and control mice (n= 3/group) were coincubated with the optical analog LLP2A-Cy5 and mouse B220, CD4, Gr1, and Mac1 antibodies and analyzed by fluorescence-activated cell sorting. Human radiation dose estimates for (64)Cu-LLP2A were extrapolated from mouse biodistribution data (6 time points, 0.78 MBq/animal, n= 4/group). Ten formalin-fixed paraffin-embedded bone marrow samples from deceased MM patients were stained with LLP2A-Cy5. (64)Cu-LLP2A and LLP2A-Cy5 demonstrated high specificity for VLA-4-positive mouse 5TGM1-GFP myeloma and nonmalignant inflammatory host cells such as T cells and myeloid/monocytic cells. Ex vivo flow cytometric analysis supported a direct effect of myeloma on increased VLA-4 expression in host hematopoietic microenvironmental elements. SUVs and the number of medullar lesions detected by (64)Cu-LLP2A PET corresponded with increased monoclonal (M) protein (g/dL) in tumor-bearing mice over time (3.29 ± 0.58 at week 0 and 9.97 ± 1.52 at week 3). Dynamic PET with (64)Cu-LLP2A and (18)F-FDG demonstrated comparable SUV in the prominent lesions in the femur. Human radiation dose estimates indicated urinary bladder wall as the dose-limiting organ (0.200 mGy/MBq), whereas the dose to the red marrow was 0.006 mGy/MBq. The effective dose was estimated to be 0.017 mSv/MBq. Seven of the ten human samples displayed a high proportion of cells intensely labeled with LLP2A-Cy5 probe. (64)Cu-LLP2A and LLP2A-Cy5 demonstrated binding specificity for VLA-4 in an immune-competent murine MM model. (64)Cu-LLP2A displayed favorable dosimetry for human studies and is a potential imaging candidate for overexpressed VLA-4.

Digital Immune Expression Profiling Coupled with Immunohistochemistry for Interrogation of Microenvironment in Formalin Fixed Paraffin Embedded Specimens of Marrow and Spleen from PMF Patients

Background: Gene expression profiling studies have demonstrated aberrant expression of inflammatory response genes in myeloproliferative neoplasm (MPN) granulocytes and/or CD34+ cells. Our understanding of the immune response to primary myelofibrosis (PMF) hematopoietic stem cells and tissue-specific microenvironments is not complete due to a limited availability of bone marrow (BM) aspirates and fresh spleen samples from PMF patients. In order to overcome this obstacle, we utilized a novel approach with mRNA enrichment analysis which utilizes formalin fixed, paraffin embedded (FFPE) specimens of BM and spleen from PMF patients to identify immune and other microenvironment cell types and to construct pathway activation patterns.Methods: We applied enzyme-free NanoString nCounter® PanCancer Immune Profiling Panel system (NanoString Technologies, Inc., Seattle, WA) consisting of 770 standard gene panel and 20 custom gene panel for identification of immune cells and assessing immunological milieus in the microenvironement of matched, archival FFPE spleen and BM samples from MPN patients.. Up to 500ng of RNA (at 100ng/ul) isolated from FFPE BM and/or spleen specimens from PMF patients was used for digital expression profiling in accordance with the manufacturer's protocol. The panel included 109 genes that define 24 immune cell types and populations, and 40 housekeeping genes that facilitate sample-to-sample normalization. Data analysis was performed using nSolver software 2.5 and the PanCancer Immune Profiling Advanced Analysis Module (v.1.0.22). Findings identified from the digital expression profiles on cells types were confirmed via immunohistochemical evaluation.Results: Twenty-six archival FFPE tissue samples (13 BM and 13 spleen) obtained from PMF patients who had undergone therapeutic splenectomy and BM biopsy at the same time, and normal tissue controls, were analyzed as described previously (Liew et al 2015). Following data normalization, genes were selected based on P < 0.05 (unpaired t -test) and fold change > 2.0 differentially expressed mRNA levels in the BM (n=208) and spleen (n=108). These genes were distributed across several functional categories including: TNF superfamily (e.g. TNFRS13C, CD70, LTB), signal ligands (cytokine, chemokine) (e.g. JAK3, IFI16, SPP1), B and T cell functions (TIGIT, CXCR5, CXCL14), and cell adhesion (e.g. ITGB3). In supervised clustering of the significantly expressed genes, the first bifurcation of the dendrogram separated controls from PMF samples in both BM and spleen. Twenty-seven genes were significantly differentially expressed by both PMF BM and spleen, compared to control specimens. Interestingly, the PMF BM samples were further separated in a second bifurcation of the dendrogram into 3 subgroups, indicating immune transcriptional diversity within PMF samples (Figure 1). Further characterization of these subgroups and potential clinical relevance are being studied in a larger number of specimens in order to achieve statistical power.Cell type analysis indicated a significant (P =<0.05) difference in activated CD4 T-cells, T helper-1 cells, CD8 T-cells, and B-cells across all BM and spleen samples. Macrophages (P =<0.001) were increased in the spleen, and neutrophils (P =0.01) were increased only in BM samples. A decrease in CD8-positive T-cells in PMF samples (p =0.009) was confirmed using immunohistochemistry with computer assisted image analysis. Heterogeneity of Tregs in PMF spleen samples (n=10) was further confirmed by immunohistochemistry (n-3).Conclusions: Digital immune expression profiling coupled with immunohistochemistry is a novel approach for characterization of tumor microenvironment in fibrotic PMF marrow and spleen. Our preliminary findings indicate a consistent decrease in cytotoxic CD8 T-cells but varying expression of Tregs. In addition, we identified several genes in various immune functional categories within PMF patients that could potentially serve as therapy targets. [Display omitted] DisclosuresHoffman:All Cells, LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding; Geron: Consultancy, Membership on an entity's Board of Directors or advisory committees. Salama:Promedior: Consultancy.

Detection of chromosomal translocations in formalin-fixed paraffin-embedded (FFPE) leukemic specimens by digital expression profiling.

Detection of chromosomal translocations in formalin-fixed paraffin-embedded (FFPE) leukemic samples is important for confirmation of histopathological findings, classification, prognostication, and therapeutic decisions. Herein, we aim to determine whether digital expression profiling could detect chromosomal translocations in FFPE leukemic samples identified by RT-PCR, FISH, and/or karyotyping. RNA was extracted from 28 FFPE bone marrow specimens from 19 patients diagnosed with leukemia. Eight patients were translocation t(9;22) positive, three inv(16)/t(16/16) positive, five translocation t(15;17) positive, and one translocation t(8;21) positive. Two patients (four specimens) were normal. The extracted RNA was hybridized to DNA reporter probes overnight at 65 °C, followed by purification of the labeled translocations. Six hundred fields of view were counted to enumerate the number of translocations. Digital expression profiling had 100% concordance with RT-PCR, FISH, or karyotyping analysis in the two normal individuals, eight translocation t(9;22) samples, five translocation t(15;17) samples, and one translocation t(8;21) sample. None of the inv(16) positive samples were detected. Digital expression profiling detected translocations with 0.014 p190 allelic burden. Digital expression profiling can be used to measure translocation t(9;22), t(15;17), and t(8;21) in FFPE samples and is useful when a confirmatory test from a FFPE sample is necessary.

Analysis of Mutational Hotspots in Routinely Processed Bone Marrow Trephines by Pyrosequencing®.

Formalin-fixed, paraffin-embedded (FFPE) bone marrow trephines are widely used in pathology, because they best preserve the morphological details of the bone marrow. However, DNA isolated from FFPE material is fragmented, limiting the size of amplification products, which is a challenge for all sequencing applications.Pyrosequencing(®) is a quantitative and sensitive method for the detection of single-nucleotide variations (SNVs) in DNA samples. Pyrosequencing can easily be performed in a 96-well-plate format with a cost-effective medium-sized throughput.This chapter provides a general outline of SNV detection in FFPE bone marrow trephines, including a detailed protocol of the Pyrosequencing procedure and guidelines for the design of new assays and evaluation of Pyrograms. The strengths of this approach are discussed using myeloproliferative neoplasms as an example.

Consequences of the JAK2V617F allele burden for the prediction of transformation into myelofibrosis from polycythemia vera and essential thrombocythemia.

Patients diagnosed with polycythemia vera (PV) or essential thrombocythemia (ET) sometimes suffer transformation of the disease into myelofibrosis (MF), which is associated with a poorer prognosis. This study investigated the prognostic value of the allele burden of JAK2V617F, a somatic driver mutation in these diseases, by comparing the allele burden between formalin-fixed paraffin-embedded bone marrow collected at initial diagnosis and peripheral blood from follow-up visits. Although the annual changes in the JAK2V617F allele burden were comparable between MF-transformed (n = 11) and untransformed (n = 23) patients, the burden was significantly increased in MF-transformed patients exhibiting a longer disease duration than untransformed patients. Furthermore, MF transformation was only observed in patients whose JAK2V617F allele burden exceeded the mean values for each disease (PV, 71.7 %; ET, 35.5 %) at initial diagnosis or during follow-up. Finally, we showed that hydroxycarbamide treatment exerted neither a preventive effect on MF transformation nor a suppressive effect on the increased JAK2V617F allele burden. In conclusion, a high JAK2V617F allele burden at initial diagnosis or during follow-up is predictive of MF transformation in PV and ET. Therefore, routine measurement of the JAK2V617F allele burden using an accurate assay system is recommended to predict MF transformation.

Dynamic Increase in JAK2V617F Allele Burden Is a Predictive Parameter for the Transformation into Myelofibrosis from Polycythemia Vera and Essential Thrombocytosis

Patients diagnosed with polycythemia vera (PV) or essential thrombocythemia (ET), a subtype of myeloproliferative neoplasms (MPN), sometimes suffer disease transformation into myelofibrosis (MF) associated with poorer prognosis. Thus, predicting which patients have a risk of MF transformation is an important task. Following the identification of a driver mutation JAK2V617F in a majority of MPN patients, several studies was performed to investigate the potential of JAK2V617F allele burden as a diagnostic marker for MF-transformation. However, the results differ between cohorts presumably due to a lack of accurate JAK2V617F allele burden measurement. Since we have previously developed alternative-binding probe competitive polymerase chain reaction (ABC-PCR) that accurately determines the JAK2V617F allele burden, we assessed the predictive value of JAK2V617F allele burden in MF-transformation in Japanese MPN cohort.In a retrospective study, we compared JAK2V617F allele burdens between formalin-fixed paraffin embedded-bone marrow (FFPE-BM) from initial diagnosis and peripheral blood (PB) from follow-up visits. We first examined whether the allele burdens in FFPE-BM and PB were comparable when they are collected at the same time. Determining the allele burdens in a set of FFPE-BM and PB taken from same patient within a 3-month period, we observed that allele burdens from these specimens are significantly correlated (n=26, R²=0.97), which is consistent with previous report with a larger cohort (Blood 122; 3784-6). Thus, in subsequent analyses, we set a base line of the mutant allele burden determined from FFPE-BM, which is then compared with allele burdens from PB during the disease duration.We examined 14 PV and 20 ET patients (mean disease duration 69.2 months) defined by WHO 2008 MPN criteria. From first diagnosis to the time when MF-transformation was first recognized, JAK2V617F allele burden was significantly increased (mean increase 19.5±17.3%, p=0.044) in patients with MF-transformation (n=11). While patients with no MF-transformation (n=23) presented limited changes (mean increase 3.9±16.1%) over a similar duration period. When subclassifying patients into three groups based on the change or the base line value of JAK2V617F allele burden, MF-transformation was more frequently (p=0.034) observed in patients whose JAK2V617F allele burden was either increased by more than 10% during the follow-up (group A) or higher at first diagnosis than the mean values for each disease (PV; 71.7%, ET; 35.5%) (group B). MF-transformation was 2 out of 4 (50%) in the group A, and 9 out of 22 (41%) in the group B. In contrast, MF transformation in the rest of the patients (group C) was 0 out of 8 (0%). Hydoxyurea-treated (n=16, 6 PV and 10 ET) and –untreated (n=18, 8 PV and 10 ET) patients do not show significant difference in frequencies of MF-transformation, confirming that Hydoxyurea has no preventative effect against MF-transformation.In conclusion, our study showed that higher JAK2V617F allele burden at first diagnosis or a dynamic increase in allele burden during the follow-up period is a predictive factor for MF-transformation. Thus, a routine measurement of the JAK2V617F allele burden by an accurate assay system is recommended to predict MF-transformation. DisclosuresNo relevant conflicts of interest to declare.

An IHC Screen For EphA3 Positive FFPE Tumors

Eph receptors are the largest subgroup of the receptor tyrosine kinases (RTK). Unlike other RTK, these function principally during development. Quiescent in postembryonic tissues, Eph expression by adult tissues is abnormal and implicated in tumor initiation, invasion and metastasis.Aberrant EphA3 expression is seen in solid and hematologic tumors, particularly advanced stage lymphoproliferative and myeloproliferative diseases. In this regard, targeting EphA3 may constitute a novel treatment for hematological malignancy.With clinical utility in mind, i.e., patient selection for anti-EphA3 therapy, a panel of commercial and proprietary (KaloBios) antibodies was screened by Western Blot for reactivity to recombinant Eph receptors (EphA3, EphA4, EphA5, EphA7, EphB2, EphB3).Those with EphA3 binding selectivity were further screened (QualTek) by immunohistochemistry (IHC) using formalin-fixed paraffin-embedded (FFPE) normal and diseased human bone marrow (NLBM, AML) as well the LK63 pre-B-ALL cell line from which EphA3 was originally isolated.Different tissue pretreatments were used for antigen/epitope retrieval of EphA3, including steam-heating in citrate-based or Tris & chelator-based buffers, subsequent/or protease digestion, or neither.Following each antigen/epitope retrieval procedure, antibody reactivity for EphA3 was assessed by light microscopy using enzymatic biotin, tyramide and polymer-based detection techniques. In each instance, the location of the EphA3/antibody complex was visualized with 3,3-diaminobenzidine that precipitates a discrete insoluble reaction product in presence of enzyme (HRP). Nuclei were counterstained with hematoxylin to assess cell/tissue morphology.From this screen, one antibody (with an epitope in the cytoplasmic CT-domain of EphA3) was chosen for further assay optimization based on its selective reactivity towards LK63 and AML and low selective reactivity towards NLBM.Assay optimization included, amongst other aspects, evaluation of EphA3 expression in a panel of NLBM and hematopoietic tumors (200+ specimens inclusive of acute & chronic leukemia, peripheral T-cell & B-cell neoplasm, Hodgkin & non-Hodgkin lymphoma, multiple myeloma, myelodysplasic syndrome, myeloproliferative neoplasm) by a board-certified hematopathologist.In NLBM, the frequency of EphA3+ immature-blast cells (CD34+ or CD117+) was insignificant. Less than 10% CD34+/CD117+ cells were EphA3+.Elevated EphA3 expression (percentage EphA3+ nucleated cells) was observed in most hematopoietic tumors.For example, in multiple myeloma, tumor cells were typically EphA3+/CD138+ plasma cells. For AML, leukemic CD34+ or CD117+ blasts/initiating cells were typically EphA3+. Some CD138- plasma cells or leukemic CD34-/CD117- cells were also EphA3+.Correlation was made between EphA3 expression and specific tumor maturation stages, differentiation status and/or tumor aggressiveness.Tumors in blast crisis presented elevated EphA3 expression, e.g., CML in accelerated or blastic crisis but not chronic phase.Elevated EphA3 expression was noted in pre-B-ALL & pro-B-ALL (early pre-B-ALL) rather than mature B-cell ALL.EphA3 expression for some peripheral B-cell neoplasms correlated well with tumor grade: high grade, poorly differentiated (typically aggressive) B-cell lymphomas or follicular lymphomas were EphA3+. A similar relationship was noted for non-Hodgkin lymphoma (Hodgkin lymphoma was EphA3-).Preclinical screening also provided evidence for EphA3 expression by stroma/fibroblast (mostly lymphoma) and vasculature/endothelium, further rationale for development of reliable tools for profiling EphA3 in hematologic tumors and other malignancies.Using well-characterized normal and diseased FFPE bone marrow biopsies, this IHC assay for EphA3 has subsequently been validated (QualTek) to provide data that is not directly available from routine histopathology review and that supports use of the assay for profiling EphA3 in specific hematologic tumors and for patient selection in early Phase clinical trial/s of an anti-EphA3 monoclonal antibody (KB004, KaloBios).Beyond this, EphA3 targeted therapy with KB004 is anticipated for treatment of solid tumors. Recent genome-wide surveys identify EphA3 amongst the most frequently overexpressed genes in pancreatic, colon and lung carcinoma, melanoma and glioblastoma. Disclosures:Locke:QualTek Molecular Labs: Employment. Bernstein:QualTek Molecular Laboratories: Employment, Equity Ownership. Lynch:QualTek Molecular Laboratories: Employment, Equity Ownership. Siami-Namini:QualTek Molecular Laboratories: Consultancy. Walling:KaloBios: Consultancy; Corcept Therapeutics: Consultancy; Prothena: Consultancy; New Gen Therapeutics: Consultancy; Valent Technologies: Consultancy; LBC Pharmaceuticals: Consultancy; Amgen: Equity Ownership; BioMarin: Equity Ownership; Crown BioScience: Membership on an entity's Board of Directors or advisory committees. Yonker:KaloBios: Employment, Equity Ownership. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; StemLine Therapeutics: Equity Ownership.

Is There Any Relationship between Human Herpesvirus-8 and Multiple Myeloma?

Background. Human herpesvirus-8 (HHV-8) is associated with some human diseases including Kaposi’s sarcoma and also some B-cell lymphoproliferative disorders. Few studies have highlighted the potential role of HHV-8 in the development of multiple myeloma (MM) which is known as a malignant proliferation of plasma cells derived from a single clone. Aims. The aim of this study was to find a relationship between HHV-8 and MM using polymerase chain reaction (PCR) method. Materials and Methods. This study was conducted on 30 formalin-fixed, paraffin-embedded (FFPE) bone marrow biopsies of multiple myeloma and 30 normal FFPE bone marrow biopsies. After the sample preparation, Deoxyribonucleic acid (DNA) was extracted by nonheating procedure. PCR for HHV-8 virus was carried out with commercial kit and the PCR products were visualized by gel electrophoresis. Finally, the statistical analysis was performed. Results. HHV-8 virus was not detected by PCR from FFPE blocks of multiple myeloma samples, while only one of the controls showed DNA band of the corrected molecular weights. Fisher’s exact test showed that no statistical differences were found between the two groups (P=0.999). Conclusion. Our report adds to the body of evidence that there is no association between HHV- 8 and MM against a major role of HHV-8 infection in the pathogenesis of clonal plasma cell proliferation.

Acute myeloblastic leukemia with associated BCR‐ABL translocation in a dog

An 8-year-old male neutered Labrador Retriever was referred to the University of Wisconsin Veterinary Medical Teaching Hospital with a presumptive diagnosis of leukemia. Hematologic abnormalities included normal neutrophil count with a left shift, monocytosis, eosinophilia, thrombocytopenia, and circulating immature mononuclear cells. Bone marrow was effaced by immature hematopoietic cells of various morphologic appearances. In addition, large multinucleated cells were observed frequently. Flow cytometric analysis of nucleated cells in blood revealed 34% CD34(+) cells, consistent with acute leukemia. By immunocytochemical analysis of cells in blood and bone marrow, some mononuclear cells expressed CD18, myeloperoxidase, and CD11b, indicating myeloid origin; some, but not all, large multinucleated cells expressed CD117 and CD42b, the latter supporting megakaryocytic lineage. The diagnosis was acute myeloblastic leukemia without maturation (AML-M1). To identify genetic aberrations associated with this malignancy, cells from formalin-fixed paraffin-embedded bone marrow were analyzed cytogenetically by multicolor fluorescence in situ hybridization (FISH). Co-localization of bacterial artificial chromosome (BAC) containing BCR and ABL was evident in 32% of cells. This confirmed the presence of the canine BCR-ABL translocation or Raleigh chromosome. In people, the analogous translocation or Philadelphia chromosome is characteristic of chronic myelogenous leukemia (CML) and is rarely reported in AML. BCR-ABL translocation also has been identified in dogs with CML; however, to our knowledge this is the first report of AML with a BCR-ABL translocation in a domestic animal.

Abstract B40: Development of a pharmacodynamic assay for the second-generation proteasome inhibitor MLN9708 in clinical trials of multiple myeloma

Abstract The ubiquitin-proteasome system processes the majority of cellular proteins and is the principal pathway by which cells regulate protein homeostasis. The successful development of VELCADE® (bortezomib) for Injection for multiple myeloma and previously treated mantle cell lymphoma has validated the proteasome as a therapeutic target for the treatment of malignancies. MLN9708 is a second-generation reversible proteasome inhibitor developed to achieve greater oral bioavailability, improved pharmacokinetics and greater antitumor activity than bortezomib. MLN9708 is currently in human clinical development for both hematological and non-hematological malignancies. MLN9708 immediately hydrolyzes to the biologically active form MLN2238 upon exposure to aqueous solutions or plasma, and MLN2238 was used for all preclinical studies described below. In vitro, MLN2238 inhibited 20S proteasome activity, preferentially binding the 20S 5 site with an IC50 of 3.4 nM and demonstrated potent activity against cultured cancer cells in cell viability assays. In vivo, MLN2238 achieved exposures that resulted in significant blood and tumor proteasome inhibition in xenograft-bearing mice and had increased plasma and tumor exposure compared to bortezomib when dosed at their respective maximum tolerated doses (MTD). MLN2238 also elicited a stronger pharmacodynamic (PD) response than bortezomib in xenograft tumors, as measured by tumor 20S 5 site-specific activity and expression levels of GADD34 and ATF-3, two genes involved in the unfolded protein response (UPR) pathway shown to be upregulated in response to proteasome inhibition. Here we describe the development of an ATF-3 IHC assay suitable for use in bone marrow samples isolated from multiple myeloma patients enrolled in Phase I trials of MLN9708. Using both quantitativeWestern blotting and IHC assays, we demonstrated that treatment of cultured myeloma cell lines with MLN2238 results in a dose-dependent increase in ATF-3 levels. Antibody specificity was confirmed by IHC analysis of HCT-116 cells following knockdown of ATF-3 by siRNA. An ATF-3 PD response is detectable by IHC in several xenograft models and in selected normal tissues from mice dosed with MLN2238. The elevation in ATF-3 is delayed by several hours compared to 20S proteasome inhibition in cells and tissues, consistent with it being a downstream effect of proteasome inhibition. To test the use of the ATF-3 IHC assay on clinical samples from multiple myeloma patients, we developed a dual staining assay with CD38, a marker expressed on multiple myeloma cells. We show baseline staining of ATF-3 and CD38 in formalin-fixed paraffin embedded bone marrow aspirates and biopsies from patients who have not received MLN9708. This assay has potential for use in evaluating the levels of CD38 and ATF-3 in pre- and post-treatment bone marrow samples from patients treated with MLN9708. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B40.

Novel Use of Molecular Inversion Probes to Interrogate Formalin-Fixed Paraffin-Embedded (FFPE) Samples of Childhood Leukemia.

Abstract 1589▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-615Genome-wide, high-resolution analyses of copy number alterations (CNAs) now play an increasingly important role in identifying new genomic loci associated with leukemia biology and prognosis. The most powerful of these studies include large numbers of patients with associated clinical features and outcome data. Molecular Inversion Probes (MIPs) analyze genetic target sequences in parallel at the highest genomic resolution and can detect both gene copy number and allelic imbalance in clinical samples, and have been demonstrated to work on archived formalin-fixed paraffin-embedded (FFPE) samples as old as 20 years. In this pilot study, we report for the first time the successful interrogation of high-resolution CNAs in archived FFPE samples in childhood leukemia. We first extracted genomic DNA from FFPE bone marrow aspirate clots from 18 pediatric patients diagnosed with precursor B-cell acute lymphoblastic leukemia (pre-B ALL) diagnosed between 2006-2008 at Primary Children's Medical Center at University of Utah. DNA from paired remission samples was also extracted for each patient, again using archived FFPE bone marrow aspirate clots. Blast percentages on pre-B ALL marrow clots ranged from 39-99% (Mean 88%, Median 94%). Genomic DNA was isolated using RecoverAll” Total Nucleic Acid Isolation Kit for FFPE Tissues (Ambion®, Applied Biosystems). Clinical features and outcome data were readily available and abstracted from the medical record. The 18 patients in the FFPE cohort included: ages 2-21 years old (Median 5.5 years old), 7 females, presenting WBC 1-75 × 103/uL (Median 3.9), CNS negative disease (n=18), no reported cytogenetic abnormalities (n=8), t(12;21) [n=6], t(9;22) [n=1], and MLL rearrangement (n=1). 7 patients were designated “High Risk” by NCI-Rome Criteria and 1 patient relapsed. The MIP assay was run using the customized 330K Cancer Panel (Affymetrix®, Santa Clara, CA), which includes both cancer-specific SNPs and genome-wide coverage with a median probe distance of 4,207 basepairs (bp). Copy number was calculated by comparing leukemia samples to pooled normal control signal intensity for each probe. CNA calls were based on 5 consecutive probes with >90% call rate, standard deviation < 20%, and copy number ' 1.2 or ≥ 2.8. MIPs revealed remarkably high-quality CNA data for each of the 18 FFPE samples, including the cytogenetically “normal” patients. Both known and novel recurring CNA loci were identified in this cohort. Deletions included: 14q11.2 (n=11 [61%], 51569 bp, no known genes), 22q11.2 (n=10 [56%], 185944 bp, VPREB1), 14q32.33 (n=10 [56%], 631377 bp, no known genes), 7q34 (n=9 [50%], 292586 bp, PRSS1, TRY6, PRSS2), and 12p13.2 (n=6 [33%], 292586 bp, ETV6). Gains included: 10p15.2 (n=10 [56%], 26481 bp, PFKP), 10q26.3 (n=10 [56%], 69691 bp, MGMT), 10p11.21 (n=8 [44%], 922257 bp, FZD8, CCNY, GJD4), and 8p23.3 (n=7 [39%], 90307 bp, ARHGEF10). Interestingly, of 52 amplified segments recurring in 35% or greater of samples, all but one were located in chromosome 10, 14, 17, 18, or 21 suggesting genetic amplification hotspots. Additionally, 100% of IKZF1-deleted samples (n=2/2) compared to 37.5% of IKZF1-normal samples (n=6/16) had M3 marrows (>25% blasts) at Day 7. These same two IKZF1-deleted FFPE samples belonged to the patients with the two highest WBC values at presentation (75.3, 22.9 × 103/uL), in addition to containing two of the six highest bone marrow blast percentages at diagnosis. This is consistent with known findings that IKZF1 deletions are associated with high-risk ALL. In this pilot study of 18 patients, we have shown that archived FFPE bone marrow aspirate specimens (standard of care for all bone marrow procedures) can be used successfully for known and novel CNA analysis in childhood leukemia. We believe this is the first time that high-resolution, genome-wide CNA data from FFPE samples in any type of leukemia have been reported. This is an important development in CNA studies of hematological disease because now it is possible to investigate an unlimited number of archived FFPE childhood leukemia samples from around the world, or to explore rarer and more difficult to find specimens such as relapse or concordant identical twins. DisclosuresNo relevant conflicts of interest to declare.

Chemotherapy‐related infections in patients with multiple myeloma: associations with mannan‐binding lectin genotypes

To study a possible association between mannan-binding lectin genotypes and severe infections in patients with multiple myeloma receiving moderate strength induction chemotherapy. Chemotherapy-related infections were identified retrospectively using clinical records and database files. Mannan-binding lectin genotypes were identified with polymerase chain reaction on stored samples of stem cells or formalin-fixed paraffin-embedded bone marrow biopsies. We included 138 myeloma patients. In five patients, data were incomplete, and 133 patients were analysed. Eighty-eight patients were homozygous for wild-type MBL2 (AA) and forty-five patients were heterozygous or homozygous for variant genotypes (AO/OO). A total of 390 chemotherapy cycles were reviewed. Common Toxicity Criteria grades 3 and 4 infections in general were seen in relation to 104 cycles and were not more common in patient with variant MBL2 (P = 0.90). Septicaemia was seen after 10% of chemotherapy cycles in AA patients vs. 15% in AO/OO patients (P = 0.15). In multi-variate analyses, we found indication of a reduced risk of septicaemia in AA patients [OR 0.27 (0.08-0.90), P = 0.03], after first chemotherapy cycle, but reduction of the risk including all cycles was not significant. A similar trend was seen for grades 3 and 4 infections in general. During induction chemotherapy in patients with multiple myeloma, a general protective effect of wild-type MBL2 against chemotherapy-related infections was not apparent in this study. However, we found indications of a reduced occurrence of septicaemia in patients with wild-type compared with variant MBL2. Further studies in larger cohorts of patients are relevant.

Primary Myelodysplastic Syndromes: Diagnostic and Prognostic Significance of Immunohistochemical Assessment of Bone Marrow Biopsies

Material from 63 cases with primary myelodysplastic syndromes (P-MDS) (French-American-British [FAB] types: refractory anemia [RA] = 21; RA with ring sideroblasts [RARS] = 8; RA with excess of blasts (RAEB) = 10; RAEB in transformation (RAEBt) = 6; chronic myelomonocytic leukemia [CMML] = 10 and unclassifiable = 8, ie, bone marrow aspiration was inadequate and stringent FAB criteria were not applicable) was analyzed for bone marrow histologic and immunohistochemical patterns. Standard Giemsa, hematoxylin and eosin (H&E) and reticulin stains were used for morphologic assessment. To identify the cell lineage precisely, chloroacetate esterase staining and an indirect immunoperoxidase technique using mouse monoclonal antibodies CD15, CD68, HLA-DR, and rabbit polyclonal CD3 and UEA-1 (lectin) was developed on formalin-fixed paraffin embedded bone marrow biopsies (BMB). The immunohistochemical assessment permitted accurate identification of dysplastic features such as mononuclear and binuclear megakaryocytes, Pelger-Huet neutrophils, and binuclear erythroblasts. Additional bone marrow histologic and immunohistochemical features observed were heterogeneity of immunohistochemical staining in various cell lineages, megakaryocytic emperipolesis, alteration of bone marrow microarchitecture, intravascular clusters of hematopoietic cells, and the types of benign lymphoid aggregates. The nature of abnormally localized immature precursors (ALIP) was discerned. Three types of clusters of immature cells were found that were difficult to distinguish on Giemsa and H&E morphology, these were erythroid aggregates (n = 18); megakaryocytic aggregates (n = 4), and immature granulocytic and monocytic aggregates (n = 32). The bone marrow histologic and immunohistologic patterns permitted the identification of four groups of clinical relevance: Group 1, cases with predominant erythroid hyperplasia and without ALIP (n = 15); group 2, cases with prominent myeloid hyperplasia and presence of ALIP (n = 32); group 3, cases with hypoplastic MDS (n = 10); and group 4, cases with hyperfibrotic MDS (n = 6). Statistical analysis showed a significant difference in survival and leukemic transformation between groups 1, 2, 3, and 4, with cases in group 2 showing the worst prognosis with early death due to increased propensity to leukemic transformation and cytopenia-related complications (P less than .0001). We conclude that immunohistochemistry is feasible on routinely processed BMB and the information obtained is of diagnostic and prognostic importance in P-MDS. The phenotype of ALIP varies with the morphologic and histologic subtypes of MDS and the term should be reserved for cases in whom the clusters in the intertrabecular region are of myeloid (granulocytic and monocytic) lineage on immunohistochemistry.