Bone Marrow Biopsy Specimens Research Articles

Niemann-Pick disease type B: HRCT assessment of pulmonary involvement.

ABSTRACTObjective:To analyze HRCT findings in patients with Niemann-Pick disease (NPD) type B, in order to determine the frequency of HRCT patterns and their distribution in the lung parenchyma, as well as the most common clinical characteristics. Methods:We studied 13 patients (3 males and 10 females) aged 5 to 56 years. HRCT images were independently evaluated by two observers, and disagreements were resolved by consensus. The inclusion criteria were presence of abnormal HRCT findings and diagnosis of NPD type B confirmed by histopathological examination of a bone marrow, lung, or liver biopsy specimen. Results:The most common clinical findings were hepatosplenomegaly and mild to moderate dyspnea. The most common HRCT patterns were smooth interlobular septal thickening and ground-glass opacities, which were both present in all patients. Intralobular lines were present in 12 patients (92.3%). A crazy-paving pattern was observed in 5 patients (38.4%), and areas of air trapping were identified in only 1 case (7.6%). Pulmonary involvement was bilateral in all cases, with the most affected area being the lower lung zone. Conclusions:Smooth interlobular septal thickening, with or without associated ground-glass opacities, in patients with hepatosplenomegaly is the most common finding in NPD type B.

Utility of 5-Methylcytosine Immunohistochemical Staining to Assess Global DNA Methylation and Its Prognostic Impact in MDS Patients

Background:DNA methylation plays a vital role in the pathogenesis of the myelodysplastic syndrome (MDS), a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders. It is reported to be an independent prognostic factor affecting overall survival (OS). Our aim was to analyze the role of global DNA methylation using an anti-5-methylcytosine (5-MC) antibody by immunohistochemistry (IHC) of bone marrow biopsy (BM Bx) specimens in MDS patients, assessing correlations with various clinical and biological prognostic factors.Material and methods:A total of 59 MDS cases, classified as per the World Health Organization (WHO) 2008 guidelines, were evaluated over a period of 4 years. Clinical data were retrieved from departmental case records and anti-5-MC expression was analyzed with formalin fixed paraffin embedded sections of BM Bx specimens of MDS patients and controls.Results:The median age at diagnosis was 52 years (15-85years). Patients were categorized into low risk (59%) and high risk (41%) according to International Prognostic Scoring System (IPSS). The median follow-up time was 10 months (1 to 37 months). We generated a methylation score (M-score) using anti-5-MC and with the derived cut-off of 30.5 from the receiver operator curve (ROC), there was a significant difference between the two groups in the percentage of BM blasts (p=0.01), WHO sub-type (p=0.01), IPSS (p=0.004), progression to AML (p=0.04) on univariate analysis. Interestingly, patients showing a high M-score (M-score ≥ 30.5) demonstrated a significantly shorter OS and progression to AML. However, on multivariate analysis, only BM blasts (p=0.01) and IPSS (p=0.02) remained independent variables for progression to AML and OS respectively.Conclusion:Immunostaining with anti-5-MC antibody with BM Bx samples is a simple and cost effective technique to detect global methylation, a powerful tool to predict overall survival in patients with MDS.

Bone Marrow Small Adipocytes in Acute Myeloid Leukemia Correlate with Prognosis

Introduction: Adipocytes have a substantial effect on the outcome and progression of certain solid tumors. However, little attention has been paid to the role of bone marrow (BM) adipocytes in acute myeloid leukemia (AML) because it is difficult to observe adipocytes through clinical BM aspiration. Although it was reported that adipocytes affected the behavior of leukemia cells in vitro, there is still no direct in vivo evidence. In the present study, we investigated the influence of adipocytes by focusing on their changing size in BM from primary AML patients.Methods: We retrospectively analyzed the biopsy specimens of BM from 70 patients with newly diagnosed AML and 70 controls with lymphoma or solid tumors without infiltration of BM. The size and type of adipocytes were analyzed for average diameter (Ad.Dm) and area (Ad.Ar) by tracing each individual adipocyte with Image-Pro Plus 5.1. Then, AML patients were further divided into 32 patients with continuous complete remission and 38 patients who were refractory based on the treatment effects. Adipocyte number (Ad.N; per square millimeter) and the percentage of adipocyte volume per tissue volume (Ad.V/TV) were compared between these two groups. Furthermore, the prognostic impact of adipocytes on the overall survival (OS) and relapse-free survival (RFS) in AML patients was analyzed by Cox regression analysis and Kaplan-Meier curves. Finally, the phenotype of adipocytes was determined by immunohistochemistry of UCP-1 and Perilipin1 to further explore the possible mechanism of the effect of adipocytes on the prognosis.Results: The Ad.Dm and Ad.Ar in BM from AML patients were 38.3±14.7 µm and 559.4±271.9 µm2, respectively, and both values were significantly smaller than those for the controls (P<0.001). The Ad.Dm exhibited no relation to the number of blasts in BM, indicating that the decreased size of adipocytes in AML cannot be attributed to extensive marrow blasts. Adipocytes were classified as small, medium and large adipocytes according to the frequency distribution of Ad.Dm in BM from controls. A significant difference was detected only in the proportion of small adipocytes (Ad.Dm<42.6 µm) between AML patients and controls (43.9% vs 25%, P=0.005). Furthermore, the Ad.V/TV and Ad.N of the adipocytes in the refractory group were 6.70±3.18% and 31.56±11.72/mm2, respectively, which were significantly higher than those of the remission subjects (P<0.001). This outcome prompted us to further analyze the role of small adipocytes in AML. Patients with Ad.V/TV of small adipocytes≤ 2.3% exhibited a longer OS than patients with Ad.V/TV of small adipocytes >2.3% (P<.001). Similarly, the subjects with Ad.N of small adipocytes <10.6/mm2 (P<.001) had a longer OS. Meanwhile, for the remission AML patients, those with Ad.V/TV of small adipocytes ≤2.5% had a shorter RFS than patients with Ad.V/TV of small adipocytes >2.5% (P<.001), and similar significant differences were also found between patients with Ad.N of small adipocytes ≥9.2/mm2 and Ad.N of small adipocytes <9.2/mm2(P<.001). In the biopsy specimens of BM, the subgroup of small adipocytes exhibited expression of Perilipin1 but not mitochondrial membrane protein UCP-1. Combination with the unilocular lipid droplets in adipocytes indicated that small adipocytes did not play a role in the conversion to brown adipose tissue.Conclusions: We first defined a subpopulation of small adipocytes in BM and demonstrated that only these cells but not all adipocytes were related to a poor prognosis in AML patients. The morphological changes of marrow adipocytes could be helpful to judge the prognosis of leukemia and could lead to other therapeutic perspectives. DisclosuresNo relevant conflicts of interest to declare.

Identification of Bone Marrow Involvement of FL: Comparison of PET-CT and Bone Marrow Biopsy.

IntroductionIn malignant lymphoma, bone marrow involvement is considered as clinical stage IV which adversely affects International Prognostic Index resulting in poor outcome. Detecting bone marrow lesion is therefore important in staging of newly diagnosed malignant lymphoma. Bone marrow biopsy (BMB) of unilateral or bilateral iliac crest has been a classical method to detect bone marrow infiltration. However, BMB in rare instance, induce some complications including excessive bleeding, infections, or lasting pain. Recently, positron emission tomography combined with computed tomography (PET-CT) became a routine tool in staging of malignant lymphoma. Although PET-CT shows high sensitivity of detecting viable nodal and extra-nodal lesions in aggressive lymphoma, its role in low-grade, indolent lymphoma such as follicular lymphoma (FL) remains controversial.The aim of this study is to retrospectively evaluate the diagnostic accuracy of PET-CT in detecting bone marrow infiltration in patients with newly diagnosed FL.Patients and Methods:We collected data of all patients who were newly diagnosed with FL from January 2005 to October 2015 at Yokohama City University Hospital and Yokohama City University Medical Center. Patients with FL who underwent both PET-CT and BMB prior to the initiation of treatments were finally included in the analysis.Results of unilateral or bilateral BMB of posterior iliac crest were collected from written reports. BMB specimens were evaluated by hemato-pathologists of each institution. The presence of lymphoma cells in the bone marrow was based on morphological and immune-histochemical findings.Written reports were used to collect PET-CT data. Interpretation of images was made by radiologists of each institution where PET-CT scans were performed. Bone marrow involvement in PET-CT was defined as greater intensity of FDG uptake in the bone marrow than those in liver or those in mediastinum.This study was approved by the Internal Review Board of Yokohama City University School of Medicine.Results:In total, 184 patients were newly diagnosed with FL from January 2005 to October 2015. Of 184 patients, 117 who underwent both PET-CT and BMB before treatment were evaluated in the further analysis. The patients included 53 males and 64 female with a median age at diagnosis of 53 years (range: 25 - 82). The distributions of histological FL grading were grade 1-2 in 3 patients, grade 1 in 41, grade 2 in 47, grade 3 in 7, grade 3a in 12, and grade unknown in 7.Bone marrow FDG uptake was elevated according to the defined criteria in 22 patients (19%), while the infiltration of lymphoma cells in the bone marrow was detected by BMB in 35 patients (30%).Of 22 patients with elevated FDG uptake in the bone marrow, 6 (32%) were diagnosed as negative for bone marrow infiltration by BMB. Among the 6 patients who were positive for PET-CT and negative for BMB, the pattern of bone marrow FDG uptake was focal in 2, diffuse in 3, and unknown in 1.Among the 35 patients positive for BMB, bone marrow FDG uptake was increased in 16 (65%). Of the16 patients positive for both BMB and PET-CT, the pattern of FDG uptake in the bone marrow was diffuse in 12, and focal in 4. The remaining 19 BMB positive patients were negative for PET-CT. Of these 19 patients positive for BMB and negative for PET-CT, the grading of FL was grade 1 or 2 in 16, and grade 3a in 3.DiscussionIn conclusion, our study revealed that significant number of patients showed discrepancy between the results of PET-CT and BMB in detecting bone marrow involvement of lymphoma. Although PET-CT is highly sensitive for detecting viable lymphoma cells and is commonly used for staging in routine practice, our data indicated that PET-CT still cannot replace BMB for identifying lymphoma cells in the bone marrow in patients with FL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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

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

Sweet's syndrome associated with clonal hematopoiesis of indeterminate potential responsive to 5-azacitidine.

Sweet's syndrome (SS) is a rare condition characterized by the abrupt appearance of painful skin lesions due to neutrophilic dermal infiltration. Hematologic neoplasms, particularly acute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs), have been commonly reported in association with SS. Clonal hematopoiesis of indeterminate potential (CHIP) is an emerging entity that is a precursor state to myeloid neoplasms. CHIP has not been previously associated with SS. We report the case of a 71-year-old man who presented with recurrent, painful edematous and erythematous papules and nodules for 18 months despite treatment with corticosteroids. He had normal blood counts, but a macrocytosis was noted (110 fl). Alternative causes of macrocytosis were ruled out. A skin biopsy confirmed a diagnosis of SS. Bone marrow biopsy specimen yielded a normal karyotype except for loss of the Y chromosome and equivocal morphologic findings. Polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) of selected genes from the peripheral blood demonstrated a mixed lineage leukemia (MLL) partial tandem duplication (PTD) and sequence variant in CCAAT/enhancer binding protein alpha (CEBPA). These findings were consistent with a diagnosis of CHIP. The patient was treated with 5-azacitidine and achieved a complete remission of his skin lesions and was able to discontinue corticosteroids. To our knowledge, this is the first report of a patient with recurrent SS associated with CHIP. In addition to other myeloid neoplasms like AML and MDS, CHIP should be considered as a potential etiology in cases of recurrent SS. Treatment with a hypomethylating agents such as azacitidine could also serve as an alternative to systemic corticosteroid therapy.

Increased Multinucleated Megakaryocytes as an Isolated Finding in Bone Marrow: A Rare Finding and Its Clinical Significance.

Multinucleated megakaryocytes are a unique morphologic form of megakaryocytes characterized by multiple, distinctly separated nuclei. We investigated whether increased multinucleated megakaryocytes (≥25%) in otherwise normal-appearing bone marrow were associated with the development of a myelodysplastic syndrome (MDS). We retrospectively reviewed the medical records and bone marrow biopsy specimens of patients evaluated at our institution from 2011 to 2015 that met all of the following criteria: (1) 25% or more multinucleated megakaryocytes, (2) no other dysplastic features, (3) absence of a myeloid neoplasm, and (4) absence of neoplastic karyotypic abnormalities. Nine cases of 19,885 bone marrow biopsy specimens were identified. The indications for bone marrow examination included plasma cell neoplasm, lymphoma, and cytopenia(s). All patients had anemia at the time the multinucleated megakaryocytes were found. No patients had thrombocytopenia or neoplastic karyotypic abnormalities. Follow-up was available for seven patients with a median of 27 months (range, 7-53 months). No patients developed progressive cytopenias or MDS. Multinucleated megakaryocytes (≥25%) as an isolated finding in the bone marrow are a rare phenomenon. These patients do not appear to be at increased risk of developing progressive cytopenia(s) or MDS. Caution should be taken to not overdiagnose these cases as MDS.

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

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

CAL2 Immunohistochemical Staining Accurately Identifies CALR Mutations in Myeloproliferative Neoplasms.

Mutations in CALR (calreticulin) have been discovered in 50% to 80% of JAK2 (Janus kinase 2) and MPL (myeloproliferative leukemia protein) wild-type patients with Philadelphia-negative myeloproliferative neoplasm (MPNs). We evaluate the performance of a monoclonal antibody for immunohistochemical detection of CALR mutations. A computerized archival search was performed for cases of non-chronic myeloid leukemia (CML) MPNs with available CALR and JAK2 V617F mutational analysis data. Bone marrow biopsy specimens were stained with monoclonal antibody CAL2, and the percentage of stained megakaryocytes was calculated. In select cases, double immunofluorescence staining was done with CAL2 and each of the following: CD61, myeloperoxidase, CD34, and glycophorin A. We studied 38 bone marrow biopsy specimens of non-CML MPNs (primary myelofibrosis, n = 21; essential thrombocythemia, n = 15; and n = 2 post-polycythemia vera myelofibrosis) from 31 patients. All eight bone marrow biopsy specimens from patients with mutant CALR showed strong cytoplasmic staining of the megakaryocytes (83.5%; range, 50%-98%; median, 87%) with the CAL2 antibody. Double immunofluorescence staining of the small mononuclear cells seen in CALR mutant cases revealed them to be myeloid blasts. Immunohistochemistry in routinely processed bone marrow biopsy specimens for mutated CALR is feasible and accurately identifies mutated cases, including rare cases with additional driver mutations.

Alterations in the bone marrow microenvironment may elicit defective hematopoiesis: a comparison of aplastic anemia, chronic myeloid leukemia, and normal bone marrow.

Hematopoiesis involves complex interactions between hematopoietic cells and the bone marrow (BM) microenvironment. The specific causes and mechanisms underlying dysregulated hematopoiesis are unknown. Here, BM biopsy specimens from patients with aplastic anemia (AA) and chronic myeloid leukemia (CML) and normal marrow were analyzed by semiquantitative immunohistochemistry to determine changes in the hematopoietic stem cell (HSC) compartment and BM microenvironment. HSC levels were lowest in AA and highest in CML. T and B lymphocytes were decreased in AA (p<0.01) and CML (p<0.01). Natural killer cells were observed in AA, but were absent in CML and healthy controls (p<0.01). Macrophages and mast cells were absent in CML. There were significant differences between AA and CML stromal cell components. No nestin+ cells were observed in CML and the mean number of stromal cell-derived factor-1-positive cells was lowest in CML. Osteopontin+ cells were higher in AA than in CML (p<0.01); osteonectin+ cells were higher in CML than in AA (p<0.01). There was no significant difference in the expression of osteocalcin between AA and CML. The number of endothelial cells was highest in CML and lowest in AA (p<0.01). Our findings suggest that changes in BM microenvironment components might be related to defective hematopoiesis leading to AA and/or CML.

A rare case of blastic plasmacytoid dendritic cell neoplasm with deletion 7q.31, in the setting of heavy pre-treatment with alkylating chemotherapy

Blastic plasmacytoid dendritic cell neoplasm is rare myeloid malignancy clinically characterized by non-pruritic, violaceous and papulo-nodular skin lesions, together with bone marrow and lymph node involvement. Histologically, there is infiltration of dermis by neoplastic mono-nuclear CD4, CD56, CD123 co-expressing cells with epidermal sparing. Most commonly blastic plasmacytoid dendritic cell neoplasm presents as a de-novo condition, and treatment-related blastic plasmacytoid dendritic cell neoplasm is a rare phenomenon. Due to rarity of the disease, there is no established standard of care treatment. Both acute myeloid leukemia and acute lymphoid leukemia type induction regimens have been used for treatment of blastic plasmacytoid dendritic cell neoplasm, with initial response rate of 50%-80%. We present a rare case of therapy-associated blastic plasmacytoid dendritic cell neoplasm in a patient with remote history alkylating agent systemic therapy. A lag period of five to seven years and presence of deletion 7q.31 seen in bone marrow biopsy specimen in our patient are consistent with a likely therapy-associated etiology of his blastic plasmacytoid dendritic cell neoplasm.

Increased Bone Marrow Iron Scores Are Strongly Correlated With Elevated Serum Ferritin Levels and Poorer Survival in Patients With Iron Overload That Underwent Allogeneic Hematopoietic Stem Cell Transplantation: A Single Center Experience.

Iron overload is one of the most significant problems as a leading cause of death in patients with leukemia and those who underwent allogeneic hematopoietic stem cell transplantation (alloHSCT). In the current study, we retrospectively evaluated the bone marrow iron scores (BMIS) in patients who underwent alloHSCT (n= 125). The first available bone marrow biopsy specimens prior to the alloHSCT procedure or date of hospitalization (control group) were assessed in a blinded fashion using a standardized scoring system. A total of 125patients were enrolled in the study. Seventy-six (60.8%) of the patients were male, and 49 (39.2%) were female. The median level of pre-transplant serum ferritin was 1023.00 ng/mL (range, 393.80-1627.50 ng/mL). The majority of the patients were diagnosed with acute leukemia (83; 66.4%) and lymphomas (20; 16.0%). The median time for neutrophil engraftment was 14.00 days (range, 13.00-16.00 days) and 11.00 days (range, 10.00-14.00 days) for platelet engraftment. The peri-transplant mortality was similar to international mortality rates (3; 2.4%). The overall survival and disease-free survival were strongly correlated with the degree of BMIS, and both were significantly poorer in patients with high bone marrow iron content (P< .001 and P= .012, respectively). The validation of BMIS for risk stratification in patients who undergo alloHSCT may predict posttransplant outcomes.

Multiparameter Analysis of Off-Target Effects of Dasatinib on Bone Homeostasis in Patients With Newly Diagnosed Chronic Myelogenous Leukemia.

We assessed patients with chronic myelogenous leukemia (CML) for serum calcium (Ca), phosphate (PO4), bone alkaline phosphatase, N-telopeptide (NTx), osteoprotegerin (OPG) levels, and trabecular bone area (TBA) in bone marrow (BM) specimens before and after treatment with dasatinib. We identified a significant increase in percentage of TBA in postdasatinib BM (P= .022). This suggests that dasatinib therapy can increase TBA without significant changes in bone and mineral metabolism. Interferences with bone homeostasis and mineral metabolism have been described in patients receiving imatinib for CML or gastrointestinal stromal tumors. Dasatinib is a potent second-generation tyrosine kinase inhibitor designed to inhibit ABL and SRC kinases while also interfering with the c-Kit, platelet-derived growth factor receptor, and STAT5 pathways. We used a multiparameter approach to examine the off-target effects of dasatinib in 30 patients with CML treated between 2009 and 2012. We recorded serum Ca and PO4 levels, analyzed markers of bone formation (bone alkaline phosphatase/bone-specific alkaline phosphatase [BAP]) and bone resorption (NTx), measured OPG levels, and digitally analyzed changes in TBA in paired BM biopsy specimens before and after treatment. We correlated all findings with each other and with the results of conventional cytogenetic and molecular analyses. We identified a significant increase in the percentage of TBA in postdasatinib BM biopsy specimens (P= .022) and noted a decrease in serum OPG levels in 75% of patients. Ca, PO4, BAP, and NTx levels remained steady, without significant changes. There was no correlation between biomarker levels, percentage of TBA, and/or cytogenetic or molecular response. These findings suggest that dasatinib therapy (within the therapeutic range) can increase trabecular bone, without causing significant changes in bone and mineral metabolism. Nonetheless, monitoring of bone health and skeletal integrity should be included into the long-term management of patients treated with dasatinib to further enhance our understanding of its safety profile and its potential role as a treatment modality for other bone diseases.

Patients With a History of Chemotherapy and Isolated del(20q) With Minimal Myelodysplasia Have an Indolent Course

Isolated deletion (20q) is relatively common in myeloid neoplasms and has been rarely reported in cases of therapy-related myelodysplastic syndrome (MDS). Our aim was to characterize cases of isolated del(20q) in bone marrow biopsy specimens from patients with a history of chemotherapy with morphologic findings insufficient for a diagnosis of MDS. In this retrospective study from one institution, we identified 22 patients with isolated del(20q) and no or minimal dysplasia and evaluated clinical and pathologic characteristics. Eleven of the patients had a history of chemotherapy for mostly lymphoproliferative disorders. There were no statistically significant differences in peripheral blood or bone marrow features between patients with a history of chemotherapy and those without. Three patients with a history of chemotherapy had died at last follow-up; cause of death was recurrent nonmyeloid neoplasm. None of the patients with a history of chemotherapy subsequently developed a high-grade myeloid neoplasm, whereas one of the patients who had not received prior chemotherapy developed refractory anemia with excess blasts 2. The presence of del(20q) as an isolated bone marrow cytogenetic abnormality in the absence of morphologic findings sufficient for a diagnosis of acute myeloid leukemia, myeloproliferative neoplasm, or MDS portends an indolent clinical course, regardless of previous exposure to chemotherapy.

Preleukemic phase of chronic myelogenous leukemia: morphologic and immunohistochemical characterization of 7 cases

Patients with chronic myelogenous leukemia (CML) present typically with an elevated white blood cell count (WBC) and cytogenetic or molecular genetic evidence of t(9;22)/BCR-ABL1 fusion gene. Rarely, CML patients may present with a normal or mildly elevated WBC and are asymptomatic, and we describe 7 patients in this study. The WBC in these patients ranged from 3.6 to 14.3 K/mm(3) with 50% to 73% granulocytes and 0% blasts. In all patients, t(9;22)(q34;q11.2) was detected by conventional cytogenetics, and BCR-ABL1 fusion was shown, supporting the diagnosis of preleukemic CML (pre-CML). We compared these patients with a group of 5 cases of CML in chronic phase (CML-CP) and 5 bone marrow specimens with a leukemoid reaction (n=5). Reticulin, CD34, and CD61 immunostains were performed on all bone marrow biopsy specimens. Peripheral blood absolute basophilia (≥200/mm(3)) was noted in only 4 of 7 pre-CML cases, whereas it was present in all CML-CP cases and absent in leukemoid reaction cases. The mean ±SD of microvascular density of pre-CML cases (10.0 ± 4.3 vessels/200× field) was twice that of leukemoid reaction cases (5.0 ± 1.0) (P=.02; Student t test) but similar to that of CML-CP cases (12.5 ± 3.6). Microvessels in pre-CML, highlighted by CD34, were tortuous with abnormal branching, although to a lesser extent than those found in CML-CP. Microvessels in leukemoid reaction were generally straight. The percentage of small, hypolobated megakaryocytes, highlighted by CD61 in pre-CML, was 40%, 3 times that found in leukemoid reaction cases (13%) but less than that of CML-CP cases (86%). We conclude that pre-CML should be suspected in patients with a normal to mildly elevated WBC and absolute basophilia. Bone marrow examination can usually distinguish pre-CML from a leukemoid reaction based on the percentage of small, hypolobated megakaryocytes; microvascular density; and morphologic features.

The Role of the Slit-Robo Family in Adult Patients with Acute Myeloid Leukemia

Introduction: SLIT-ROBO is newly discovered ligand-receptor family of neuronal guidance molecules. Recently, it has been proved that these proteins are involved in both, physiologic and pathologic angiogenesis. In animal models, it was shown both, pro-and antiangiogenic of SLIT-ROBO signaling. Moreover, the interaction of SLIT ligands with their roundabout receptors (ROBO) results in promotion of apoptosis, adhesion and blocking of cell cycle. There is evidence that SLIT-ROBO proteins are involved in pathogenesis of solid tumors, both in angiogenesis dependent and independent way. The role of SLIT-ROBO proteins in biology of acute myeloid leukemia (AML) remains unknown.The only two hitherto published studies considering ROBO4 expression in AML have revealed its increased expression in the blasts cells.The aim of the study was to evaluate the role of SLIT-ROBO proteins in AML. The expression of SLIT ligands, and their receptors ROBO was assessed in bone marrow of newly diagnosed AML patients and in the control group. The expression level of the proteins was correlated with known prognostic factors, response to treatment and overall survival (OS), as well as angiogenesis activity.To our knowledge, it has been the first study investigating the whole family of SLIT-ROBO proteins in AML.Methods: Expression SLIT-ROBO proteins was assessed in bone marrow biopsy specimens of 79 newly diagnosed AML patients with median age 59 years [18-87]. The paraffin-embedded tissue blocks were retrieved and subjected to immunohistochemistry for SLIT ligands (SLIT1, SLIT2, SLIT3), and their receptors ROBO1, ROBO2, ROBO3, and ROBO4. The positive blasts cells were semi-quantitatively analyzed according to previously published methods (Perrone et al, 2006). For the purpose of analysis the patients were divided into "low-expressers" and "high-expressers". Concurrently, all samples were immunostained for CD34 to calculate microvessel density (MVD) as an equivalent of angiogenesis. The control group was composed of 23 BM biopsies form patients with newly diagnosed lymphoma without bone marrow involvement.Results:Expression of ROBO receptors and SLIT ligands in AML patients and in the control group.In our study higher expression of ROBO1, ROBO2, and ROBO3 was observed more often in AML patients compared to the control group (p<0.0001, p<0.001, and p=0.09, respectively, Fig 1.). In contrast, low expression of SLIT1, SLIT2, and SLIT3 ligands has been shown more often in AML than in control BM samples (p<0.0001, p=0.003, and p=0.001, respectively,Fig.2.). Higher expression of ROBO1, ROBO2, and ROBO3 was more often in AML patients ≥60 years (p=0.04, p=0.008, and p=0.02, respectively).Conversely, low expression of ROBO4 was more often observed in elderly AML (p=0.06). The majority of patients with de novo AML had low expression of SLIT1 and SLIT2 (p=0.053 and p=0.055, respectively). As to ROBO, higher expression of ROBO2 in the group with secondary AML was more frequent (p=0.09). No significant correlations between the SLIT-ROBO proteins' expression,neither cytogenetic risk group nor clinical stage parameters such as WBC, hemoglobin level, proportion of leukemic blasts in BM, or LDH activity were found. Similarly, neither of the SLIT-ROBO proteins influenced the complete remission rate (CR) and overall survival (OS).Relationship between SLIT-ROBO expression and angiogenesis activity in AML patients and control group.Significantly higher MVD in BM of AML patients than in control group (Me 51 [9-140] vs 16 [4-78], p<0.0001) has been observed. ROBO4was the only protein that expression correlated significantly with MVD. Higher expression of ROBO4 was associated with higher MVD in both, AML and the control group (p=0.05 and p=0.01, respectively).Conclusions:SLIT-ROBO family members play a role in biology of AML. ROBO4 is involved in both, physiologic and pathologic angiogenesis A better understanding of SLIT-ROBO signaling pathway in leukemic blasts may create new optionsfor AML therapy.Acknowledgments:AG and DJ-K both equally contributed to the study.This work was supported by grants from Medical University of Lodz, Lodz, Poland (502-03/1-093-01/502-14-077 and 503/1-093-01/503-11-001). [Display omitted] [Display omitted] DisclosuresRobak:Eisai Inc: Research Funding. Wierzbowska:Janssen, Celgene: Consultancy.

Presence of PD-1 Expressing T Cells Predicts for Inferior Overall Survival in Newly Diagnosed Multiple Myeloma

Background: Programmed cell death 1 (PD-1) protein downregulates T cell activation and is related to immune tolerance. PDL1 up regulation, T cell infiltration, and T cell exhaustion are features, which suggest susceptibility to PD-1 blockade antibodies. Blockade of PD-1 or its ligand PD-L1 has shown promising responses in several malignancies. Although little clinical activity has been seen in patients with relapsed multiple myeloma (MM), the role of the PD-1 pathway and T cell exhaustion in newly diagnosed MM has not been explored.Objective: To determine whether T-cell infiltrate or expression of PD-1 correlates with clinical features and prognosis among patients with newly diagnosed multiple myeloma.Methods: We screened a clinically annotated database of 341 patients seen at MSKCC between 1998 and 2012 that had Multiple Myeloma, received a bone marrow transplant and were consented to a biospecimen research protocol for availability of pre-treatment bone marrow specimens. A total of 64 bone marrow biopsy specimens were identified. Immunohistochemistry (IHC) was performed in formalin-fixed paraffin-embedded specimens using an anti human CD3 monoclonal antibody (mAb) (Dako, Clone F7.2.38) and an anti human PD-1 mAb (Cell Marque, catalog #315M-95). CD3 and PD1 IHC staining were graded as negative (<5% for CD3, < 1% for PD1), or positive (≥5% for CD3, ≥1% for PD1). Correlative analyses were performed between CD3/PD1 expression and clinical outcome using the following parameters: International Staging System (ISS), cytogenetic risk, progression free survival (PFS), overall survival (OS), and response to treatment. Groups were compared by Fisher's exact test. OS and PFS were assessed by Cox regression and estimated by Kaplan-Meier methods.Results: 23 specimens (36%) were CD3 positive and 10 specimens (16%) were PD-1 positive. All PD-1 positive specimens were CD3 positive. 41 specimens (64%) were CD3 negative (<5%) and PD1 negative (<1%). Based on these results, specimens were divided into three groups: Exhausted T-cell infiltrate (CD3+/PD1+), non exhausted T-cell infiltrate (CD3+/PD1-) and no T-Cell infiltrate (CD3-/PD1-). In the exhausted T-cell infiltrate group 30% of patients had ISS stage 3 and 40% had high risk cytogenetics. In the non-exhausted T-cell group 15% of patients had ISS stage 3 and 15% high cytogenetic risk. In the no T-cell infiltrate group 10% had ISS stage 3 disease and 22% high cytogenetic risk. These proportions were not significantly different across the 3 groups. Median OS from 1st auto infusion was 7 years while median PFS was 2.3 years. On univariate analysis, there was no significant difference in PFS between the 3 groups. The presence of CD3 and PD1 T-cells were significantly associated with OS (p-value = 0.04). Median OS from 1st auto infusion was 43 months for the exhausted T-Cell infiltrate group followed by 83 months for the no T-Cell infiltrate group. The non exhausted T-cell group had the highest OS, median not reached; OS by 7-years was 75%. Cytogenetic risk at diagnosis was significantly associated with OS (p-value = 0.03). In a multivariable model, CD3/PD1 staining continued to trend toward an association with OS (p-value = 0.08) and cytogenetic risk remained significant (p-value = 0.05).Conclusions: The presence of T-cells with PD-1 expression was not associated with higher risk disease at MM diagnosis based on cytogenetics and ISS stage. The presence of PD-1 expressing CD3+ T cells trends toward an association with poorer overall survival in newly diagnosed MM, especially compared to non exhausted T-cell infiltrate, suggesting the possibility that T cell exhaustion represents a novel high risk disease characteristic. Further investigation is necessary to assess if the presence of CD3+PD-1+ T cells is an independent prognostic feature in newly diagnosed MM. [Display omitted] DisclosuresGiralt:JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; TAKEDA: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding. Landgren:International Myeloma Foundation: Research Funding; Onyx: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Medscape: Honoraria; Bristol-Myers Squibb: Honoraria; Medscape: Consultancy; BMJ Publishing: Consultancy; Celgene: Honoraria; Onyx: Honoraria; BMJ Publishing: Honoraria; Onyx: Research Funding. Hassoun:Novartis: Consultancy; Takeda: Research Funding; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Lesokhin:Genentech: Research Funding; Aduro: Consultancy; Janssen: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Efranat: Consultancy.

Morphologically occult systemic mastocytosis in bone marrow: clinicopathologic features and an algorithmic approach to diagnosis.

Bone marrow (BM) biopsy specimens involved by systemic mastocytosis (SM) typically show multifocal, compact, dense aggregates of spindled mast cells (MCs). However, some cases lack aggregate formation and fulfill the World Health Organization 2008 criteria for SM, based on minor criteria. We identified 26 BM cases of KIT D816V-mutated, morphologically occult SM in the BM. All patients had some combination of allergic/MC activating symptoms. Peripheral blood counts were generally normal. BM aspirates showed 5% or less MCs, which were only occasionally spindled. BM biopsy specimens showed no morphologic classic MC lesions. Tryptase immunohistochemistry (IHC) demonstrated interstitial, individually distributed MCs (up to 5%) with prominent spindling, lacking aggregate formation. MCs coexpressed CD25 by IHC and/or flow cytometry. Spindled MCs constituted more than 25% of total MCs in all cases and more than 50% in 20 of 26 cases. Morphologically occult involvement of normal-appearing BM by SM will be missed without appropriate clinical suspicion and pathologic evaluation by tryptase and CD25 IHC and KIT D816V mutation analysis. On the basis of these findings, we propose a cost-effective, data-driven, evidence-based algorithmic approach to the workup of these cases.

Increased SYK activity is associated with unfavorable outcome among patients with acute myeloid leukemia.

Recent discoveries have led to the testing of novel targeted therapies for the treatment of acute myeloid leukemia (AML). To better inform the results of clinical trials, there is a need to identify and systematically assess biomarkers of response and pharmacodynamic markers of successful target engagement. Spleen tyrosine kinase (SYK) is a candidate therapeutic target in AML. Small-molecule inhibitors of SYK induce AML differentiation and impair leukemia progression in preclinical studies. However, tools to predict response to SYK inhibition and to routinely evaluate SYK activation in primary patient samples have been lacking. In this study we quantified phosphorylated SYK (P-SYK) in AML cell lines and establish that increasing levels of baseline P-SYK are correlated with an increasing sensitivity to small-molecule inhibitors targeting SYK. In addition, we found that pharmacological inhibition of SYK activity extinguishes P-SYK expression as detected by an immunohistochemical (IHC) test. Quantitative analysis of P-SYK expression by the IHC test in a series of 70 primary bone marrow biopsy specimens revealed a spectrum of P-SYK expression across AML cases and that high P-SYK expression is associated with unfavourable outcome independent of age, cytogenetics, and white blood cell count. This study thus establishes P-SYK as a critical biomarker in AML that identifies tumors sensitive to SYK inhibition, identifies an at-risk patient population, and allows for the monitoring of target inhibition during treatment.

Human herpesvirus 6 infection in a bone marrow biopsy specimen after allogeneic stem cell transplantation.

A 70-year-old woman with therapy-related acute myeloid leukaemia underwent a haploidentical umbilical cord stem cell transplant (SCT). Twenty-eight days later, she presented with altered mental status and diarrhoea. Bone marrow biopsy showed maturing trilineage haematopoiesis with no evidence of residual disease. There were scattered enlarged cells with large eosinophilic cytopathic intra-nuclear inclusion bodies and abundant foamy cytoplasm (image, haematoxylin and eosin). The inclusions were round, centrally situated, and surrounded by a clear halo. Immunohistochemical staining for cytomegalovirus (CMV), Epstein–Barr virus (EBV), BK virus, human herpesvirus 8, herpes simplex virus 1, herpes simplex virus 2 and parvovirus B19 was negative. Quantitative real-time polymerase chain reaction assays and serological tests for CMV, EBV, parvovirus B19, BK virus and Toxoplasma gondii were all unremarkable. However, very high copy numbers of human herpesvirus 6 (HHV-6) were detected (~300·103 copies/ml). The patient was started on foscarnet and subsequently switched to valganciclovir. As HHV-6 levels decreased to <200 copies/ml, the patient's clinical status quickly improved. Two follow-up bone marrow biopsies at 3 and 6 months post-SCT showed no intra-nuclear inclusions. Currently the patient remains in complete remission. Initial exposure to HHV-6 occurs in early childhood and results in a life-long latency in peripheral blood mononuclear cells, salivary glands and central nervous system. HHV-6 reactivation is noted in 30–70% of patients undergoing allogeneic SCT, typically 2–4 weeks after transplantation. Although HHV-6 reactivation is generally asymptomatic, it can present with fever, rash, diarrhoea, bone marrow suppression or encephalitis. HHV-6 infection is known to cause nuclear and/or cytoplasmic inclusion bodies in peripheral blood mononuclear cells and T-lymphocytes in skin. The exceptionally high copy numbers of HHV-6 and lack of laboratory or immunophenotypic evidence of any other infectious process lead us to postulate that the intra-nuclear inclusions represent evidence of HHV-6 infection. In summary, reactivation of HHV-6 should be considered in the differential diagnosis of cytopathic viral inclusions seen in a biopsy of a post-transplant patient. This diagnosis is clinically relevant, because HHV-6 reactivation has been associated with serious transplantation-related morbidity and mortality.