Paraffin-embedded Bone Marrow Biopsies Research Articles

Multiplex imaging reveals spatially resolved DNA-damage response neighborhoods in TP53-mutated myelodysplastic neoplasms.

While increased DNA damage is a well-described feature of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), it is unclear whether all lineages and all regions of the marrow are homogeneously affected. In this study, we performed immunohistochemistry on formalin-fixed, paraffin-embedded whole-section bone marrow biopsies using a well-established antibody to detect pH2A.X (phosphorylated histone variant H2A.X) that recognizes DNA double-strand breaks. Focusing on TP53-mutated and complex karyotype MDS/AML, we find a greater pH2A.X+ DNA damage burden compared to TP53 wild-type neoplastic cases and non-neoplastic controls. To understand how double-strand breaks vary between lineages and spatially in TP53-mutated specimens, we applied a low-multiplex immunofluorescence staining and spatial analysis protocol to visualize pH2A.X+ cells with p53 protein staining and lineage markers. pH2A.X marked predominantly mid- to late-stage erythroids, whereas early erythroids and CD34+ blasts were relatively spared. In a prototypical example, these pH2A.X+ erythroids were organized locally as distinct colonies, and each colony displayed pH2A.X+ puncta at a synchronous level. This highly coordinated immunophenotypic expression was also seen for p53 protein staining and among presumed early myeloid colonies. Neighborhood clustering analysis showed distinct marrow regions differentially enriched in pH2A.X+/p53+ erythroid or myeloid colonies, indicating spatial heterogeneity of DNA-damage response and p53 protein expression. The lineage and architectural context within which DNA damage phenotype and oncogenic protein are expressed is relevant to current therapeutic developments that leverage macrophage phagocytosis to remove leukemic cells in part due to irreparable DNA damage. © 2024 The Pathological Society of Great Britain and Ireland.

The Novel Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) Platform Identifies Synergistic Activity of Lanraplenib and Ruxolitinib in Hematological Malignancies

Background: Investigation of drug combinations across different contexts can provide useful insights on the anti-cancer mechanism and can ultimately lead to new treatments. However, conventional drug combination screening methods are limited by throughput. Efforts to systematically identify the most effective active combinations and the optimal molecular contexts in a high throughput screening (HTS) format could greatly accelerate the development of combination treatments. Spleen Tyrosine Kinase, SYK, is a non-receptor tyrosine kinase known to regulate intracellular signaling, including FLT3, AKT/mTOR and STAT5 pathways, via its immunoreceptor tyrosine-based activation motif (ITAM). Deregulated SYK signaling has been reported to play a central role in pathogenesis of allergic and autoimmune diseases as well as hematological malignancies. Lanraplenib (LANRA) is a next-generation SYK inhibitor currently being evaluated in combination with gilteritinib, a FLT3 inhibitor, in patients with relapsed or refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) (NCT05028751). Given its critical role in intracellular signaling and interaction with receptor tyrosine kinases(RTKs), we hypothesized that LANRA could synergize with ruxolitinib, a JAK inhibitor. To address this hypothesis, we performed a high throughput drug combination screen using the Broad Institute's Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) platform, which enables rapid screening of thousands of compounds in a 930-cell line panel across 45 different lineages. Methods: In this study, we performed a combination screen using the PRISM platform with ruxolitinib as the test compound, with a 10 μM top dose (7 dose concentrations with 3-fold dilutions), in combination with LANRA at two anchor doses, 2 μM and 10 μM. PRISM is a pooled, multiplexed cell viability assay that provides 7-point dose response curves, IC 50, AUC values, and relative abundance of unique cell line barcodes. To understand the drug synergy landscape across different lineages, we developed a bioinformatics pipeline which uses PRISM viability data to calculate synergy scores across all the cell lines and drug combinations. Secondary validation studies of the combinations used CTG viability measurements. Phospho-SYK expression was evaluated in archival formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with myeloid proliferative neoplasm (MPN) by immunohistochemistry (IHC). RNA-seq was performed to evaluate differential changes in gene expression in response to LANRA. Gene set enrichment analysis (GSEA) was performed to evaluate perturbation in leukemogenic signaling pathways. Results: In the PRISM cell line panel, ruxolitinib in combination with LANRA demonstrated synergistic activity in hematological malignancy cell lines. Among AML cell lines in the panel, OCIAML5, OCIM1, HL60, HEL, EOL1, MONOMAC6, NB4, U937, PL21, and TF1 showed the highest synergy scores. The most sensitive cell lines to the combination showed up-regulation of JAK-STAT and inflammatory signaling pathways in a gene set enrichment analysis (GSEA) prior to treatment. Consistent with this, phosphorylated SYK was associated with inflammatory megakaryocytes and fibrosis in primary samples from patients with MPN. LANRA down-regulated JAK-STAT signaling in a reporter cell line and repressed gene expression associated with dysregulated inflammatory pathways in AML cells. Additionally, the combination of LANRA and ruxolitinib showed synergistic antiproliferative activity across a broad range of concentrations in FLT3-ITD primary AML cells and a panel of hematological malignancy cell lines, confirming the PRISM results. Conclusion: These studies demonstrate the utility of PRISM as a platform to rapidly identify rational combination agents. Importantly, LANRA is effective in combination with ruxolitinib in AML and other hematological malignancy preclinical models. This finding is consistent with the observation that SYK can regulate STAT signaling and cooperate with other RTKs like FLT3. Given the central role of SYK in regulation of oncogenic and inflammatory signaling, SYK inhibition with LANRA in combination with ruxolitinib may be a promising strategy for patients with myeloid malignancies.

Cellular Interactions within Clonal Dendritic Cell Aggregates Drive Immune Tolerance in Chronic Myelomonocytic Leukemia Bone Marrow Microenvironment

Introduction: Clonal dendritic cell (DC) aggregates in chronic myelomonocytic leukemia (CMML) are seen in approximately 20-30% patients at diagnosis. Previously, these were thought to be comprised of CD123+ plasmacytoid DCs but our data has suggested that these are heterogenous and comprise of myeloid DCs, monocytes, and myeloid-derived suppressor cells. Furthermore, these aggregates uniformly express immune checkpoints such as indoleamine 2,3-dioxygenase-1 (IDO), associate with an IDO-specific systemic impact (increased trytophan catabolism), and expanded regulatory T cell population in CMML, suggesting a role in inducing immune tolerance. DC aggregates are also associated with increased risk of acute myeloid leukemia (AML) transformation and resistance to hypomethylating agents, highlighting that they are important for disease progression in CMML (Mangaonkar A et al. Blood Adv 2020). We performed this study to decipher the architecture of a CMML DC aggregate to understand the complex inter-cellular interactions, and assess their impact on the adaptive immune system. Methods: In this study, we used archival fresh-frozen paraffin-embedded (FFPE) bone marrow biopsy specimens obtained from CMML patients, and isolated monocytes (CD14+ magnetic bead sorting) and lymphocytes (CD14-ve fraction) from untreated CMML and age-matched normal control (NC) peripheral blood mononuclear cells (PBMCs). DCs were cultured from monocytes using a commercial protocol. Spatial architecture of the CMML BM microenvironment was interrogated through multiplex imaging of FFPE samples by co-detection by indexing (CODEX) using 30 markers (CCR6, CD107a, CD11c, CD20, CD21, CD31, CD3e, CD4, CD44, CD45, CD45RO CD56, CD68, CD8, E-Cadherin, FoxP3, HLA-DR, CD14, CD16, CD11b, CD33, CD15, CD66b, CD123, CD303, CD34, CD163 and IDO). Single-cell transcriptomic signatures were analyzed using the 10X single cell Fixed RNA profiling platform, and validated using bulk RNA seq (Illumina stranded mRNA prep) from isolated and cultured cells. Impact of DCs on lymphocytes was assessed via autologous mixed DC-T culture experiments. Results: BM FFPE samples from 5 CMML cases (3 with DC aggregates) were included for multiplex imaging via CODEX. Results confirm the significant interactions of CD8+ and CD4+ T cells with CD123+/HLA-DR+ DCs within a CMML DC aggregate (Fig. 1A). Upon quantitative assessment of cell-cell interactions, IDO+/CD8+T ( P=0.006), IDO+/MDSC ( P=0.005), and IDO+/macrophage ( P=0.001) interactions were significantly increased in CMML samples with versus without DC aggregates . Specific findings were validated using single-stain immunohistochemistry and multiplex immunoflourescence staining. Next, in order to assess transcriptomic signatures within BM at the single-cell level, we chose two cases; CMML (BM aspirate blast% 1, WBC 49.6 x 10 9/L, ASXL1/GATA2/SETBP1/SRSF2 mutations) and AML transformed from CMML (BM aspirate blast% 81, WBC 152 x 10 9/L, NPM1/DNMT3A/TET2 mutations). Approximately, 10,000 cells were obtained and analyzed with cell type annotation through human PBMC reference (Azimuth). Results show an expanded regulatory T cell population in AML compared to CMML (UMAP plot, Fig. 1B), suggestive of immune tolerance. In order to validate findings and perform functional experiments, we used sorted monocytes and cutured DCs from CMML and NC. DC phenotype was confirmed both through visual inspection and flow cytometry, and clonality was confirmed through Sanger sequencing (for previously identified mutations). Differential gene expression analysis through bulk RNAseq showed significant differences between CMML and NC lymphocytes with immunoinhibitory receptor, LILRB2 (log2fold change=7.05, adjusted P=0.0002) being one of the top five significantly upregulated genes, further confirming immune tolerance. Finally, mixed autologous DC-lymphocyte reaction (1:4 ratio) experiments demonstrated that DCs induce T cell tolerance in CMML (expansion of exhausted PD1/TIM3/LAG3+ CD8 T cells) when compared to NC. Conclusion: Clonal dendritic cell aggregates within the CMML bone marrow microenviroment interact with T cells and induce immune tolerance. This represents a novel mechanism of disease progression in CMML with potential therapeutic targets (IDO, LILRB2, and/or CD123).

P392: PHARMACOLOGICAL INHIBITION OF SYK CONFERS ANTI-PROLIFERATIVE AND NOVEL ANTI-TUMOR IMMUNE RESPONSES IN AML

Background: Spleen tyrosine kinase (SYK) acts as a key integrator of signals from cell surface receptors containing an immunoreceptor tyrosine-based activation motif to boost cellular proliferation. In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network linked to NPM1, HOXA9 and MEIS1. The selective, orally bioavailable SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity and tolerability in HOXA9/MEIS1-driven AML. ENTO is currently being investigated in a global Phase 3 trial, AGILITY (NCT05020665), in combination with intensive induction/consolidation chemotherapy in patients (pts) with treatment-naive NPM1-mutated (NPM1m) AML. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency and selectivity to ENTO but with more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in pts with relapsed or refractory FLT3-mutated AML (NCT05028751). Aims: To investigate the effects of ENTO and LANRA in T-cell responses in AML. Methods: All pt-derived bone marrow (BM) and peripheral blood samples were obtained in accordance with IRB (OHSU#004422) approval. Results from gene expression profiling studies (RNA sequence [RNA-seq]) in bone marrow/peripheral blood mononuclear cells (MNCs) derived from 152 pts with AML, for which matching NPM1 status and ex vivo sensitivity to ENTO was available, previously published by Tyner et al (2018), were reanalyzed using trimmed mean of M values normalization and differential expression using edgeR. ENTO/LANRA sensitivity was assessed ex vivo after 72 hours in culture at 37°C by MTS assay. T-cell functional assays were performed by culturing MNCs with anti-CD3 and treating with ENTO or LANRA, singly and in combination with anti-PD1. CD3+ T-cell proliferation as measured by Ki67 expression and pSYK activation were assessed by quantitative single-cell immunofluorescence microscopy. Six formalin-fixed, paraffin-embedded BM biopsies from newly diagnosed AML pts were assessed for spatial expression of mRNA using a digital spatial profiling method. Results: In AML, mutations in NPM1 with co-expression of HOXA9/MEIS1 at baseline predicted anti-proliferative activity to ENTO in ex vivo drug sensitivity studies. Accordingly, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. Pathway analysis of archival RNA-seq data from pts enrolled in the Leukemia and Lymphoma Society BEAT AML Master Protocol (NCT03013998 [BAML-16-001-S6]) revealed gene expression signatures at baseline associated with the observed sensitivity to ENTO, including significant enrichment in the expression of genes associated with leukemogenesis, myeloid differentiation and immune regulation. This was supported by T-cell functional studies, which demonstrated that ENTO and LANRA, singly as well as in combination with anti-PD1, could restore T-cell proliferation in primary AML pt samples that exhibited suppression. Lastly, Cancer Transcriptome Atlas analysis on archival BM biopsies obtained from these AML pts showed a strong correlation between immune checkpoint response and gene expression associated with immune pathway signaling. Summary/Conclusion: Inhibition of SYK is a promising therapeutic approach in NPM1m AML. Our studies illustrate that ENTO and LANRA may restore T-cell proliferation in a subset of AML pts with dysfunctional T-cell responses, suggesting a novel mechanism of action. Additional studies are required to fully understand the mechanism of SYK-mediated antitumor immune responses in AML.

Frequency of Nucleophosmin 1 Expression by Immunohistochemistry in Acute Myeloid Leukemia.

Nucleophosmin (NPM1) mutation is one of the most common recurring genetic abnormalities seen in acute myeloid leukemia (AML). Immunohistochemistry serves as a cost effective and simple surrogate testing method for detection of NPM1 mutation. This study was conducted to evaluate the frequency of aberrant cytoplasmic nucleophosmin 1 expression in leukemic blast cells on formalin fixed bone marrow trephine biopsy (BMB) sections and also to correlate this data with the reference molecular method (reverse transcriptase-polymerase chain reaction; RT-PCR and gene sequencing), where available. Immunostains were performed using mouse anti-NPM1 monoclonal antibody on 71 paraffin embedded bone marrow biopsies (BMB) of patients with AML of any French-American-British (FAB) subtype. Results of immunohistochemistry (IHC) were then compared with the reference molecular method. The proportion of NPM1 expression by immunostaining in AML cases was found to be 17%. Twelve of the total 71 cases demonstrated cytoplasmic nucleophosmin (NPMc+) on immunostaining. Eleven of the positive cases that were correlated with the molecular standard demonstrated mutation in exon 12 of NPM1 gene. Cytoplasmic nucleophosmin expression by immunostaining was found to be in complete agreement with the standard molecular method. In a resource restricted setup, the information from this study might help in providing an inexpensive and accurate detection method to facilitate introduction of this marker in diagnostic and prognostic workup of AML especially in patients showing normal karyotype and no common recurrent translocations.

Rational Targets in Systemic AL Amyloidosis

IntroductionDysproteinemia refers to a spectrum of gammopathies in which an aberrant clone(s) of late stage B lymphoid cells or plasma cells produces monoclonal immunoglobulins and/or fragments. The spectrum includes incidentally noted benign conditions like monoclonal gammopathy of undetermined significance (MGUS) to life threatening diseases such as multiple myeloma (MM) and systemic light chain (AL) amyloidosis. Exploitation of normal plasma cell biology forms the basis of treatments used in both MM and AL amyloidosis. In recent years, immunotherapeutic strategies targeting plasma cells have redefined the treatment landscape of MM and could be particularly useful in AL amyloidosis which is distinguished by a low burden of less proliferative disease and a paucity of high risk genetics. We sought to characterize the expression of potentially clinically relevant and/or immunotherapeutic targets in amyloidogenic plasma cells.MethodsAll patients diagnosed with systemic AL amyloidosis at Memorial Sloan Kettering Cancer Center, NY between January 1, 2012, and December 31, 2018, who had unstained bone marrow samples were identified. Decalcified formalin fixed paraffin embedded bone marrow biopsy sections were stained for G protein-coupled receptor, class C group 5 member D (GPRC5D), Cyclin D1 (CCND1), B-cell lymphoma 2 (BCL2), and B-cell maturation antigen (BCMA) expression using standard laboratory developed Immunohistochemistry (IHC) assays in a CLIA compliant setting. We scored the biopsies for percentage expression, and intensity of staining. We also obtained the demographic details, staging, and cytogenetic information for the patients from whom these samples were obtained.ResultsDuring the queried period, 32 unstained samples were available for testing from 27 unique patients. There were 27 diagnostic and 5 patients had additional samples from the time of relapse. The median age was 63 years (range 41-73). 64% of patients were male and 75% had lambda restricted plasma cells. Cytogenetic abnormalities using fluorescence in situ hybridization (FISH) were reviewed, t(11;14) was seen in 38% samples. The median clonal PC burden in BM at diagnosis was 10% (range 2-80%) and 36% had >10% plasma cells. In clonal PCs, the median BCL2 expression was 100% (range 80-100%) with a staining intensity of 2 (range 1-3). 84% (27) samples had BCL2 expression meeting threshold used in the Bellini study. CCND1 expression could be tested in 16 samples median expression 100% (range 20-100%) and median staining intensity 2 (range 1-3). Patients with CCND1 expression also had 100% BCL2 staining. GPRC5D expression was available in 18 samples and all samples tested expressed GPRC5D with median 80% (range 30-100%) with median intensity 1 (range 1-3). BCMA expression was available for 25 samples, with median expression 80% (range 50-100%) with a median staining intensity 2 (range 1-3). Among the five patients with samples from diagnosis and relapse, samples retained their expression of BCL2, BCMA, and GPRC5D. Among samples with t(11;14) by FISH, 92% expressed BCL2 (per Bellini study) and 58% expressed CCND1.ConclusionCell surface expression of novel targets has enabled the development of several efficacious therapeutic strategies in MM. Amyloidogenic plasma cells express GPRC5D, BCMA and BCL2. Our data provides the rationale for careful investigation and development of targeted agents (BCL2 inhibitors, e.g.venetoclax) and immunotherapeutic strategies directed at GPRC5D and BCMA in AL amyloidosis. [Display omitted] DisclosuresDogan: Roche: Consultancy, Research Funding; Peer View: Honoraria; Seattle Genetics: Consultancy; Takeda: Consultancy, Research Funding; Physicians' Education Resource: Honoraria; EUSA Pharma: Consultancy. Hassoun: Celgene, Takeda, Janssen: Research Funding. Mailankody: Fate Therapeutics: Research Funding; Allogene Therapeutics: Research Funding; Bristol Myers Squibb/Juno: Research Funding; Plexus Communications: Honoraria; Legend Biotech: Consultancy; Evicore: Consultancy; Jansen Oncology: Research Funding; Physician Education Resource: Honoraria; Takeda Oncology: Research Funding. Giralt: SANOFI: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Landau: Takeda: Research Funding; Genzyme: Honoraria; Takeda, Janssen, Caelum Biosciences, Celgene, Pfizer, Genzyme: Membership on an entity's Board of Directors or advisory committees.

Evaluation of an innovative new method for quantitation of plasma cells on CD138 immunohistochemistry

AimTo compare the frequently used CD138 immunohistochemistry-based method of plasma cell quantitation, to a proposed new method, using interobserver and intraobserver concordance parameters.MethodsArchival CD138 immunohistochemically stained slides made from paraffin-embedded...

Restricted Immunoglobulin Joining Chain (IgJ) Protein Expression in B Lymphoblastic Leukemia (B-ALL) Based on B-ALL Subtype

Background Philadelphia-like (Ph-like) B Lymphoblastic Leukemia (B-ALL) is a high-risk subtype of B-ALL that lacks the BCR-ABL1 fusion but has a gene expression profile similar to Philadelphia positive (Ph+) B-ALL. Gene expression profiling has previously identified Immunoglobulin Joining chain (IgJ) overexpression at the mRNA level in Ph-like B-ALL. This is surprising since IgJ is normally expressed by mature or maturing B cells. The normal function of IgJ protein is to concatenate monomers of immunoglobulin IgM or IgA into the mature pentameric and dimeric forms of these molecules respectively. IgJ also plays a crucial role in transport of IgA protein across mucosal epithelium to facilitate mucosal humoral immunity. In hematopathology, J chain immunohistochemistry (IHC) has been used to identify neoplastic cells in nodular lymphocyte predominate Hodgkin lymphoma (NLPHL) and can be used in distinguishing this disease from morphologic mimickers. It does not have known diagnostic utility outside of this context. Lymphoblasts do not typically express immunoglobulins at the protein level. Therefore, we sought to determine the protein expression of IgJ in B-ALL and to determine whether IgJ immunohistochemistry may be employed in identifying particular subtypes of B-ALL. Methods We selected a total of 46 B-ALL cases diagnosed from a bone marrow sample at our institution from 2016-2019 with adequate diagnostic material for IHC. This included 5 cases of Ph-like B-ALL, all with a CRLF2 rearrangement and overexpression, 7 de novo Ph+ B-ALL and 34 cases representing the other most commonly recognized WHO subtypes of B-ALL, determined based on cytogenetic studies performed at the time of diagnosis. No cases of B-ALL with t(5;14) and B-ALL with iAMP21 was represented. Our cohort included 23 pediatric cases and 24 adult cases and the patients ranged from 1 to 82 years old at the time of initial diagnosis. A total of 8 normal bone marrow cases (negative staging bone marrow biopsies for diffuse large B cell lymphoma, neuroblastoma or classic Hodgkin lymphoma) were used as controls. IgJ IHC was performed on B-plus fixed paraffin embedded bone marrow biopsy specimens using a commercially available and validated anti-IgJ monoclonal antibody (clone OTI3B3). Staining of bone marrow samples was performed at 2 different dilutions; tonsil secondary follicles and neoplastic cells from NLPHL were used as technical controls. Cellular staining in the lymphoblasts was scored in a blinded manner by a board certified hematopathologist and a pathologist in training as diffusely positive, partially positive, or negative. Results Cellular staining was distinguishable from background staining due to circulating immunoglobulins and there was almost perfect inter-observer concordance in identifying positive and negative cases (agreement of 98%, kappa test 0.94). All normal bone marrow controls cases were negative for IgJ cellular staining. A total of 11/46 (23%) B-ALL cases demonstrated partial or diffuse cellular staining for IgJ in the lymphoblasts. This included 4/5 (80%) Ph-like cases, 5/7 (71%) Ph+ cases, 1/3 MLL rearranged cases and 1/6 ETV6-RUNX1. All TCF3-PBX1 (0/4), hyperdiploid B-ALL (0/10), hypodiploid B-ALL (0/2), and B-ALL, NOS cases (0/9) were negative for IgJ. Diffuse IgJ staining was restricted to Ph-like (2/4) or Ph+ (2/5) B-ALL subtypes; the positive MLL rearranged and ETV6-RUNX1 B-ALL cases only showed weak partial staining. IgJ protein was significantly expressed in Ph+/Ph-like B-ALL (p<0.0001) and in our cohort, detected these cases with a 75% sensitivity, 95% specificity, 82% positive predictive value and 92% negative predictive value. Conclusion We conclude that IgJ protein expression occurs in a subset of B-ALL, predominantly restricted to Ph+ and Ph-like cases. Although, these findings will need to be validated in larger studies, our results suggest that IgJ IHC, in concert with routine standard cytogenetics studies may be a rapid and cost effective method in identifying Ph-like B-ALL. Disclosures No relevant conflicts of interest to declare.

PDL1 Positivity By FISH in Patients Not in Complete Remission at Transplantation

Introduction Failure to achieve morphological complete remission occurs in 30% AML patients resulting in dismal outcomes. Data addressing influence of pre-transplant PDL-1 status on post-transplant outcomes is limited. We hypothesize that PDL-1 expression by immunohistochemistry (IHC) or Fluorescent in situ hybridization (FISH) prior to transplantation may predict a poor outcome. Methods This was a retrospective, observational single center pilot study involving refractory AML patients who underwent allogeneic stem cell transplantation (ASCT) at Karmanos Cancer Institute from 01/01/2012 to 01/01/2018. A planned sample size of 24 patients were selected and assigned to two cohorts based on their respective responses to ASCT: a) poor responders: persistent leukemia by bone marrow biopsy within 30 days of ASCT, and b) good responders: no evidence of leukemia at 1-year post-transplant. Descriptive analyses evaluated differences in demographic, disease and treatment variables. FISH assay was used to evaluate PDL-1 alterations in the pre-transplant archival, formalin-fixed, paraffin-embedded bone marrow biopsy specimens including frequency and magnitude of 9p24.1 alterations— low-level disomy, polysomy, copy gain, and amplification. The expression of PD-L1 was evaluated by IHC in a semi-quantitative (0 through +3) manner. Results Of the total 24 patients, 12 patients were poor responders and 12 were good responders; among poor responders, median age was 58 years (range, 31- 72) and gender distribution was 1:1. Seven patients (58%) had prior MDS, Myelofibrosis or CMML, while five patients had de-novo AML. Treatment information is provided in table 1. At pre-transplant bone marrow biopsy assessment, median number of bone marrow and peripheral blood blast counts were 8.1% (1.7-37) and 2% (0-37), respectively. Bone marrow cytogenetics assessed prior to transplant included intermediate risk in 2 (17%) and high risk disease in 9 (75%) patients and one patient without cytogenetic evaluation. PD-L1 expression was negative by IHC in all samples, but FISH testing was positive in 3 of 12 (25%) poor responders, characterized by copy gain. In good responders, median age was 59 years (range, 43-70) with a 3:2 male predominance. Four patients (25%) had prior MDS or Myelofibrosis and 8 had de-novo AML. Five patients (42%) had intermediate risk and 7 (58%) had high-risk cytogenetics. At pre-transplant bone marrow biopsy assessment, median number of bone marrow and peripheral blast counts were 6% (1-40) and 1% (0-7), respectively. PD-L1 expression was negative by both IHC and FISH examination in this cohort. Conclusion Our study demonstrates that PDL-1 expression by FISH was specifically detected in 25% of patients with poor responders following ASCT, which could possibly provide an insight into the poor outcome. A larger sample will be required to validate this observation in this population.

Distinct Bone Marrow Microenvironment Abnormalities in MDS and MPN with Fibrosis

Background: Bone marrow (BM) fibrosis is common in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN), its pathogenesis remains unclear. It is also unknown whether and how the pathophysiology of BM fibrosis in MDS and MPN are similar or different, while it is anticipated that altered BM microenvironment plays a key role. Methods: To visualize various BM microenvironmental cells in MDS or MPN patients as compared to those in control subjects, we utilized multicolor immunostaining using a tyramide signal amplification system and a fluorescence spectrum analyzer. Paraffin-embedded BM biopsy samples from 47 patients with MDS (26 without and 21 with fibrosis), 10 with primary myelofibrosis or myelofibrosis secondary to essential thrombocythemia (MPN-F), and 17 with non-Hodgkin lymphoma without BM involvement (as control) were immunostained with antibodies for mesenchymal/neuronal cell markers. Gene mutations were analyzed by targeted sequencing. Results: In control samples, C-X-C motif ligand 12 (CXCL12) was stained mainly in cells at perivascular areas and the periphery of a minor proportion of adipocytes. These patterns did not differ in MDS without fibrosis from control. However, CXCL12-stained non-adipose cells were significantly increased in MDS with fibrosis (MDS-F) [median (range), 5190 (2117-14299) μm2/mm2 as compared to 628 (464-1331) μm2/mm2 in control; p=0.00453]. These cells tended to be increased in also MPN-F though statistical significance was not reached [6096 (1098-24458) μm2/mm2; p=0.06304]. The localization of these cells was not confined to the perivascular areas in both diseases. There were no apparent differences in the degree or the pattern of staining for CXCL12 between MDS-F and MPN-F. We also focused on Schwann cells and Nestin-expressing stromal cells because these cells have been independently reported to function as an important hematopoietic stem cell niche. Neurofilament heavy chain (NFH) or neuron-specific class III beta-tubulin-positive nerve fibers (Tuj1) surrounded by glial fibrillary acidic protein (GFAP)-positive Schwann cells were rarely observed in control samples. This observation was true of MDS without fibrosis. However, GFAP-positive Schwann cells were significantly increased in MDS-F and MPN-F [median (range), 39457 (1325-60654) um2/mm2 and 75951 (19879-326216) um2/mm2, as compared to 6258 (1325-14842) um2/mm2 in control; p=0.0017 and 0.0034, respectively]. When the section was stained for Nestin, Schwann cells in MPN-F did not show positive signals except for one case. In contrast, Nestin was frequently stained in the GFAP-positive Schwann cells of MDS-F (9 of 21 MDS-F, 42.9%); particularly 5 of 7 cases (71.4%) in which fibrosis was judged as severe (MF-3), showed positive signals. One exceptional case of MPN-F that showed Nestin-positivity in increased Schwann cells was a triple-negative case (no mutations in JAK2, CALR, or MPL); instead, mutations were found in NRAS, TET2, BCOR, and SMC1A, indicating that this case may have a feature of MDS. Conclusion: Abnormal increases in CXCL12-expressing mesenchymal cells and nerve fiber-surrounding Schwann cells were visualized by multicolor fluorescence in MDS-F and MPN-F. Schwann cells in MDS-F and MPN-F showed a remarkable difference in the expression of Nestin. It is to be elucidated whether the increase in the Schwann cells has a role in the pathogenesis/pathophysiology, and if so, whether those Schwann cells with distinct phenotypes have differential roles in these two disease conditions. Disclosures Ogawa: ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; RegCell Corporation: Equity Ownership; Qiagen Corporation: Patents & Royalties; Asahi Genomics: Equity Ownership.

PS1327 PRO‐NECROPTOTIC RIPK3 AS A STAGE‐DEPENDENT MARKER IN THE BONE MARROW OF PATIENTS WITH MDS AND CMML

Background:Chronic inflammation plays a central role in the development of hematological neoplasms. In Myelodysplastic syndromes (MDS) pro‐inflammatory microenvironment is suspected to drive disease development. Several studies have shown increased rates of programmed cell death in marrow cells of patients with early stage MDS. In these patients improvement of cytopenia is a major therapeutic approach. Overcoming resistance to programmed cell death is a central therapeutic aim in patients with high‐risk MDS/sAML. RIPK3 is a crucial player of regulated necrosis (necroptosis). Necroptosis has a strong inflammatory capacity and pro‐inflammatory processes trigger necroptosis.Aims:The aim of this project is to identify pro‐necroptotic proteins in the bone marrow of patients with MDS, CMML and sAML and to correlate the necroptotic capacity with the stage of the disease. By pharmacological modulation of necroptosis we would like to evaluate the effects of necroptotic signaling on inflammation, cell death and cell differentiation.Methods:We therefore analyzed the impact of pro‐necroptotic signaling on bone marrow bulk and the subset of CD34+stem/progenitor cells of patients with MDS and chronic myelomonocytic leukemia (CMML) in vitro. Bone marrow mononuclear cells (BMMNCs) were isolated from BM aspirates of 5 patients with early MDS (MDS‐MLD, MDS‐RS‐SLD and MDS‐RS‐MLD), 5 patients with late MDS (MDS‐EB1 and MDS‐EB2), 5 patients with CMML‐1, 7 patients with CMML‐2 and 4 patients with sAML after a history of MDS and 4 patients with sAML after a history of CMML. Bulk of BMMNCs and purified CD34+stem/progenitor cells were stained intracellular for RIPK3 and analyzed by flow cytometry. The patient samples were compared to 12 age‐matched BM samples obtained from hip replacement surgery from otherwise healthy individuals. Furthermore, immunohistochemistry was performed in an enlarged cohort of patients. Therefore paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression. Additionally BMMNCs of patients with MDS were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitors for 72 h. Short term viability analysis by flow cytometry were performed and long‐term analysis using growth factor enriched methylcellulose.Results:Expression of RIPK3 was significantly increased in sAML after a history of MDS compared to late MDS and compared to healthy controls. Furthermore patients with sAML after a history of CMML showed a significantly higher expression of RIPK3 than patients with CMML‐1, CMML‐2 and healthy controls. These findings were confirmed by immunohistochemistry in an enlarged cohort of patients. Here paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression.Additionally BMMNCs of patients with MDS or CMML were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitorsfor 72 h. Short term viability analysis by flow cytometry showed no impact of inhibitor treatment. However after culture in growth factor enriched methylcellulose for 10–14 days colony forming capacity was significantly increased in low‐risk MDS patients after inhibition of pro‐necroptotic signaling. Here increase in colony numbers was not restricted to a specific lineage.Summary/Conclusion:We conclude that RIPK3 plays a critical stage‐dependent role in patients with MDS and CMML. Disease progression to sAML is associated with an increased RIPK3‐Expression. We further hypothesize that pro‐inflammatory processes like necroptosis have an impact on aberrant myeloid homeostasis.

Immunophenotypic and Genetic Overlap between JMML and CMML

Introduction:Juvenile myelomonocytic leukemia (JMML) is a rare hematological malignancy of early childhood with characteristics of both myeloproliferative neoplasms and myelodysplastic syndromes. JMML shares pathological features and diagnostic criteria with chronic myelomonocytic leukemia (CMML), a malignancy predominantly affecting the elderly. While 85% of patients with JMML have somatic or germline mutations in RAS pathway genes (NF1, NRAS, KRAS, PTPN11, and CBL), the most frequently mutated genes in CMML include TET2, SRSF2, ASXL1, and RAS and are generally somatic-only. The extent to which histone modification genes (ASXL1, EZH2) or spliceosome machinery genes (SF3B1, SRSF2, U2AF1, ZRSR2) play a role in JMML pathogenesis is unclear. Despite mutational differences, both JMML and CMML manifest as myelomonocytic proliferation with varying amounts of dysplasia in the bone marrow. Clusters of clonally-related CD123+ plasmacytoid dendritic cells (PDCs) have been observed in the bone marrow of patients with CMML but have not been investigated in JMML. Here, we report the mutation profiles and immunophenotypic characteristics of JMML specimens from children treated at our institution.Methods:The pathology archives (1987-2017) at the Children's Hospital of Philadelphia (CHOP) were searched to identify JMML cases (n=21) and included formalin fixed paraffin-embedded diagnostic bone marrow biopsies and splenectomy tissue obtained prior to hematopoietic stem cell transplant. JMML diagnosis was confirmed in all cases by clinicopathological review. Cytogenetic analysis and whole genome SNP array were performed at initial clinical presentation. Genomic DNA and RNA were extracted from JMML patients' bone marrow (n=8) and spleen tissue (n=10) for next-generation sequencing analysis of 118 cancer genes for sequence and copy number variants and 110 genes for known and novel fusions via our custom CHOP Hematologic Cancer Panel.CD123 immunohistochemical (IHC) staining was performed on bone marrow and spleen tissues from children with JMML. Presence of CD123+ PDC clusters was evaluated manually and by digital image analysis. CD123 staining was enumerated using the Aperio Image Scope quantitation of membranous staining v9 with the analysis parameters set such that normal endothelial staining was quantified as 1+, and true CD123 staining cells were quantified as 2+ or 3+. The percentage of CD123+ cells (out of total cellularity) was calculated. Bone marrow from patients with non-JMML myeloid malignancies (n=6) and splenectomy tissue from patients with sickle cell anemia (n=8) were used as controls for the CD123 IHC analysis.Results:We confirmed canonical JMML-associated somatic or germline NF1 (n=3), NRAS (n=4), KRAS (n=2), PTPN11 (n=6), or CBL (n=2) mutations in 16 of the 17 (94%) patients with sequencing data. Interestingly, both PTPN11A72T and NF1R2637* mutations were detected in one patient. In addition, we found potential variants in genes affecting histone modifications (ASXL1, DNMT3A, KDM6A, SETD2), spliceosomal processes (SF3B1, U2AF1), transcription (BCOR, RUNX1, ETV6), or cellular growth (SETBP1, BRAF) in 8/17 patients (47%). While mutations in these genes have been well-characterized in other myeloid disorders, many of these alterations have not been reported to date in children with JMML or are currently of unclear biologic and prognostic significance.We also observed increased clustering of CD123+ PDCs in bone marrow and spleens from patients with JMML compared to IHC staining of control tissues. 2.2 ± 0.42% and 1.8 ± 0.74% of cells expressed CD123 in the spleen and bone marrow specimens, respectively. Control bone marrow and spleen samples did not show significant CD123+ staining.Conclusions:Our study demonstrates frequent variants in histone modification, splicing, and transcription-associated genes in JMML specimens in addition to known pathogenic RAS pathway mutations. We further report histopathologic CD123+ PDC clustering in JMML specimens analogous to that observed in CMML, which may aid in the workup of this often difficult-to-diagnose disease. Our findings of genetic and immunophenotypic overlap between JMML and CMML suggest similarities in pathogenesis despite typical presentation at extremes of age. DisclosuresTasian:Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

Abnormal Increase in a Distinct Subset of Nestin-Expressing Cells in the Bone Marrow of Myelodysplastic Syndromes

Background: Nestin-expressing cells (NeC) have been characterized to consist of hematopoietic stem cell (HSC) niche in the mouse bone marrow (BM). Decreases of BM NeC have been reported in myeloproliferative neoplasms (MPN) in humans and in the mouse model of MPN. These lines of information further emphasize the importance of the NeC for the maintenance of normal hematopiesis. Nevertheless, NeC appear to be heterogenous; nestin is reported to be expressed in multiple types of BM stromal cells distinct from each other, with regard to the anatomical localization and the cell-surface antigen expression pattern. One type is reported to be localized adjacent to sinusoids and another type surrounding arterioles. A subset of endothelial cells also appears to be a candidate of NeC in the BM. It is thus critical to define the identities of distinct subsets of BM NeC. Furthermore, each subset of NeC needs to be studied in the human BM from normal subjects and those with BM diseases to understand pathophysiologic significance of NeC in patients. Myelodysplastic syndromes (MDS) are a clonal disease characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. In this disease, anormalities of BM microenvironment have been repeatedly reported; however, consensus in detail has not been reached.Purpose: To define the identities of distinct subsets of NeC in the BM from normal human subjects and to explore their abnormalities in MDS.Methods:Formalin-fixed paraffin-embedded BM biopsy samples from lymphoma patients without BM involvement (designated normal) and from MDS patients were immunostained with antibodies against six markers: nestin, CD34, laminin, α-smooth muscle actin (αSMA), glial fibrillary acidic protein (GFAP), and neurofilament heavy chain (NFH). Immunohistochemistry (IHC) and immunofluorescence (IF) staining were performed. The microscopic analysis of IHC-stained samples involved 10 randomly selected fields of view at 400× magnification, where the numbers of NeC and CD34-positive spindle-shaped cells were counted for quantitative analysis, as well as the association of these two types of cells was evaluated. IF samples were analyzed by a confocal laser scanning microscope using 10 randomly selected fields of view at 63× magnification. Then, nestin-, GFAP-, and NFH-stained areas were measured using the confocal LAS AF software for quantitative analysis.Results:NeC were found at multiple locations in distinct contexts in the normal human BM. A majority of NeC were present in association with the arterior/arteriolar structures. These artery/arteriole-associated NeC were distributed at each of the three layers; the intimal layer inside the laminine-stained basement membrane, the tunica media epressing αSMA, and the adventitial layer outside the αSMA-stained structure. The NeC located at the intimal layer expressed the highest level of nestin. The NeC were present in a close proximity with the CD34-expressing endothelial cells, although whether the endothelial cells co-expressed nestin and CD34 was unclear. The NeC at the other layers showed relatively lower levels of nestin expression. We identified NeC which did not associate with the vascular structures, albeit at a low frequency and with weak nestin staining in the normal human BM. In MDS BM, there was a significant increase in the NeC that were unassociated with the vascular structures. A portion of these increased NeC co-expressed GFAP. These cells potentially represented Schwann cells, because some of them surrounded the NFH-stained structure. Consistent with this, GFAP- and NFH-stained areas were increases in the MDS BM, together with the nestin-stained areas when measured by the confocal LAS AF software.Discussion: Multiple subsets of NeC were identified in the normal human BM as well as in the MDS BM. It is yet elusive whether each subset of NeC has a HSC niche function. In MDS BM, there was an increase in a distinct subset of NeC. The origin of these cells was elusive, but the Shwann cells normally present along with the arterial/arteriolar structures could be a candidate, because in the normal BM, a portion of GFAP-expressing cells along with the vascular structures expressed nestin. It should be elucidated whether the increased sympathetic nervous structure is involved in the pathophysiology of MDS. DisclosuresObara:Alexion Pharmaceuticals: Honoraria, Research Funding.

Multiplex Quantitative Imaging Analysis of Bone-Remodeling Cells in Patients with Myelofibrosis

Background: Bone marrow (BM) fibrosis is a major cause of morbidity and mortality in patients with myeloproliferative neoplasms. Its progression correlates with thickening and distortion of the bone trabeculae, a phenomenon commonly referred to as osteosclerosis. The physiological process of bone remodeling depends on a delicate interplay between monocyte-derived bone-resorbing osteoclasts and mesenchymal cell-derived bone-forming osteoblasts. Whereas impairment in bone resorption is believed to be the underlying cause of inherited sclerosing bone dysplasias, little is known about the pathogenesis of osteosclerosis associated with myelofibrosis (MF). Phenotypically complex bone-remodeling cells are attached to the bone surface and as such are difficult to study by conventional techniques. Here, we present a novel method for visualization and quantification of these cells in situ and assess their role in inducing osteosclerotic changes in the BM of patients with MF.Methods: Formalin-fixed paraffin-embedded BM biopsy specimens were obtained from the iliac crest of patients with advanced stages of MF (MF-2 and MF-3) and healthy controls. Staining of each sample was performed using a series of previously validated monoclonal antibodies directed against osteoclast- (TRAP and cathepsin K) and osteoblast-specific antigens (CD56 and RUNX2). To achieve non-interfering identification, we employed a tyramide-based fluorescent immunohistochemistry approach (PerkinElmer Opal). Specimens were imaged in toto using an automated multispectral system at 0.5 µm resolution and segmented to individual cells via a pattern recognition algorithm. Cells were assigned to one of the two phenotypes based on specific signal intensities and size. Finally, cells lying within 20 µm of the bone perimeter were quantified utilizing methods of spatial statistics.Results: Samples from 10 MF patients (age 63 ± 8 years, median ± MAD) were compared to 3 healthy controls (age 48 ± 7 years). Area of examined tissue ranged from 8.2 to 38.4 mm2 with the median BM to bone ratio of 1.6:1 and 3.5:1, in MF and controls respectively. Discrimination of bone-remodeling subsets by the assay demonstrated high sensitivity and specificity, estimated both visually (Figure 1) and quantitatively. In MF patients we detected 7.1 ± 3.1 osteoclasts per 100 mm of bone perimeter (mean ± SEM) versus 2.4 ± 1.9 per 100 mm in healthy controls (p < 0.0001). Frequency of observable osteoblasts per bone perimeter also showed a significant increase in MF compared to healthy controls (p < 0.0001). Quantification using area of the bone as denominator was in concordance with the densities found per bone perimeter and similar statistical significance was observed.Conclusions: Our analytical method offers a feasible approach for in situ investigation of complex cellular subsets in the BM. This analysis demonstrates that both osteoclasts and osteoblasts are pathologically affected in advanced stages of MF. Expansion of osteoblastic population supports the involvement of cells of mesenchymal lineage in the progression of osteosclerosis. By contrast, an overwhelmingly increased density of osteoclasts suggests a functional loss of these cells of monocytic origin. This is particularly intriguing in view of the emerging role neoplastic monocyte-derived fibrocytes play in the pathogenesis of MF. Further studies exploiting the interaction between these cells will likely shed light on the pathogenesis of osteosclerosis in MF. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunohistochemical loss of 5-hydroxymethylcytosine expression in acute myeloid leukaemia: relationship to somatic gene mutations affecting epigenetic pathways.

Genes affecting epigenetic pathways are frequently mutated in myeloid malignancies, including acute myeloid leukaemia (AML). The genes encoding TET2, IDH1 and IDH2 are among the most commonly mutated genes, and cause defective conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), impairing demethylation of DNA, and presumably serving as driver mutations in leukaemogenesis. The aim of this study was to correlate 5hmC immunohistochemical loss with the mutation status of genes involved in epigenetic pathways in AML. Immunohistochemical staining with an anti-5hmC antibody was performed on 41 decalcified, formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with AML. Archived DNA was subjected to next-generation sequencing for analysis of a panel of genes, including TET2, IDH1, IDH2, WT1 and DNMT3A. TET2, IDH1, IDH2, WT1 and DNMT3A mutations were found in 46% (19/41) of the cases. Ten of 15 cases (67%) with TET2, IDH1, IDH2 or WT1 mutations showed deficient 5hmC staining, whereas nine of 26 cases (35%) without a mutation in these genes showed loss of 5hmC. It is of note that all four cases with TET2 mutations showed deficient 5hmC staining. Overall, somatic mutations in TET2, IDH1, IDH2, WT1 and DNMT3A were common in our cohort of AML cases. Immunohistochemical staining for 5hmC was lost in the majority of cases harbouring mutations in these genes, reflecting the proposed relationship between dysfunctional epigenetic pathways and leukaemogenesis.

Collagen I and collagen III expression in fibrotic bone marrow

Objective: The aim of this study was to assess the expression of collagen I and III in fibrotic bone marrow (BM) in different pathologies. Background: A wide variety of benign conditions and malignant disorders are associated with BM fibrosis. BM fibrosis can be demonstrated by histochemical staining using silver impregnation or by trichrome stains. Immunohistochemical stains, especially anticollagen I and III, are also used for more accurate assessment of BM fibrosis. This study demonstrates the expression of collagen I and III in fibrotic marrow in different pathologies. No clinical data, prognosis , survival were mentioned in this study and so the impact was not investigated. Materials and methods: In this study 50 formalin-fixed paraffin-embedded BM biopsy samples were selected from archived cases at the Clinical Pathology Department, Cairo University. These 50 cases included seven cases with myeloproliferative neoplasms, 14 with lymphoproliferative disorders, three with acute leukemia, four with metastasis, and 22 with reactive BM changes. Cases were divided into two groups according to the Masson's trichrome staining pattern: the fibrotic group, which included 13 cases, and the nonfibrotic group, which included 37 cases. The included cases were subjected to reticulin and trichrome stains, in addition to assessment of collagen I and III expression by immunohistochemistry. Results: As regards collagen I and III staining grades, grade 4 was significantly higher in the fibrotic group, whereas grade 0 was significantly higher in the nonfibrotic group. Conclusion: Collagen I and III are markedly expressed in fibrotic marrow and marrow fibrosis; in particular, collagen fibrosis appears to be more evident in myeloproliferative neoplasms and in tumor metastasis to the BM.

Nucleophosmin mutation analysis in acute myeloid leukaemia: Immunohistochemistry as a surrogate for molecular techniques.

Background & objectives:Mutation of nucleophosmin (NPM1) gene in the absence of FLT3-ITD (FMS related tyrosine kinase 3 - internal tandem duplications) mutation carries a good prognosis in cytogenetically normal acute myeloid leukaemia (AML). NPM1, a multifunctional nucleolar phosphoprotein that shuttles between nucleus and cytoplasm, gets trapped in the cytoplasm when mutated. Immunohistochemical (IHC) demonstration of its aberrant cytoplasmic location (NPMc+) has been suggested as a simple substitute for the standard screening molecular method. This study was aimed to assess the diagnostic utility of IHC on formalin fixed bone marrow biopsies in comparison with the reference molecular method (allele specific oligonucleotide - polymerase chain reaction; ASO-PCR) to predict NPM1 mutation status in AML patients.Methods:NPM protein IHC was performed using mouse anti-NPM monoclonal antibody on 35 paraffin-embedded bone marrow biopsies of patients with primary AML of any French-American-British (FAB) subtype. Results of IHC were compared with those of ASO-PCR.Results:Of the 35 AML patients, 21 (60%) were positive for NPM1 exon 12 gene mutation by ASO-PCR, 19 (90.47%) of these 21 were NPMc+. Thirteen of the 35 patients were negative by both the methods. One NPMc+ patient was not detected by ASO-PCR. IHC had a sensitivity and specificity of 90 and 93 per cent, respectively, compared to the molecular screening gold standard.Interpretation & conclusions:Mutation of NPM1 determined by the widely available and inexpensive IHC agrees closely with results of the standard molecular methods. Thus, technically and financially not well endowed laboratories can provide the prognostically and potentially therapeutically important information on NPM1 mutation using IHC.

FOXM1 Binds Nucleophosmin in AML and Confers Resistance to Chemotherapy

Background: The nucleo-cytoplasmic shuttle protein nucleophosmin (NPM1) is mutated in 50% of cytogenetically normal AML cases (CN-AML) resulting in the nuclear export of this protein and a favorable prognosis. The mechanism by which mutated NPM1 (NPM1mut) confers this advantage in CN-AML is unclear. We have previously demonstrated that FOXM1, an oncogenic transcription factor, co-localizes with NPM1 in cancer cells and NPM-binding is required for sustaining the level of FOXM1. In the NPM1mut AML cell line OCI-AML3, as detected by immunofluorescence, NPM1 and FOXM1 co-localize in the cytoplasm where FOXM1 is transcriptionally inactive. We hypothesize that improved outcomes in the subset of NPM1mut AML is partly attributable to the cytoplasmic relocalization and functional inactivation of FOXM1.Methods: Patient samples were identified retrospectively using the University of Illinois Cancer Center Registry (IRB #: 2013-1245). Clinical, laboratory and molecular data were collected by chart review. Formalin fixed paraffin embedded bone marrow biopsies were retrieved to prepare slides. Patients with favorable or unfavorable karyotypes were excluded and only biopsies with >70% blasts were included. We performed a 2-target staining protocol with fluorescent antibodies to visualize NPM1 and FOXM1 in the same cells using the Vectra multispectral tissue imaging system. This system combines machine learning capability (inForm® software) with acquisition of spectral data from each pixel in the image, so that multiple targets can be quantified in the same image. Leukemia cell lines with stable knockdown of FOXM1 were generated using lentiviral transduction of HL60 cells (NPM1wt) transduced with PLKO.1-shFOXM1 or PLKO.1-empty vector. In parallel, OCI-AML3 cells (NPM1mut) were transduced with pBabe control or pBabe-FOXM1 expressing retrovirus to induce overexpression of FOXM1.Results: FOXM1 is a well-characterized oncogenic transcription factor. In this study we examined its role in AML. In a series of AML cell lines, cellular fractionation followed by immunoblotting revealed strong nuclear expression of FOXM1 in HL-60 and KG1, both of which express the wild-type NPM1 gene. In comparison the OCI-AML3 cell line, carrying the mutated NPM1 gene, showed predominantly cytoplasmic expression of FOXM1 where it is inactive. To better understand the role of FOXM1 in mediating chemoresistance in AML, we inhibited FOXM1 expression in the HL60 leukemia cell line. Stable knockdown of FOXM1 in this cell line resulted in increased sensitivity to the chemotherapeutic agent cytarabine compared to empty vector transduced cells. This was demonstrated by increased caspase-3 cleavage as detected by immunoblotting and decreased cell viability by Trypan blue exclusion after 24 hour exposure to the drug. We then proceeded to validate our observations of the FOXM1-NPM interaction in primary AML samples. Using quantitative data generated from the Vectra imaging of 20 AML pre-treatment bone marrow biopsies we show a strong correlation between the nuclear: cytoplasmic ratios of FOXM1 and NPM, Pearson correlation co-efficient r=0.87, which is indicative of cellular co-localization. When the patients are stratified in 2 groups based on NPM1 mutation status, mean cytoplasmic expression of FOXM1 is 2-fold higher in NPM1mut patients (36% versus 18%).Conclusion: We demonstrate that FOXM1, an oncogenic transcription factor is predominantly cytoplasmic and thereby inactive when NPM1 is mutated in AML. We show the functional significance of FOXM1 in mediating chemoresistance in this disease using AML cell lines. Using a novel quantitative imaging modality we establish for the first time the cellular co-localization of FOXM1 and NPM1 in AML primary blast cells. Studies are ongoing to manipulate FOXM1 expression in additional leukemia cell lines and xenograft models of AML to confirm our findings that FOXM1 inhibition in AML leads to increased sensitivity to chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Epstein–Barr Virus MicroRNAs are Expressed in Patients with Chronic Lymphocytic Leukemia and Correlate with Overall Survival

Although numerous studies highlighted the role of Epstein–Barr Virus (EBV) in B-cell transformation, the involvement of EBV proteins or genome in the development of the most frequent adult leukemia, chronic lymphocytic leukemia (CLL), has not yet been defined. We hypothesized that EBV microRNAs contribute to progression of CLL and demonstrated the presence of EBV miRNAs in B-cells, in paraffin-embedded bone marrow biopsies and in the plasma of patients with CLL by using three different methods (small RNA-sequencing, quantitative reverse transcription PCR [q-RT-PCR] and miRNAs in situ hybridization [miRNA-ISH]). We found that EBV miRNA BHRF1-1 expression levels were significantly higher in the plasma of patients with CLL compared with healthy individuals (p<0·0001). Notably, BHRF1-1 as well as BART4 expression were detected in the plasma of either seronegative or seropositive (anti-EBNA-1 IgG and EBV DNA tested) patients; similarly, miRNA-ISH stained positive in bone marrow specimens while LMP1 and EBER immunohistochemistry failed to detect viral proteins and RNA. We also found that BHRF1-1 plasma expression levels were positively associated with elevated beta-2-microglobulin levels and advanced Rai stages and observed a correlation between higher BHRF1-1 expression levels and shorter survival in two independent patients' cohorts. Furthermore, in the majority of CLL cases where BHRF1-1 was exogenously induced in primary malignant B cells the levels of TP53 were reduced. Our findings suggest that EBV may have a role in the process of disease progression in CLL and that miRNA RT-PCR and miRNAs ISH could represent additional methods to detect EBV miRNAs in patients with CLL.

MYD88 L265P and CXCR4 mutations in lymphoplasmacytic lymphoma identify cases with high disease activity.

Recurrent mutations in MYD88 have been identified in >90% of lymphoplasmacytic lymphoma (LPL). Recently, WHIM (warts, hypogammaglobulinaemia, infections, myelokathexis) syndrome-like mutations in CXCR4 have been described in 28% of LPL cases, and seem to impact clinical presentation and response to therapy. We investigated the presence of the MYD88 L265P mutation in 90 decalcified, formalin-fixed, paraffin-embedded (FFPE) bone marrow (BM) biopsies, including 51 cases of LPL, 14 cases of B-cell chronic lymphocytic leukaemia (CLL), 13 cases of marginal zone lymphoma (MZL) and 12 normal controls. In addition, the C-terminal domain of CXCR4 was sequenced in LPL cases. MYD88 L265P was found in 49/51 (96%) LPL cases and in 1/13 (7·6%) MZL (splenic type), whereas all CLL samples remained negative. The two MYD88 wild type LPL cases were associated with cold agglutinin disease. Mutations in CXCR4 were detected in 17/47 (36·2%) LPL cases, which showed a higher extent of BM infiltration and lower leucocyte counts (P=0·02), haemoglobin (P=0·05) and platelet counts (P=0·01). In conclusion the detection of MYD88 L265P mutation in FFPE samples is reliable and useful for subtyping small B-cell lymphomas in BM biopsies. In addition, the presence of CXCR4 mutations identifies a subgroup of LPL patients with higher disease activity.