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T-cell lymphomas (TCLs) are a heterogenous group of diseases that show histologic and immunophenotypic features overlapping with reactive lymphoid proliferations and often require the use of ancillary testing for accurate diagnosis. The oncoprotein, bcl-2, is expressed in various types of lymphoma. At present, expression of this protein is useful for distinguishing several B-cell lymphomas. Although there are some anecdotal reports that the lack of bcl-2 expression by T cells might also be a useful marker for the diagnosis of TCL, there are no focused studies to address this hypothesis. Another antigen with value in TCL diagnosis is programmed death-1 (PD-1), a marker of follicular helper T cells, which has been reported to be sensitive in the detection of angioimmunoblastic TCL and peripheral T-cell lymphoma, unclassified. However, several reports have also shown that PD-1-positive cells may be increased in a number of settings other than TCL, including reactive and atypical lymphadenopathies. Finally, lymphoma cells express a variety of cytokine receptors and signaling molecules that are current or potential targets for immunomodulatory therapy. One such target is the interleukin (IL)-2 receptor (CD25), which is acted on by denileukin diftitox/ONTAK, a recombinant diphtheria toxin-IL-2 fusion protein. Selection of suitable patients for therapy often includes pretreatment assessment of CD25 expression in tumor cells. In order to further assess the diagnostic and therapeutic utility of these antigens, we compared the expression of the CD25, PD-1, and bcl-2 in 119 cases of T-cell non-Hodgkin lymphoma using immunohistochemical techniques applied to routinely processed and paraffin-embedded tissues. We show that lack of expression of bcl-2 was observed in 52% cases of TCL and may aid in identification of neoplastic T-cell populations. In combination, bcl-2, CD25, and PD-1 provide diagnostic utility and may aid in selecting appropriate patients for immunomodulatory therapy.

Context: Immunoglobulin G4 (IgG4)-related disease is a recently described entity that presents as mass-forming lesions in soft tissue, exocrine glands, and in lymph nodes as IgG4-related lymphadenopathy. The underlying pathologic mechanism of IgG4-related disease is unclear; however, rituximab (an anti-CD20 monoclonal antibody) has been shown to have clinical efficacy., Objective: To look for the presence or absence of CD20 on the IgG4-expressing plasma cells in IgG4-related lymphadenopathy., Design: Twelve flow cytometry cases were identified through a retrospective review from the authors' institutions files. Cases were selected by the presence of a lymph node biopsy specimen with increased IgG4 plasma cells by immunohistochemistry and a histologic diagnosis compatible with IgG4-related lymphadenopathy., Results: We report dim CD20 expression on plasma cells in all cases for which a plasma cell population was clearly identified by flow cytometry. These cases were from patients with lymph node biopsy specimens that met published criteria for IgG4-related lymphadenopathy., Conclusions: This finding may be one potential explanation for the clinical efficacy of rituximab in IgG4-related disease.

Kikuchi-Fujimoto disease, Kimura disease, Rosai-Dorfman disease and IgG4 related lymphadenopathy may present with enlarging masses clinically mimicking lymphoma. A combination of clinical and histologic findings is necessary to diagnose these important rare entities, which may occasionally have aggressive clinical behavior. The recognition of these disorders is important in order to avoid misdiagnosis of malignancy, other systemic diseases such as systemic lupus, and to institute correct management and therapy, such as steroid treatment for IgG4 related lymphadenopathy. The underlying etiologies of these diseases are not completely clear at present, however, their recognition has become more common as diagnostic techniques improve. Their diagnosis and recognition may help to elucidate their underlying pathobiology., (Copyright © 2013 Elsevier Inc. All rights reserved.)

IgG4-related sclerosing disease encompasses a family of disorders associated with increased numbers of IgG4 plasma cells and mass forming lesions in various tissues. Lymphadenopathy is a common finding, seen in up to 80% of cases. In the largest series of cases to date, we describe histologic, immunohistochemical, special stain and flow cytometric findings in 29 cases of enlarged lymph nodes with increased IgG4 plasma cells. Lymph node biopsies showed all resection specimens; no needle core biopsies of tissue were evaluated. Cases were considered to have increased numbers of IgG4 plasma cells using the histological criteria outlined by Cheuk and Chan (2010): IgG4 plasma cells >50 cells in a high-power field and >40% of IgG-positive plasma cells positive for IgG4. Additionally, increased intrafollicular plasma cells were a common finding. The lymph nodes showed a variety of reactive histological features including follicular hyperplasia, progressive transformation of germinal centers, interfollicular expansions, variable degrees of fibrosis, increased histiocytes and occasionally an appearance similar to that of plasma cell Castleman disease.

Dimeric pyrrole-imidazole alkaloids represent a rich and topologically unique class of marine natural products. This full account will follow the progression of efforts that culminated in the enantioselective total syntheses of the most structurally ornate members of this family: the axinellamines, the massadines, and palau'amine. A bio-inspired approach capitalizing on the pseudo-symmetry of the members of this class is recounted, delivering a deschloro derivative of the natural product core. Next, the enantioselective synthesis of the chlorocyclopentane core featuring a scalable, catalytic, enantioselective Diels-Alder reaction of a 1-siloxydiene is outlined in detail. Finally, the successful divergent conversion of this core to each of the aforementioned natural products, and the ensuing methodological developments, are described.

Introduction: High-resolution array comparative genomic hybridization (aCGH) is a method of evaluating chromosomal alterations over the entire genome. We compared aCGH with routine cytogenetics and FISH in detecting genetic alterations in chronic lymphocytic leukemia (CLL)., Methods: Array comparative genomic hybridization testing was performed on 55 cases of CLL in addition to a standard panel of FISH probes (ATM on 11q22, trisomy 12, 13q14, p53 on 17p13). The frequency of detecting abnormalities was compared, and discordant results between methodologies were compared., Results: Fifty-five CLL cases [male to female ratio of 2.2:1 and a mean age of 71 (52-90)] were analyzed by both aCGH and FISH. This group of CLL cases showed genetic abnormalities by FISH (60%; 27/45). In contrast to FISH, aCGH detected genetic abnormalities in 82% (45/55) of CLL cases; aCGH identified genetic abnormalities not detected by FISH studies in 16% (7/45) of cases, whereas FISH identified abnormalities not detected by aCGH in only 7% (3/45) of cases. Rare recurring genetic alterations were detected by aCGH including losses in 6q, 8p, 10q, 14q32, and 18q and gains in 10q., Discussion: Our findings suggest aCGH is an effective technique for evaluating recurring genetic abnormalities in CLL and improves on standard FISH in detecting genetic abnormalities in CLL., (© 2010 Blackwell Publishing Ltd.)

Rosai-Dorfman disease and Langerhans cell histiocytosis are both disorders of accessory immune cells. Two cases have been previously reported of concurrent Langerhans cell histiocytosis and Rosai-Dorfman disease. In this report, we characterize the findings and selected molecular studies in nine additional cases. Histology was reviewed. Immunohistochemical stains were performed on all cases in which slides or blocks were available. A combination of CD1a, S-100, CD3, CD20, langerin, CD68, CD163, CD21, CD35 and CD123 immunohistochemical stains were performed. High-resolution array comparative genomic hybridization was performed on six samples from five cases. In these cases, seven were female and two male, with an average age of 25 years (15 months-59 years). A majority of the cases were identified in lymph node. Areas of Langerhans cell histiocytosis had a typical appearance with the existence of bland 'coffee-bean' nuclei, clear cytoplasm and associated eosinophils. The immunophenotype was typical, including expression of CD1a, S100, CD68 and langerin. In areas of Rosai-Dorfman disease, there was emperipolesis seen in all cases. Cells were intermediate-large in size with large round nuclei and ample clear or pale cytoplasm. The lesional cells were positive for S100, CD68, CD163, without expression of langerin or CD1a. Array comparative genomic hybridization showed gains and/or losses in four of the six samples. One case showed no gains or losses and one additional case showed gains and losses in the Langerhans cell histiocytosis, while no abnormalities were discovered in the Rosai-Dorfman disease component. These findings are comparable to those seen in previous studies of Langerhans cell histiocytosis. We report the clinical and pathologic findings of the combination of Langerhans cell histiocytosis and Rosai-Dorfman disease. Furthermore, we suggest on the basis of evidence from our cases that, when simultaneous, the two entities may be pathophysiologically related.

Sceptrin, a natural compound produced by various marine sponges, was tested for its effect on cell motility. We report for the first time that sceptrin inhibits cell motility in several cancer cell lines. The compound shows no toxicity at concentrations that are double the amount of sceptrin required for maximal inhibitory effect. Both random and factor-induced migration were impaired, suggesting that sceptrin targets a central process of cell motility machinery. Activity of de novo synthesized sceptrin was indistinguishable from sceptrin purified from Agelas nakamurai, and the inhibitory activity was found to be, at least partially, due to sceptrin's capability to inhibit cell contractility. Additionally, sceptrin was found to bind to monomeric actin, further suggesting a mechanism involving the actin cytoskeleton. Close analogues of sceptrin were synthesized, tested for their effect on cell motility, and found to be either equimolar or less potent compared to the parental compound. Inadvertent cell motility is a key contributing factor in various human diseases, including cancer and chronic inflammation. Marine compounds isolated from sponges have been proven to be an excellent source of metabolites that show biological activities. Given the recently achieved total synthesis of sceptrin in multigram quantities, sceptrin could prove to be an attractive lead molecule for further preclinical testing and development for therapeutic purposes, as well as a useful research tool to elucidate the mechanisms involved in cell motility.

The recent development of inhibitors of key immune response proteins has revolutionized the therapy of autoimmune diseases; these immunomodulator agents include monoclonal antibodies and receptor antagonists. However, as with all therapies, these new agents are not without side effects and complications. In particular, anti-tumor necrosis factor alpha (TNFalpha) agents have been reported to be associated with an increased incidence of lymphoproliferative disorders, infections, and vasculitis. We evaluated the clinicopathological features of 18 cases of immunomodulator agent-related lymphoproliferative disorders (IAR-LPD) from several institutions. These included 6 cases of B-cell lymphoma, 2 cases of T-cell lymphoma, 3 cases of classical Hodgkin lymphoma, and 7 atypical lymphoid proliferations that did not fulfill diagnostic criteria for lymphoma; two of the latter regressed after discontinuation of the immunomodulator agent therapy. All eight lymphoma patients with available information had also received prior chemotherapy (methotrexate or 6-mercaptopurine). EBV was strongly associated with the B-cell and classical Hodgkin lymphomas. This case series illustrates that a broad range of lymphoid proliferations can occur after immunomodulator agent therapy and that these immunomodulator agent-related lymphoproliferative disorders have considerable overlap with other well-defined lymphoproliferative diseases associated with iatrogenic immunosuppression. Further study is warranted to evaluate how these therapies interact with other immunosuppressive agents and the underlying abnormal immune system to enhance the development of lymphomas and atypical lymphoid proliferations.

IUPAC defines chemoselectivity as "the preferential reaction of a chemical reagent with one of two or more different functional groups", a definition that describes in rather understated terms the single greatest obstacle to complex molecule synthesis. Indeed, efforts to synthesize natural products often become case studies in the art and science of chemoselective control, a skill that nature has practiced deftly for billions of years but man has yet to master. Confrontation of one or perhaps a collection of functional groups that are either promiscuously reactive or stubbornly inert has the potential to unravel an entire strategic design. One could argue that the degree to which chemists can control chemoselectivity pales in comparison to the state of the art in stereocontrol. In this Account, we hope to illustrate how the combination of necessity and tenacity leads to the invention of chemoselective chemistry for the construction of complex molecules. In our laboratory, a premium is placed upon selecting targets that would be difficult or impossible to synthesize using traditional techniques. The successful total synthesis of such molecules demands a high degree of innovation, which in turn enables the discovery of new reactivity and principles for controlling chemoselectivity. In devising an approach to a difficult target, we choose bond disconnections that primarily maximize skeletal simplification, especially when the proposed chemistry is poorly precedented or completely unknown. By choosing such a strategy--rather than adapting an approach to fit known reactions--innovation and invention become the primary goal of the total synthesis. Delivery of the target molecule in a concise and convergent manner is the natural consequence of such endeavors, and invention becomes a prerequisite for success.

The myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) occasionally demonstrate overlapping morphological features including hypercellularity, mild/nonspecific dysplastic changes and variable bone marrow fibrosis. Thus, when the associated bone marrow fibrosis results in a suboptimal specimen for morphological evaluation, the descriptive diagnosis "fibrotic marrow with features indeterminate for MDS versus MPN" is often applied. The JAK2 ( V617F ) mutation was recently shown to be frequently identified in MPN, but it is rarely present in other myeloid disorders. However, the diagnostic utility of JAK2 ( V617F ) screening in hypercellular bone marrow specimens with fibrosis has not been previously investigated. Using a real-time polymerase chain reaction melting-curve assay capable of detecting JAK2 ( V617F ) in archived fixed materials, we retrospectively studied JAK2 ( V617F ) in 45 cases with fibrotic hypercellular bone marrow at initial presentation, including 19 cases initially described as "with features indeterminate for MDS versus MPN". These 19 cases were reclassified into more specific categories of MDS (n = 14) or MPN (n = 5) based on the availability of subsequent clinical data and/or bone marrow examinations. The JAK2 ( V617F ) allele was identified in 17 out of 18 BCR/ABL gene-negative MPN cases with marrow fibrosis, whereas only wild-type alleles were identified in the remaining non-MPN cases. Importantly, JAK2 ( V617F ) alleles were seen in all five cases of "with features indeterminate for MDS versus MPN" at initial presentation that were later determined to be MPN, but they were absent in the 14 cases later determined to be MDS. Our results suggest that JAK2 ( V617F ) allele evaluation can be a useful ancillary test for discriminating MDS from MPN in specimens with bone marrow fibrosis.

Primary mediastinal large B-cell lymphoma (PMBCL) has a characteristic clinical presentation, morphology, and immunophenotype, representing a clinically favorable subgroup of diffuse large B-cell lymphoma (DLBCL). By gene expression profiling (GEP), PMBCL shares features with classical Hodgkin lymphoma (cHL). Of further interest, BCL6 gene mutations and BCL6 and/or MUM1 expression in a number of PMBCLs have supported an activated B-cell (ABC) origin. Several studies, including GEP, have failed to detect BCL2 gene rearrangements (GRs) in PMBCL. An index case of t(14; 18)+ PMBCL prompted our study of the incidence of BCL2 GRs in PMBCL by polymerase chain reaction (PCR)/fluorescence in situ hybridization (FISH) analyses and its possible clinical impact. Twenty-five retrospectively identified, well-defined PMBCLs (five with cytogenetics) from three institutions were analyzed for a BCL2 GR by PCR/FISH analyses. The formalin-fixed, paraffin-embedded tissue blocks of 24 available cases were also analyzed by BCL2 immunohistochemistry (IHC). Of the five with cytogenetics, two had a t(14; 18) (q32; q21). Of the 25 analyzed by PCR, 2 had no amplifiable DNA (aDNA), including 1 t(14; 18)+ case. Of those with aDNA, two showed a BCL2 GR; by FISH analysis, three demonstrated a BCL2 GR. BCL2 protein expression by IHC analysis was variably detected in 21 out of 24 (strongly, uniformly expressed: 6, including all with a t(14; 18) or a BCL2 gene rearrangement; moderately weakly expressed in a subset of the malignant cells: 15). Available clinical follow-up of this BCL2+ subset showed a similar course to the other PMBCL cases. Our results imply that a subset of PMBCL [(4 out of 24 analyzed) in our series] may be of GC origin. A larger study is necessary to determine any clinical significance.

A 21-year-old pregnant woman presented with a rapidly growing >2 cm nodule on her right leg, involving dermis and subcutaneous tissue. Histologically, the tumor was composed of sheets and nests of neoplastic cells with variable cytomorphology, including typical round to ovoid glomus cells with clear cytoplasm and well-defined cell borders, small cells and spindle cells. Numerous medium to large vessels were present throughout the tumor. Moderate- to high cellularity, nuclear atypia and frequent mitotic figures (42 MF/50 High power field (HPF)) were noted. Immunohistochemistry showed cytoplasmic and membranous expression of actin (HHF-35) and membranous expression of type IV collagen. The histologic features and immunoprofile were consistent with the diagnosis of malignant glomus tumor, a rare soft tissue neoplasm that typically arises on the extremities. Histologic features that infer malignancy in glomus tumors include the combination of large size (>2 cm) and deep location, or atypical mitotic figures, or moderate to severe cytologic atypia with high mitotic activity (>5 mitoses /50 HPF). Although our case was superficially located, the nuclear atypia and mitotic rate, as well as the large size, fulfilled the criteria for a malignant glomus tumor.

Background: The histologic diagnosis of myeloid leukemia cutis (LC) can be difficult, requiring confirmatory immunohistochemical stains., Objective: We reviewed 21 biopsy-proven cases of LC with emphasis on the use of immunohistochemistry in the diagnosis., Materials and Methods: Clinical and histologic features were reviewed on 21 cases of biopsy proven LC. Immunohistochemical stains for CD4, CD34, CD56, CD68, CD117, CD123, TdT, lysozyme and myeloperoxidase were performed on 12 with available tissue blocks., Results: Ages ranged from 24 to 88 years (mean = 57), with 12 men: 9 women. Primary hematologic diagnoses included acute myeloid leukemia (n = 14), myelodysplastic syndrome (n = 3), essential thrombocythemia (n = 1) and myeloid leukemia, NOS (n = 3). Monocytic myeloid LC was most common (35%). There was 100% positivity with CD68 and lysozyme. Myeloperoxidase, CD117 and CD34 immunostains were less sensitive in myeloid LC (58%, 33% and 17%, respectively). CD4 was positive in 67%. CD56 was positive in 33%., Conclusion: Myeloid leukemia with monocytic differentiation more commonly involves the skin than other types of myeloid leukemia. CD68 and lysozyme immunostains, although not lineage specific for monocytes/macrophages, are the most sensitive immunostains in the detection of myeloid LC. Myeloperoxidase immunostains are useful, but immunostains for CD117 and CD34 are insufficiently sensitive. CD4 expression is common, but CD56 expression is not.

This study evaluates changes in genetic loci of chronic myeloid disorders using loss of heterozygosity (LOH) techniques. We present the combined results of three experiments. First, examination of a panel of genetic loci in groups of myeloproliferative disorders was evaluated. The second experiment involved microdissection of megakaryocytes from myeloproliferative disorders and comparison of their genetic changes to surrounding neoplastic marrow elements. Finally, we compared results of LOH studies of myeloproliferative disorders to those of myelodysplastic syndromes and chronic myelomonocytic leukemia. A total of 41 bone marrow biopsies were evaluated. Twenty-seven were myeloproliferative disorders (11 chronic idiopathic myelofibrosis, 11 essential thrombocythemia, 5 polycythemia vera). The remaining cases consisted of myelodysplastic syndromes (total=5; RAEB-1=2; RAEB-2=2; MDS, not otherwise specified=1) and chronic myelomonocytic leukemia (n=8). The abnormalities in myeloproliferative disorders were distributed as follows: D7S2554-4/14 (5/14); D8S263-4/15 (5/15); D9S157-5/15 (5/15); D9S161-7/17 (6/17); D13S319-5/14 (4/14); TP53-5/16 (5/16); D20S108-4/15 (4/15). In 75% cases diagnosed as essential thrombocythemia (6/8), both cases of polycythemia vera (2/2), and 29% cases of chronic idiopathic myelofibrosis (2/7), there were genetic differences between the megakaryocytes and the surrounding marrow. These results suggest that in some cases, megakaryocytes have different clonal abnormalities than surrounding hematopoietic tissue. The genetic profiles of myeloproliferative disorders had several differences from those of myelodysplastic syndromes. Although different from both, chronic myelomonocytic leukemia appeared more similar to myeloproliferative disorders using these techniques.

Mycosis fungoides (MF) exhibits a variety of underlying molecular defects. Loss of heterozygosity (LOH) is a technique used to detect chromosomal imbalances in neoplastic disorders using archival tissue. We analyzed skin biopsies of MF in different stages for the presence of LOH at specific loci to evaluate underlying genetic aberrations involved in MF and its progression. Twenty-five skin biopsies (15 plaque stage and 10 tumor stage) from 19 patients were evaluated. LOH was examined at 1p22 (D1S2766), 9p21 [IFNA, p15 (D9S1748), p16 (D9S171)], 10q23 [PTEN (D10S185, D10S541, D10S2491)], and 17p13 [p53 (TP53)]. Abnormal lymphocytes were microdissected from formalin-fixed, paraffin-embedded tissue sections. Sixteen of the 25 (64%) specimens evaluated had at least one abnormal LOH locus and LOH was identified in 7 of 15 (47%) plaque and in 9 of 10 (90%) tumor stage lesions, respectively. All 3 patients with sequential biopsies (plaque followed by tumor lesions) had additional LOH abnormalities in tumor specimens compared with plaque stage lesions. LOH most frequently involved chromosome 10, including 7 of 10 (70%) tumor stage lesions. Loss of multiple alleles was only identified in tumor stage cases, with 3 tumors undergoing allelic losses at 3 separate loci. Our results suggest that LOH studies are a robust method for evaluating genetic abnormalities in MF. Tumor stage lesions manifest increasing allelic losses compared with plaque stage. Further, in this series, several loci associated with the tumor suppressor gene PTEN on chromosome 10 appear to be associated with progression from plaque to tumor stage.

Extramedullary hematopoiesis (EMH) in the spleen is a characteristic feature of the chronic myeloproliferative disorders (CMPDs) and various other neoplastic or reactive myeloid conditions. However, the origin of these hematopoietic precursor cells and the molecular mechanisms underlying their development in the spleen is uncertain. The V617F mutation in the Janus Kinase 2 gene (JAK2(V617F)) was recently shown to be frequently and preferentially present in the peripheral blood and bone marrow cells of CMPD patients, and the resulting dysregulation of its downstream targets is important to CMPD pathogenesis. To determine the occurrence and potential role of JAK2(V617F) in splenic EMH cells, we studied splenectomy specimens from 47 patients with significant EMH. JAK2(V617F) was detected by real-time PCR melting curve analysis in 22 specimens, including 11/17 chronic idiopathic myelofibrosis, 7/7 polycythemia vera, 1/1 essential thrombocythemia, 1/3 CMPD unclassifiable, 1/5 chronic myelomonocytic leukemia, 0/5 chronic myelogenous leukemia, 1/3 myelodysplastic syndrome and 0/6 acute myeloblastic leukemia cases, whereas only the JAK2 wild-type allele was detected in the other 25. Nineteen of 20 cases with adequate bone marrow samples available for molecular examination demonstrated concordant JAK2 genotypes. Laser-capture microdissection was then used to enrich the EMH and non-EMH splenic cell fractions, confirming that the mutant alleles specifically originated from the EMH cells. Furthermore, megakaryocytes in the JAK2(V617F)-positive splenectomy specimens expressed higher levels of Bcl-xL, an antiapoptotic protein and downstream target of the JAK2/STAT5 pathway. Thus, JAK2(V617F) is frequently present in splenic EMH cells associated with CMPD, but it is rarely identified in splenic EMH cells associated with other myeloid disorders. Our results indicate that the precursor cells leading to extramedullary hematopoietic expansion in CMPD most likely originate from the transformed bone marrow clone. Also, dysregulation of downstream pathways such as Bcl-xL may be important to CMPD disease pathogenesis in the spleen.

Background: Accurate bone marrow (BM) blast counts (BCs) are essential for diagnosis (dx) of myelodysplasia (MDS), MDS/myeloproliferative (MDS/MPD) disease, or acute myeloid leukemia (AML), and may be difficult in hemodiluted bone marrow aspirates (BMAs). Erythroid precursors (EPs) may be indistinguishable from myeloblasts in BM sections (aspirate clots/cores). We compare the usefulness of immunohistochemistry (IHC) [ie, CD34, CD117, myeloperoxidase (MPO), Hemoglobin A1 (HbA1), and terminal deoxynucleotidyl transferase (TdT)] of BM sections (IHC-BM) with BMA, bone marrow touch preparation (BMTP), and flow cytometry (FC) BCs., Design: The initial BC (48), percentage (%) of Eps (38) (both based on initial 100 to 600-cell counts), and FC expressions of CD34, CD117, Glycophorin A(GLY A), and TdT (44) were tabulated from 50 BMs (MDS, MDS/MPD, or AML). BMAs (48) and BMTPs (25) subsequently received 500-cell counts. IHC-BM was performed (45:formalin, 5:B5-fixed) [CD34 (46), CD117 (45), HbA1 (45), TdT (42), and MPO (45)]., Results: Retrospective BMA BCs revealed a 31% (15/48) discrepant rate between the original/retrospective BMA BCs; 80% revealed an underestimated initial BC. There was a 28% discordance rate between the retrospective BMA and BMTP reviews; 77% showed a higher BMTP BC. IHC showed significantly higher BCs in 19% (9/47), resulting in a different dx (5). However, CD34 and CD117 IHCS revealed lower BCs in 38% and 48%, respectively. The CD34 IHC results were primarily due to CD34-negative blasts by FC. The CD117 IHC results were largely unexplained. EPs were CD34 and CD117-negative., Conclusions: (1) Evaluation for MDS/AML requires 500-cell counts of BMAs and/or BMTPs. (2) CD34 and/or CD117 blasts by FC indicate IHC-BM may increase BC accuracy. (3) CD34 is more reliable than CD117 by IHC; however, in combination, they are most reliable and should be performed on BM clots/cores due to variable reactivity.

Session 9 of the 2005 Society for Hematopathology/European Association for Haematopathology Workshop focused on large granular lymphocyte (LGL) leukemias and related disorders. T-cell LGL (T-LGL) leukemias, discussed herein, account for 2% to 3% of cases of small lymphocytic leukemia. T-LGL diseases cover a heterogeneous spectrum of disorders that include reactive conditions, typically associated with autoimmune disease, to outright leukemia. These disorders are found in older people, with an average age at initial examination of approximately 60 years and a median survival of more than 10 years in T-LGL leukemia. Systemic symptoms and neutropenia are common at initial examination. Lymphocytosis, composed of small mature lymphocytes with increased cytoplasm, is common. The spleen and bone marrow are involved in T-LGL leukemia, although morphologic findings may be subtle. The immunophenotype is typically that of CD3+/CD8+ cytotoxic T cells. Some cases may be due to chronic immune stimulation, with subsequent clonal escape and proliferation of a neoplastic population of lymphocytes.

The dimeric pyrrole imidazole natural products are a growing class of alkaloids with exotic connectivity, unique topologies, high nitrogen content, and exciting bioactivities. This full account traces the evolution of a strategy that culminated in the first total syntheses of several members of this family, including sceptrin, ageliferin, nagelamide E, nakamuric acid (and its methyl ester), and oxysceptrin. Details on the fascinating conversion of sceptrin to ageliferin, which has been used to produce gram quantities of this sensitive natural product, are provided. In addition, the first enantioselective total synthesis of sceptrin and ageliferin are reported by programming the fragmentation of an oxaquadricyclane. A hallmark of our approach to this family of alkaloids is the minimal use of protecting groups despite the presence of 10 nitrogen atoms in the target compounds. Thus, the fundamental chemistry of the 2-aminoimidazole heterocycle was explored without masking its innate reactivity. Insights gained during these explorations led to total syntheses of oxysceptrin and nakamuric acid and a successful construction of the carbon skeleton of axinellamine.

Extramedullary proliferations of bone marrow elements are infrequently encountered in routine pathology practice. On occasion, they can present diagnostic difficulties when seen in unusual or unanticipated sites. This review serves to cover aspects of underlying embryogenesis of myeloid elements, as well as sites and circumstance of benign proliferations of myeloid elements along with their occasional confusion with neoplastic myeloid proliferations. Benign proliferations associated with hematologic disorders and hematopoietic growth factors are discussed. Immunohistochemical evaluation of myeloid proliferations is considered as well.

Background: The clinical course of Epstein-Barr virus (EBV)-associated smooth muscle tumors is variable and there are no reports in patients with mixed T-cell chimerism after bone marrow transplantation (BMT)., Observations: A child with X-linked severe combined immunodeficiency disease developed multiple renal and pulmonary leiomyomata 8 years after haploidentical BMT. Epstein-Barr viral DNA was detectable in the blood and in situ hybridization for EBV-encoded RNAs was positive in the tumor. The tumors have been radiographically stable, chimerism remains mixed, and plasma EBV DNA has been repeatedly negative for over 2 years after donor lymphocyte infusion., Conclusions: EBV-associated smooth muscle tumors may occur in patients who are partially reconstituted after BMT for severe combined immunodeficiency and may not require surgery or chemotherapy.

We report a case of a 75-year-old man with a cutaneous CD4+CD56+ hematodermic neoplasm. CD4+CD56+ hematodermic neoplasms are rare and commonly present as cutaneous lesions. This is an important diagnosis in the differential diagnosis of cutaneous hematologic malignancies because of the extremely poor prognosis.

The immunohistochemical evaluation of hematopoietic tissues has expanded our knowledge of both the function and pathological processes in a wide range of lymph nodal, extranodal tissues, and bone marrow. It is impossible to cover in detail the full range of immunohistochemical markers, which are available to study hematopoietic cells and their malignant counterparts. This chapter attempts to provide an overview of the antibodies, which are commonly used in studying the hematopoietic and lymphoid tissues and to diagnose hematological disorders. In addition, an immunohistology-based diagnostic approach to identify specific neoplastic entities is described in some length.

The World Health Organization criteria for diagnosing chronic myelomonocytic leukemia (CMML) are largely based on findings observed in the peripheral blood and bone marrow aspirate. A specific diagnostic role for the bone marrow biopsy has not been adequately explored. We examined whether bone marrow biopsy supplemented by immunohistochemistry may be helpful in distinguishing CMML from cases of chronic myelogenous leukemia and atypical chronic myeloid leukemia (aCML). We immunostained 25 cases of CMML with paraffin reactive antibodies which included CD68 (KP1), CD68R (PG-M1), and CD163, and compared the results with those observed in six cases of chronic myelogenous leukemia and in three cases of atypical CML. In addition, we examined whether CD34 immunohistochemistry could be useful in separating cases of CMML with less than 10% blasts (type-1) from cases of CMML with blasts accounting for 10-19% (type-2), and cases of CMML in acute transformation to acute myeloid leukemia (blasts > or = 20%). The presence of nodules of plasmacytoid monocytes was investigated by CD123 staining. CD42b was used to highlight abnormal megakaryocytes. Our results demonstrate significant differences between the groups. CD34 analysis allowed separating CMML type-1 from type-2 and the former from CMML in acute transformation. CD123-positive plasmacytoid monocyte nodules were found only in CMML and not in the other two disease groups. Overlap between CMML and the other two groups were observed with CD68 immunostaining. CD68R was more restricted to bone marrow macrophages and monocytes than CD68, but the differences between CMML and chronic myelogenous leukemia or atypical CML were still not significant. Although CD42b immunostaining facilitated the detection of dwarf megakaryocytes often present in CMML, the distinction between those and the small forms seen in chronic myelogenous leukemia was still problematic.

Myelolipomas are defined as mature fat associated with hematopoietic elements, often found in the adrenal gland. The question of whether the hematopoietic cells are truly "normal" has not been evaluated extensively. In this study, we evaluated histologic, immunohistochemical features and comparisons of X-chromosome inactivation patterns in 19 myelolipomas. Formalin-fixed, paraffin-embedded tissue from 19 myelolipomas was stained with hematoxylin and eosin and immunostained with monoclonal antibodies against CD138, CD34, CD117, CD42a, hemoglobin, myeloperoxidase, collagen IV, and nerve growth factor receptor. Histologic evaluation included estimates of overall cellularity of hematopoietic tissue, estimates of cellularity in the areas of highest concentration of hematopoietic tissue, myeloid to erythroid ratio, and numbers of megakaryocytes. X-chromosome inactivation analysis was performed on myelolipomas from 11 female patients by polymerase chain reaction. Myelolipomas showed wide variation in cellularity within the lesion (5% to 90%) with no correlation with the patient's age. All the myelolipomas demonstrated normal trilineage hematopoiesis and cellular morphology, with few early myeloid precursors, as evidenced by negativity for CD117 and only rare positivity for CD34 antibodies. Most of the myelolipomas (14/18) had markedly increased megakaryocytes compared with normal marrows. The majority of myelolipomas also had a stromal composition and vascular patterns that were different from those of normal bone marrow. X-chromosome inactivation studies demonstrated nonrandom X-chromosome inactivation in 8/11 myelolipomas from female patients. Myelolipomas are morphologically different from the normal bone marrow. The majority of myelolipomas also have nonrandom X-chromosome inactivation, suggesting a clonal origin for these tumors.

Aims: To evaluate a group of lymphoid proliferations using only Ki67-stained slides to determine the value of the pattern of proliferating cells in diagnosis. Ki67 immunohistochemistry allows evaluation of the distribution of proliferating cells in addition to simply determining the proliferation rate of cells., Methods and Results: Three observers, using a Ki67-stained slide only, studied 149 cases from five diagnostic groupings: follicular hyperplasia, mixed pattern hyperplasia, localized Castleman's disease, follicular lymphoma and marginal zone lymphoma. The sensitivity for benign lesions varied from 94% to 97% among the three observers. Follicular lymphomas were recognized as neoplastic with a sensitivity of 96% and 100% by two of the observers. Marginal zone lymphoma was recognized as neoplastic in 67-73% of cases., Conclusions: The Ki67 stain alone is a powerful tool for distinguishing benign from malignant proliferations within the selected groups. Nuances and pitfalls in the interpretation of Ki67 staining pattern are discussed.

Neoplastic myeloid proliferations are seen in the spleens of some patients with acute and chronic myeloproliferative disorders. Both acute myeloid leukemia (AML) and chronic myeloproliferative disorders have a variety of underlying cytogenetic defects that can be evaluated by loss of heterozygosity (LOH) studies. LOH studies have advantages over conventional cytogenetics by allowing the use of archival tissues. We evaluated the spleens in AML and chronic myeloproliferative disorders with neoplastic myeloid proliferations for the presence of LOH at several chromosome loci, and X-chromosome inactivation. A total of 17 spleens were evaluated (chronic myelogenous leukemia = 6; chronic idiopathic myelofibrosis = 6; essential thrombocythemia = 1; AML arising from previous chronic myeloproliferative disorders = 4). We examined LOH loci 7q (D7S2554), 8q (D8S263), 9p (D9S157, D9S161), 13q (D13S319), common sites of genetic abnormality in chronic myeloproliferative disorders, and TP53. In six cases, spleen LOH findings were compared to those of concurrent or preceding bone marrow biopsies. Five spleens of female patients were evaluated for the presence of clonality using X-chromosome inactivation. Of the 16 cases analyzed, 14 (88%) had at least one abnormal LOH locus, with 6/16 with two abnormal loci. The abnormalities were distributed as follows: D9S161-7/15 (47%), TP53-6/16 (38%), D7S2554-5/16 (31%), D9S157-5/15 (33%), D8S263-3/14 (21%), and D13S319-2/14 (14%). Of the six bone marrows, 4/6 showed concordance in bone marrow and spleen specimens, with additional LOH abnormalities being identified in the spleen specimens of all four cases. X-chromosome inactivation studies were showed nonrandom (clonal) patterns in two cases. Our results show that allelic losses were common in the neoplastic extramedullary hematopoiesis found in spleens of chronic myeloproliferative disorders and AML. Comparison of spleen and bone marrow specimens by LOH demonstrated additional abnormalities in the spleen compared to the marrow.

Spleen is a common site of extramedullary hematopoiesis. Extramedullary hematopoiesis seen in non-neoplastic conditions can occasionally be extensive and raise concerns for a myeloid neoplasm. We compared the morphologic and immunohistochemical features of splenic hematopoietic proliferations seen in neoplastic myeloid disorders (eg chronic myeloproliferative disorders, myelodysplastic/myeloproliferative disorders and acute myeloid leukemias) to extramedullary hematopoiesis seen in a variety of reactive conditions. In all, 80 spleen specimens were reviewed. The presence of each marrow-derived lineage, dysplasia and immunohistochemical results were evaluated (CD34, CD117, myeloperoxidase, CD68, p53, TdT, CD42b and hemoglobin). Neoplastic hematopoietic proliferations in chronic myeloproliferative disorders are characterized by trilineage hematopoiesis with significant dysplasia in all cell lineages. Acute myeloid leukemia showed an increase in immature forms, which were highlighted by immunohistochemistry. Reactive extramedullary hematopoiesis showed variability in histologic features. Post-bone marrow transplant and thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome spleens showed extramedullary hematopoiesis with some morphologic features of immaturity, which could simulate chronic myeloproliferative disorder. However, they lacked characteristic immunohistochemical features of neoplastic myeloid disorders such as positivity for CD34 or CD117.

The WHO criteria for diagnosing acute panmyelosis with myelofibrosis are somewhat distinct from those for acute megakaryoblastic leukemia. However, clinical and hematopathologic findings partially overlap. This has raised questions as to whether these are indeed separate, definable entities. To determine the potential importance of bone marrow biopsy supplemented by immunohistochemistry in distinguishing between these two conditions, we studied 17 bone marrow biopsies of well-characterized cases of acute panmyelosis with myelofibrosis (six cases) and acute megakaryoblastic leukemia (11 cases). We compared blast frequency, reticulin content, CD34 expression, and the degree of megakaryocytic differentiation of the blast cells in these two conditions. Our results demonstrate important differences. Acute panmyelosis with myelofibrosis is characterized by a multilineage myeloid proliferation with a less numerous population of blasts than acute megakaryoblastic leukemia (P<0.01). In the former condition, blasts are always positive with CD34, while in acute megakaryoblastic leukemia they express CD34 in 60% of the cases. The blasts in acute panmyelosis with myelofibrosis only rarely express megakaryocytic antigens. By contrast, acute megakaryoblastic leukemia has a significantly higher proportion of blasts expressing megakaryocytic antigens (P<0.01 with CD42b). Our results confirm that histology supplemented by immunohistochemistry permits the distinction of these conditions in routinely processed bone marrow biopsies.

Gifted with novel chemical features and extraordinary biological activity, sceptrin has remained a prominent unanswered synthetic challenge since its characterization in 1981 by Faulkner and Clardy. A concise and practical solution to the myriad of chemical challenges posed by sceptrin is reported in this Communication. Thus, through a sequence involving rearrangement of an oxaquadricyclane, a new method for chemo- and regioselective halogenation, a mild sequence for 2-aminoimidazole formation, and careful synthetic choreography, (+/-)-sceptrin is obtained in a minimum of steps and in 24% overall yield from dimethyl acetylenedicarboxylate without a single use of chromatography.

The chromosome abnormalities observed in a dedifferentiated chondrosarcoma are reported. A new molecular cytogenetic technique, spectral karyotyping, was used to identify and confirm structural rearrangements in this case. A review of the literature revealed that nine cases have been reported, in eight of which a complete description of the cytogenetic abnormalities was described. Structural aberrations were most frequently reported in chromosomes 1 and 9, and chromosomes 7 and 19 were most frequently observed to be involved in numerical aberrations (trisomy and tetrasomy). In chondrosarcomas, structural aberrations in chromosomes 1 and 9 and trisomy or tetrasomy of chromosome 7 are among the more frequently observed aberrations.

A detailed ultrastructural description of the cellular variant of congenital mesoblastic nephroma (CMN) is presented and compared to the classical form. Studied were 9 cases of the cellular variant, 6 mixed (cellular/classical) tumors, and 1 classical CMN. The occurrence of a broad selection of ultrastructural features was assessed using a semiquantitative scoring system. The results indicate that cellular CMNs are composed mainly of primitive mesenchymal cells, but also usually contain varying numbers of differentiating fibroblasts and myofibroblasts. This entity thus bears a closer resemblance at the ultrastructural level of organization to infantile fibrosarcoma than to conventional fibrosarcoma. Electron microscopy can be useful in distinguishing this relatively benign entity from the several malignancies with which it is sometimes confused.

We have defined the chromosomal location of the human MAGE (melanoma antigen) gene family by polymerase chain reaction amplification of several segments of the MAGE genes from somatic cell hybrids containing well-defined groups of human chromosomes and hybrids containing human derivative chromosomes. The data show that the three known MAGE family members (MAGEs -1, -2, & -3) are syntenic and map to the human X chromosome region q27-qter. Because males and females are hemizygous for most X-linked genes, the frequency of antigen-loss variants for the MAGE system is expected to be greater in comparison to antigens encoded by somatic chromosomes. In this regard, we believe that patients enrolled in MAGE-specific immunotherapy trials should be carefully monitored for the presence of MAGE antigen-loss variants.

A case of pre-B cell acute lymphoblastic leukemia (pre-B ALL) with a dir ins(11;4)(q23;q21q31) chromosome rearrangement is presented. The patient's clinical findings and history were similar to those described for the t(4;11)(q21;q23) subgroup of childhood ALL. These findings suggest that the interfacing of the distal breakpoint at band 4q21 to the proximal breakpoint of band 11q23 represents the primary cytogenetic change observed in this subgroup of ALL.

Two cases of childhood neuroblastoma are presented. Case 1 was diagnosed as Stage IV with metastasis to the bone marrow. During remission, histologic studies of bone marrow aspirate and biopsy showed a normocellular marrow with no evidence of malignant cells. Concurrent cytogenetic studies of the bone marrow showed the majority of the cells to contain double minute chromosomes (DM). The chromosome findings indicated the presence of neuroblastoma cells in the marrow prior to histologic evidence of relapse. Case 2 was diagnosed as Stage I neuroblastoma with no metastasis to the bone marrow. Subsequent cytogenetic studies showed DM present in a small number of cells and a deletion of chromosome 1 (1p-) in a single cell. The chromosome findings indicated an advanced stage of malignancy which was not evident with histologic techniques. These findings suggest that cytogenetic analysis of bone marrow can be a valuable aid to the early diagnosis, prognosis, and treatment of neuroblastoma.