Clot Sections Research Articles

Improving adequacy rates of bone marrow aspirate and core biopsies through training and a bedside X-ray device.

e23228 Background: Bone marrow (BM) evaluation is crucial to the diagnosis of hematolymphoid disorders. WHO requires an adequate number of well-spread-out spicules in aspirates and defines an adequate BM core biopsy as ≥1.5 cm with at least 10 inter-trabecular areas (ITA). Gross examination of the core biopsy at the bedside can be misleading due to attached clot, thick periosteum, and cortical bone, or tangential biopsies, which histologically results in biopsy without adequate ITAs. Suboptimal BM biopsies hinder appropriate diagnosis, delay patient care, and may require the patient to undergo additional BM biopsies. Bedside image analysis via a cabinet x-ray system can be utilized as a point-of-care tool to assess the amount of evaluable marrow permitting re-biopsy at the time of the procedure. Methods: The study was conducted over 5 months at Westchester Medical Center. We retrospectively estimated BM adequacy rates by analyzing the aspirate quality and measuring the BM core biopsy length. Subsequently, a training demonstration to the proceduralist (fellows in training) by an experienced attending was performed and the adequacy rates were reassessed. BM biopsies were analyzed with a cabinet x-ray system, Faxitron, soon after biopsy, then subjected to routine histology and processing. High-resolution images of the core biopsy were obtained. The length of the core biopsy and the number of ITAs were calculated from the x-ray images. Measurement of the gross biopsy length and microscopic assessment of evaluable marrow were compared to the measurements obtained by x-ray imaging. Results: Our adequacy rates which met aspirate quality were 5% (1/20) pre-intervention and 65% (13/20) post-intervention. The aspirate smear quality improved after the demonstration technique to 65%. Only 13/53 core biopsies exceeded the WHO standard of 1.5 cm by microscopy, while 34/53 were grossly estimated to be > 1.5 cm. There was general concordance between the number of ITAs and the length of core biopsies by microscopy. In approximately 20% of cases, the x-ray images highlighted suboptimal core biopsy ( < 10 ITA/cortical bone/cartilage), which weren’t apparent by gross examination alone at the bedside. Utilizing x-ray image measurements, core biopsy fragment length was shown to be in concordance with microscopic estimation of length and number of ITAs. X-ray imaging readily identified clot sections (non-radiopaque), sclerotic cortical bone (absence of trabeculae), and periosteum (soft tissue shadow) in all 10 sub-1 cm biopsies. Conclusions: We conclude that a standard instruction technique of slide preparation can improve the quality of bone marrow aspirate. Core biopsy length and number of ITAs can easily be estimated using bedside point-of-care x-ray analysis to ensure adequate BM evaluations. We propose training and the routine use of bedside x-ray machine to improve the adequacy rates of BM biopsies.

Enhancing Bone Marrow Biopsy Sample Quality: A Comprehensive Quality Improvement Study at UNM Cancer Center

Introduction Adequate samples from bone marrow biopsies are required for pathologists to diagnose and monitor hematologic conditions accurately. These biopsies involve the extraction of bone marrow samples in two main forms: an aspirate and a core biopsy. In some cases, additional molecular testing may be conducted for the purposes of diagnosis and classification. The journey of a bone marrow sample from the patient's bedside to the laboratory is a complex process that involves the collaboration of numerous participants and interconnected systems to ensure the integrity and accuracy of the results. At our cancer center, we initiated a quality improvement (QI) project to assess the baseline success rate of bone marrow sample collection, identify areas for improvement in collection, form and implement targeted interventions, and then measure the project's success. Methods In this QI study, we utilized an A3 model which included 1) assessing the current state of bone marrow biopsy samples at our institution 2) reviewing the current process of collection 3) analysis to identify the root causes affecting quality 4) Identifying our goal state 5) implementing interventions to improve the quality and 6) follow up to evaluate the effectiveness of our interventions against the stated goal. To identify the current state, we assessed the quality of 101 bone marrow biopsy samples collected from May to October 2022, focusing on the aspirate, clot, and biopsy sections. Two pathologists reviewed 20 of these samples at random to re-read them and defined inadequate aspirate and clots as those which were ‘aspicular’ or ‘paucispicular’ and inadequate trephine biopsies as those that were reported as ‘limited’ or ‘inadequate’ for interpretation. This limited analysis was hypothesis generating and we relied on descriptions of aspirate quality and core length in the biopsy reports for the remaining samples. We observed different providers and technicians performing bone marrow biopsies to understand the current process of collection across our institution. Our system analysis identified the following issues: Non-standardized collection technique, communication errors between technicians and providers in terms of core length and aspirate adequacy, and technician proficiency in identifying spicules and core length To address these issues, we wrote a guideline outlining the recommended standard technique and performed a training for all providers. This guideline included a standard communication guideline between technicians and providers. Technicians underwent additional training in terms of identifying spicules and measuring core length. We set a pre-defined goal of success as 70% adequacy for bone marrow biopsy aspirates and 90% adequacy for core biopsies. Following our interventions, we started collecting post-intervention bone marrow biopsies in May 2023 and plan to evaluate the adequacy of 100 bone marrow samples Results Samples were categorized as those performed by interventional radiology (IR) providers and non-IR providers (advanced practice providers, attendings, and fellows).At baseline, the adequacy rates for aspirate, clot, and biopsy sections were 35%, 36%, and 87%, respectively, for IR providers, and 63%, 70%, and 67%, respectively, for non-IR providers. (Table 1)We plan to evaluate 100 samples post-intervention and to date, we have evaluated 20 samples. Among IR cases, adequacy rates for aspirate, clot, and biopsy sections were 69%, 85%, and 92%, respectively. For non-IR cases, adequacy rates were 86%, 100%, and 86%, respectively. (Table 2) Although additional samples are needed to confirm our results, there is thus far a noticeable improvement in the rate of adequate sample collection for both IR and non-IR providers. While not included in the current analysis, we plan to add patient factors that may affect sample collection such as Body Mass Index or use of sedation in our final analysis to address potential confounders. Conclusion Through our QI study, we aimed to enhance the quality of bone marrow biopsy samples at our institution. Preliminary results demonstrate improvements in the rate of adequate sample collection for aspirate and core biopsies following our quality interventions. Future studies will explore the impact of patient and disease factors on sample quality and also assess whether the improved outcomes are sustained at a later point in time with additional follow up.

Marrow Hypercellularity Does Not Exclude the Diagnosis of Essential Thrombocythemia (ET)

Background: The 2022 WHO criteria do not list bone marrow cellularity as a criterion for ET diagnosis (dx) 1, but a review discussing the International Consensus Classification criteria states that the marrow should be normocellular for age, with rare cases of mild hypercellularity 2. The ELN also cited normocellularity as a key factor in differentiating ET from polycythemia vera (PV) or prefibrotic myelofibrosis (MF), further suggesting that normocellularity is the expected standard in ET 3. However, we encountered a number of cases in our hematology practice with increased cellularity in bona fide ET patients (pts). Therefore, we decided to more systematically review the marrow cellularity and its prognostic impact. Methods: Medical records were queried for pts with ET per the ICD, versions 9-10. The dx of ET per WHO 2022 was confirmed by manual review. Cases lacking marrow biopsy were excluded. Structured query language was used for automated extraction of clinical, lab, and molecular findings. We utilized our previously described natural language processing pipeline to extract cellularity information from marrow reports 4. All hematopathology reports of hypercellular marrows were manually reviewed by 3 observers for confirmation and a randomly selected subset of the core and clot sections were reviewed for histomorphology and cellularity by 2 expert hematopathologists and 1 histomorphologist, blinded to pt age and clinical information prior to their review. Quantitative cellularity was determined independently and then congruently for consensus. Qualitative cellularity was defined using (100-age) ± 20% as the normal range with increases above this range considered hypercellular for age. Disease progression was defined as either progression to post-ET MF (PETMF) or direct transformation to acute myeloid leukemia. Transformation to PV was defined as pts with a JAK2 mutation for whom PV was thoroughly excluded at the time of ET dx but who developed absolute erythrocytosis and met the PV WHO diagnostic criteria later in their disease course. We performed univariable and multivariable analysis of progression, PV transformation, thrombosis and mortality risk using Cox proportional-hazards models. Overall (OS), progression-free (PFS), PV-free (PVFS) and thrombosis-free (TFS) survival were estimated using Kaplan-Meier methods. Results: 382 pts met WHO defined ET with marrow dx, of which 145 had pre-treatment diagnostic marrows available for review at Weill Cornell. In the other 237, the diagnostic biopsies had either been performed after treatment was initiated or only the report was available. Thirty-three (23%) were described as hypercellular and 112 (67%) were not. Review of 20 randomly selected marrows from the hypercellular group confirmed age-adjusted hypercellularity ranging from 30 to 80%. The hypercellular pts were significantly older at dx compared to those without hypercellularity (59 vs 50 yrs, p <0.001, Table 1). We identified no other significant differences in blood counts at dx, sex, driver mutation or thrombosis history between the groups. Univariate and multivariate analyses demonstrated no significant difference between the groups in PFS (HR 1.29, p=0.871), TFS (HR 1.39, p=0.671) or OS (HR 0.50, p=0.487) using age, thrombosis history and sex as covariates. A Cox model could not be generated for PVFS due to the low number of events. The 15-year PFS for pts with hypercellularity vs those without was 97% and 94% respectively ( p=0.56, Figure 1), 15-year PVFS was 100% and 67% respectively ( p=0.12), 15-year TFS was 79% and 87% respectively ( p=0.53) and 15-year OS was 84% and 81% respectively ( p=0.74). Conclusion: Our data to date suggests that hypercellularity is of importance diagnostically but not prognostically. Nearly a quarter of our pts with WHO ET had marrow that was hypercellular for age at dx. Pts with increased marrow cellularity tended to be older but were clinically indistinguishable from the remainder in terms of driver mutation, blood counts and thrombosis history. Univariate and multivariate analyses demonstrate that hypercellularity does not confer increased risk of progression, thrombosis or mortality. Thus, hypercellularity alone should not be used as a factor for excluding ET dx.

High Prevalence of Iron Deficiency Among Daratumumab-Treated Newly Diagnosed Multiple Myeloma Patients

Background: Iron deficiency (ID), a common cause of anemia, is often overlooked among patients with hematologic malignancies given alternative etiologies. Daratumumab is an anti-CD38 monoclonal antibody used in the management of multiple myeloma (MM). Approximately 45% of patients on daratumumab are anemic. In a study of 22 patients with relapsed refractory AL amyloidosis treated with daratumumab, 40% developed iron deficiency requiring IV iron replacement. It is unclear whether iron deficiency in this setting is specific to AL amyloidosis, or whether it is universally seen with daratumumab treatment. In this study we evaluated iron deficiency parameters, including bone marrow iron stores, in MM patients treated with daratumumab. Methods: We conducted a retrospective study to evaluate the prevalence of ID among newly diagnosed MM patients who were treated uniformly in a phase II study with the combination of daratumumab, ixazomib, lenalidomide and dexamethasone conducted at our center. Laboratory values of ferritin, hemoglobin and MCV were extracted from the medical records at trial enrollment and at 1-year from treatment initiation. ID was defined as ferritin <30 mcg/L. Bone marrow iron stores were assessed at pre-trial and 1-year from therapy initiation by Prussian blue stain of bone marrow clot sections, with grade 0 (absent iron) or grade 1 (trace to low iron) considered as depleted iron storage. Results: Among 54 included patients, 2 (3.7%) had ferritin <30 mcg/L at trial-enrollment, while 27 (50%) developed ID within 1 year and 33 (61%) became iron deficient during their treatment course. All patients (100%) had lower ferritin at 1 year compared to pre-trial value. The median reduction of ferritin at 1-year was 109 (IQR: 41-239 mcg/L), a decrease by 75% (IQR:56- 86%) from baseline value. Fifteen patients (28%) received IV or oral iron replacement, and 4 were already on iron-repletion before the 1-year landmark assessment. The median ferritin improved from 16 to 84 mcg/L (p<.0001) following iron replacement. Forty patients (74%) had improved hemoglobin at 1-year compared to baseline correlated to disease response to therapy. The median increment in hemoglobin was 1.1 (IQR: -0.3 - 2.7 g/dL). Hemoglobin at 1-year was higher in patients having ferritin ≥ 30 mcg/L compared to the ID group: 13.5 (IQR: 12.3-14.1 g/dL) vs 12.8 (IQR: 12.3-13.4 g/dL), p=.05. BM samples from pre-trial (n=37) and 1-year on-trial (n=36) were available for iron staining. Depleted iron storage was more frequent at1-year compared to baseline (63.9% vs 40.5%, p<.05). The median ferritin was lower in patients with iron depleted versus non-iron depleted BM: 58 (IQR: 31-120 mcg/L) vs 243 (IQR: 125-486 mcg/L) at baseline, p=.0002; 27 (IQR: 16-41 mcg/L) vs 59 (IQR: 19-245 mcg/L) at 1-year landmark, p=.07. Comparisons of laboratory markers at initiation and after 1-year on daratumumab trial are listed in Table 1. Conclusions: Our study demonstrated a high prevalence of ID among patients receiving daratumumab-based treatment for newly diagnosed MM. While some decrease in ferritin over treatment course may be caused by myeloma disease control and improved systemic inflammation, this study should increase awareness for ID among MM patients treated with daratumumab. Future studies are needed to better understand the possible role of daratumumab in the pathogenesis of ID in MM and other plasma cell disorders.

Assessment of Diagnostic Values and Comparative Evaluation of Bone Marrow Aspiration and Bone Marrow Clot Section in Various Hematological Disorders

Assessment of Diagnostic Values and Comparative Evaluation of Bone Marrow Aspiration and Bone Marrow Clot Section in Various Hematological Disorders

Evaluation of laboratory diagnostic tests for light-chain clonality and bone marrow findings in AL amyloidosis.

Light-chain amyloidosis (AL) is the most common form of systemic amyloidosis. This study aimed to evaluate the usefulness of laboratory tests for light-chain clonality and bone marrow (BM) findings in AL amyloidosis. We retrospectively enrolled patients newly diagnosed with AL amyloidosis on pathological examination who underwent a BM biopsy. Laboratory test data for light-chain clonality were collected and compared. Amyloid deposits were identified with H&E, Congo red, and PAS stains. We reviewed 98 patients with AL amyloidosis. Light chain clonality (λ, 64 cases; κ, 34 cases) was detected by serum immunofixation electrophoresis (IFE) (63.3%), urine IFE (70.8%), serum protein electrophoresis (PEP) (44.9%), urine PEP (44.8%), serum free light chain (SFLC) ratio (79.5%), and BM immunohistochemistry (IHC) (85.7%). Flow cytometric (FCM) assay identified aberrant BM plasma cells in 92.9% of cases. BM amyloid deposits were identified in 35 of the 98 cases (35.7%); 71.4% (25/35) were Congo red-positive, and 100.0% (35/35) were PAS-positive. Laboratory tests for detecting light-chain clonality in AL amyloidosis in order of sensitivity include FCM assay for aberrant plasma cells, IHC for light chains on BM biopsy or clot section, SFLC ratio, and serum and urine IFE. Congo red staining of BM samples remains an important tool for identifying amyloid deposits in BM. Periodic acid-Schiff (PAS) staining can be useful in diagnosing some cases of Congo red-negative amyloidosis.

Comparison of DNA Sequencing and Multispecific Monoclonal Antibody (Msmab-1) based Immunohistochemical Detection of IDH1/2 Mutation in Acute Myeloid Leukemia Cases.

Background: The IDH1/2 mutation is an important epigenetic modifier involved in the pathogenesis of AML. It is associated with variable prognosis in AML cases. Lack of proper molecular diagnostic infrastructure and high cost limits the routine use of PCR with sequencing as routine diagnostic methodology. The aim of this study was to find the prevalence of IDH mutation in AML cases using both PCR with sequencing and Immunohistochemistry method.
 Methods: We evaluated 60 patients registered at KGMU, Lucknow for diagnosis and treatment of AML. PCR followed by sequencing was done. IHC staining of the IDH1/2 mutation was performed on all cases using bone biopsy or clot section (in cases of pediatric AML cases).
 Results: Out of the total 60 patients of AML 4(6.7%) patients had IDH1R312 mutation and 5(8.3%) Patients had IDH2R172 mutation. IDH2 R140 mutation was not detected in any sample. On immunohistochemistry analysis 10 cases showed positive staining against anti IDH1/2 mutant (R132/R172) antibody, clone MsMab-1 with a sensitivity of 77.8% and specificity of 94.1%.
 Conclusion: IHC could be an alternative method to direct Sanger sequencing for IDH1/2 mutation detection in AML cases. However, the antibody used in the study is not effective for individual assessment of IDH1 and IDH2 mutation.
 Keywords: Immunohistochemistry, Sanger sequencing, IDH1/2 Mutation, Acute Myeloid Leukemia

Abstract 132: Clot Radiomics Features From Ct Imaging Predict Treatment Outcomes From Stroke Thrombectomy

Background: Radiomics features (RFs) extracted from CT images have the potential to provide valuable biological and structural information about ischemic stroke blood clots, which may inform mechanical thrombectomy (MT) outcome. Hypothesis: We aimed to identify RFs that predict MT outcome and interpret these findings using paired histological data. Methods: We extracted 293 RFs from co-registered non-contrast CT (nCCT) and CT angiography (CTA) images. RFs predictive of revascularization outcome (defined by first pass effect [FPE], which is near complete removal of occlusive clot in one pass of MT) were selected. We then trained and cross-validated (5-fold) a balanced logistic regression model to assess the RFs’ ability in FPE prediction. In a subset of cases, we performed digital histology on clots retrieved during MT. We computed 227 engineered clot histomic features from whole slide histopathology that represented clot structure and texture. Results: Following univariate feature selection and multi-variate feature ranking, we identified 6 RFs significantly associated with FPE (Figure, top rows). Continuity in lower pixel intensities, scattered higher intensities, and intensities with abrupt changes in texture were associated with successful revascularization outcome. The multi-variate model trained using the 6 RFs could predict FPE well, with AUC=0.832±0.031 and accuracy=0.760±0.059 in training and AUC=0.787±0.115 and accuracy=0.787±0.127 in cross-validation. Each of the 6 RFs were related to clot component organization in terms of red blood cell (RBC) and fibrin/platelet (FP) distribution. Clots with more diversity of components and with varying sizes of RBC and FP regions in the clot section were positively associated with RFs predictive of FPE. Conclusion: Clot RFs are potential structurally- and biologically-interpretable candidate biomarkers for accurate FPE prediction.

Detection of focal lesions in the clot section with negative bone marrow aspirate and trephine biopsy-A series of 5 cases.

Bone marrow aspiration and trephine biopsies are commonly used procedures in clinical practice. The practice of making a clot section by using the leftover blood from the bone marrow aspirate material is not a commonly followed practice across centers. A clot section has the advantage of studying the added material with an increased possibility of detecting focal lesions such as myeloma, lymphoma, granuloma, and metastasis in the bone marrow. Bone marrow aspirate, trephine biopsy, and clot section were compared for the detection of focal lesions in a series of 5 patients, 3 of who presented with a history of fever and 2 were already diagnosed cases of Hodgkin lymphoma. Focal lesions were detected in the 5 cases in the clot section alone, whereas bone marrow aspirate and trephine biopsy did not show any focal lesion. Granulomatous infiltration was detected in 3 patients, and lymphomatous infiltration was detected in 2 patients in the clot section, whereas bone marrow aspirate and trephine biopsy were negative for any focal lesion in all 5 cases. A clot section is particularly useful in the detection of bone marrow lesions with a focal distribution. Hence, it must be studied alongside bone marrow aspirate smears, touch smears, and trephine biopsy to increase the diagnostic yield.

Deep Learning Accurately Quantifies Plasma Cell Percentages on CD138-Stained Bone Marrow Samples

The diagnosis of plasma cell neoplasms requires accurate, and ideally precise, percentages. This plasma cell percentage is often determined by visual estimation of CD138-stained bone marrow biopsies and clot sections. While not necessarily inaccurate, estimates are by definition imprecise. For this study, we hypothesized that deep learning can be used to improve precision. We trained a semantic segmentation-based convolutional neural network (CNN) using annotations of CD138+ and CD138- cells provided by one pathologist on small image patches of bone marrow and validated the CNN on an independent test set of image patches using annotations from two pathologists and a non-deep learning commercial software. On validation, we found that the intraclass correlation coefficients for plasma cell percentages between the CNN and pathologist #1, a non-deep learning commercial software and pathologist #1, and pathologists #1 and #2 were 0.975, 0.892, and 0.994, respectively. The overall results show that CNN labels were almost as accurate as pathologist labels at a cell-by-cell level. Once satisfied with performance, we scaled-up the CNN to evaluate whole slide images (WSIs), and deployed the system as a workflow friendly web application to measure plasma cell percentages using snapshots taken from microscope cameras.

Relapse of tagraxofusp treated blastic plasmacytoid dendritic cell neoplasm with loss of CD123 expression

Tagraxofusp, a CD123-based-targeted immunotherapy, was recently approved to treat blastic plasmacytoid dendritic cell neoplasm (BPDCN) with excellent response. Also, a subset of BPDCN shows resistance to tagraxofusp. These resistant cases continue to express CD123, which forms the basis of the continued utility of tagraxofusp in newer combination chemotherapies to overcome resistance in BPDCN. Herein, we report a case of an elderly male with BPDCN that achieved complete remission on initial primary treatment with tagraxofusp. However, BPDCN relapsed after 1.5 years while on treatment, with loss of CD123 expression. At relapse, the neoplasm was comprehensively immunophenotyped by flow cytometry (performed on both peripheral blood and bone marrow specimen) and by immunohistochemical evaluation of the bone marrow clot section. The neoplasm at relapse was diagnostic of BPDCN with a lack of CD123 expression. This case highlights a potential limitation of current and upcoming tagraxofusp-based multidrug therapies, at least in a subset of refractory BPDCN. We believe our report will serve as a sentinel to incite future investigations involving alternate resistance mechanisms in BDPCN.

Comparative study of bone marrow clot section with bone marrow aspiration smears in various haematological conditions

Comparative study of bone marrow clot section with bone marrow aspiration smears in various haematological conditions - IJPO- Print ISSN No: - 2394-6784 Online ISSN No:- 2394-6792 Article DOI No:- 10.18231/j.ijpo.2021.045, Indian Journal of Pathology and Oncology-Indian J Pathol Oncol

Bone Marrow Clot Section a Useful Tool for Diagnosis of Haematological Diseases- A Case Series

Bone marrow examination is required for diagnosis of various haematological disorders. It includes both neoplastic and non-neoplastic disorders. Usually, bone marrow examination includes Bone Marrow Aspiration (BMA), bone marrow biopsy and bone marrow imprints. Bone marrow clot sections can also be an adjuvant of bone marrow examination. The bone marrow clot sections are prepared from the left-over blood after aspirate smears have been prepared. An adequate bone marrow clot section can be valuable for diagnosis of various diseases, especially in cases in which the aspirate and core biopsy material are inadequate or non contributory or it can be an adjuvant procedure. Little has been published about usefulness of bone marrow clot section. Studies are going on to evaluate the role of bone marrow clot section as an adjuvant or a diagnostic tool. This study includes a series of three cases in which bone marrow clot section was diagnostic.

Atypical Crystal Morphology in Crystal-Storing Histocytosis in Multiple Myeloma

Crystal-storing histiocytosis with massive accumulation of particulate immunoglobulin fragments is a rare phenomenon accompanying multiple myeloma and B-cell dyscrasias. The pathogenesis is not fully understood. A 68-year-old female was hospitalized for pyelonephritis, possible pancreatitis, anemia, and 20-pound weight loss. Abnormal blood work included elevated serum IgG kappa (4,129 mg/dL) on SPEP. A final diagnosis of stage II kappa-restricted IgG multiple myeloma was made. Bone marrow aspirate smears, touch prep, clot sections, and core biopsy showed atypical clusters and sheets of a CD138-positive plasmacellular infiltrate comprising 65% of the marrow cellularity with numerous multinucleated forms. The infiltrate was kappa light chain restricted by ISH and IHC. Approximately 35% of the marrow space was replaced by crystalline deposits that was faintly positive for CD68 and CD163 and contained iron on the Prussian blue stain. Crystalline deposits were negative for CD20, CD138, kappa, and lambda, using immunohistochemical PAS, Congo red, and thioflavin T. Immunofluorescence on Pronase kappa- and lambda-digested paraffin sections was negative. Electron microscopy (EM) revealed two distinct types of intra- and extracellular crystalline deposits. These varied in size and shape; one was electron dense rectangular and, less commonly, rhomboidal. The other was delicate, less electron dense, and fibrillary. Molecular genetic studies results revealed an abnormal FISH panel with aneuploidy (gain of chromosomes 9, 13, and 17) and loss of chromosome 15 and IgH/14q. Although the ultrastructure of crystalline inclusions in plasma cell neoplasms has been previously described, to the best of our knowledge, this is the first report of such inclusions with two distinct ultrastructural features.

Frequency of Lymphomatous Infiltration of Hodgkin’s & Non-Hodgkin’s Lymphoma in Bone Marrow by Bone Marrow Aspiration, Trephine Biopsy and Clot Section

Frequency of Lymphomatous Infiltration of Hodgkin’s & Non-Hodgkin’s Lymphoma in Bone Marrow by Bone Marrow Aspiration, Trephine Biopsy and Clot Section

Transformation of Follicular Lymphoma to a High Grade B-Cell Lymphoma with MYC and BCL2 Translocations and Overlapping Features of Burkitt Lymphoma and Acute Lymphoblastic Leukemia

Most commonly, histologic transformation (HT) from follicular lymphoma (FL) manifests as a diffuse large B cell lymphoma, not otherwise specified (DLBCL, NOS). Less frequently, HT may result in a high-grade B cell lymphoma with MYC and BCL2 and/or BCL6 gene rearrangements, also known as "double-hit" or "triple-hit" lymphomas. In the 2016 revision of the WHO classification of lymphoid neoplasms, the category B-cell lymphoma, unclassifiable was eliminated due to its vague criteria and limiting diagnostic benefit. Instead, the WHO introduced the high-grade B-cell lymphoma (HGBL) category, characterized by MYC and BCL2 and/or BCL6 rearrangements. Cases that present as an intermediate phenotype of DLBCL and Burkitt lymphoma (BL) will fall within this HGBL category.Very rarely, HT results in both the intermediate DLBCL and BL phenotype, and exhibits lymphoblastic features, in which case the WHO recommends that this morphological appearance should be noted. In comparison to de-novo patients with DLBCL, NOS, those with MYC and BCL2 and/or BCL6 gene rearrangements have a worse prognosis.A 63-year-old female presented with left neck adenopathy. Lab assessment including complete blood count (CBC), complete metabolic panel (CMR), serum lactate dehydrogenase (LDH) and B-2 microglobulin were all normal. A whole body computerized tomography (CT) scan revealed diffuse adenopathy above and below the diaphragm. An excisional node biopsy showed grade 3A nodular FL. The Ki-67 labeling index was 40-50%. A bone marrow biopsy showed a small focus of para-trabecular CD20+ lymphoid aggregates.She received 6 cycles of bendamustine (90 mg/m2 days +1 and + 2) and rituximab (375 mg/m2 on day+2) with each cycle delivered every 4 weeks. A follow up CT scan at completion of therapy showed a partial response with resolution of axillary adenopathy and a dramatic shrinkage of the large retroperitoneal nodes.18 months later she had crampy abdominal pain in the absence of B symptoms. Positron emission tomography with 2-deoxy-2-[fluorine-18] fluoro- D-glucose integrated with CT (18F-FDG PET/CT) scan showed widespread adenopathy, diffuse splenic involvement and substantial marrow involvement. Biopsy of a 2.4 cm right axillary node (SUVmax of 16.1) showed involvement by grade 3A FL with a predominant nodular pattern of growth. A bone marrow biopsy once again showed only a small focus of FL.She received idelalisib (150 mg twice daily), and rituximab (375 mg/m2, monthly) beginning May, 2015. After 4 cycles, a repeat CT scan showed a complete radiographic response. Idelalisib was subsequently held while she received corticosteroids for immune-mediated colitis. A month later she restarted idelalisib with a 50% dose reduction. Two weeks later she returned to clinic complaining of bilateral hip and low lumbar discomfort but no B symptoms. A restaging 18F-FDG PET/CT in January of 2016 showed dramatic marrow uptake. A bone marrow aspirate showed sheets of tumor cells representing a spectrum from intermediate sized cells with lymphoblastic features to very large atypical cells with multiple nucleoli. Two distinct histologies were present; one remained consistent with the patient's known FL with a predominant nodular pattern and the other consistent with HT (The large atypical cells expressed PAX5, CD10, BCL2, and cMYC, and were negative for CD20, MPO, CD34, CD30, and BCL6). Focal areas showed faint, heterogeneous expression of terminal deoxynucleotidyl transferase (TdT) best seen on the clot section. Ki67 proliferation index was high (4+/4). FISH analysis showed two populations with MYC amplification and/or rearrangement, and no evidence of BCL6 rearrangement; a karyotype analysis showed a complex abnormal female karyotype with t (14; 18) and multiple structural and numerical abnormalities.She started dose-adjusted rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (DA-R-EPOCH) with concomitant prophylactic intrathecal methotrexate and cytarabine. She had but a short lived response before dying in hospice from progressive lymphoma.Whether idelalisib could provide a microenvironment for selection of more aggressive clones needs to be addressed. Our patient's clinical course is confounded by the incorporation of idelalisib while being further complicated by the complexity of HT and the mechanisms in which first-line chemotherapy regimens impact DHL. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Bone Marrowmmorphology in Addition to JAK2 Allele Burden Is Essential for Assessing Disease Status in Recombinant Interferon Alpha-Treated Polycythemia Vera Patients

IntroductionPolycythemia vera (PV) is a myeloproliferative neoplasm (MPN) associated with the JAK2 V617F mutation. Recombinant interferon alfa-2b (rIFNα-2b) or pegylated rIFN2α (pegrIFNα-2a) treatment in PV is effective in inducing hematologic and molecular responses. Reduction in JAK2V617Fallele burden (%V617F) has been used increasingly as a surrogate marker of disease response to treatment and as a unique criterion for discontinuation of therapy. To date, no studies have evaluated the relationship between changes in %V617F and established indicators of disease activity such as bone marrow cellularity and degree of fibrosis. Having observed persistent marrow hypercellularity or progressive fibrosis in PV patients despite a significant reduction in %V617F, we therefore systematically examined the correlation of %V617F with marrow morphology. We also assessed immunohistochemical expression of pSTAT5 as a marker of JAK2 activation.MethodsThe diagnosis of PV was based on the WHO criteria which include presence of erythrocytosis and/or an increased Cr51 red cell mass, JAK2V617F mutation and typical bone marrow findings such as hypercellularity due to panmyelosis and megakaryocyte morphology consistent with PV. Patients with two marrow examinations and quantitative JAK2 levels measured at a median of 1.2 months from the biopsy date were eligible for inclusion. The JAK2V617Fallele burden in peripheral blood samples was determined by pyrosequencing. Clinical, hematologic and molecular responses were graded according to ELN criteria. Marrow fibrosis was scored according to the WHO three-tiered semi quantitative grading system. Immunohistochemical staining for p-STAT5 was performed on clot sections in a subset of patients.ResultsWe identified 15 patients for inclusion. The median %V617F at the time of the initial biopsy was 62.2% (18.2-100%), which decreased to a median of 33% (0-93.7%) on subsequent evaluation (p=0.02). Patients were initially treated with peg rIFNα-2a 45-90 mcgm weekly (n=13) or rIFNα-2b (n=2), 1x106units thrice weekly, and gradually increased based upon response and tolerance. The median duration of treatment between biopsy was 4.2 years (0.8 - 6.6). All patients achieved either a complete (CHR) (n=8) or a partial (PHR) (n=7) hematologic response. Of these 15 patients, 3 achieved a complete molecular response (CMR), 4 a partial molecular response (PMR), and 8 patients no molecular response (NMR). We observed no correlation between clinical and molecular response: of the 8 patients who achieved CHR, 2 achieved a CMR and 6 did not. There was no correlation between clinical response and fibrosis or cellularity; of 8 patients who achieved CHR, 5 showed persistent/increased hypercellularity and 6 had persistent/increased fibrosis.We also found no correlation between marrow morphologic changes and molecular response. Of the 3 patients with CMR, all had persistent hypercellularity (range: 60-90%) and increased/persistent fibrosis. Among the 4 patients who achieved a PMR, 2 had increased cellularity and 2 decreased cellularity. All had increased or persistent fibrosis. Among the 8 patients with NMR, 2 had an increase in marrow cellularity, 2 no change, and 4 an insignificant decrease. Fibrosis increased in 2, was unchanged in 3, and decreased in 3. Thus, there were no significant correlations between changes in %V617F and cellularity (κ=0.02) and fibrosis (κ=0.04). Immunohistochemical staining for p-STAT5 expression in megakaryocyte nuclei showed no correlation between degree of positivity and %V617F.ConclusionsAlthough we observed a statistically significant decrease in %V617F after treatment with rIFNα, there were no parallel changes in marrow morphology. In all patients with CMR, we observed persistent or worsening marrow disease, suggesting to us a need for continued therapy. In patients who achieved either CHR or PHR, we observed persistent hypercellularity and persistent/progressive fibrosis. Our results question the use of JAK2V617F allele burden as the sole criterion for discontinuation of rIFNα therapy. The prognostic significance of failing to attain a morphologic response in patients who have achieved a hematologic and/or molecular response remains unresolved. Resolution of these issues with longer follow-up is important, as premature discontinuation of rIFNα may allow the pathogenic mechanisms of the disease to progress unfettered. DisclosuresOrazi:Novartis: Honoraria.

A rare case of acute promyelocytic leukemia with focal bone marrow involvement presenting as a paraspinal myeloid sarcoma

Acute promyelocytic leukemia (APL) accounts for 5–8% of acute myeloid leukemias (AMLs). Myeloid sarcomas (MS) are rare extramedullary tumors consisting of immature myeloid cells. These tumors may develop de novo or concurrently with AML and may precede or coincide with the occurrence of AML. The occurrence of MS in an APL patient is a rare clinical event. In these cases, it usually occurs as a sign of relapse. Focal bone marrow involvement by APL is also rare; the majority of cases of APL demonstrate a diffusely hypercellular bone marrow throughout the axial skeleton due to expansion of neoplastic blasts/promyelocytes. We describe a 23-year-old female who presented with gait problems, including multiple falling episodes, and lower extremity weakness. An MRI demonstrated a paraspinal soft tissue mass. Histopathologic analysis demonstrated a myeloid sarcoma expressing CD45, myeloperoxidase, CD117, BCL2, focal weak CD34, and weak CD99 and negative for CD30, TdT, CD20, CD3, and CD10. The bone marrow aspirate demonstrated blasts/promyelocytes accounting for approximately 26% of the total bone marrow aspirate cellularity. The bone marrow biopsy and clot section demonstrated normocellularity with a few blast/promyelocyte foci on the clot section accounting for approximately 5% of total bone marrow cellularity. These foci expressed MPO and CD117 and were negative for CD34. Flow cytometry demonstrated blasts accounting for approximately 31% of all CD45+ events expressing MPO, CD33, CD13, CD117, CD64, CD2, CD38, and CD71 and negative for CD34, HLA-DR, CD11b, CD14, CD15, CD16, CD19, CD22, CD10, CD56, CD61, and surface and cytoplasmic CD3. Fluorescence in situ hybridization (FISH) analysis was positive for the t(15;17)(q22;q12) promyelocytic leukemia/retinoic acid receptor alpha (PML/RARA) translocation. The patient was induced with arsenic trioxide and subsequently underwent consolidation chemotherapy with daunorubicin and all-trans-retinoic acid. Her post-chemotherapy bone marrow biopsy was negative for residual disease. Flow cytometry and FISH analysis were also negative. This is a rare case of APL involving both the initial presentation as myeloid sarcoma and focal bone marrow involvement.

Paraffin embedded marrow clot sections as an adjuvant procedure in the diagnosis of bone marrow diseases.

Objectives: This study was conducted to evaluate the role of histopathological examination of paraffin embedded marrow clot sections in the diagnosis of different neoplastic and non-neoplastic marrow diseases, and whether these sections can replace the need for performing bone marrow trephine biopsy. Methods: Patients included in the study are those with clinical manifestations of hematological disorders who were subjected to peripheral blood examination, marrow aspirate cytology with complimentary bone marrow trephine and marrow clot biopsy. Along with marrow aspiration, the blood left behind after preparing marrow smears, was used to prepare paraffin embedded histological clot sections. The results for 114 patients were analyzed simultaneously for their concordance to highlight the usefulness of marrow clot sections in the diagnosis of different diseases affecting the bone marrow. Immunohistochemical stains were used whenever indicated to obtain additional diagnostic information. Results: Marrow clot sections were diagnostic and concordant with the complete blood count, marrow aspirates and trephines in 63% of the cases. While trephines were unconvincing in another 15.7% of the cases, the clot sections were diagnostic. In 1.7% of the cases, the clot sections and trephines were diagnostic while marrow aspirates were inconclusive, thus in 81% of the cases, marrow clot sections provided the diagnosis and replacing the need for performing trephine biopsy. However, trephine biopsy is still considered necessary in 19 % of the cases in whom the clot sections are of poor quality and unconvincing Conclusion: In most of the cases clot sections can replace the need for trephine biopsy, but trephine biopsy is still required in other cases. The clot sections are useful as an adjuvant procedure to increase the diagnostic yield in marrow studies. Because it is difficult to expect which patients will be diagnosed by clot sections and which will need trephine biopsy, it is advisable to perform both trephine and clot biopsy together with the marrow aspirates.

Double minute chromosomes in acute myeloid leukemia, myelodysplastic syndromes, and chronic myelomonocytic leukemia are associated with micronuclei, MYC or MLL amplification, and complex karyotype

Double minute chromosomes (dmin) are small, paired chromatin bodies that lack a centromere and represent a form of extrachromosomal gene amplification. Dmin are rare in myeloid neoplasms and are generally associated with a poor prognosis. Most studies of dmin in myeloid neoplasms are case reports or small series. In the current study, we present the clinicopathologic and cytogenetic features of 22 patients with myeloid neoplasms harboring dmin. These neoplasms included acute myeloid leukemia (AML) (n = 18), myelodysplastic syndrome (MDS) (n = 3), and chronic myelomonocytic leukemia (CMML) (n = 1). The AML cases consisted of AML with myelodysplasia-related changes (n = 13) and therapy-related AML (n = 5). Dmin were detected in initial pre-therapy samples in 14 patients with AML or CMML; they were acquired during the disease course in 8 patients who had AML or MDS. The presence of dmin was associated with micronuclei (18/18; 100%), complex karyotype (17/22; 77.3%), and amplification of MYC (12/16; 75%) or MLL (4/16; 25%). Immunohistochemical staining for MYC performed on bone marrow core biopsy or clot sections revealed increased MYC protein in all 19 cases tested. Except for one patient, most patients failed to respond to risk-adapted chemotherapies. At last follow up, all patients had died of disease after a median of 5 months following dmin detection. In conclusion, dmin in myeloid neoplasms commonly harbor MYC or MLL gene amplification and manifest as micronuclei within leukemic blasts. Dmin are often associated with myelodysplasia or therapy-related disease, and complex karyotypes.