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Cardiac amyloidosis is often preceded by orthopedic manifestations such as carpal tunnel syndrome, and 10% of patients who underwent idiopathic carpal tunnel release surgery will have biopsy-confirmed amyloid deposits in the tenosynovial sheath. Trigger finger is also commonly reported in patients with amyloidosis and involves the same tendon sheath as carpal tunnel syndrome, but the prevalence of amyloid deposition is unclear. This prospective cross-sectional study enrolled 100 patients aged ≥50 years at the time of surgery for idiopathic trigger finger. Patients underwent release surgery, and a sample of the tenosynovium of the affected finger was excised, stained with Congo red, and subtyped with mass spectrometry if amyloid was demonstrated. Further cardiac evaluation was performed in patients with amyloid deposition. Of the 100 patients (mean age 65.5 ± 8.1 years) enrolled, only 2 demonstrated amyloid deposits on Congo red staining. One patient with previous proteinuric kidney disease had fibrinogen A α-chain amyloidosis, and the other patient had untyped amyloidosis. Neither patient had cardiac involvement. A total of 13 of the 100 patients underwent concomitant carpal tunnel release surgery, and 2 of these patients had amyloid deposits in the carpal tunnel with “false-negative” samples from the trigger finger tenosynovium. In conclusion, biopsy during trigger finger release surgery demonstrated a 2% yield for amyloidosis, which is significantly lower than the previously published yield of 10% during carpal tunnel release surgery. This observation has important implications for the development of diagnostic algorithms to screen patients for amyloidosis during orthopedic operations.

AIMS Mammary amyloid is an uncommon and easily overlooked pathological diagnosis with ambivalent presentation. Herein, we delineate the clinicopathological and radiographic characteristics of mammary amyloid. METHODS AND RESULTS The Department of Pathology database was searched from 1993 to 2019 for keywords 'breast' and 'amyloid', yielding 32 cases from 23 patients, including consultation cases. All patients were female, age range = 52-81 (mean = 67.4 years). The left breast was involved more than the right (43 versus 33%, respectively); bilateral amyloid involvement was also present (24%). Amyloid was most often associated with a benign histopathological diagnosis (57%), lymphoma in 39% [all B cell lymphomas; five of nine were mucosa-associated lymphoid tissue (MALT) lymphoma] and rarely with a concurrent epithelial malignancy (invasive lobular carcinoma, 4%). Of the 14 patients with available clinical history, amyloid presented as a mass clinically or radiographically (six patients, 43%), as microcalcifications (five patients, 36%), and only occasionally as an asymmetry (14%) or fibroglandular density (7%). Microscopic examination detected microcalcifications in an additional nine cases (total 14 patients; 44% of the cohort). Interestingly, one patient had concurrent epithelial and haematological malignancy and amyloid within an axillary lymph node. Co-morbidities included autoimmune diseases and multiple myeloma. CONCLUSION The majority of mammary amyloid cases are associated with benign histopathological findings, while imaging most frequently noted microcalcifications or mass lesions. To avoid overlooking amyloid as simply fat necrosis or fibroelastotic stromal change, a low threshold for performing ancillary stains should be considered in elderly women with benign core needle findings performed for mass lesions or microcalcifications.

Objectives To assess bone marrow (BM) sampling in academic medical centers. Methods Data from 6,374 BM samples obtained in 32 centers in 2001 and 2011, including core length (CL), were analyzed. Results BM included a biopsy (BMB; 93%) specimen, aspirate (BMA; 92%) specimen, or both (83%). The median (SD) CL was 12 (8.5) mm, and evaluable marrow was 9 (7.6) mm. Tissue contraction due to processing was 15%. BMB specimens were longer in adults younger than 60 years, men, and bilateral, staging, and baseline samples. Only 4% of BMB and 2% of BMB/BMA samples were deemed inadequate for diagnosis. BM for plasma cell dyscrasias, nonphysician operators, and ancillary studies usage increased, while bilateral sampling decreased over the decade. BM-related quality assurance programs are infrequent. Conclusions CL is shorter than recommended and varies with patient age and sex, clinical circumstances, and center experience. While pathologists render diagnoses on most cases irrespective of CL, BMB yield improvement is desirable.

Intravascular T-cell lymphomas are rare, poorly characterized lesions. We discuss the clinical, radiologic and especially the laboratory characteristics of a lesion which presented in a 62-year-old woman with a history of progressive CNS abnormalities. Throughout the course of the disease, radiologic findings consisted mainly of multifocal mixed areas of ischemia and vasogenic edema involving cortical and subcortical regions. A brain biopsy identified an abnormal T-cell population confined to lumens of vessels. These T-cells were abnormal cytotoxic cells, positive for CD3, CD8, and negative for CD2, CD4, CD5, CD7 and CD30. While flow cytometry and immunohistochemistry failed to identify a similar population in the blood or bone marrow, molecular studies showed a clonal T-cell population in both the brain and the bone marrow. No other organs were involved. In spite of aggressive treatment, the patient's medical condition continued to progress and she passed away. In conclusion, this abnormal population of cytotoxic T-cells with intravascular localization probably represents a specific type of T-cell lymphoma with specific clinical, radiologic, molecular and immunophenotypic characteristics.

The characteristics of the IgA plasma cell neoplasms are not clearly reported in the literature. The main goal of this study is to examine IgA plasma cell neoplasms (PCN) and to compare them to IgG lesions. After at least 5 years from the identification of an M protein, 98 cases were selected, the presentation and clinical evolution of 45 IgA neoplasms were compared to 43 cases of IgG gammopathies. The classification at presentation as monoclonal gammopathy of undermined significance (MGUS)-22 of 45 IgA and 20 of 43 IgG (49 vs 46%), plasma cell myeloma (PCM)-22 of 45 IgA and 22 of 43 IgG (49 vs 51%) and smoldering PCM (SPCM)-1 each (2% for both) was essentially identical. No solitary plasmacytomas were identified. At presentation, IgA patients were younger (66.5 ± 11.3 vs. 69.2 ± 10.7 years), less likely to have bone lesions (12/45 vs 18/43, p 0.14) or immunoparesis (51% vs. 63%), differences statistically insignificant. Cases with normal fluorescence in-situ hybridization (FISH) results, 27% for IgA vs 61% for IgG (p 0.037) were statistically different. The IgA patients had worse survival (80 vs 108 months median IgA vs IgG, p 0.013), difference not detectable in the first 5 years, but substantial after 10. In conclusion, poorer long-term survival and increased genomic complexity by FISH are characteristics of IgA PCNs.

Multiple myeloma cells secrete more disulfide bond–rich proteins than any other mammalian cell. Thus, inhibition of protein disulfide isomerases (PDI) required for protein folding in the endoplasmic reticulum (ER) should increase ER stress beyond repair in this incurable cancer. Here, we report the mechanistically unbiased discovery of a novel PDI-inhibiting compound with antimyeloma activity. We screened a 30,355 small-molecule library using a multilayered multiple myeloma cell–based cytotoxicity assay that modeled disease niche, normal liver, kidney, and bone marrow. CCF642, a bone marrow–sparing compound, exhibited a submicromolar IC50 in 10 of 10 multiple myeloma cell lines. An active biotinylated analog of CCF642 defined binding to the PDI isoenzymes A1, A3, and A4 in MM cells. In vitro, CCF642 inhibited PDI reductase activity about 100-fold more potently than the structurally distinct established inhibitors PACMA 31 and LOC14. Computational modeling suggested a novel covalent binding mode in active-site CGHCK motifs. Remarkably, without any further chemistry optimization, CCF642 displayed potent efficacy in an aggressive syngeneic mouse model of multiple myeloma and prolonged the lifespan of C57BL/KaLwRij mice engrafted with 5TGM1-luc myeloma, an effect comparable to the first-line multiple myeloma therapeutic bortezomib. Consistent with PDI inhibition, CCF642 caused acute ER stress in multiple myeloma cells accompanied by apoptosis-inducing calcium release. Overall, our results provide an illustration of the utility of simple in vivo simulations as part of a drug discovery effort, along with a sound preclinical rationale to develop a new small-molecule therapeutic to treat multiple myeloma. Cancer Res; 76(11); 3340–50. ©2016 AACR.

Aims Marked thrombocytosis is uncommon in chronic myelogenous leukaemia (CML) but may be associated with mutation of JAK2 V617F, calreticulin ( CALR ) and/or phospho-STAT5 (p-STAT5) activation in other myeloproliferative neoplasms (MPNs), particularly essential thrombocythaemia (ET). We investigated the JAK2 V617F, CALR and STAT5 activation status in patients with CML and thrombocytosis (CML-T) that mimicked ET, trying to identify a common mechanism for thrombocytosis in MPN. Methods Blood and bone marrow morphological findings were reviewed from seven CML-T, four otherwise typical CML and one CML in blast phase. All cases were analysed for BCR-ABL1 , JAK2 V617F and CALR exon 9 mutation and p-STAT5 expression. Results Four of seven cases of CML-T had marked thrombocytosis (>1000×10 9 /L). Eleven of 12 cases had megakaryocyte morphology typical for CML. All cases were BCR-ABL1 positive. Eleven of 12 cases were negative for JAK2 V617F, while STAT5 was activated in six of seven CML-T and in four of five CML cases. No case had a detectable CALR exon 9 mutation. One case of CML developed ET-like morphology and had JAK2 V617F detected while in molecular remission for CML. Conclusions Detection of BCR-ABL1 is critical in the distinction of ET from CML. Thrombocytosis and STAT5 activation in CML-T are not consistently associated with CALR exon 9 or JAK2 V617F mutation.

Cotta C V, Coleman J F, Li S & Hsi E D (2011) Histopathology 59, 1194–1203 Nodular lymphocyte predominant Hodgkin lymphoma and diffuse large B-cell lymphoma: a study of six cases concurrently involving the same site Aims: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a slowly progressing neoplasm with a favourable prognosis. However, in a minority of cases (3–12%) it progresses to a clonally related diffuse large B-cell lymphoma (DLBCL), diagnosed between 6 months and 24 years after NLPHL. This study investigated six cases of NLPHL and DLBCL at the same location. Methods and results: The patients were five men and one woman. In four cases, the site was an axillary lymph node, and in two it was inguinal. In all cases, NLPHL areas had typical morphological and immunophenotypic features. DLBCL involvement was multifocal, diffuse, and characterized by large centroblastic and anaplastic cells. Immunohistochemical studies showed DLBCL cells to be positive for CD20, CD45, and BCL6. In one case, DLBCL cells were positive for BCL2, and in two cases they were positive for MUM-1. There were no networks of follicular dendritic cells (FDC) associated with DLBCL. Rosettes of PD-1-positive and CD57-positive cells surrounding malignant cells in NLPHL were absent in DLBCL. All the cases were negative for Epstein–Barr virus. No translocations involving MYC were identified in DLBCL. Treatment and outcome were known in four cases. All of these patients were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), and this was followed by clinical remission (CR). Conclusions: In adequately sampled tumors, DLBCL can be associated with NLPHL at diagnosis. Diffuse architecture, loss of FDC networks, sometimes immunophenotype shift are characteristics of DLBCL associated with NLPHL. Treatment with R-CHOP usually leads to CR.

Acute myeloid leukemia (AML) with inv(3) (q21q26.2) or t(3;3)(q21;q26.2) is a distinct subtype in the World Health Organization classification. The natural history of myelodysplastic syndrome (MDS) associated with these cytogenetic aberrations is poorly understood. We studied 17 MDS (11 de novo and 6 therapy related) and 3 chronic myelomonocytic leukemia (CMML) cases associated with inv(3) (q21q26.2) or t(3;3)(q21;q26.2). The de novo cases were further classified as refractory cytopenia with multilineage dysplasia (n = 8) and refractory anemia with excess blasts (n = 3). Isolated inv(3)/t(3;3) was identified in 4 cases, whereas –7/7q (n = 13) and –5/5q (n = 6) were common additional aberrations. Nineteen patients died, including 13 in whom the disease progressed to AML after a median of 7 months. Median survival for patients with de novo disease was similar to that for patients with therapy-related MDS (13 vs 17.5 months). MDS or CMML with inv(3)/t(3;3) are aggressive diseases with a high risk of progression to AML. The inv(3)(q21q26.2) or t(3;3)(q21;q26.2) is present in approximately 1% of patients with myelodysplastic syndrome (MDS) and in a subset of patients with acute myeloid leukemia (AML) and the blast phase of myeloproliferative neoplasms. 1-4 Patients with inv(3)/t(3;3)-associated myeloid neoplasms often have anemia. The platelet count may be normal or increased. The bone marrow typically shows increased numbers of small hypolobated megakaryocytes and

The c-kit receptor is expressed in 95% of relapsed acute myeloid leukemias (AMLs) and mediates leukemic proliferation. We conducted a Phase 1 study of the c-kit inhibitor, imatinib mesylate (IM), in combination with cytarabine and daunorubicin (7+3) in c-kit+ relapsed AML. IM was dose escalated using a 3 by 3 design. Phosphorylated STAT5 was absent to minimally present in residual blasts on day 14 bone marrows. The maximum tolerated dose of IM was 300 mg. The dose-limiting toxicity was Grade 3-4 hepatic toxicity. The CR/CRp rate was 57%. Cytotoxic therapy that includes IM for relapsed AML is well-tolerated and effective.

NPM1 is the most frequently mutated gene in acute myeloid leukemia (AML). Unfortunately, there are no 'precision' or rational treatments for this subtype of AML.To elucidate molecular mechanisms of pathogenesis, we performed the first comprehensive, unbiased analysis of the endogenous NPM1 protein-interactome using mass-spectrometry (LC-MS/MS). This approach identified abundant amounts of the master transcription factor driver of monocyte lineage-differentiation PU.1 (SPI1). The NPM1/PU.1 interaction causes PU.1 functional deficiency when NPM1 is mutated, because mutant-NPM1 dislocates PU.1 into the cytoplasm with it.This was confirmed using six different methods: (i) Immunoprecipitation (IP)-LC-MS/MS from nuclear and cytoplasmic fractions of wildtype (wt) and NPM1 -mutated AML cell lines (n=2); (ii) IP-Western blot (WB) from nuclear/cytoplasmic fractions of NPM1 -mutated/wt AML cell lines (n=2); (iii) WB of nuclear/cytoplasmic fractions of NPM1 -mutated/wt AML cell lines (n=5); (iv) immunofluorescence microscopy (IF) of NPM1 -mutated/wt AML cell lines (n=5); (v) IF of NPM1 -mutated/wt primary AML cells (n=6); and (vi) cotransfection of HEK293 cells to express PU.1 + mutant vs wt-NPM1 followed by IF. Re-introduction of Pu.1 into the nucleus of Pu.1-null myeloid precursors which are differentiation-arrested and exponentially proliferating repressed key myeloid precursor genes (e.g., Hoxa9) and triggered terminal monocytic differentiation. Even though primary AML cells (n=900) express the PU.1/RUNX1/CEBPA monocyte differentiation-driving master transcription factor circuit at levels comparable to or exceeding that in normal monocytes, the expression of ~300 monocyte terminal-differentiation genes was suppressed. Importantly, the genes affected are strongly positively correlated (avg. rho 0.7) with PU.1 expression in normal hematopoiesis, consistent with functional disruption of PU.1 in AML. To translate these observations into a treatment option for NPM1 -mutated AMLs, we were guided by additional observations. First, the NPM1/PU.1 protein complex is exported by the nuclear export protein XPO1. XPO1-mediated nuclear export is inhibited by the small molecule selinexor. We found that sub-cytotoxic /low nanomolar concentrations of selinexor locked mutant-NPM1/PU.1 in the nucleus, releasing terminal monocytic differentiation of NPM1 -mutated AML cells both in vitro and in vivo . Briefly, NSG mice were xenotransplanted with NPM1 / FLT3 -mutated primary AML cells and observed until AML engraftment (≥20%) was confirmed. Selinexor was then administered by oral gavage at 2 mg/kg 4X/week (Fig1A), which is 10-fold lower than the usual in vivo cytotoxic dose (15-20 mg/kg) - low doses are well-tolerated and sufficient to promote non-cytotoxic differentiation of AML cells. After 50 days of treatment, bone marrow (Fig1B) and spleen (Fig1C) AML burden was significantly lower in selinexor vs vehicle treated mice. In addition, selinexor treated mice preserved murine hematopoiesis (Fig1D) and IF confirmed the partial nuclear restoration of PU.1 (Fig1E). Terminal monocytic differentiation of AML cells was evident by Giemsa-stained morphology (Fig1F) and flow cytometry (Fig1G). RUNX1 and CEBPA remain in NPM1 -mutated AML cell nuclei at high levels - PU.1 usually cooperates with these master transcription factor partners to exchange corepressors for coactivators and activate differentiation genes. Accordingly, IP-LC-MS/MS of endogenous nuclear CEBPA demonstrated enrichment for corepressors. Depletion of one of these corepressors, DNMT1, using non-cytotoxic concentrations of decitabine or 5-azacytidine (clinical DNMT1-depletors), also induced terminal-differentiation. Moreover, the granulocytic direction of differentiation naturally downregulated NPM1, an event inherent to CEBPA-driven granulocytic (but not monocytic) differentiation. This approach readily induced terminal-differentiation in NPM1 -mutated AML cells selected over 52 weeks of culture for resistance to selinexor, shown by exponential growth in selinexor 20 nM. The mechanisms by which mutant-NPM1 creates leukemic self-replication (proliferation uncoupled from differentiation) are thus reversed by non-cytotoxic molecular targeted clinical drugs. We are evaluating the combination of selinexor with non-cytotoxic DNMT1-depletion in vivo and clinical trials are planned. Disclosures Landesman: Karyopharm Therapeutics: Employment.

A previously healthy 31-year-old man was seen at our institution for evaluation of severe fatigue, night sweats, and supraclavicular lymphadenopathy. About 9 months earlier, he experienced symptoms...

The introduction of molecular techniques in the study of myeloid neoplasms has resulted in the identification of numerous genetic abnormalities with diagnostic and prognostic significance. The impact of these recent discoveries cannot be understated, as the definitions of several myeloid neoplasms have been changed to include new, molecular criteria. Also, the presence of molecular abnormalities with prognostic significance can impact the therapeutic decisions in many cases. This review is directed to an audience of clinical and laboratory professionals involved in the diagnosis and management of myeloid neoplasms, and it aims to succinctly present the most important genes that have been shown to be important in these disorders. In addition to the information about mutations, their significance, and the methods used in their diagnosis, the normal patterns of expression of these genes, their normal functions, and their structures are also discussed.

BACKGROUND. Inhibitor of DNA binding 2 (Id2), a helix-loop-helix protein of the inhibitor of differentiation (ID) family, is involved in hematopoiesis, mainly through interaction with the E-family of transcription factors. Recent studies have shown that Id2 is overexpressed in some types of B-cell lymphoma, including classical Hodgkin lymphoma. The authors of the current study hypothesized that Id2 also is overexpressed in T-cell lymphomas. METHODS. By using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analyses, high Id2 messenger RNA (mRNA) and protein levels, respectively, were detected in all 4 T-cell anaplastic large cell lymphoma (ALCL) cell lines that were tested. RESULTS. Immunohistochemistry results indicated that Id2 was expressed strongly in 21 of 27 (78%) ALCL tumors (79% anaplastic lymphoma kinase [ALK]-negative and 75% ALK-positive), in 5 of 10 (50%) extranodal natural killer/T (NK/T)-cell lymphomas of the nasal type, in 2 of 8 (25%) cutaneous ALCLs, in 2 of 9 (22%) enteropathy type T-cell lymphomas, in 1 of 5 (20%) peripheral T-cell lymphomas not otherwise specified, and in 1 of 8 (13%) T-cell prolymphocytic leukemias. Other types of T-cell lymphoma were negative for Id2. Because it is known that myc is expressed in ALCL, myc was inhibited selectively in 2 ALCL cell lines, resulting in a concentration-dependent decrease in Id2 mRNA and protein levels. Conversely, forced expression of myc in HEK 293T cells using an myc/green fluorescent protein adenoviral vector resulted in Id2 up-regulation. CONCLUSIONS. Taken together, the current data suggest that Id2 commonly is overexpressed in highly proliferative T-cell lymphomas, and its expression may result from transcriptional activation of myc in these tumors. Cancer 2008. © 2007 American Cancer Society.

BackgroundLight chain deposition disease (LCDD) is usually a systemic disorder characterised by non-amyloid monoclonal immunoglobulin light chain deposition in tissues. Localised nodular pulmonary (NP) LCDD is a rare and poorly characterised entity and, owing to the difficulties in diagnosis, limited data are available.MethodsWe investigated the clinical, radiological and pathological characteristics of a series of six confidently diagnosed cases of NPLCDD.ResultsThere were three men and three women with ages ranging from 33 to 74 years. In all cases there were single or multiple pulmonary nodules, in one case associated with cysts. Two patients had no previous history of a lymphoproliferative or autoimmune disorder, two had Sjögren syndrome (SS) and two had extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). Lung biopsies led to diagnoses of MALT lymphoma in four patients, including both of those with a previous history of lymphoma and one with SS. In five cases the diagnosis was confirmed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and in one by electron microscopy. There was no evidence of systemic LCDD in any of the cases. Five patients had an indolent course in spite of limited therapeutic intervention while, in the patient who died, the cause of death was related to the spread of the lymphoma and was not due to the pulmonary lesions.ConclusionsNPLCDD is an indolent disease, in most cases associated with MALT lymphoma or autoimmune disease.

The nonhematopoietic tumors most often diagnosed in the bone marrow are metastatic. Currently, accurate diagnosis of tumor metastasis requires integration of the clinical findings; morphological features; and results of immunohistochemical stains, cytogenetics, and molecular studies. This review focuses on a practical approach to the diagnosis of metastatic tumors in the bone marrow according to current standards of practice and discusses recent advances in understanding of tumor metastasis and the interaction between tumors and the bone marrow environment.

Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF-dependent block in B cell development occurs near the onset of B-lineage commitment, which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted, multipotential progenitor cells. To test this hypothesis, we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF-expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were >95 and 90% GFP+EBF+ mature B cells, respectively. Both NK and lymphoid-derived dendritic cells were also significantly reduced compared with control-transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid-derived cell pathways.

In the study published in this issue, Louzao Martinez et al1 investigate the significance of increased IgG4-positive plasma cells in lymph nodes. The accurate interpretation of this finding is important as awareness of IgG4-related disease (IgG4-RD) is growing among both clinicians and pathologists, and in consequence, IgG4-RD is included in the differential diagnosis of an increasing number of specimens. From a historical perspective, until recently, no common pathophysiologic features were identified among cases diagnosed as Mikulicz syndrome, Riedel thyroiditis, Kuttner tumor, inflammatory pseudotumor, Ormond syndrome, and autoimmune pancreatitis. The discovery of IgG4 and the realization that some cases of sclerosing pancreatitis were associated with local accumulation of IgG4-positive plasma cells, extra-pancreatic involvement,2 and increased serum levels of IgG43 opened a new avenue of investigation that led to the recognition of IgG4-RD as a systemic disease that can involve virtually any organ. This and the fact that treatment with corticosteroids or with rituximab is often effective increased the importance of an accurate diagnosis of IgG4-RD. Confident identification of cases of IgG4-RD led to a better morphologic, immunohistochemical, and serologic characterization of this entity. Indeed, several morphologic features have been consistently described in cases of IgG4-RD: dense lymphoplasmacytic infiltrate, fibrosis with at least a focal storiform pattern, obliterative phlebitis, nonobliterative phlebitis, increased eosinophils, increased numbers of IgG4-positive plasma cells per high-power field (hpf), and increased IgG4/IgG plasma cell ratio (>40%). In addition, most patients have elevated serum IgG4 levels.4 After cases with varying presentations were analyzed, an interesting development was the realization that while features such as fibrosis, dense lymphoplasmacytic infiltrate, obliterative phlebitis, and elevated serum IgG4 are shared by most cases, there is significant variability in the morphologic manifestations of …

Acute myeloid leukemia (AML) is self-renewal by immature myeloid precursors that fail to differentiate. An influential 'leukemia stem cell' model thus proposes that leukemogenic proteins augment or introduce a stem cell-like self-renewal program into cells, e.g., by upregulating signaling or transcription factors (TF) emblematic of stem cells (e.g., HOX). We investigated how the most recurrently mutated protein in AML, mutant nucleophosmin (mNPM1), causes leukemic cell expansion. The results challenge this model, but most importantly, open the door to rational targeted therapy for mNPM1 AML. One way of examining for stem cell programs in AML cells is to look at expression patterns of master TF that regulate expression of hundreds of genes and dictate cell fates. Of these select TF, the master TF that create hematopoietic stem cells (HLF etc.) are minimally or not expressed. Instead, there are very high levels of the master TF that drive monocyte and granulocyte lineage fates, PU.1 (SPI1) and CEBPA. Clearly, however, the lineage-programs intended by PU.1/CEBPA are inefficiently executed if at all - mNPM1 AML patient bone marrows had 85-97% cells with a granulocyte-monocyte progenitor phenotype, accumulated at the expense of downstream mature cells (Quek et al, JEM 2016). This aggregation at a lineage-committed, intermediate, naturally proliferative level of the hematopoietic hierarchy suggests an alternative model - instead of introducing a poorly-defined stem cell self-renewal program, mutant proteins disable differentiation programs which usually quench MYC-driven proliferation intrinsic to lineage-progenitors. To better understand how mNPM1 interacts with cellular machinery, we used mass-spectrometry to comprehensively document the protein interactions of endogenous NPM1 in AML cell nuclear and cytoplasmic fractions, the first analysis of this kind. Notably, the NPM1 protein interactome was enriched for PU.1. Critically, wild-type (wt) NPM1/PU.1 was in the nucleus of wtNPM1 AML cells, but mNPM1/PU.1 was in the cytoplasm of mNPM1 AML cells. This was evident clearly also by Western blot of cell fractions and by IF microscopy of primary AML cells and cell lines. Is cytoplasmic dis-location of PU.1 sufficient to explain persistent hematopoietic precursor proliferation? We used murine Pu.1 knock-out hematopoietic precursors transduced to express Pu.1 fused with the estrogen receptor (Pu.1-ER) to answer this question - Pu.1 relocation from the cytoplasm to the nucleus by tamoxifen triggered monocytic differentiation that terminated proliferation. Moreover, Pu.1-ER cells, like mNPM1 AML cells, highly express Hox genes, rapidly suppressed upon Pu.1 relocation to the nucleus. Thus, Pu.1 dominantly controls Hox and proliferation, as befitting of a master TF, and persistent HOX expression, like persistent progenitor proliferation, can be caused by Pu.1 loss-of-function. Protein macromolecules like NPM1 require transport factors to exit (exportins) the nucleus. A specific exportin, XPO1, was the major exportin found in the NPM1 interactome. XPO1 interactions with transported cargo can be inhibited by the small molecule drug KPT330. KPT330 10-20 nM rapidly re-located mNPM1 and PU.1 to the nucleus, downregulated MYC, upregulated p27/CDKN1B, upregulated monocyte but not granulocyte differentiation markers, induced morphologic changes of monocyte differentiation, and terminated proliferation of mNPM1 AML cells. The same low nanomolar treatment did not induce differentiation of wtNPM1 AML cells (THP1). Moreover, these KPT330 levels are not toxic to normal hematopoiesis (also shown by others). Thus, rather than gain-of-function of elusive stem cell-like self-renewal, the most frequently mutated protein in AML creates self-renewal by disabling a differentiation program that quenches intrinsic MYC-driven proliferation of lineage-progenitors. These observations are a mechanistic rationale to select refractory/relapsed mNPM1 AML patients for treatment with low well-tolerated doses of KPT330, with a defined molecular pharmacodynamic objective of returning PU.1 to the nucleus, to produce cell cycle exits by differentiation rather than p53-mediated apoptosis (to address chemotherapy resistance), to spare precious normal HSC (good therapeutic index), and directly reverse the basis for leukemic self-renewal (proliferation without differentiation). Figure. Figure. Disclosures Landesman: Karyopharm Therapeutics Inc: Employment, Other: stockholder. Saunthararajah:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine.

This chapter is an overview of epidemiologic, diagnostic, and prognostic features of mature NK- and T-cell lymphomas. In the introduction, there is a brief presentation of the epidemiology of these neoplasms and of the factors that may impact their geographical distribution. The discussion of the general principles of the 2008 World Health Organization classification of lymphoid tumors is followed by a review of specimen handling that ideally should allow a reliable diagnosis. The bulk of the chapter consists of the presentation of each type of lymphoma, with an emphasis on the morphologic, immunophenotypic, molecular, and genetic characteristics of each diagnostic category.

This chapter covers hematopoiesis and the methodologies used to study maturation of bone marrow precursors into mature blood elements. The roles of particular genes and signaling pathways are highlighted.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) has clinicopathologic, immunophenotypic, and molecular features that are distinct from classical Hodgkin lymphoma (cHL). Distinguishing these entities is usually straightforward, but unusual variants of NLPHL have been described that can be problematic diagnostically and potentially have clinical significance. We describe a case of NLPHL showing a variant histologic pattern characterized by small and large clusters of lymphocyte predominance cells within nodules defined by follicular dendritic cell meshworks, associated with numerous neutrophils and areas of internodular fibrosis. Focally, the neoplastic cells had lacunar cell-like cytologic features. The resulting combination, not previously reported, closely mimicked the syncytial variant of nodular sclerosis cHL. After chemotherapy the patient was free of disease 14 years later. In our review of the literature we identified rare cases of NLPHL that had similar histologic features, but lacked the neutrophilic infiltration. Recognition of this rare variant, facilitated by immunohistochemical studies in this case, further expands the morphologic spectrum of NLPHL.

The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFβ-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFβ-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFβ-SMMHC/FLT3-ITD–expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFβ-SMMHC–expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFβ-SMMHC/FLT3-ITD–reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFβ-SMMHC in an animal model of inv(16)-associated AML.

BACKGROUND Bone marrow examination is essential in diagnosis, staging and monitoring of various hematologic disorders. The aspirate smears and core biopsy are complementary samples; current clinical benchmarks recommend an optimal core sample length of at least 15-20 mm. We assessed the core length in 2 cross-sectional cohorts from 2001 and 2011 in 32 academic medical centers from the US and Canada, the first such study to date. METHODS After IRB approval, participants collected data from pathology reports (including the preprocessing length) and measured aggregate postprocessing and evaluable marrow length using a uniform validated methodology on 100 consecutive marrow samples in 2001 and 2011 at each institution. Deidentified data was centralized at Roswell Park Cancer Institute (RPCI) and centers were anonymized. A total of 6374 samples were accepted for statistical analysis, performed using SAS (v. 9.4 or higher; SAS Institute, Cary, NC) at RPCI. Relationship between core length and NCCN status, geographic location, gender, age, and staging was assessed using the PROC MIXED and PROC GLIMMIX procedure using a random center effect and a nominal significance level of 0.05. RESULTS The study cohort included 56% men and 44% women, mean age 51 (range, 1-102) years, 88% adults (³18) and 12% children ( A core biopsy was obtained in 90% of 2001 and in 95% of 2011 samples. Most cores were unilateral (85%); bilateral sampling decreased from 10% to 4% between 2001 and 2011. An aspirate specimen was received in the pathology department in 86% of the cases; however, a clot section was prepared in only 56% of the cases. Preprocessing core length (PreCL; n=3141) documentation in pathology reports was missing in 9 centers for both years; in 3 centers it was available only for one year; its mean (standard deviation) was 16.9 (9.9) mm, decreasing from 2001 to 2011 [17.7 (11.9) to 16.2 (8); p=0.002]. Postprocessing core length (PostCL; n=5742) mean (SD) was 14.2 (8.5) mm, significantly shorter in women than in men (p Evaluable marrow space length (EML; n=5617) mean (SD) was 10.7 (7.5) mm and decreased from 2001 to 2011 [10.9 (8.5) to 10.6 (6.6); p=0.002]. Ninety-one samples (1.6%) with measureable PostCL were entirely devoid of evaluable marrow. PostCL was 15% shorter than PreCL. The EML measured 24% less than the PostCL and 33% less than the PreCL. Lymphoma staging samples (n=1222) were obtained from adults (96%) and children (4%); of these, only 45% and 28% reached the PostCL of 15 mm and 20 mm, respectively. Lymphoma involvement rate was 16%, 25%, 32%, 30%, and 23% when PostCL was There was no significant difference of PostCL mean based on geographic region or NCCN status. PostCL mean ranged from 8.8 to 29.6 mm and its median was ³15 mm in only 12 of 23, 9 of 32, and 2 of 32 centers for PreCL, PostCL, and EML, respectively (Fig 3). When compared to current benchmarks, only 53%, 38%, and 22% of all samples were ³15 mm and only 30%, 19%, and 10% were ³20 mm for PreCL, PostCL, and EML, respectively. CONCLUSIONS Current benchmarks for bone marrow core biopsy adequacy are met in only a minority of cases. Samples from women were shorter than from men. Bilateral sampling decreased and PreCL and EML diminished significantly between 2001 and 2011, while PostCL showed only minimal decrease. This discrepancy may be due to technical differences. Although staging cores were longer than non-staging ones, most did not meet the benchmarks. The wide variability among different centers suggests significant institutional differences and lack of standardization in bone marrow sampling. Over a third of all centers did not record preprocessing length, an important parameter which may guide operator assessment of adequacy at the bedside. PostCL and/or EML can be measured by the pathologist and integrated in the report to document adequacy. In staging samples, a minimal marrow space sampling should be required to avoid understaging. Consensus definition of an inadequate core biopsy is currently lacking but would be desirable for uniform pathology reporting, clinical protocols and management. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Taking a mechanistically unbiased approach to the discovery of new myeloma drugs we developed a three organ system assay that selected anti-myeloma compounds from a primary 30,000 small molecule ATP-based screen in one “sandwich” setup for tolerability by normal bone marrow, stability towards liver enzymes, and activity in the context of cell barriers, bone marrow stromal support, and short, kidney-clearance-like exposure (fig.1). The most promising compound (CCF642) emerging from this test had IC50s around 500 nM in all 8 MM cell lines tested including a proteasome inhibitor refractory line while the IC50 was not reached in 5 healthy bone marrow samples at doses up to 6750 nM. Similar sensitivity as in MM was seen in 7 lymphoma cell lines. Investigation of its mechanism of action revealed that CCF642 immediately stimulates protein ubiquitination which is followed by degradation of major myeloma survival factors (c-MYC, IRF4, NF-κB) and apoptosis, while normal bone marrow cells increase ubiquitination to a much lesser degree and only transiently without undergoing cell death. Experiments with an active biotinylated analog showed that it preferentially enters myeloma cells with cytoplasmic accumulation and over 10-fold lower entry into normal bone marrow. Immunoblots revealed that it becomes covalently attached to proteins in a time- and distribution pattern that parallels ubiquitination responses. In vitro, it bound to ubiquitin activating enzyme UBA1 at the active site cysteine in a similarly stable and reversible way as ubiquitin. Selective alkylation of cysteine sulfhydryl groups inhibited binding of biotinylated CCF642 to UBA1, while addition of ATP and ubiquitin led to its dissociation from UBA1. CCF642 may therefore at least in part act as a small molecule ubiquitin analog. Accordingly CCF642 binding to target proteins of UBA1 containing HeLa fraction II was increased by addition of ATP and blocked in the presence of EDTA. Injected twice a week IP at 30mg/kg it suppressed systemically engrafted luciferase expressing 5TGM1 mouse myeloma cells in syngeneic mice comparable to bortezomib given at the MTD for this mouse strain (0.5mg/kg SC twice a week) with equal prolongation of survival. Results support use of our “sandwich” assay to select myeloma drug candidates for in vivo testing and reveal a new UBA1 interacting small molecule ubiquitination enhancer with promise for clinical translation. Figure 1: Sandwich Three Organ System Assay Figure 1:. Sandwich Three Organ System Assay Disclosures No relevant conflicts of interest to declare.

Homeobox (Hox) gene mutations and their altered expressions are frequently linked to human leukemia. Here, we report that transforming growth factor beta (TGFbeta)/bone morphogenetic protein (BMP) inhibits the bone marrow transformation capability of Hoxa9 and Nup98-Hoxa9, the chimeric fusion form of Hoxa9 identified in human acute myeloid leukemia (AML), through Smad4, the common Smad (Co-Smad) in the TGFbeta/BMP signaling pathway. Smad4 interacts directly with the homeodomain of Hoxa9 and blocks the ability of Nup98-Hoxa9 to bind DNA, thereby suppressing its ability to regulate downstream gene transcription. Mapping data revealed that the amino-terminus of Smad4 mediates this interaction and overexpression of the Hoxa9 interaction domain of Smad4 was sufficient to inhibit the enhanced serial replating ability of primary bone marrow cells induced by Nup98-Hoxa9. These studies establish a novel mechanism by which TGFbeta/BMP regulates hematopoiesis and suggest that modification of Hox DNA-binding activity may serve as a novel therapeutic intervention for those leukemias that involve deregulation of Hox.

PU.1 is a member of the ETS family of transcription factors and is required for the development of multiple hematopoietic lineages. PU.1-/- mice die from hematopoietic failure at about embryonic day 18.5 (e18.5) and show a complete absence of B cells, mature T cells, and macrophages. This phenotype suggests that PU.1 may function at the level of the hematopoietic stem cell (HSC) or a multilineage progenitor. To investigate the role of PU.1 in the regulation of HSCs, PU.1-/- embryos were analyzed at various stages of embryonic development. The absolute number and frequency of HSCs were determined by flow cytometric analysis of c-Kit+Thy-1.1loLin-Sca-1+ (KTLS) cells. We found that KTLS cells were absent or severely reduced in PU.1-/- fetal liver from e12.5 to e15.5. Progenitor cells with a c-Kit+Lin-AA4.1+ and c-Kit+Lin-CD34+ phenotype were also severely reduced. In addition, PU.1-/- fetal liver at e14.5 lacked common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) but retained megakaryocyteerythroid progenitors (MEPs). Consistent with the loss of HSC activity, a 10-fold reduction in erythroid progenitors (mature erythroid burst-forming units [BFUEs]) was observed between e14.5 and e16.5. These data suggest that PU.1 plays an important role in the maintenance or expansion of HSC number in murine fetal liver. (Blood. 2004;104:3894-3900)

Since the mid-1980s, murine retroviral vectors have been used extensively by a number of investigators to clonally mark and genetically modify primitive hematopoietic stem cells (HSC) (1,2). During this period, both vectors and packaging systems used to generate virus have undergone considerable modification. This has led to increased production of high-titer, replication-defective retrovirus that is more resistant to gene inactivation following integration into hematopoietic cells. Current approaches to murine HSC transduction have become increasingly more standardized, although there remain numerous variations on a theme (see Chapter 15). This "classical" method utilizes preconditioned bone-marrow cells (typically from 5-fluorouracil [5-FU]-treated animals) and coculture of these cells with virus-producing packaging cells in the presence of exogenous cytokines. This approach generally yields high proportions of transduced cells that can repopulate lethally irradiated recipient mice for long periods of time, indicating that self-renewal activity is maintained-at least to some extent-in conditions that promote stem-cell cycling. With this approach, it is difficult to re-isolate transduced cells from packaging cells and from nontransduced bone marrow, which would be desirable in some clinically relevant cases.

Progenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B- versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression.

Abstract 2736 Background: Epigenetic instability and aberrant repression of tumor suppressor genes are fundamental aspects of acute myeloid leukemia (AML) biology. Current prognostic assays for AML focus on the genetic aspects of disease biology, for example, chromosome abnormalities and recurrent mutations. We hypothesized that a simple assay for an epigenetic abnormality can address the epigenetic domain of AML, with implications for prognostication and for treatment selection with chromatin relaxing drugs. We focused on patients with intermediate risk cytogenetics (CG) and evaluated the association between histone (H4) acetylation and complete remission (CR), relapse-free survival (RFS), and overall survival (OS) in newly diagnosed AML patients with intermediate risk CG. Methods: We assessed histone H4 acetylation status in bone marrow biopsies from newly diagnosed AML patients with intermediate risk CG treated at our institution between the years 2003–2005. CG risk group was ascribed per Cancer and Leukemia Group B criteria. Association with CR, RFS and OS was assessed using logistic regression and Cox proportional hazard regression models. Variables used in the multivariate models included: age, WBC at diagnosis, gender, and history of antecedent hematologic disorder (AHD). B5-fixed bone marrow core biopsies were reviewed for areas with the highest blast concentration. Immunohistochemistry was performed for acetyl-H4 (1:200 dilution; polyclonal; Upstate Biotech, Lake Placid, NY) using automated stainers and heat-induced epitope retrieval. In each case, five hundred blasts were counted and only strong nuclear staining was classified as positive. Based on the distribution of cell counts, cases were classified as strongly positive if nuclear staining occurred in > 80% of the blasts. Results: Forty-two patients had intermediate risk CG and received cytarabine-based induction chemotherapy. The median age was 56.5 years (range 17–79) and median WBC count at diagnosis was 10.9 × 109/L (range 0.8– 227.9). Twenty two percent of patients had an AHD. Thirty three patients (78%) had normal cytogenetics. Chromosomal abnormalities +8,+21, and 11q23 abnormalities were present in 3 (7.1%), 1 (2.3%) and 4 (9.4%) patients, respectively. FLT3 status was available on only 9 patients; with 7 patients having no FLT3 mutations. Twelve patients (28.5%) received an allogeneic bone marrow transplant in first remission. The median follow-up time was 22.4 months (range 0.6–65.3). Thirty-four patients (81%) achieved CR. Using the 80% cut-off, 15 patients (35.7%) were positive for histone H4 acetylation. Histone (H4) acetylation was not associated with achievement of CR (OR=3.0, 95% CI 0.3–25.3, p=0.3). However, on multivariate analysis, a significant association was found between histone acetylation and risk of relapse (HR 0.34, 95% CI 0.11–0.99, p=0.05) and overall survival (HR=0.34, 95% CI 0.15–0.80, p=0.01). Inclusion of allogeneic transplant in the multivariate models did not change these estimates. Conclusion: Histone H4 acetylation is associated with improved RFS and OS in newly diagnosed AML patients with intermediate risk CG. These results will need to be validated in a larger population of uniformly treated patients. However, histone H4 acetylation has the potential as a simple assay to assess for the contribution of abnormal epigenetics to AML biology, with prognostic and potentially therapeutic implications, given the increasing role of epigenetically targeted therapy in myeloid malignancy management. Disclosures: No relevant conflicts of interest to declare.

Abstract 4667 Current concepts of the pathophysiology of primary immune thrombocytopenia (ITP) reflect a complex interplay between antiplatelet antibodies, cytotoxic T-lymphocytes (CTL), T regulatory cells, and impaired bone marrow megakaryopoiesis. The mechanisms of ITP in the presence of monoclonal gammopathy of unknown significance (MGUS), a form of secondary ITP, are less well understood, yet treatment has been essentially the same as for primary ITP. Inhibition of antiplatelet CTL responses through apoptosis induction in clonal antiplatelet-antibody producing plasma cells and proteasome inhibition in CTLs may be achieved with bortezomib. However, bortezomib may cause transient isolated thrombocytopenia via presumed inhibition of marrow thrombopoiesis or enhanced platelet clearance peripherally. We used bortezomib in a 55 year-old female with IgG lambda monoclonal gammopathy and ITP refractory to previous treatment with corticosteroids, intravenous immunoglobulin, splenectomy, danazole, and rituximab, none of which induced a durable increase in her platelet counts to hemostatic concentrations. She suffered from repeated episodes of prolonged epistaxis, and when the platelet count fell below 10K/mm3, she experienced a life-threatening gastrointestinal hemorrhage. Otherwise, she had no anemia, her creatinine and calcium remained normal throughout, and skeletal surveys showed no evidence for myeloma. Repeated bone marrow examinations, reviewed at our institution, demonstrated lambda monotypic plasma cells always below 10%, and a normal female karyotype on metaphase cytogenetics. Her thrombocytopenia and bleeding episodes improved transiently on dexamethasone/IMiD combinations (thalidomide followed by lenalidomide), with concurrent reduction in total IgG by almost 50% (M-spike was not measured prior to IMiDs), however she became intolerant to thalidomide due to peripheral neuropathy and experienced diarrhea on lenalidomide. Her platelets decreased and her epistaxis worsened as her m-spike increased on dexamethasone single agent. We thus focused our treatment approach on more robust suppression of the monoclonal protein and initiated a course of weekly bortezomib (1.3 mg/m2), with dexamethasone (20 mg) on the day of and the day after bortezomib. After one month of therapy, the platelet count normalized, and she had no further bleeding episodes. The bortezomib dose was reduced to 1 mg/m2 and dexamethasone reduced to 20 mg weekly after 2 months, with persistently normal platelet counts thereafter. Her ITP resolution on bortezomib was accompanied by a decrease of her serum M-protein from 1.04 to 0.39 g/dL. This case illustrates that bortezomib can be remarkably effective in refractory ITP with concurrent monoclonal gammopathy. Assuming similar pathophysiology, it might also have efficacy in primary refractory ITP. This case also raises the question of whether refractory ITP in the presence of a clonal plasma cell disorder should be considered as a myeloma defining symptom. Disclosures: No relevant conflicts of interest to declare.