Bone Marrow Core Biopsies Research Articles

Topographic Analysis of Low-Grade Myeloid Neoplasms By Multiparametric in Situ Imaging of Human Bone Marrow Core Biopsy Tissues

Diagnosis of low-grade myelodysplastic syndromes (LG-MDS) is one of the most challenging in hematopathology as it relies predominantly on morphologic assessment of dysplasia. Prior studies have demonstrated poor interobserver agreement among pathologists. Histomorphological evaluation of bone marrow core biopsy samples remains the gold standard for diagnostic workup of LG-MDS, including myelodysplastic syndromes (MDS) and other myeloid neoplasms. However, this approach may be subjective, and cannot quantitatively assess subtle differences in marrow topography and the cellular microenvironment. Multiparametric in situ imaging (MISI) through various techniques enables multiple biomarker detection in a single tissue. BostonGene has developed an AI-based image analysis platform to reveal spatial information and subtle histomorphologic features in an objective, quantitative fashion. Here, we demonstrate the potential for automated AI-based imaging analysis of MISI to assist in the differentiation of LG-MDS samples from normal marrow tissues (NBM).Decalcified human bone marrow core biopsy tissues from LG-MDS (n=6) and uninvolved staging marrows (NBM, n=4) were first prepared by immunofluorescence-based MISI via staining with DAPI and CD34, CD38, CD117, CD71, CD15, and CD61 antibodies. BostonGene analyzed the resulting images (fig.1) using a proprietary AI-based imaging platform to identify cells by segmentation performed with the pre-trained instance segmentation neural network. Cell types were identified with mean marker expression values using an accelerated version of BostonGene's phenograph clustering algorithm. Pathologists manually masked fat and bone trabeculae.Using a combination of cell size/shape parameters and antigen expression levels, the following unique cell types were identified: myeloblasts, proerythroblasts, erythroid normoblasts, maturing granulocytes, megakaryocytes, mast cells, plasma cells, and B-cell precursors (hematogones). Data revealed differences in the cellular content of NBM and LG-MDS samples, and separation of LG-MDS samples with the del(5q) subtype (n=2). While linear slender islet-like small clusters of erythroid normoblasts were detected in NBM, we observed a chaotic arrangement of them in LG-MDS samples. In LG-MDS samples, we found an increase in the total number of erythroid normoblasts from 17% to 31%, LG-MDS-del(5q) had 14%.The ratio of maturing granulocytes to erythroid normoblasts (M:E ratio) was significantly lower in LG-MDS (0.63) than in NBM (1.95). The M:E ratio generated by MISI strongly correlated to the M:E ratio produced by manual differential count of bone marrow aspirate samples (R=0.83, p < 0.003). Additionally, fewer hematogones were identified in LG-MDS marrows compared to NBM samples, as reported by others using orthogonal methods.Topographic analysis showed the fat to cellular tissue area ratio was higher in NBM (0.73) than LG-MDS (0.41), but the ratio of trabecular area to total tissue area was higher in LG-MDS (1.67) than NMB (0.74). Spatially, myeloblasts and megakaryocytes were found closer to trabeculae in NBM than LG-MDS;12 different cell communities were identified;2 of them (cluster 3 - erythroid normoblasts enriched, cluster 5 - erythroid normoblasts contacting proerythroblasts) were distributed statistically significantly differently in NBM and LG-MDS samples, indicating the use of MISI with AI-based imaging to distinguish LG-MDS from NBM. Patients of MDS-del(5q) subtype differ significantly from other MDS samples and are more similar to NBM.AI-based image analysis applied to MISI of bone marrow tissue revealed multiple cell types in single tissue sections, along with histologically subtle differences in topography between NBM and LG-MDS samples. These results highlight the importance of integrating in situ tissue analysis with techniques that examine single cell characteristics for a more comprehensive picture of the differences between normal tissue and tumor samples. Coupling sophisticated imaging analytics with this imaging method may provide a more powerful tool for novel biomarker discovery of prognostic and therapeutic significance in the management of MDS and other marrow-based disorders. [Display omitted] DisclosuresSvekolkin: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Varlamova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Galkin: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Akaeva: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Smirnova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Ovcharov: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Polyakova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Tabakov: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Postovalova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Koltakova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Gunn: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Bagaev: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene.

Analysis of Hematopoietic Niche Components Post Allogeneic Transplantation

Timely and effective reconstitution of the hematopoietic niche is imperative post-allogeneic stem cell transplantation (alloHCT) for prevention of life-threatening opportunistic infections and relapses. Efficient engraftment is dependent on the ability of donor hematopoietic stem cells (HSCs) to expand and provide long-term multi-lineage progenitors in the recipient. Mesenchymal stem cells (MSCs) are essential in providing a conducive environment for HSC reconstitution and maintenance. Conditioning chemotherapy with or without total body irradiation is required to confer a competitive advantage to donor HSCs. However, myeloablative conditioning (MA) regimens also destroy the specialized stromal niches which support HSC self-renewal and maintenance (Acar et al., 2015), providing a plausible explanation for the long term cytopenias observed post allogeneic transplantation (Domenech et al., 1995). Previous studies in humans showed CD146-expressing stromal cells support the establishment of the hematopoietic cell microenvironment (Sacchetti et al., 2007). Expression of markers of stromal/progenitor cells (CD146, CD31 and CD34) was evaluated pre and post alloHCT at day 30 in bone marrow biopsies obtained from patients treated with either MA or reduced intensity conditioning (RIC) approaches. CD146 and CD31 expression was evaluated by immunohistochemistry performed on formalin-fixed, paraffin-embedded bone marrow core biopsies. Whole slide imaging (Aperio) was used to digitize the histology and positive cells were scored using image analysis software (strong positive pixel count; ST PPC). In bone marrow, CD146 is expressed on vascular endothelial cells, pericytes on sinusoids, adipose and bone marrow stromal cells. CD31 positive cells include vascular endothelial cells, sinusoidal cells, megakaryocytes, and myeloid cells (Fig. 1). The percent of CD34 positive stem cells was determined by flow cytometry in concurrent bone marrow aspirate cells.The indications for an alloHCT were AML (5/10), ALL (2/10) or MDS (3/10). Median age was 62 (59-69) years in the RIC group and 34 (25-46) years in the MA group. Average HCT CI score was higher in the RIC group (2.5 versus 1.2) (Table 1). 3/10 patients received total body irradiation (TBI) based conditioning. All patients received peripheral blood stem cell alloHCT and 7/10 were MUDs. Chronic GVHD was present (grade 2 or 3) in 3/10 patients in the RIC groups and 1 patient (grade 1) in the MA cohort. Chimerism studies showed optimum engraftment of donor cells in 2 patients in the RIC group and 1 patient in the MA (Table 1).On average, pretransplant bone marrow cellularity was lower in the RIC group. The average overall cellularity on D30 was similar in the two groups (Fig.2). The average percent of CD34 positive cells was similar pretransplant and lower at D30 in the RIC cohort compared to the MA group (0.96% CD34 pre alloHCT versus 0.30% D30 post alloHCT RIC; pre alloHCT 0.96% CD34 versus D30 post alloHCT 0.92% MA). The mean number of CD31 positive cells was higher in the RIC group compared to the MA cohort both pre-transplant and at D30 (Fig 3). On average, an increase in CD146 positive cells on D30 was noted in the alloHCT RIC group compared to the MA cohort (Fig. 4).In conclusion, the RIC cohort showed a pre-post-transplant increase in average overall bone marrow cellularity, more numerous CD31 and CD146 positive cells on average and an increase in CD146 positive cells at D30 post alloHCT compared to the MA group. Despite the modest number of patients, this is the first study to date comparing pre and post alloHCT niche components which may provide important insights in the reconstitution process and impact of conditioning. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Biopsy T Cell Aggregates and Cytokine Transcriptional Signatures Predict Sustained Response to CAR T-Cell Therapy in B-ALL

BackgroundCAR T-cell therapies directed against CD19 (CAR19) are used to treat relapsed/refractory (R/R) B-acute lymphoblastic leukemia (B-ALL) and show high complete remission (CR) rates compared to salvage chemotherapy. However, subsets of patients do not respond or relapse after initial CR. While previous work has demonstrated the importance of CAR T cell immunophenotype and vector integration site in long term CAR19 responders, there are currently no clinically applicable predictors of response. Given the importance of the B-ALL tumor microenvironment in mediating chemoresistance, inflammatory signaling and recruitment of immune effectors to the leukemic bone marrow, we hypothesized that the pre-CAR19 infusion bone marrow biopsy immune microenvironment may predict CAR19 responses.MethodsPatients treated with CAR19 for R/R B-ALL from 2012-2017 were retrospectively identified. Marrow core biopsies were collected immediately prior to CAR19 infusion (pre-CAR) and at one month post-infusion (post-CAR). Bone marrow biopsy slides immunohistochemically stained for CD3 were digitally scanned, analyzed algorithmically for T-cell infiltration and assessed qualitatively for interstitial T cell aggregates of >5 cells (Leica Aperio ImageScope). B-ALL involvement was determined by bone marrow aspirate counts and minimal residual disease (MRD) flow cytometry. Targeted RNA sequencing-based gene expression profiling (EdgeSeq Immuno-Oncology Panel, HTG Diagnostics) was performed on pre- and post-treatment biopsies, with differential expression assessed by DESeq2 within HTG Reveal software, and further analyses conducted using Metascape.ResultsWe identified 143 bone marrow core biopsies (84 pre-treatment, 59 post-treatment) from 84 R/R B-ALL patients treated with CAR19. Pre-CAR B-ALL marrow involvement ranged from 0-95% (median: 67.5%), and the dataset included patients with relapses occurring between 1-33 months following CAR therapy (median: 6 months).Pre-CAR CD3+ T cell aggregates were significantly increased in CAR19 Sustained Responders (SR) compared to Non-Responders (NR) (92.3% versus 50%, p=0.007) and eventual relapses (CD19+: 40%, p= 0.002 and CD19-: 61.5%, p=0.019) (Figure 1A-B). A lack of T cell aggregates in pre-CAR marrow biopsies showed a sensitivity of 45.8% and a specificity of 92.3% in distinguishing NRs and eventual relapses from SRs. In one month post-CAR marrow biopsies, T cell aggregates continued to show higher frequency in SRs compared to eventual CD19+ relapses (100% versus 42.9%, p=0.007) and NRs (50%, p=0.04).Pre-CAR marrow T cell infiltration was also significantly greater in SRs compared to NRs (2146 CD3+ T cells per biopsy versus 850, p=0.038) and eventual CD19- relapses (1086, p=0.022), and trended toward a significant increase compared to eventual CD19+ relapses (1056, p=0.063) (Figure 1C). Pre-CAR marrow T cells of < 920 per biopsy showed a sensitivity of 45.8% and a specificity of 92.3% in distinguishing NRs and relapses from SRs (Figure 1D).RNAseq differential gene expression analysis of pre-CAR marrow biopsies revealed a chemokine axis of CXCL9, CXCL13, CXCR5, CXCL5, CXCL2 and CCL1 in SR that was deficient in patients with eventual relapse (Figure 1E). By contrast, NRs and eventual relapses showed a pre-CAR transcriptional signature reflecting Th2 (IL5RA, IL13) and Th17 (IL17A, IL23R) skewing, in addition to higher IL2, IFNG and IL6 transcripts.ConclusionThese immunohistochemical and transcriptional findings support a pivotal role for the peri-infusion bone marrow microenvironment in long-term responses to CAR19 therapy in B-ALL. A deficiency of marrow T cell aggregates and marrow-infiltrating T cells pre-CAR shows high specificity in distinguishing NRs and eventual relapses from SRs to CAR19, and marrow T cell aggregates are a persistent feature of SRs at one month post-CAR infusion. SR to CAR19 is also associated with elevated marrow expression of a suite of immune chemoattractants which may enhance the ability of infused CAR T cells and other effectors to infiltrate the B-ALL microenvironment. Combined assessment of bone marrow T cell infiltrates and transcriptional profiling could be used to improve the sensitivity and specificity of predictive algorithms for responses to CAR19 therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Colony-stimulating factor 3 receptor gene (CSF3R) T618I mutated chronic myelomonocytic leukemia: A proliferative subtype with a distinct mutational profile

Abstract Introduction/Objective Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm characterized by sustained monocytosis, ranging from cytopenia with a dysplastic subtype to leukocytosis with a proliferative subtype, with a typical mutational profile involving TET2, ASXL1, and SRSF2. Mutation in colony-stimulating factor 3 receptor gene (CSF3R) is commonly associated with chronic neutrophilic leukemia (CNL) but exceedingly rare in CMML, particularly CSF3R T618I (~10 cases described, ~30 cases of CSF3R non-T618I mutations). We report a case of CSF3R T618I mutated CMML and compare the clinicopathologic features to reported CMML cases with and without CSF3R T618I mutations. Methods/Case Report A 27-year-old woman presented for evaluation of leukocytosis, sustained monocytosis, and anemia. Peripheral blood (PB) revealed leukocytosis (white cell count 35x109/L), left-shifted and dysplastic neutrophils (myelocytes and metamyelocytes, 5%), absolute and relative monocytosis (7x109/L, 29%), anemia (Hgb 4.3 g/dL), and thrombocytopenia. Bone marrow aspirate and core biopsy demonstrated a hypercellular marrow with increased myeloblasts (~3%, immunophenotypically aberrant by flow cytometry), increased myelomonocytic cells, and multilineage dysplasia, including ring sideroblasts and hypolobated megakaryocytes. Cytogenetic and molecular studies revealed a normal karyotype and mutations in CSF3R T618I, ASXL1, SETBP1, BCORL1, KRAS, and PTPN11. Despite the presence of a CSF3R T618I mutation, CMML was diagnosed given marked monocytosis, left- shifted neutrophils in PB, multilineage dysplasia, and immunophenotypically aberrant myeloblasts. Results (if a Case Study enter NA) NA Conclusion Our case demonstrates clinicopathological features similar to those of reported CSF3R T618I mutated CMML, i.e., a proliferative subtype and less likely to have co-occurring mutations in TET2 or SRSF2, which is distinct from CSF3R non-T618I mutated CMML; the latter often has a dysplastic subtype and mutational profile of frequent TET2 and SRSF2 mutations, similar to CSF3R unmutated CMML. While additional cases with this unusual mutation need to be studied to arrive at a more definitive conclusion, the CSF3R T618I mutation seems to define a unique proliferative subtype CMML with a distinct mutational profile.

Programmed necroptosis is upregulated in low-grade myelodysplastic syndromes and may play a role in the pathogenesis

Myelodysplastic syndrome (MDS) is characterized by persistent cytopenias and evidence of morphologic dysplasia in the bone marrow (BM). Excessive hematopoietic programmed cell death (PCD) and inflammation have been observed in the bone marrow of patients with MDS, and are thought to play a significant role in the pathogenesis of the disease. Necroptosis is a major pathway of PCD that incites inflammation; however, the role of necroptosis in human MDS has not been extensively investigated. To assess PCD status in newly diagnosed MDS, we performed immunofluorescence staining with computational image analysis of formalin-fixed, paraffin-embedded BM core biopsies using cleaved caspase-3 (apoptosis marker) and necroptosis markers (receptor-interacting serine/threonine-protein kinase 1 [RIPK1], phospho-mixed lineage kinase domain-like protein [pMLKL]). Patients with MDS, but not controls without MDS or patients with de novo acute myeloid leukemia, had significantly increased expression of RIPK1 and pMLKL but not cleaved caspase-3, which was most evident in morphologically low-grade MDS (<5% BM blasts) and in MDS with low International Prognostic Scoring System risk score. RIPK1 expression highly correlated with the distribution of CD71+ erythroid precursors but not with CD34+ blast cells. We found that necroptosis is upregulated in early/low-grade MDS relative to control participants, warranting further study to define the role of necroptosis in the pathogenesis of MDS and as a potential biomarker for the diagnosis of low-grade MDS.

CLL-140: Chronic Lymphocytic Leukemia Presenting as a Solitary Breast Mass

An 80-year-old woman with a past medical history of hypertension and no prior history of malignancy underwent a screening mammogram followed by a breast ultrasound that showed a right breast mass. She remained asymptomatic. The physical exam did not reveal any palpable lymphadenopathy nor hepatosplenomegaly. Peripheral blood smear showed absolute lymphocytosis of 69% with a normal total white blood cell count of 8.1 without dysplastic cells, blasts, anemia, or thrombocytopenia. Breast ultrasound showed an irregular hypoechoic mass measuring 0.7 x 0.5 x 0.5 cm. Subsequent US-guided core biopsy showed non-Hodgkin B-cell lymphoma positive for cluster designation (CD)20 and CD5. Bone marrow aspirate and core biopsy revealed clusters of small-to-medium atypical lymphocytes positive for CD20, CD79a (weak), with a co-expression of CD5 (strong) and CD23CD138, findings that are compatible with Small Lymphocytic Lymphoma (SLL)/Chronic Lymphocytic Leukemia (CLL). Positron Emission Tomography scan showed an uptake within the right breast only. She is being managed with observation and close follow-up. Breast lymphoma (BL) represents around 0.5% of malignant breast neoplasms. Diffuse Large B-Cell Lymphoma (DLBCL) and follicular lymphoma are the predominant subtypes. SLL and CLL are subtypes of non-Hodgkin lymphoma originating from a B-cell lineage. While there have been reported cases of CLL and SLL manifesting as BL, the incidence still is very low and limited to case reports. Moreover, they have been reported to be found concomitantly with breast carcinoma. Most BL are asymptomatic when found incidentally on screening imaging, as in our patient. A hypoechoic round mass is the most common reported appearance. There are no current established guidelines for the treatment of BL. In our patient, we followed the recommendations for managing CLL. Not all breast masses are adenocarcinoma. It is critical to keep in mind the differential diagnosis lymphoproliferative disorders, melanoma, lung cancer, and gynecological malignancies.

T Cells Genetically Modified to Express an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor with a CD28 Costimulatory Moiety Cause Remissions of Poor-Prognosis Relapsed Multiple Myeloma

▪B-cell maturation antigen (BCMA) is expressed by normal plasma cells and the malignant plasma cells of multiple myeloma (MM). We developed the first anti-BCMA chimeric antigen receptor (CAR-BCMA). CAR-BCMA includes an anti-BCMA single-chain variable fragment, a CD28 costimulatory domain, and a CD3-zeta T-cell activation domain (Carpenter et al. Clinical Cancer Research 2013. Vol. 19. 2048-2060). We used a gamma-retroviral vector to genetically modify autologous T cells to express CAR-BCMA. We are conducting a phase I clinical trial of CAR-BCMA T cells. On this trial, patients receive a chemotherapy regimen of 300 mg/m2 of cyclophosphamide and 30 mg/m2 of fludarabine on days -5 to -3 followed by an infusion of CAR-BCMA T cells on day 0. Chemotherapy is administered to increase the activity of CAR T cells by depleting endogenous leukocytes. Twenty-three patients have received CAR-BCMA T cells on one of 4 dose levels, 0.3x106, 1x106, 3x106, and 9x106 CAR+ T cells/kg of bodyweight. All patients treated on the lower dose levels and 2 patients treated on the highest dose level have been reported previously (Ali et al. Blood 2016. 128(13). 1688-1700). This abstract exclusively covers the 13 patients treated to date on the highest dose level of 9x106CAR-BCMA T cells/kg.The median age of the 13 patients was 54 years (range 43-66). The patients had a median of 11 prior lines of therapy. Five patients (12, 19, 20, 23, and 25), had a history of the poor-prognosis chromosome 17p-deletion prior to protocol enrollment. The monoclonal protein produced by each patient's MM and the number of prior therapies of each patient are shown in the table.Toxicities were consistent with prior reports of cytokine-release syndrome (CRS) after infusions of CAR T cells. The most prominent, signs of CRS were fever, tachycardia, hypotension, and reduced cardiac function. CRS grades are in the table. Of thirteen patients, 4 received the interleukin (IL)-6-receptor antagonist tocilizumab to treat CRS; 2 of these 4 patients also received corticosteroids. While CAR-BCMA T-cell toxicity was severe in some cases, the toxicities were mainly limited to the first 2 weeks after CAR-BCMA T-cell infusion. Because of Grade 3 and 4 CRS experienced by some patients with high bone marrow myeloma burdens, we modified the clinical protocol to only allow enrollment of patients with lower myeloma burdens defined as MM making up 30% or less of bone marrow cells. Two patients (16 and 18) experienced delayed neutropenia and thrombocytopenia that started approximately 1 month after CAR T-cell infusion when blood counts had recovered from the chemotherapy administered before CAR-BCMA T-cell infusions. These patients were treated with filgrastim, eltrombopag, and prednisone based on the hypothesis that the cytopenias were caused by CAR T cells in the patient's bone marrow. At the time of delayed cytopenias, 15 percent of Patient 16's bone marrow T cells were CAR+, and 16.5 percent of Patient 18's bone marrow T cells were CAR+. In both cases, cytopenias resolved after approximately 1 month.CAR-BCMA T cells exhibited clear anti-myeloma activity. Nine of 11 evaluable patients obtained objective anti-myeloma responses with 2 stringent complete responses, 5 very good partial responses, and 2 partial responses; the duration of responses varied. The longest response to date is 66 weeks (Table). Eight of 10 evaluable patients obtained minimal residual disease negative status by bone marrow flow cytometry. Consistent with BCMA-specific T-cell activity, plasma cells were reduced on bone marrow core biopsies in all 8 evaluated patients and absent in 4 of these patients 2-3 months after CAR-BCMA T-cell infusion.CAR+ cell levels have been quantified in the blood of patients. CAR T cell levels peaked in the first 2 weeks after infusion and persisted at lower levels for many months in some cases. Cytokines were measured in the serum of all patients. In patients with CRS, multiple cytokines including interferon-gamma, IL-6, IL-8, and IL-17A were consistently elevated in the serum.These results demonstrate that CAR-BCMA T cells can induce responses in poor-prognosis MM. Accrual to this trial continues. Toxicity was significant but limited in duration and controllable. A compelling atribute of CAR T-cells is that they have a mechanism of action different from other MM therapies. The results reported here should encourage research to improve CAR T-cell therapies for MM. [Display omitted] DisclosuresWang:Acerta Pharma: Consultancy, Research Funding; Asana Biosciences: Research Funding; BeiGene: Research Funding; Celgene: Honoraria, Research Funding; Dava Oncology: Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; June Therapeutics: Research Funding; Kite Pharma: Research Funding; Onyx: Research Funding; Pharmacyclics: Research Funding; Proteolix: Honoraria, Research Funding. Kochenderfer:Bluebird bio: Research Funding; Kite Pharma: Research Funding; N/A: Patents & Royalties: I have multiple patents in the CAR field.

Versican (VCAN) Proteolysis Predicts T-Cell Infiltration in Myeloma Bone Marrow Post- Autologous Stem Cell Transplant (ASCT)

Background/ Hypothesis:Versican (VCAN), a large matrix proteoglycan, accumulates in myeloma lesions (Hope et al., Blood 123(21): 3305-3315, 2014). VCAN sustains tumor-promoting inflammation but also generates immunosuppression through IL-6 and IL-10-mediated antigen-presenting cell dysfunction (Tang et al., Cell Rep 13(12): 2851-2864, 2015). VCAN proteolysis generates bioactive fragments (VCAN-derived matrikines, e.g., versikine) that oppose the functions of the parent macromolecule, intact VCAN (Hope et al., Blood 128(5): 680-685, 2016). VCAN-matrikines promote “T-cell inflammation” through co-ordinated effects on antigen-presenting and myeloid regulatory cells. In particular, we have shown that VCAN-matrikines promote Batf3-lineage dendritic cell (DC) differentiation from Flt3L-mobilized bone marrow progenitors and induce IL-12 but not IL-10 production by myeloid cells (Hope et al., J Immunol, in press, 2017). Batf3-DC constitute an intratumorally sparse DC subset with critical roles in tumor antigen cross-presentation and effector T-cell homing. Recently, VCAN-producing macrophages were shown to expand post-ASCT in myeloma and correlate with minimal residual disease (MRD) persistence (Arana, Zabaleta et al., Blood 128(22): 482-482, 2016). However, it is unknown whether VCAN proteolysis predicts T-cell infiltration post-ASCT for myeloma at a timepoint (Day 90-100 post-ASCT) when MRD is routinely quantitated and immunotherapies to control MRD are often instituted.Specific Aims:1.To detect VCAN proteolysis in paraffin-embedded bone marrow tissue obtained post-ASCT for myeloma2.To investigate the relationship between T-cell infiltration and VCAN proteolysis in post-ASCT myeloma bone marrowStudy Design:Bone marrow core biopsies from 40 patients who underwent ASCT for myeloma were analyzed at day 90-100 post-ASCT. ADAMTS protease-mediated VCAN proteolysis was analyzed by immunohistochemistry (IHC) through detection of neo-epitope DPEAAE as previously shown (Hope et al., J Immunol, in press, 2017). DPEAAE staining intensity was scored in the following scale: 0=none, +=patchy, ++= diffuse /weak, +++= diffuse/ strong. T-cell infiltration was scored in the following scale: rare, dispersed and intense. Clusters of T-cells (>5 CD8+ cells clustered together) were independently scored. Granzyme B staining status by IHC was scored using a scale similar to CD8+ status.Results:We found that prior de-calcification procedures did not prevent optimal evaluation and scoring of VCAN proteolysis status by IHC. Intense CD8+ T-cell infiltration and/or CD8+ clustering were only seen in the presence of intense (++ or +++) VCAN proteolysis. 14/14 marrows classified as displaying intense CD8+ infiltration plus CD8+ clusters were also scored as VCAN proteolysis ++/+++. 17/17 marrows with intense CD8+ infiltration were scored as VCAN-proteolysis ++/+++. 18/18 marrows with at least one CD8+ cluster detected in the specimen were VCAN proteolysis ++/+++. 67% of marrows (n=12/18) with +++ VCAN proteolysis had intense CD8+ infiltration. 83% (n=15/18) of marrows with +++ VCAN proteolysis had at least one CD8+ cluster detected. Intense staining for granzyme B was only seen in the presence of ++/+++ VCAN proteolysis (n=10/10).Conclusions/ Future implications:Intense VCAN proteolysis correlated with evidence of adaptive immune activation post-ASCT. T-cell infiltration in myeloma bone marrow post-ASCT was exclusively seen in the presence of intense VCAN proteolysis. Mechanistically, we hypothesize that T-cell infiltration is the result of enhanced Batf3-DC density in VCAN proteolysis-rich myeloma tumor microenvironments. We are currently investigating VCAN proteolysis-detection as an immune biomarker to predict efficacy of anti- myeloma immunotherapies as well as in relationship with MRD status post ASCT. There is a dearth of immune biomarkers that can be analyzed on standard de-calcified, paraffin-embedded bone marrow core specimens. For example, PD-L1 detection is problematic using de-calcified bone marrow specimens. By contrast, detection of VCAN proteolysis was unaffected by the de-calcification process. VCAN-matrikines (e.g., versikine) can also be therapeutically used to boost Batf3-DC whose paucity is often the limiting factor for efficacy of checkpoint inhibition and other immunotherapies. DisclosuresDhakal:Celgene: Honoraria.

Deconvolution of the Immune Microenvironment Can Predict the Outcome of Myeloma Patients and Inform Potential Intervention Strategies

IntroductionThe microenvironment of cells around multiple myeloma (MM) tumors strongly influences patient outcomes, as evidenced by the success of immunomodulatory therapies. To develop precision immunotherapeutic approaches, it is essential to identify and enumerate pro- and anti-myelomatous cell types within the bone marrow.MethodsThe population of immune and other non-tumor cell types was estimated from RNA microarray data derived from paired whole bone marrow (WBM) core biopsies and CD138-enriched (CD138+) bone marrow aspirates at presentation from 405 newly diagnosed MM patients who underwent similar treatments. Expression profiles from CD138+ cells were used to infer the residual transcriptome contribution of immune and stromal cells in the WBM array data. We performed unsupervised clustering of the estimated microenvironment gene expression profiles and calculated the statistical significance of cluster splitting using the R packages ConsensusClusterPlus and sigclust, respectively. Individual cell type composition of these microenvironments was estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) that we augmented with phenotypic profiles of 5 bone marrow-specific cell types (myelomatous plasma cells, plasma memory cells, osteoclasts, osteoblasts, and adipocytes).ResultsThis deconvolution approach accurately estimated the percent of tumor cells in WBM core biopsy as estimated by both microscopy and flow cytometry (PCC = 0.57, RMSE = 10.9%). Unsupervised consensus clustering of whole bone marrow microenvironment gene expression data revealed 5 significantly different subtypes. The two largest subtypes, Cluster 1 (N=136) and Cluster 2 (N=128), had the most divergent outcomes with median PFS values of 78 months and 53 months, respectively. Cluster 1 was significantly enriched for ISS-I patients (FDR < 0.02) while Cluster 2 was enriched for ISS-III patients (FDR < 7x10^-5). Cluster 2 showed a significant increase in estimated M1 macrophages, CD8+ T cells, adipocytes, and dendritic cells with a decrease in estimated osteoclasts versus all other clusters combined (FDRs < 0.05). Cluster 5 (N=63) had the second worst PFS at 55 months, however, it had 44% fewer than expected ISS-III patients (FDR = 0.04). Cluster 5 had fewer estimated T cells and adipocytes, and it was the only cluster with a significant (3-fold) increase in estimated NK cells compared to the other clusters combined (FDRs < 0.05). No clusters were significantly enriched for older individuals, hyperdiploidy or common chromosomal translocations (t(4;14), t(11;14), t(14;16)). No clusters were significantly enriched for CD138+ gene expression-defined high risk patients (UAMS GEP-70) or CD138+ gene expression-based molecular subtypes (Zhan et al., 2006).ConclusionsWe demonstrated the technical feasibility to accurately delineate CD138+ expression profiles from a bulk transcriptome admixture and to use the residual microenvironment transcriptome to cluster patients into distinct microenvironment subtypes. Within these subtypes, the clinical outcomes are not explained by CD138+ molecular features nor ISS stage. For example, Cluster 5 has patients with poor prognosis compared to the rest of the cohort, yet it is not significantly enriched for high risk translocations, ISS-III patients, nor GEP-70 high risk patients. Cluster 5 patients show significant decrease in both CD8+ T cell infiltrate and activated dendritic cells, perhaps reflective of a high risk immune state that would not be responsive to checkpoint inhibition. Moreover, Cluster 5 patients are the only ones to have an increase in NK cells, suggesting one could try to stimulate these NK cells to reinvigorate their anti-tumor immunity. Cluster 2, with the poorest prognosis patients and highest tumor burden (by ISS stage), shows increased CD8+ T cells and dendritic cells (where all other clusters are neutral or decreased in these cell types). Cluster 2 patients may thus benefit from checkpoint blockade, and we plan to further explore relevant checkpoint-related pathways in this cohort. Adding this layer of immune-microenvironment to the analysis of myeloma patient samples enables us to formulate biological hypotheses independent of CD138+ biology and may eventually guide more tailored therapeutic interventions and improve outcomes for patients. [Display omitted] DisclosuresDanziger:Celgene Corporation: Employment. Dervan:Twinstrand Biosciences: Equity Ownership; Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment. Copeland:Celgene Corporation: Employment. Fitch:Celgene Corporation: Employment. Newhall:Celgene Corporation: Employment, Equity Ownership. Barlogie:Celgene Corporation: Consultancy, Research Funding; Millenium Pharmaceuticals: Consultancy, Research Funding. Foy:Celgene Corporation: Employment, Equity Ownership. Trotter:Celgene Corporation: Equity Ownership; Celgene Institute for Translational Research Europe: Employment. Hershberg:Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Fraizer Healthcare Partners: Consultancy; NanoString Technologies: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Employment, Equity Ownership. Ratushny:Celgene Corporation: Employment. Morgan:Takeda: Consultancy, Honoraria; Bristol Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Programmed Necrosis Correlates with Development and Progression of Myelodysplastic Syndromes

Myelodysplastic syndrome (MDS) is characterized by persistent cytopenias and evidence of morphologic dysplasia in the bone marrow. Lower-grade MDS (bone marrow blasts <5%) is challenging to diagnose, as the morphologic dysplasia may be subtle and many patients lack characteristic cytogenetic abnormalities.Multiple studies of the bone marrow in MDS have demonstrated excessive hematopoietic cell death, and this increased cell death is thought to play an important role in the pathogenesis of the disease. Early studies of MDS revealed increased in situ end labeling and increased TUNEL staining which led to the conclusion of apoptotic cell death, but these methods cannot properly distinguish between necroptosis and apoptosis. Apoptosis is driven by caspases that break down the cytoskeleton to cause collapse of the cell in a predominantly immune silent process. Recently, necroptosis has been identified as a new pathway of programmed cell death, which is driven by Rip kinases and is characterized by premature rupture of the plasma membrane, resulting in an immune response. Necroptosis has been shown to play a role in inflammatory diseases as well as host response to infection, and tumors.Methods: To assess cell death status in normal bone marrow as compared to lower-grade MDS (RCUD, RA, RARS, RCMD) and higher-grade MDS (RAEBI/RAEBII, RAEB-t), we evaluated a total of 37 de novo MDS cases and 36 normal control cases. The MDS specimens included 20 cases of lower-grade MDS and 17 cases of higher-grade MDS. All cases were reviewed and the diagnosis confirmed by board-certified hematopathologists. Necroptosis was assessed by measuring Rip1 and phosphorylation of Mixed Lineage Kinase domain-like protein (MLKL) and apoptosis was measured by cleaved-caspase 3.Formalin fixed paraffin embedded bone marrow core biopsies were immunostained with the apoptosis marker (caspase 3) and necroptosis markers (Rip1, MLKL) and visualized by immunofluorescence. Whole slide images were obtained using Aperio Versa 200 (Leica Microsystems). We developed a pipeline for statistical analysis to evaluate the immunofluorescence of stained and scanned images using CellProfiler (Broad Institute) image analysis software. For each slide over 10,000 cells were analyzed.Results: We found that the proportion of RIP1 positive cells was significantly different between normal controls (6.91±1.32%) and lower-grade MDS (32.80±6.19%, p value 0.0042). Likewise, RIP1 staining in higher-grade MDS was less common than in lower-grade MDS (15.99±4.76% vs 32.90±6.19%, p value 0.0506). There was no significant difference between normal controls and high-grade MDS. In lower-grade MDS samples, RIP1 positive cells were mononuclear cells arranged in clusters, morphologically consistent with erythroid progenitors. Similar trends were seen with MLKL staining.To evaluate apoptosis, we performed cleaved caspase 3 staining on the same patient samples. There was no difference in the proportion of caspase 3 positive cells among the groups: normal controls (2.02±1.08%), lower-grade MDS (2.77±0.86%) or higher-grade MDS (1.99±0.71%). As apoptotic and necroptotic cells have markedly different cellular ultrastructural morphologies, we performed electron microscopy on fresh bone marrow cells to define cell death. After scoring cells with a nucleus as either being live, apoptotic (shrinkage of cell membrane and pyknotic nucleus), or necrotic (swelling of cell membrane and nucleus), we found that there was a significant increase in the number of necrotic cells in the bone marrow of the lower-grade MDS patients relative to the normal control or higher-grade MDS patients.We then performed over-representation analysis of the publicly available MDS microarray data (GEO GSE15061) from the international multi-institutional Microarray Innovations in Leukemia (MILE) research program. Although the study was underpowered to identify differences between normal controls and early MDS, we found that RIP1, RIP3 and MLKL had significantly increased expression in the MDS group vs the de novo AML group, suggesting that genes in the necroptotic pathway may be upregulated in MDS as compared to AML.In summary, our findings reveal that the necroptosis cell death pathway is upregulated in early MDS relative to normal controls and warrants further study to define its role in the pathogenesis of MDS and as a potential biomarker for the diagnosis of lower-grade MDS. [Display omitted] DisclosuresSavona:Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Incyte Corporation: Consultancy, Research Funding; Karyopharm: Consultancy, Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.

IRF8 is a Reliable Monoblast Marker for Acute Monocytic Leukemias.

Blast evaluation in patients with acute monocytic leukemias (AMoL) is notoriously difficult due to the lack of reliable surface markers and cytologic subtleties on the aspirate smears. While blasts of most nonmonocytic acute leukemias express CD34, available immunohistochemical antibodies to monocytic blasts also mark normal background mature monocytes. We searched for a potential biomarker candidate by surveying specific gene expression profiles of monocyte progenitors. Our investigations led us to IRF8, which is a lineage-specific transcription factor critical for the production of monocytic and dendritic cell progenitors. In this study, we tested and validated a monoclonal antibody to IRF8 as a novel immunohistochemical stain for trephine core biopsies of human bone marrow. We assessed the expression of IRF8 in 90 cases of AMoL, including posttherapy staging bone marrows, 23 cases of chronic myelomonocytic leukemia, 26 cases of other acute myeloid leukemia subtypes, and 18 normal control marrows. In AMoL, there was high correlation of IRF8-positive cells to aspirate blast count (R=0.95). Comparison of IRF8 staining to aspirate blast percentage in chronic myelomonocytic leukemia also showed good correlation (R=0.86). In contrast, IRF8-positive cells did not correlate with blast count in other subtypes of acute myeloid leukemia (R=0.56) and staining was <5% in all normal control marrows, even those with reactive monocytosis. We found that IRF8 was also weakly reactive in B cells and hematogones, with the latter accounting for rare cases of discrepancies. When IRF8 was used to categorize cases as AMoL, positive for residual leukemia or negative, the sensitivity was 98%, specificity was 82%, positive predictive value was 86%, and negative predictive value was 98%. These results demonstrate that IRF8 may serve as a clinically useful immunostain to diagnose and track AMoLs on bone marrow core biopsies. This can be particularly impactful in the setting of poor aspiration and focal blast increase. In the era of new targeted therapies that have been reported to induce monocytic outgrowths of leukemia, a marker for malignant monoblasts may prove even more critical.

Histopathological Parameters of Canine Bone Marrow in Cell-Block Preparations

Studies using the cell-block technique for bone marrow (BM) analysis are limited in the veterinary literature. This work assessed whether the histopathological analysis of canine BM was feasible using cell-block cytoinclusions prepared from fine-needle sternal aspirate samples. Eight clinically healthy young-to-middle-aged dogs underwent both fine-needle sternal aspiration for BM cell-block (BM-Cb) processing and iliac-crest BM core biopsy (BM-B). Histopathologic parameters were compared between the 2 methods. There were no statistically significant histopathological differences between hematopoietic tissue areas (P = .6294) in the BM-Cb and BM-B sections, and they had similar microscopic characteristics and microarchitecture. Cellularity and reticulin-fiber staining were equivalent in the BM-Cb and BM-B preparations in 87.5% (7/8) and 100% (8/8) of the sections, respectively. However, the quantitative results of the megakaryocytic series differed between BM-Cb and BM-B in 37.5% (3/8) of the sections, and the myeloid:erythroid (M:E) ratios differed between the 2 methods in 25% (2/8). These preliminary data indicate that cell-block preparations made from sternal fine-needle aspiration samples warrant continued evaluation in a larger number of animals, including those with various diseases affecting the bone marrow.

CLINICOPATHOLOGICAL STUDY OF MULTIPLE MYELOMA WITH EXPRESSION OF CD56

Background: Multiple myeloma (MM) is a plasma cell neoplasm with spectrum of clinical presentation and multisystem involvement. CD56 (a neural cell adhesion molecule, NCAM) is a membrane glycoprotein belonging to the immunoglobulin superfamily. Normal plasma cells do not express CD56, but it is frequently expressed by malignant plasma cells in patients with MM. The present study was conducted to evaluate the expression of CD56 in MM and its association with disease characteristics. Methods: We analysed CD56 expression in 34 newly diagnosed MM cases using immunohistochemistry in bone marrow core biopsies processed routinely. Results: CD56 expression was found in 70.97% cases. The clinicopathological disease characteristics such as age, serum creatinine level, serum β2-microglobulin level and plasmablastic morphology showed no statistically signicant difference between CD56 positive and negative groups. Bone lesions were signicantly higher in positive cases than in negative cases. Conclusion: CD56 expression dene a unique clinicopathological entity in MM. Expression of CD56 correlate well with lytic bone lesion and may correlate with its pathogenesis.

A Challenging Diagnosis of Acute Myeloid Leukemia with Myelodysplasia Related Changes in the Setting of Ayurvedic Supplements and B12 Deficiency

Abstract Introduction/Objective Bone marrow findings of myelodysplastic changes can be associated with vitamin or mineral deficiencies (B12, folic acid or copper), infections, inflammatory disorders, exposure to toxins, heavy metals, zinc excess, recent radio- or chemotherapy, or myeloid neoplasms including myelodysplasia (MDS) and acute myeloid leukemia (AML). We present a case of a 32 year old Indian woman who presented with fatigue and weakness. She has a history of vitamin B12 deficiency and has been taking Ayurvedic supplements. This clinical history is very important because studies have shown that vitamin B12 deficiency has led to incorrect diagnoses of AML and/or MDS. Additionally, Ayurvedic supplements can contain a variety of heavy metals which can contribute to myelodysplastic changes. Results A complete blood count and peripheral smear analysis showed pancytopenia with rare blasts. A bone marrow core biopsy with ancillary testing was performed to evaluate for a hematologic malignancy. Vitamin B12 testing and heavy metal analysis was necessary to reach a final diagnosis. Flow cytometric analysis of the marrow identified 19.5% CD34 positive myeloblasts. The aspirate and core are hypercellular with increased blasts (17% on aspirate and 20-25% on core biopsy) with trilineage dyspoiesis. AML panel by fluorescence in situ hybridization showed no abnormalities. Heavy metal analysis shows no increase in lead, cadmium, mercury and arsenic levels. Recent vitamin B12 tests show results within normal range secondary to successful replacement. Ultimately, chromosome analysis identifies t(3;12)(q26,p13) involving ETV6/MECOM characteristic of a very poor prognosis in MDS or AML. Molecular AML panel identified BCOR and PTPN11 gene mutations which are associated with more aggressive disease and a poor prognosis in myeloid neoplasms. Conclusion This case had multiple possible causes of myelodysplastic changes and it was necessary to exclude these factors before yielding the final diagnosis of Acute Myeloid Leukemia with myelodysplastic related changes which demonstrated a rare translocation t(3;12) ETV6/MECOM which has a poor prognosis.

Clinical laboratory validation of the MCL35 assay for molecular risk stratification of mantle cell lymphoma

Mantle cell lymphoma (MCL) is a clinically heterogeneous B cell malignancy for which a variety of prognostic factors have been proposed. Previously, a digital gene expression profiling “proliferation signature” capable of risk stratifying MCL was identified and subsequently developed into a multi-analyte prognostic assay, known as the “MCL35” assay. In this study, we sought to explore the performance characteristics of the MCL35 assay in a clinical laboratory and compare results with the Ki67 proliferation marker. The results describe the clinical validation of the MCL35 assay for molecular risk stratification of MCL including accuracy, sensitivity, specificity, use in acid-decalcified bone marrow core biopsies, fixatives, lower limit of RNA input, quality metrics, and other laboratory parameters. The resulting data indicate that this is a robust technique with outstanding reproducibility. Overall, the data support the concept of molecular signatures, as assessed with digital gene expression profiling, for improved standardization and reproducibility for proliferation assessment in MCL.

Chromosomal instability upregulates interferon in acute myeloid leukemia.

Chromosome instability (CIN) generates genetic and karyotypic diversity that is common in hematological malignancies. Low to moderate levels of CIN are well tolerated and can promote cancer proliferation. However, high levels of CIN are lethal. Thus, CIN may serve both as a prognostic factor to predict clinical outcome and as a predictive biomarker. A retrospective study was performed to evaluate CIN in acute myeloid leukemia (AML). Chromosome mis-segregation frequency was correlated with clinical outcome in bone marrow core biopsy specimens from 17 AML cases. Additionally, we induced chromosome segregation errors in AML cell lines with AZ3146, an inhibitor of the Mps1 mitotic checkpoint kinase, to quantify the phenotypic effects of high CIN. We observed a broad distribution of chromosome mis-segregation frequency in AML bone marrow core specimens. High CIN correlated with complex karyotype in AML, as expected, although there was no clear survival effect. In addition to CIN, experimentally inducing chromosome segregation errors by Mps1 inhibition in AML cell lines causes DNA damage, micronuclei formation, and upregulation of interferon stimulated genes. High levels of CIN appear to be immunostimulatory, suggesting an opportunity to combine mitotic checkpoint inhibitors with immunotherapy in treatment of AML.

Efficacy and safety of bone marrow aspiration and biopsy using fluoroscopic guidance and a drill-powered needle: clinical experience from 775 cases.

To evaluate the efficacy and safety of performing a fluoroscopically guided bone marrow aspiration and biopsy (BMAB) using a drill-powered needle in a large patient population. This retrospective study received institutional review board approval with a waiver of patient informed consent. We identified all BMAB procedures from August 2012 through December 2016 performed at our institution using fluoroscopic guidance and a drill-powered needle. Clinical diagnosis, patient age, patient gender, biopsy site, biopsy needle gauge, bone marrow aspirate volume, bone marrow core biopsy length, patient platelet count, conscious sedation details, complications, and diagnostic adequacy were investigated for each case and summarized. A total of 775 BMAB procedures were performed and analyzed. These were performed in 436 female patients and 339 male patients ranging in age between 16 and 91 years (average age of 53 years). Samples obtained from the procedures in our series were diagnostic in 95.0% of cases. The complication rate for our series was 0.3%. The use of fluoroscopic guidance and a drill-powered needle for bone marrow aspiration and biopsy is a safe and efficacious procedure. • Fluoroscopy can be utilized for imaging guidance during bone marrow aspiration and biopsy. • The use of fluoroscopic guidance and a drill-powered needle for bone marrow aspiration and biopsy has a high diagnostic yield. • The procedure has an excellent patient safety profile.

Investigating macrophage function as a mechanism of resistance to daratumumab in relapsed refractory multiple myeloma patients.

e20547 Background: Daratumumab (Dara), an anti-CD38 antibody, has a variety of Fc-dependent immune effector mechanisms of action including macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). However, it remains unclear which mechanism, when disrupted, drives in vivo resistance. Given widespread use of this agent for treatment of multiple myeloma, identifying how to overcome Dara resistance has become an urgent clinical need. We hypothesized that inhibiting macrophage activity, via increased plasma cell surface expression of the CD47 “don’t eat me” signal, could be a mechanism of acquired Dara resistance. Methods: We retrospectively identified 11 patients with relapsed refractory multiple myeloma who progressed on Dara and had sequential bone marrow core biopsies before and after Dara with both samples containing &gt; 20% plasma cells. Nine patients had sufficient material for analysis. Core biopsy slides were stained and analyzed for expression of CD47 (H-scoring for total intensity on plasma cells, as well as annotation of membrane vs. cytosolic localization), CD68 (cell counts of macrophages), and CD4/CD8/FOXP3 triple stain (cell counts of CD4 and CD8 effector and regulatory T-cells). Results: Median age was 57 years. Median R-ISS stage was 2. Two patients had high-risk cytogenetics at diagnosis. Median time from diagnosis to progression on Dara was 56.3 months (95% CI 37.43 – 88.92). Median overall survival was 69.5 months (95% CI: 46.5 – 97.1). Plasma cells in all samples expressed CD47 (H-score median = 96, range 43 - 290). However, in contrast to our hypothesis, there was no significant change in CD47 H-score at progression on Dara (H-score median = 100, range 29 – 140). Furthermore, we observed no significant change in CD68+ macrophage counts nor CD68+ localization at Dara progression. We did note a trend toward CD47 expression shifting from membrane to cytosol at Dara resistance ( p = 0.14). Consistent with prior studies, in our cohort we observed no influx in Tregs but a markedly increased CD8/CD4 ratio at resistance ( p = 0.01). Conclusions: In vitro and murine studies have proposed ADCP as an important mechanism of Dara efficacy. However, we did not observe signatures of resistance in patients being driven by this pathway. Our work suggests that anti-CD47 therapy, leading to activation of macrophages to eliminate tumor cells, will potentially be efficacious for both the Dara-naïve and Dara-refractory settings.

Materials of the II Conference “Current Issues of Diagnosis and Treatment of Ph-Negative and Ph-Positive Myeloproliferative Neoplasms” (March 15–16, 2019; National Research Center for Hematology, Moscow)

The publication contains materials of the reports presented at the II Conference “Current Issues of Diagnosis and Treatment of Ph-Negative and Ph-Positive Myeloproliferative Neoplasms” held from 15 to 16 March 2019 at the National Research Center for Hematology (Moscow). The conference was organized to enable professional communication of the clinicians specializing in the treatment of myeloproliferative neoplasms (MPN), and the researchers in the related fields as well as to allow the exchange of views on the implementation of current diagnosis and treatment methods in Ph-negative and Ph-positive MPNs. Reports covered a wide range of rare and non-standard settings. Of particular importance was the opportunity to debate them in detail at panel discussions and interactive sessions. This format of the conference allowed to provide expert opinions in the present publication. It emphasizes the importance of complex diagnosis in MPN using morphological examination of bone marrow core biopsy samples and molecular genetic testing. Accordingly, the second day of the conference was devoted to a thorough analysis of the morphological characteristics of the cases presented and based on bone marrow core biopsy samples.

Diagnostic value of bone marrow core biopsy patterns in lymphoplasmacytic lymphoma/Waldenström macroglobulinaemia and description of its mutational profiles by targeted NGS

AimsThe aim of this study was to describe the characteristics of the bone marrow infiltration found in a series of clinically defined lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinaemia (WM) and IgM-monoclonal gammopathy...