Shared clonal cytogenetic abnormalities in aberrant mast cells and leukemic myeloid blasts detected by single nucleotide polymorphism microarray-based whole-genome scanning.

Systemic mastocytosis (SM) is characterized by a clonal proliferation of aberrant mast cells within extracutaneous sites. In a subset of SM cases, a second associated hematologic non-mast cell disease (AHNMD) is also present, usually of myeloid origin. Polymerase chain reaction and targeted fluorescence in situ hybridization studies have provided evidence that, in at least some cases, the aberrant mast cells are related clonally to the neoplastic cells of the AHNMD. In this work, a single nucleotide polymorphism microarray (SNP-A) was used to characterize the cytogenetics of the aberrant mast cells from a patient with acute myeloid leukemia and concomitant mast cell leukemia associated with a KIT D816A mutation. The results demonstrate the presence of shared cytogenetic abnormalities between the mast cells and myeloid blasts, as well as additional abnormalities within mast cells (copy-neutral loss of heterozygosity) not detectable by routine karyotypic analysis. To our knowledge, this work represents the first application of SNP-A whole-genome scanning to the detection of shared cytogenetic abnormalities between the two components of a case of SM-AHNMD. The findings provide additional evidence of a frequent clonal link between aberrant mast cells and cells of myeloid AHNMDs, and also highlight the importance of direct sequencing for identifying uncommon activating KIT mutations.

A retrospective comparative outcome analysis following systemic therapy in Mycosis fungoides and Sezary syndrome.

Cutaneous T-cell lymphomas (CTCL), with few exceptions, remain incurable and treatment is largely palliative. We performed a retrospective analysis of systemic treatment outcomes of patients diagnosed with MF/SS. We identified 223 patients with MF/SS evaluated at a single institution from 1997 to 2013. Disease stage at diagnosis, time of treatment, and treatments received were retrospectively analyzed using our CTCL database. The primary endpoint was time to next treatment (TTNT). Treatment outcomes were analyzed using Kaplan-Meier method and comparisons among groups were made using log-rank analysis. A superior TTNT was associated with retinoid or interferon therapies when compared with HDAC inhibitors or systemic chemotherapy. Retinoids and interferon were associated with superior TTNT in both limited-stage and advanced stage disease. Extracorporeal photophoresis (ECP) had a superior TTNT in Sezary Syndrome. HDAC inhibitors and chemotherapy were associated with inferior TTNT in both limited stage disease and advanced stage disease. With the exception of interferon, retinoids, or ECP, durable responses are rarely achieved with systemic therapies in MF/SS patients, particularly those with advanced-stage disease. Therefore, clinical trial participation with novel agents should be encouraged. Am. J. Hematol. 91:E491-E495, 2016. © 2016 Wiley Periodicals, Inc.

Fourier Transform Mass Spectrometry and Nuclear Magnetic Resonance Analysis for the Rapid and Accurate Characterization of Hexacosanoylceramide.

Ceramides are a central unit of all sphingolipids which have been identified as sites of biological recognition on cellular membranes mediating cell growth and differentiation. Several glycosphingolipids have been isolated, displaying immunomodulatory and anti-tumor activities. These molecules have generated considerable interest as potential vaccine adjuvants in humans. Accurate analyses of these and related sphingosine analogues are important for the characterization of structure, biological function, and metabolism. We report the complementary use of direct laser desorption ionization (DLDI), sheath flow electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) and high-field nuclear magnetic resonance (NMR) analysis for the rapid, accurate identification of hexacosanoylceramide and starting materials. DLDI does not require stringent sample preparation and yields representative ions. Sheath-flow ESI yields ions of the product and byproducts and was significantly better than monospray ESI due to improved compound solubility. Negative ion sheath flow ESI provided data of starting materials and products all in one acquisition as hexacosanoic acid does not ionize efficiently when ceramides are present. NMR provided characterization of these lipid molecules complementing the results obtained from MS analyses. NMR data was able to differentiate straight chain versus branched chain alkyl groups not easily obtained from mass spectrometry.

Mycosis fungoides with CD20 expression: report of two cases and review of the literature.

CD20 expression is exceedingly rare in T-cell lymphomas. Most published cases have been diagnosed as peripheral T-cell lymphomas, not otherwise specified. Only 18 cases of CD20-positive mycosis fungoides (MF) have been previously reported. Here, we describe two cases of CD20-positive MF. Patient 1 was an 84-year-old woman who presented with a 5-year history of multiple pruritic erythematous papules coalescing into thin plaques over 80% of her body surface area. She expired after developing tumors and large cell transformation. Patient 2 was a 67-year-old woman with a long-standing history of tumor stage MF with large cell transformation. She developed a nodular plaque while receiving topical and systemic therapy. In both cases, the neoplastic T-cells demonstrated a CD4-positive immunophenotype with loss of pan-T-cell markers and a monoclonal T-cell receptor gamma gene rearrangement. CD20 was expressed by a significant population of the neoplastic T-cells, but these T-cells lacked expression of other B-cell markers, including CD79a, CD19 and PAX5. This report adds to and summarizes the small body of literature describing CD20-positive MF, and discusses diagnostic and clinical implications.

Myelodysplasia Cutis.

CONTEXT.­: Myelodysplasia cutis is an emerging concept in cutaneous neoplasia. Many of these cases were previously included under the umbrella of histiocytoid Sweet syndrome. However, with the advent of next-generation sequencing, cutaneous involvement by myelodysplastic syndrome is being increasingly recognized. OBJECTIVE.­: To review histiocytoid Sweet syndrome and myelodysplasia cutis and discuss our current understanding of these entities. Additionally, to discuss how next-generation sequencing can be applied in the evaluation of cutaneous infiltrates of immature histiocytoid cells. DATA SOURCES.­: The English-language literature from 2005 to 2023 on the topic of histiocytoid Sweet syndrome and myelodysplasia cutis was reviewed. CONCLUSIONS.­: Biopsy specimens showing infiltrates of histiocytoid, immature myeloid cells may represent cutaneous involvement by myelodysplastic syndrome. Close clinical correlation is recommended in these cases. Recent studies suggest that next-generation sequencing is useful in separating myelodysplasia cutis from true histiocytoid Sweet syndrome. This distinction has important implications for patients.

Pediatric extranodal marginal zone B-cell lymphoma presenting as amyloidosis in minor salivary glands: a case report and review of the literature.

We report an unusual case of an extranodal marginal zone B-cell lymphoma (EMZL) arising in the labial minor salivary gland in an immunocompetent 11-year-old boy. The initial histopathologic review favored localized amyloidosis. However, further evaluation supported the diagnosis of low-grade B-cell lymphoma with plasmacytic differentiation, surrounded by deposits of AL κ-type amyloid. Clinical management consisted of excision with no recurrence at 1-year follow-up. This case demonstrates that a diagnosis of lymphoma must be considered in cases of amyloidosis associated with minor salivary gland involvement, even in children. In addition, we provide a literature review of extranodal marginal zone B-cell lymphoma arising in salivary glands.

Perifosine plus lenalidomide and dexamethasone in relapsed and relapsed/refractory multiple myeloma: a Phase I Multiple Myeloma Research Consortium study.

The combination of lenalidomide-dexamethasone is active in multiple myeloma (MM). Preclinical data showed that the Akt inhibitor, perifosine, sensitized MM cells to lenalidomide and dexamethasone, providing the rationale for this Phase I, multicentre, single-arm study to assess the safety and determine the maximum-tolerated dose (MTD) of perifosine-lenalidomide-dexamethasone in relapsed and relapsed/refractory MM. Patients received escalating doses of perifosine 50-100 mg daily and lenalidomide 15-25 mg once daily on days 1-21 of each 28-d cycle, plus dexamethasone 20-40 mg weekly thereafter, as indicated. Thirty-two patients were enrolled across four dose cohorts. MTD was not reached, with 31 patients evaluable for safety/tolerability. The most common all-causality grade 1-2 adverse events were fatigue (48%) and diarrhoea (45%), and grade 3-4 neutropenia (26%), hypophosphataemia (23%), thrombocytopenia (16%), and leucopenia (13%). Among 30 evaluable patients, 73% (95% confidence interval, 57·5-89·2%) achieved a minimal response or better, including 50% with a partial response or better. Median progression-free survival was 10·8 months and median overall survival 30·6 months. Response was associated with phospho-Akt in pharmacodynamic studies. Perifosine-lenalidomide-dexamethasone was well tolerated and demonstrated encouraging clinical activity in relapsed and relapsed/refractory MM.

Synthesis of 5-fluoro- and 5-hydroxymethanoprolines via lithiation of N-BOC-methanopyrrolidines. Constrained Cγ-exo and Cγ-endo Flp and Hyp conformer mimics.

Proline derivatives with a C(γ)-exo pucker typically display a high amide bond trans/cis (K(T/C)) ratio. This pucker enhances n→π* overlap of the amide oxygen and ester carbonyl carbon, which favors a trans amide bond. If there were no difference in n→π* interaction between the ring puckers, then the correlation between ring pucker and K(T/C) might be broken. To explore this possibility, proline conformations were constrained using a methylene bridge. We synthesized discrete gauche and anti 5-fluoro- and 5-hydroxy-N-acetylmethanoproline methyl esters from 3-syn and 3-anti fluoro- and hydroxymethanopyrrolidines using directed α-metalation to introduce the α-ester group. NBO calculations reveal minimal n→π* orbital interactions, so contributions from other forces might be of greater importance in determining K(T/C) for the methanoprolines. Consistent with this hypothesis, greater trans amide preferences were found in CDCl(3) for anti isomers en-MetFlp and en-MetHyp (72-78% trans) than for the syn stereoisomers ex-MetFlp and ex-MetHyp (54-67% trans). These, and other, K(T/C) results that we report here indicate how substituents on proline analogues can affect amide preferences by pathways other than ring puckering and n→π* overlap and suggest that caution should be exercised in assigning enhanced pyrrolidine C(γ)-exo ring puckering based solely on enhanced trans amide preference.

Synthesis of conformationally constrained 5-fluoro- and 5-hydroxymethanopyrrolidines. Ring-puckered mimics of gauche- and anti-3-fluoro- and 3-hydroxypyrrolidines.

N-acetylmethanopyrrolidine methyl ester and its four 5-syn/anti-fluoro and hydroxy derivatives have been synthesized from 2-azabicyclo[2.2.0]hex-5-ene, a 1,2-dihydropyridine photoproduct. These conformationally constrained mimics of idealized C(ß)-gauche and C(ß)-anti conformers of pyrrolidines were prepared in order to determine the inherent bridge bias and subsequent heteroatom substituent effects upon trans/cis amide preferences. The bridgehead position and also the presence of gauche(syn)/anti-5-fluoro or 5-hydroxy substituents have minimal influence upon the K(T/C) values of N-acetylamide conformers in both CDCl(3) (43-54% trans) and D(2)O (53-58% trans). O-Benzoylation enhances the trans amide preferences in CDCl(3) (65% for a syn-OBz, 61% for an anti-OBz) but has minimal effect in D(2)O. The synthetic methods developed for N-BOC-methanopyrrolidines should prove useful in the synthesis of more complex derivatives containing α-ester substituents. The K(T/C) results obtained in this study establish baseline amide preferences that will enable determination of contributions of α-ester substituents to trans-amide preferences in methanoprolines.

5(6)-anti-Substituted-2-azabicyclo[2.1.1]hexanes: a nucleophilic displacement route.

Nucleophilic displacements of 5(6)-anti-bromo substituents in 2-azabicyclo[2.1.1]hexanes (methanopyrrolidines) have been accomplished. These displacements have produced 5-anti-X-6-anti-Y-difunctionalized-2-azabicyclo[2.1.1]hexanes containing bromo, fluoro, acetoxy, hydroxy, azido, imidazole, thiophenyl, and iodo substituents. Such displacements of anti-bromide ions require an amine nitrogen and are a function of the solvent and the choice of metal salt. Reaction rates were faster and product yields were higher in DMSO when compared to DMF and with CsOAc compared to NaOAc. Sodium or lithium salts gave products, except with NaF, where silver fluoride in nitromethane was best for substitution by fluoride. The presence of electron-withdrawing F, OAc, N(3), Br, or SPh substituents in the 6-anti-position slows bromide displacements at the 5-anti-position.

131I-tositumomab therapy as initial treatment for follicular lymphoma.

BACKGROUND: Advanced-stage follicular B-cell lymphoma is considered incurable. Anti-CD20 radioimmunotherapy is effective in patients who have had a relapse after chemotherapy or who have refractory follicular lymphoma, but it has not been tested in previously untreated patients. METHODS: Seventy-six patients with stage III or IV follicular lymphoma received as initial therapy a single course of treatment with 131I-tositumomab therapy (registered as Tositumomab and Iodine I 131 Tositumomab [the Bexxar therapeutic regimen]). This consisted of a dosimetric dose of tositumomab and 131I-labeled tositumomab followed one week later by a therapeutic dose, delivering 75 cGy of radiation to the total body. RESULTS: Ninety-five percent of the patients had any response, and 75 percent had a complete response. The use of polymerase chain reaction (PCR) to detect rearrangement of the BCL2 gene showed molecular responses in 80 percent of assessable patients who had a clinical complete response. After a median follow-up of 5.1 years, the actuarial 5-year progression-free survival for all patients was 59 percent, with a median progression-free survival of 6.1 years. The annualized rate of relapse progressively decreased over time: 25 percent, 13 percent, and 12 percent during the first, second, and third years, respectively, and 4.4 percent per year after three years. Of 57 patients who had a complete response, 40 remained in remission for 4.3 to 7.7 years. Hematologic toxicity was moderate, with no patient requiring transfusions or hematopoietic growth factors. No cases of myelodysplastic syndrome have been observed. CONCLUSIONS: A single one-week course of 131I-tositumomab therapy as initial treatment can induce prolonged clinical and molecular remissions in patients with advanced follicular lymphoma.

Comprehensive analysis of copy number and allele status identifies multiple chromosome defects underlying follicular lymphoma pathogenesis.

PURPOSE: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. The clinical course is variable and only in part explained by known tumor-intrinsic or -extrinsic factors. FL carries the hallmark chromosomal translocation t(14;18), deregulating the expression of Bcl-2, but this is not sufficient to explain either FL biology or clinical behavior. EXPERIMENTAL DESIGN: We have employed high-density genomic profiling technology using the Affymetrix 50K-XbaI oligonucleotide single nucleotide polymorphism-chip platform to interrogate the genomes of 58 fluorescence-activated cell-sorted (FACS) FL specimens for chromosomal copy number changes and 46 specimens for loss of heterozygosity (LOH). RESULTS: We report (a) previously unknown high-frequency copy-neutral LOH (uniparental disomy) in FL on chromosomes 1p (approximately 50%) and 6p (approximately 30%); (b) that del6q is complex, as reported, with at least two regions of minimal common loss at 6q13-15 and 6q23-24, and that in addition, approximately 8% of FL specimens contain a homozygous deletion at 6q23.3-24.1 that spans the negative NFkappaB regulator A20 and the p53 apoptosis effector PERP; (c) that combined analysis of chromosome 17p for LOH, copy number, and p53 mutations shows that most p53 mutations in FL do not involve del17p. Finally, we map high-frequency LOH with and without copy loss on chromosomes 9p, 10q, and 16p and genomic gains on 2p15-16 and 8q24.22-24.3. CONCLUSIONS: This comprehensive description of the pathologic anatomy of the FL genome uncovers novel genetic lesions and should aid with identification of genes relevant to FL biology and clinical behavior.

Philadelphia Chromosome-like Mixed-Phenotype Acute Leukemia Demonstrating P2RY8-CRLF2 Fusion and JAK1 Mutation.

OBJECTIVES: Philadelphia chromosome-like (Ph-like) genetic alterations define a subset of B lymphoblastic leukemia/lymphoma (B-ALL), which represents a separate provisional entity in the World Health Organization 2016 updated classification. However, these alterations have not been described outside the context of B-ALL. METHODS: Cytogenomic array and molecular analysis identified a Ph-like signature in a mixed-phenotype acute leukemia (MPAL), B/myeloid, confirmed using conventional immunophenotypic and cytochemical analysis. RESULTS: Flow cytometry identified a blast population demonstrating a B-cell lineage and myeloperoxidase positivity. A P2RY8-CRLF2 fusion and JAK1 mutation were detected, both of which are associated with Ph-like features. CONCLUSIONS: To our knowledge, this is the first report of Ph-like MPAL, which may represent a new diagnostic entity. We emphasize the need for refinement of diagnostic criteria for MPALs and highlight an opportunity for expansion of inclusion criteria in ongoing clinical trials studying the use of tyrosine kinase inhibitor therapy to include cases of Ph-like MPAL.

ALK-positive anaplastic large cell lymphoma with primary bone involvement in children.

We describe the clinical, radiologic, and pathologic features of primary bone anaplastic large cell lymphoma (ALCL) in 3 boys. Radiologic imaging showed lytic lesions involving sacrum, femur, or rib. Bone was the only site of disease in 2 cases; an associated partial lymph node was involved in case 3. Differential diagnoses included osteomyelitis and small round cell tumors of childhood, particularly Ewing sarcoma. Preoperatively, ALCL was not a diagnostic consideration in any case. Two cases showed classic large pleomorphic cells; 1 showed a composite pattern with a distinct small cell component and the more typical large cell type. Neoplastic cells in all cases showed strong CD30 and anaplastic lymphoma kinase expression with relatively weak epithelial membrane antigen positivity. Cytotoxic granule protein was expressed in 2 cases. All cases showed unusually strong expression of neuron-specific enolase (NSE). Two patients were disease-free at last follow-up (15 months and 11 years); 1 patient died of disseminated disease within a year of diagnosis. ALCL should be considered a diagnostic possibility when evaluating neoplastic bone lesions in children. Although expression of NSE in ALCL has not been emphasized in the literature, it is worth noting because it may pose a diagnostic pitfall.

A novel compact mass detection platform for the open access (OA) environment in drug discovery and early development.

A new 'compact mass detector' co-developed with an instrument manufacturer (Waters Corporation) as an interface for liquid chromatography (LC), specifically Ultra-high performance LC(®) (UPLC(®) or UHPLC) analysis was evaluated as a potential new Open Access (OA) LC-MS platform in the Drug Discovery and Early Development space. This new compact mass detector based platform was envisioned to provide increased reliability and speed while exhibiting significant cost, noise, and footprint reductions. The new detector was evaluated in batch mode (typically 1-3 samples per run) to monitor reactions and check purity, as well as in High Throughput Screening (HTS) mode to run 24, 48, and 96 well plates. The latter workflows focused on screening catalysis conditions, process optimization, and library work. The objective of this investigation was to assess the performance, reliability, and flexibility of the compact mass detector in the OA setting for a variety of applications. The compact mass detector results were compared to those obtained by current OA LC-MS systems, and the capabilities and benefits of the compact mass detector in the open access setting for chemists in the drug discovery and development space are demonstrated.

Subcutaneous panniculitis-like T-cell lymphoma with bone marrow involvement.

OBJECTIVES: To describe a rare case of subcutaneous panniculitis-like T-cell lymphoma (SPTCL) with morphologic and immunophenotypic evidence of bone marrow involvement. METHODS: Biopsy specimens of skin and subcutis and bone marrow were examined using H&E-stained sections. Immunohistochemical studies for CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30, CD56, and granzyme B were reviewed. In addition, T-cell receptor γ gene rearrangement studies were performed. RESULTS: A bone marrow core biopsy demonstrated several lymphohistiocytic aggregates containing atypical, cytotoxic T cells that rimmed adipocytes and were associated with karyorrhexis. These T cells were morphologically and immunophenotypically identical to a concurrent SPTCL, expressing CD2, CD3, CD7, CD8, and granzyme B but with diminished CD5 expression. CONCLUSIONS: SPTCL may rarely involve the bone marrow. Bone marrow infiltrates show a similar morphologic and immunophenotypic appearance to those in the subcutaneous fibroadipose tissue, including rimming of adipocytes by neoplastic lymphocytes.

Nodular lymphocyte predominant Hodgkin lymphoma. An immunophenotypic reappraisal based on a single-institution experience.

In our experience, certain commonly cited immunophenotypic features of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) are not encountered in day-to-day practice. We reviewed 60 cases of NLPHL (18 women, 42 men; median age, 34 years) to discern immunophenotypic features from a large, single-institution cohort. All cases contained lymphocytic and histiocytic (L&H) cells. These cells expressed CD20 in 98% (59/60), CD79a (usually faint) in 87% (27/31), CD30 in 7% (4/59), epithelial membrane antigen in 21% (12/56), bcl-2 in 5% (2/41), and bcl-6 in 83% (30/36) of cases. CD10 was negative in all 36 cases studied; 100% of cases (55/55) demonstrated CD3+ rosettes. Although CD57+ T cells were common within the background infiltrate, CD57+ rosettes were seen in only 48% of cases (15/31) and were rare when encountered. Based on these patterns, we conclude that bcl-2 and bcl-6 may be useful additions to the immunophenotypic analysis of NLPHL, but that the diagnostic usefulness of epithelial membrane antigen and CD57 rosettes may have been overemphasized in previous reports.

Histopathologic features of splenic small B-cell lymphomas. A study of 42 cases with a definitive diagnosis by the World Health Organization classification.

We studied 42 cases of splenic small B-cell lymphoma (SBL) (21 women, 21 men; aged 32-82 years; median, 65 years) with a definitive diagnosis by the World Health Organization classification: chronic lymphocytic leukemia (CLL), 8; mantle cell lymphoma (MCL), 9; follicular lymphoma (FL), 12; marginal zone lymphoma, 13 (splenic [SMZL], 12; extranodal [EMZL], 1). Splenectomy was performed for diagnosis or therapy; splenic weights were 0.2 to 3.8 kg (median, 1.4 kg). In general, splenic SBLs showed white pulp (WP) expansion; morphologic features of the nodules recapitulated the corresponding lymph node histopathologic features. "Marginal zones" were observed commonly in SMZL and FL, may be present in MCL involving the spleen, and may be seen in hilar lymph nodes (HLNs) in SBLs other than SMZL. FL may simulate SMZL and can be distinguished by the presence of neoplastic follicles and HLN morphologic features. Extracellular hyaline deposits (EH) are common in FL and SMZL. MCL typically shows WP expansion by a monotonous small lymphocytic infiltrate, without diffuse red pulp (RP) infiltration or EH; leukemic MCL may show RP infiltration. Splenic morphologic features in CLL vary in WP or RP dominance; marginal zones usually are not observed in CLL.