Blastoid Cells Research Articles

Growth-Inhibitory Effects of a Class I PI3K Inhibitor GDC-0941 Are Modulated by Hypoxia-Induced mTOR Pathway Activation in Mantle Cell Lymphoma

Abstract 1424Hypoxia confers pro-survival signals to tumor cells via multiple mechanisms including activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. Constitutive activation of PI3K/Akt signaling pathway is documented in aggressive blastoid variants of mantle cell lymphoma (MCL) and is implicated in the pathogenesis of MCL. mTOR signaling, one of the important downstream targets of Akt, positively regulates protein translation by phosphorylating p70 ribosomal S6 kinase (S6K) or eukaryotic initiation factor 4E-binding protein-1 (4E-BP1). We have reported that the Class IA PI3K isoforms, mainly p110α and partially p110δ, are responsible for PI3K/Akt/mTOR signaling activation in blastoid MCL cells (Zhou et al., ASH abstract 2010). In this study, the activity and molecular mechanisms of action of a selective pan-isoform class I PI3K inhibitor GDC-0941 (Genentech) were investigated in the context of hypoxic microenvironment in MCL cells. For hypoxia experiments, MCL cells were cultured under 1% O2 for at least 14 days to assure their sustained proliferation and survival. Under hypoxia, more cells accumulated in G0/G1 phase, indicating that hypoxic conditions promote cell cycle quiescence in MCL cells. GDC-0941 treatment in normoxia resulted in a reduction of cell proliferation in a dose-dependent manner (IC50 at 48 hrs; 0.7 μM for Granta519, 0.5 μM for JVM2, 1.0 μM for MINO, 0.7 μM for Jeko-1, MTT test). In hypoxia, higher doses of GDC-0941 were required to cause cell growth inhibition compared to normoxic conditions in Granta519 (IC50; 1.7 μM), JVM2 (3.5 μM) and MINO (1.2 μM). Further, GDC-0941 failed to inhibit growth of Jeko-1 cells under hypoxia. GDC-0941 (0.5 μM) induced G0/1 cell cycle arrest under both normoxic and hypoxic conditions in Granta519, JVM2 and MINO cells. No significant induction of apoptosis by GDC-0941 (0.5 μM) was observed in any of the tested MCL cells. GDC-0941 completely inhibited phosphorylated (p-Ser473) Akt in all cell lines analyzed under both normoxic and hypoxic conditions, which resulted in decreased expression of p-GSK-3β and its downstream target cyclin D1. GDC-0941 further downregulated the mTOR downstream targets p-S6K and p-4EBP1 in Granta519, JVM2 and MINO cells under both, normoxia and hypoxia. On the contrary, GDC-0941 failed to affect hypoxia-induced p-S6K and p-4EBP1 expression levels in Jeko-1 cells, whereas potently suppressed these proteins at ambient oxygen level. These results suggest that in Jeko-1 cells hypoxia activates mTOR through PI3K-independent pathways. To test this, we examined consequences of mTOR blockade in Jeko-1 cells. mTOR inhibitor rapamycin (100 nM) moderately diminished cell growth under normoxic conditions causing accumulation of cells in G0/1 cell cycle phase but no apoptosis. In turn, under hypoxia rapamycin profoundly inhibited cell growth, inducing >80% of MCL cells to undergo cell death.These findings indicate that in certain MCL cells hypoxia activates mTOR through alternative, PI3K-independent pathways, which facilitate cell survival under hypoxia. Hence, blockade of class I PI3K may eliminate circulating MCL cells but not tissue-resident cells in hypoxic areas of the bone marrow or lymph nodes. In turn, simultaneous therapeutic ablation of PI3K/Akt and mTOR pathway may represent a promising strategy to target the aggressive blastoid variants of MCL cells in the hypoxic microenvironments. Disclosures:No relevant conflicts of interest to declare.

Continuous CD4+ T Cell Lines Derived From the Classical Hodgkin Lymphoma Microenvironment: A Challenge to the Assumption of Anergy,

Abstract 3645 Introduction:The bulk of the tumour infiltrate in classical Hodgkin lymphoma (CHL) is composed of immune cells, predominantly CD4+ T cells, with the malignant Hodgkin Reed Sternberg cell (HRS) representing <1% of cells. The lymphoid microenvironment has been described as anergic and hypoproliferative with suppressive properties (Marshall et al. Blood 2004 103:1755–62) but the functional significance of this is unclear. This study set out to examine the proliferative capacity and phenotype of T cells derived from CHL-diagnostic lymph node tissue taken at diagnosis. Method:Frozen single cell suspensions (SCS) from 6 patients were selected from the tissue bank of our Institute. T cell growth-augmenting and/or Th2 polarising cytokines were added in various combinations (IL2, IL4 only, IL2+4 or no added cytokine) to SCS-derived cells in 96 well plates at 0.3 × 106 cells per well in 200mcl of optimized lymphocyte culture media. No CD4+ enrichment step was carried out: all recovered cells were plated at baseline to maintain potential interactions between CD4+ cells and other cells, and no mitogen or T-cell receptor-stimulating or costimulating agents were added at any point. As controls, SCS derived from normal tonsil, and ÔreactiveÕ lymph nodes (n=4) (confirmed by histological report at the time of diagnosis) were also plated. Plates were examined daily for cell/colony morphology to estimate growth and split with fresh media and cytokines once every 7 days, with estimated proliferation (by haemocytometry) plotted. Cultures were assessed at baseline, 10 days, 28 days, 50 days and 100 days. Results:Proliferation, based on formation of discrete colonies and blastoid cell morphology, occurred in the majority of wells by day 7 in all CHL-derived cultures, and in a minority of wells, and to a lesser extent in all control cultures. CHL-derived T cells from one patient continue to expand after 200 days, doubling every 3–5 days, while the other 5 continue after 50–100 days. In contrast, no tonsil or reactive node-derived T cells survived beyond 50 days and none showed a net expansion in cell numbers. Growth was superior in the IL2+4 and IL2-only conditions, with no growth in the media-only or IL4-only conditions. The most favorable condition was with the addition of IL2+4. By day 21 a net increase in CHL-derived T cells was apparent, but not in any control T cell populations (Figure). At baseline, composition of the CHL-derived cells revealed a majority of CD3+ cells as expected, of which 60–80% were CD4+ and the remainder CD8+. By D21 the CD4+ component had outgrown all other cells in the CHL-derived cultures, being CD3+CD4+CD45RO+ consistent with antigen-experienced T helper cells, while all tonsil and reactive node-derived cells were CD8+CDRO+. Markers of central memory (CCR7 & CD62-L), Th2 (CCR4 and IL4), Treg (FOXP3 and CD25) and anergy (CD57) were absent after expansion, while markers of activation were upregulated (CD28, CD27, CD69, CD40L, CD30 & CD95). This phenotype persisted in the ongoing T cell lines. [Display omitted] Conclusions:The CD4+ compartment of the CHL microenvironment contains a primed subset of cells capable of massive expansion without further mitogenic stimulation and of generating cytokine-dependent continuous cell lines with an antigen-experienced, activated phenotype. This challenges the assumption of T cell anergy and hypoproliferation in the tissue microenvironment of CHL. We are currently assessing the function and anti-tumor specific or tumor supportive nature of these T cells. Disclosures:Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Platelet Functions and Risk Prognosis in Myelodysplastic Syndromes,

Abstract 3806▪▪This icon denotes a clinically relevant abstractThe myelodysplastic syndromes (MDS) are a heterogenous group of clonal stem cell disorders with peripheral cytopenias and increased incidence of leukemic transformation.The prognosis of MDS is determined by several factors, including the presence of specific cytogenetic abnormalities, the percentage of blastoid cells in bone marrow and peripheral blood, the number of affected cell lineages, and transfusion dependency. The most commonly used risk stratification system is the International Prognostic Scoring System (IPSS). This score divides patients into a lower risk subset (low and intermediate-1) and a higher risk subset (intermediate-2 and high).Patients with MDS may have hemorrhagic complications with serious outcomes that are among the major causes of death in this population. These bleeding episodes that are often related to thrombocytopenia also occur in MDS patients with normal platelet count.The aim of this work was to study functional characteristics of platelets in MDS patients and their relationship to risk evaluated as indicated by IPSS.Eighty diagnosed MDS patients risk-stratified according to IPSS were included: 40 with low-risk, 29 with intermediate-1-risk (I-1), 8 with intermediate-2-risk (I-2) and 3 with high-risk. Eighty healthy donors were included as control group.Platelet-related primary haemostasis was evaluated with an automated platelet function analyzer (PFA-100®, Siemens Healthcare Diagnostics). Samples of citrated blood were aspirated under a shear rate of 4,000–5,000/s through a 150-μm aperture cut into a collagen-ADP (COL-ADP) or collagen-epinephrine (COL-EPI) coated membrane. The platelet haemostatic capacity is indicated by the time required for the platelet plug to occlude the aperture (closure time, CT), which is expressed in seconds.Platelet activation was determined through FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 μM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (αIIb and β3 subunits) was determined by flow cytometry with specific mAbs.Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane phosphatidylserine (PS) exposed in basal conditions.I-2 and high-risk patients were gathered together in a high-risk group in order to analyze experimental results. Statistical analysis was performed with one-way ANOVA and Tukey test.CTs obtained with COL-EPI and COL-ADP cartridges in controls and low risk patients were similar and significantly shorter than CTs observed in I-1-risk and high-risk MDS patients (p<0.05).Platelets from all MDS patients showed a reduced capability for being activated by 100 μM TRAP. This impairment was more evident in I-1-risk and high-risk patients: PAC-1 binding, in arbitrary units (AU), was 11368±1017 in controls; 7849±789 in low-risk MDS (p<0.05); 4161±591 in I-1-risk MDS (p<0.01 versus control and p<0.05 versus low-risk) and 492±184 in high-risk MDS (p<0.01 versus control and p<0.05 versus low-risk). The platelet surface expression of P-selectin induced by 100 μM TRAP was also reduced: 5102±340 AU in controls, 3318±400 AU in low-risk MDS (p<0.05); 1880 ±197 AU in I-1-risk MDS (p<0.05 versus control and versus low-risk), and 1211±130 AU in high-risk MDS (p<0.05 versus control and versus low-risk). Diminished responses to TRAP were not due to a reduction in surface expression of fibrinogen receptor in platelets from MDS patients.Platelets from MDS patients expressed more PS than controls under basal conditions. Mean fluorescence values for FITC-annexin binding were: 383±16 in controls; 444±21 in low-risk (p<0.05); 575±52 in I-1-risk MDS (p<0.05 versus control and versus low-risk); 611±17 in high-risk MDS (p<0.05 versus control and versus low-risk).Our results indicated that platelets from MDS patients had less ability to be activated and were more apoptotic than control ones. These dysfunctions were more pronounced when the risk of the disease was higher according to IPSS. Disclosures:No relevant conflicts of interest to declare.

Mantle cell leukemia as a cause of leukostasis

Mantle cell leukemia as a cause of leukostasis Daniel Smith1, Christian Cable2, Cary Chisholm1, Walter Linz1, William Koss1, Sheila Dobin1, Edward Rappaport11Department of Pathology, 2Internal Medicine, Scott and White Healthcare/Texas A &amp; M Health Science Center College of Medicine, Temple, TX, USAAbstract: A 72-year-old man was admitted with hypoxemic respiratory distress. Given a white blood cell count of 600 &times; 109/L and symptoms of leukostasis, emergency leukapheresis was initiated. The white blood cell count immediately after the first leukapheresis was paradoxically increased to over 700 &times; 109/L. Peripheral blood smear findings showed morphologically immature mononuclear cells and numerous circulating mitotic figures. Initial flow cytometry results showed a lambda light chain-restricted B lymphoid population positive for CD20, CD19, CD5, and FMC-7, and negative for TdT, CD10, CD23, CD34, CD117, and myeloid markers, suggesting classification as a blastoid variant of mantle cell lymphoma in a leukemic phase. Subsequent testing using DNA fluorescence in situ hybridization was positive for t(11;14), confirming the diagnosis of mantle cell leukemia. Although mantle cell lymphoma occasionally transforms or can even present as leukemia (leukocytes &gt;40 &times; 109/L), it is rare for it to present with such profound leukocytosis and an overwhelming number of pleomorphic/blastoid forms. Although morphology suggested acute lymphoblastic leukemia, a more specific diagnosis of blastoid variant mantle cell lymphoma was obtained in 12 hours by applying complementary techniques of flow cytometry and rapid cytogenetics.Keywords: mantle cell lymphoma, chemotherapy, leukapheresis, lymphocytic leukemia

Establishment of a Novel Cell Line of Plasmablastic Lymphoma with Loss of p16 Tumor Suppressor Protein and Overexpression of MDR

AbstractAbstract 4451Plasmablastic lymphoma is characterized by diffuse proliferation of large blastoid cells with immunophenotype of plasma cells. It frequently presents as a mass in the oral cavity, but it may also be encountered in other extranodal sites. It is clinically aggressive with a median survival of 6 months. Immunodeficiency, mainly by HIV, is the major risk factor for this disease, however, in some Asian countries like Japan and Korea, more numbers of HIV-negative cases of plasmablastic lymphoma has been reported, with fewer Epstein Birr Virus (EBV)-associated cases. Thus, it needs to be further determined whether any ethnic differences in the occurrence of this disease exists.We have experienced a rare case of plasmablastic lymphoma of the uterus in an otherwise healthy young Japanese female. Although treated with multiple chemotherapy regimens such as CHOP, hyper CVAD and VAD, the patient died of the disease within three months after the diagnosis. The clinical course of the disease was unique with de novo onset in the uterus, progressing to systemic lymph node swelling. Immunophenotype of the tumor was positive for CD138, VS38c and EMA but negative for CD20 and EBER. Karyotyping of the tumor showed two novel chromosome translocations, t(4;7)(q35;q22) and t(9;13)(p22;q22), either as a single translocation of t(9;13) or both. Southern blot analysis of the tumor was negative for EBV or c-myc translocation, which has recently been reported to have causal relationship with this type of lymphoma. Written informed consent was obtained from the patient and the family, and tissue sample obtained by lymph node biopsy was used for further analysis.The lymph node specimen was transplanted subcutaneously in immunodeficient NOG mice and was stably transplantable up to over five passages. The tumor was also transplantable subcutaneously to nude mice. We also succeeded in maintaining these newly established plasmablastic lymphoma cell lines in vitro. Two cell lines, one with a single translocation of t(9;13) and another with double translocation of t(4;7) and t(9;13) were established. Both cell lines were identical to the original patient's tumor in terms of immunophenotype.Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) was performed to identify the precise chromosomal breakpoints in these translocations. FISH analysis using approximately 50 probes identified consistent result with CGH analysis, which lead to the identification of lack of tumor suppressor p16 protein expression (t(9;13)) and upregulation of multidrug resistant protein (MDR) (t(4;7)).The expression of cell surface MDR and its function were also confirmed using flow cytometry and dye efflux method. Comparison among the established cell lines with or without the t(4;7) translocation confirmed increased resistance to chemotherapeutic drugs such as adriamycin and cisplatinum in t(4;7) positive, MDR overexpressing cell line. This result is in accordance with the clinical course of the patient, where adriamycin was initially the key drug, but lost its responsiveness with disease progression.We believe that this is the first report of the establishment of cell line for plasmablastic lymphoma that has identified genetic lesions involved in the pathogenesis of the disease. These newly established cell lines and experimental animal models of plasmablastic lymphoma will be a useful tool to understand the pathogenesis of the disease including development of the typical plasmablastic feature of this type of lymphoma, and to identify novel effective treatment strategy for this highly aggressive lymphoma. Disclosures:No relevant conflicts of interest to declare.

Primary cutaneous blastic plasmacytoid dendritic cell neoplasm without extracutaneous manifestation: Case report and review of the literature

Blastic plasmacytoid dendritic cell (BPDC) neoplasm is a rare, highly aggressive hematopoietic malignancy with involvement of bone marrow and peripheral blood. We present 2 cases of primary cutaneous BPDC neoplasm without extracutaneous manifestation during the course of disease. A 36-year-old and a 51-year-old male presented with erythematous patches, purple plaques, and nodules on the head, trunk, and extremities. Skin biopsies revealed that the lesions of both cases were composed of diffusely medium-sized monomorphic blastoid cells infiltrating into the dermis and subcutis. The neoplastic cells were strongly positive for CD4, CD56, CD123, and TdT, whereas other T-cell markers and EBV markers were not expressed. The patients underwent polychemotherapy with hyper-CVAD regimen and obtained a remarkable clinical response with regression of skin lesions. No sign of recurrence and extracutaneous manifestation was found during the period of follow-up. We presume that the favorable prognosis of our cases might result from the presentation only with a skin lesion, diffuse TdT expression in tumor cells, and aggressive chemotherapy with hyper-CVAD regimens. Laboratory examination for blood and bone marrow should be performed every 3–6 months during the first period of follow-up to monitor the progression of disease even if the patients had complete remission at initial chemotherapy.

Plasmablastic lymphoma of the oral cavity in immunocompetent patients: report of two cases

Plasmablastic lymphoma (PBL) is a diffuse large B-cell lymphoma, a new entity, the incidence of which is increasing, predominantly in the oral cavity of immunocompromised patients, most of whom are infected with human immunodeficiency virus (HIV); only four cases have been reported in non-HIV-infected patients. This report details two cases of PBL of the oral cavity in immunocompetent patients. In both cases, the tumour manifested as a solitary, firm mandibular swelling with dense infiltration of plasma and blastoid cells in the stroma with strong positivity for CD 138 and negativity for CD 20. The patients received radiotherapy and chemotherapy, which resulted in complete remission of the lesion. This is the only case report of two cases of PBL in the oral cavity in immunocompetent patients in which both patients received treatment and showed complete remission of the lesion.

Cutaneous Manifestations of Blastic Plasmacytoid Dendritic Cell Neoplasm—Morphologic and Phenotypic Variability in a Series of 33 Patients

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a neoplasm derived from precursors of plasmacytoid dendritic cells. Cutaneous involvement represents often the first manifestation of the disease. We studied 45 skin biopsies from 33 patients (M:F=7.25:1; median age: 71 y; age range: 30 to 89) with BPDCN to delineate histopathologic and immunophenotypic features of this disease. Patients presented with generalized (n=18), localized (n=6), or solitary (n=9) macules, plaques, and/or tumors. Staging investigations at presentation were negative in 20 patients. Unusual histologic features included a perivascular/periadnexal pattern (6 biopsies from 4 patients) and the presence of pleomorphism of neoplastic cells with blastoid cells admixed with elongated, twisted, or hyperchromatic cells (observed in 24 specimens). Negativity of 1 among the 4 markers CD4, CD56, CD123, and TCL-1 was seen in 11 biopsies, and of 2 markers in 4 biopsies. Staining for CD68 revealed positivity of the majority of cells in 1 and of scattered cells in 24/37 stained cases. Terminal deoxynucleotidyl transferase was observed in 22/37 stained cases. Staining for Bcl-6, MUM-1 and FOX-P1 revealed positivity of a variable proportion of neoplastic cells in 16/30, 19/29, and 21/23 cases, respectively. Our study shows that cutaneous lesions of BPDCN display a greater variability of morphologic and phenotypic features than recognized earlier. Discrete perivascular infiltrates, pleomorphic morphology of neoplastic cells, and unusual phenotypic profiles may be the source of diagnostic pitfalls. These atypical variants should be recognized to make an early diagnosis and to manage properly patients with this aggressive hematological disorder.

Over-expression of CCL3 MIP-1α in a blastoid mantle cell lymphoma with hypercalcemia

We analyzed a case with the blastoid variant of mantle cell lymphoma (MCL-BV), a rare subtype of B-cell lymphoma, presenting with marked hypercalcemia at diagnosis. Enzyme-linked immunosorbent assay (ELISA) showed elevated serum levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), and type I collagen telopeptide, but not parathyroid hormone, calcitriol or parathyroid hormone-related peptide at diagnosis, suggesting local osteoclastic hypercalcemia in this case. By reverse transcription polymerase chain reaction (RT-PCR) analysis, we found predominant expression of mRNA for MIP-1alpha in addition to those for receptor-activator of nuclear-factor kappa B ligand (RANKL), TNF-alpha, and IL-6 in lymphoma cells obtained from the patient. Furthermore, recombinant MIP-1alpha significantly stimulated (3)H-thymidine uptake by isolated MCL cells in vitro. Treatment with intravenous fluids, bisphosphonate, and methylprednisolone followed by combination chemotherapy promptly corrects the hypercalcemia and successfully induced complete remission, which was accompanied by a decrease of these cytokines in the serum, including MIP-1alpha. In the present case, MIP-1alpha, an osteoclast-activating factor produced by mantle lymphoma cells, may contribute to the development of hypercalcemia. It likely acts through RANKL expression in tumor cells and/or stroma cells, as indicated in multiple myeloma (MM) and adult T-cell leukemia/lymphoma (ATLL). Furthermore, MIP-1alpha is also involved in the development of an aggressive phenotype on MCL by stimulating proliferation of these lymphoma cells. In summary, the present study demonstrated that MIP-1alpha is an important factor in the development of both hypercalcemia and an aggressive phenotype in some types of B-cell lymphoma.

Diffuse blastoid B-cell lymphoma: a histologically aggressive variant of t(14;18)-negative follicular lymphoma

Among the diffuse lymphomas of B-cell origin, we have encountered one variant displaying blastoid features that morphologically mimic lymphoblastic lymphoma, the blastoid variant of mantle cell lymphoma, and the so-called blastoid transformation of follicular lymphoma. To better characterize this entity, we studied eight cases morphologically, immunohistochemically, and by fluorescence in situ hybridization (FISH) for cytogenetic abnormalities commonly associated with follicular lymphoma and B-cell lymphomas exhibiting high-grade histological features. All eight cases were presented as de novo neoplasms, and displayed an entirely diffuse (five cases) or only minimal follicular (three cases) growth pattern. The neoplastic lymphoid cells were of medium size with round nuclei, fine chromatin, inconspicuous nucleoli, and high mitotic rate; they expressed CD10, BCL6, and BCL2—a phenotype consistent with follicle center cell origin. A proportion of cases expressed MUM1. Their lack of TdT and CYCLIN D1 distinguished them from lymphoblastic lymphoma and the blastoid mantle cell lymphoma, respectively. The neoplastic lymphoid cells consistently expressed CD43 (seven of eight cases) and occasionally other T-cell-associated antigens, including CD5, CD7, CD8, and CD57. Although all cases overexpressed BCL2, t(14;18) was not detected in any of the five cases examined by FISH; instead, extra copies of chromosome 18 were found in four of five cases. Finally, other cytogenetic abnormalities, including structural abnormalities of BCL6 (allelic loss/gain, rearrangement), monosomy 7, del(13)(q14), and MYC allelic loss, were frequently detected. The combination of a B-cell CD10+ BCL6+ BCL2+ phenotype in the presence of structural abnormalities of BCL6 is consistent with a follicular center cell derivation for our cases. The lack of t(14;18) seen in our cases, although rare in most cases of follicular lymphoma, has been nevertheless reported in cases of follicular lymphoma with a predominantly diffuse growth pattern. The molecular pathogenesis, clinical manifestations, and prognostic significance of these lesions remain to be elucidated.

A Case of Natural Killer Cell Leukemia Misdiagnosed as Tuberculous Lymphadenopathy

Natural killer (NK) cell neoplasms are a group of rare but highly malignant tumors. We report here one case of NK cell leukemia. A 54-yr-old woman presented with a 2-month history of progressive left neck mass. Based on the positive result of tissue PCR for Mycobacterium tuberculosis, she was at first diagnosed with tuberculous lymphadenopathy. After two weeks, she developed generalized lymphadenopathy, hepatosplenomegaly, fever and anemia. Subsequent evaluation was performed including bone marrow aspiration and biopsy. Peripheral blood smear showed leukoerythroblastic features with 31% blasts. Bone marrow was packed with agranular blastoid cells, which were periodic acid-Schiff (PAS) positive and myeloperoxidase (MPO) negative. Immunophenotyping showed that these cells were positive for CD45 and HLA-DR, whereas negative for CD3, CD5, CD7, CD10, CD13, CD14, CD19, CD20, CD22, CD33, CD34, and CD61. Because of the absence of the markers of T-cell, B-cell, and myeloid lineage-specific antigens, we added CD16/56 for the immunophenotyping and the blasts were positive (94%). The tumor cells of biopsied lymph node were only positive for CD56, consistent with NK cell lymphoma. Epstein-Barr virus (EBV) was not detected by RNA in situ hybridization. Culture for M. tuberculosis was negative. Thus this patient was diagnosed with blastic NK cell lymphoma/leukemia involving bone marrow and lymph node.

Primary Cutaneous Blastoid Mantle Cell Lymphoma-Case Report

Mantle cell lymphoma (MCL) commonly involves extranodal sites, usually as a manifestation of disseminated disease. In rare cases, MCLs may arise as a primary tumor in the skin. Blastoid mantle cell lymphoma (BV-MCL) is a rare variant and has a more aggressive clinical course. The phenotype of BV-MCL is characterized as CD20+, CD5+, cyclin D1+, CD23-, and CD10-. Interphase fluorescence in situ hybridization shows a characteristic t(11;14) fusion pattern. We report a case of a BV-MCL arising in skin as primary cutaneous MCL with the characteristic immunophenotype and translocation.

Cutaneous CD30+ pseudolymphoma: report of a case highlighting the importance of attention to basic cytomorphological details and clinico-pathological correlation

Distinction between a cutaneous lymphoma and pseudolymphoma can sometimes be exceedingly difficult. An increasing number of reactive inflammatory conditions with large numbers of CD30-positive atypical cells are being recognized. Some of these cases may be potentially misdiagnosed as a cutaneous lymphoma (CD30-positive lymphoproliferative disorder) on morphology alone. Cutaneous herpesvirus infection, particularly when developing in immunosuppressed individuals, can present as a pseudolymphoma with numerous CD30-positive blastoid cells. A careful search for viral cytopathic changes in the epidermis and adnexal epithelium, along with correlation with the overall clinical picture and appropriate ancillary investigations, may be necessary to establish a correct diagnosis.

下顎骨骨折を契機に発見された急性骨髄性白血病の1例

We report the rare case of a mandibular fracture in a patient with acute myeloid leukemia（AML）. A 69-year-old man was referred to us because of a mandibular fracture. On admission, the white cell count was 14100/μl with 69.5 ％ blastoid cells, the hemoglobin level was 6.9 g/dl, and the platelet count was 23000/μl. Bone marrow aspiration revealed hypercellular marrow containing many peroxidase-positive blastoid cells（85.4 ％）. A mandibular fracture with AML was diagnosed, and we initially planned an operation for the mandibular fracture. However, because infection is known to be the most common cause of death in patients with leukemia, antibiotics were administered in accordance with the guidelines for the use of antimicrobial agents in neutropenic patients with cancer. Subsequently, fixation of the mandible and drainage of the intermandibular space were performed, with no evidence of severe infection in the perioperative period. On postoperative day 13, the patient received induction therapy with cytarabine and idarubicin. Although the leukemia responded well to chemotherapy, the patient died of an intracerebral hemorrhage after consolidation therapy.

Dose Escalated Radioimmunotherapy with Yttrium-90-Ibritumomab Tiuxetan as Part of a Reduced Intensity Conditioning Regimen for Allogeneic Hematopoietic Cell Transplantation in Patients with Advanced Aggressive Non-Hodgkin Lymphoma: An Interim Analysis of a Phase I/II Study

Allogeneic hematopoietic cell transplantation (HCT) using reduced intensity conditioning (RIC) regimens offers a potential curative therapy to patients with advanced aggressive NHL. RIC HCT induces potent graft-versus-lymphoma effects with best results in patients with low tumor burden at time of HCT. Combined use of radioimmunotherapy (RIT) with RIC may increase anti-lymphoma activity of RIC. In addition to the graft versus lymphoma effect, HCT provides rescue from hematologic toxicity of RIT which may allow dose escalation of RIT. A multicenter phase I/II dose escalation study of RIT using yttrium-90-ibritumomab tiuxetan (Y90-CD20, Zevalin®) at two dose levels (0,6 (22 MBq) and 0,8 (30 MBq) mCi/kg) in 10 patients each, combined with RIC for treatment of patients with aggressive NHL has been initiated. Patients received dosimetry with In111-CD20 on day −21 and RIT with an escalated dose of Y90–CD20 (0,6–0,8 mCi (22–30 MBq)/kg) on day −14 followed by RIC using fludarabine (30 mg/m2 day −8 to −4), melphalan (140 mg/m2 day −3) and alemtuzumab (20–30 mg day −3 to −2). For postgrafting immunosuppression cyclosporine is administered. Primary objective of this study was the evaluation of treatment related mortality and engraftment after RIT-RIC HCT. Secondary objectives were disease response, relapse rate, disease free and overall survival, GVHD and immune reconstitution.Twenty patients have been enrolled on the study. Diagnoses were diffuse large B-cell lymphoma (n=13), transformed chronic lymphocytic leukaemia (n=4), blastoid mantle cell lymphoma (n=2) and follicular lymphoma grade 3 (n=1). Median age was 51 (range, 30–69) years. PBSC grafts were either from matched related (n=5) or matched unrelated donors (n=15). All patients were “high risk” with refractory disease or relapse after preceding autologous HCT. Disease status at time of HCT was CR=4, PR=15 and SD=1. No additional toxicity due to RIT was observed in comparison to our previous experience with the same RIC even after dose escalation to 0.8 mCi (30 MBq)/kg. Engraftment was rapid and sustained with no graft rejections. Median time to >500 granulocytes/μL was 14 (range, 9–32) days and to >20000 platelets/μL 11 (range, 9–56) days. TRM at day +100 was 0% and overall 10% with two deaths due to infection. Incidence of grade II–IV GVHD was 45% (II=8, III=1). To date, chronic extensive GVHD occurred in 3 patients. Causes of death were relapse=6, suspected suicide=1 and infection=2. 11/20 patients (55%) are alive with a median follow-up of 119 (range, 28–363) days. Disease status of patients alive is CR=8, PR=2 and Relapse=1.In conclusion, dose escalation of RIT and combined use with RIC is feasible with no additional toxicity due to RIT and with stable engraftment in all patients. Preliminary response data suggest that this strategy may improve early post-transplant disease control, but long-term disease-free survival remains to be determined.

A Case Report of Aggressive Adult Neuroblastoma Mimicking Acute Leukemia with Fulminant Course and Fatal Outcome

Neuroblastoma is an embryonic neoplasm of the sympathetic nervous system derived from the primitive neural crest cells. It is the second most common extra cranial malignant tumor of childhood and the most common solid tumor of infancy, comprising 8 to 10 percents of pediatric cancers. Forty percent of patients who present with clinical symptoms at diagnosis are under 1 year of age, and less than 5% with clinical symptoms are over the age of 10 years. The tumor very rarely occurs in adult patient (defined as 20 years of age and older) and usually has an indolent course with poor survival. Up until June 2008, no more than 65 neuroblastomas in adults had been reported in the literature. Majority of the reports describe an indolent and longer course of the disease among adults with different biological characteristics when compared to children, but ultimately poor outcome regardless of the initial stage of the disease. Here, we report a case of aggressive adult neuroblastoma mimicking acute leukemia with fulminant course and fatal outcome. The patient, a previously healthy 38 years old Caucasian male, presented at our department with a 4 months history of hip pain, headaches, malaise, weakness, persistent diarrhea, poor appetite and weight loss. On admission he was febrile, prostrated, pale, fixed in bed. His abdomen was distended with present bowel movements. The liver and spleen were 3 cm and 5 cm below the costal margins respectively. No abdominal mass was palpable. Neurological examination showed right Horner's syndrome, reduction of the superficial sensitivity on the Th1 and Th2 level and loss of the tendon-muscle reflexes of the lower limbs. He had paraplegia, indicating possible spinal cord compression. The complete blood counts revealed pancytopenia and presence of 11 % peculiar circulating blastoid cells. The LDH was 3552U/l. Under suspicion of acute leukemia bone marrow aspirate and biopsy was performed. Immunophenotyping of the bone marrow mononuclear cells showed presence of around 50% CD45negative, CD56 and CD9 positive cells, indicating a possible involvement of the bone marrow with neuroblastoma. A routine second opinion from a reference pathologist was also interpreted as neuroblastoma and confirmed with positive immunohistochemical staining for NSE, chromogranin, synaptophysin and negative for LCA. A work-up for neuroblastoma was done and investigations confirmed disseminated stage 4 neuroblastoma. In spite of the appropriate treatment with intensive chemotherapy regimen, the patient failed to improve and died of progressive disease. Our case report illustrate the usefulness of including CD56 marker in flow-cytometry acute leukemia assays, especially in cases when CD45 expression is absent, as initial screening tool for recognizing cases of neroblastoma which simulate acute leukemia.

In-111 Ibritumomab Scintigraphy (Planar and SPECT) and FDG PET/CT Before Y-90 Ibritumomab Treatment in a Patient With Mantle Cell Lymphoma

We report the case of a 75-year-old man, in whom Y-90 ibritumomab was requested because of relapse of blastoid variant mantle cell lymphoma diagnosed in 1995. Before Y-90 ibritumomab treatment, FDG PET and In-111 ibritumomab scintigraphy with planar views at 24 hours, 48 hours, and 5 days, including SPECT, were performed. Discordant information between both examinations was observed as, in addition to the lesions detected by In-111 ibritumomab imaging, FDG PET detected lesions that did not take up the ibritumomab. The discrepancy shown by both radiotracers has to be kept in mind before planning treatment with Y-90 ibritumomab, and for the correct evaluation of treatment response.

Safety and Tolerability of Intrathecal Liposomal Cytarabine during CNS Prophylaxis in Patients with Non Hodgkin Lymphoma and Acute Lymphoblastic Leukemia.

Meningeal recurrence in aggressive NHL and ALL occurs in up to 20% of patients and mostly depends on intensity and efficacy of front-line CNS prophylaxis. Liposomal cytarabine (lip araC) is a sustained release formulation of araC with a homogeneous distribution in the neuraxis and a prolonged half life, maintening cytotoxic concentrations in the CSF for more than 14 days. The present study aims to evaluate the safety and tolerability of lip araC in the CNS prophylaxis of NHL and ALL meningeal recurrences. Forty-four patients aged 16–77 years (median 43,9) have been preventively treated with a total of 159 (range: 1–8) doses of lip araC 50 mg. Diagnosis consisted on 33 NHL: 14 high risk-CNS DLBCL (involvement of testes, paranasal sinuses, hard palate, orbit, paravertebral masses and bone marrow or IPI ≥2 with high level of LDH and ≥1 extranodal site involvement), 7 BL, 2 blastoid mantle cell, 7 lymphoblastic, 1 gastric marginal zone lymphoma, 1 anaplastic, 1 follicular, and 11 ALL: 7 B-ALL, 3 T-ALL, 1 hybrid cells. Five patients (1 DLBCL, 1 BL, 2 T-ALL, 1 B-ALL) received lip araC at their 1st systemic recurrence; of them, the DLBCL did not receive previous prophylaxis, instead of the T-ALLs had standard treatment with IT MTX; BL and B-ALL received not specified prophylactic therapy. All patients were treated according to the standard protocols in use for their disease; in particularly, NHL received RCHOP-like treatments, a part of 5 BL (RCODOX-M/R-IVAC-like therapy) and 5 lymphoblastic (hyperC VAD/HD MTX/araC); ALL patients received standard treatments according with GIMEMA, NILG and BMF protocols. Five patients (2 DLBCL, 2 BL and 1 B-ALL) underwent autotransplantation. Four patients received IT or HD systemic MTX during treatment with lip araC. Seven patients received RT, as a part of prophylaxis program. All patients received lip araC 50 mg every 2, 3, 4 or 8 weeks, excepted 2 receiving 30 mg. All patients had corticosteroids for prevention of chemical aracnoiditis. A part of an episode of G2 headache and 18 cases of G1 headache, 8 episodes of G1 nausea/vomiting and 2 cases of localized or diffuse bone pain, no severe toxicity has been noted. So far, after a medium observation period of 9 months (range 1–26) only 1 patient, affected by mantle cell lymphoma, showed CNS recurrence 10 months after diagnosis while in systemic relapse and died. None of the patients developed neurological symptoms or unexpected long term neurological side effects. IT lip araC therapy with concomitant corticosteroids appears to be feasible and well tolerated in the prophylactic setting. Because of only few patients received CNS-directed concomitant therapy, lip araC appears effective towards CNS recurrence in the high risk NHL and ALL. More randomized studies are warranted.

CD4+/CD56+ Hematodermic Tumor

Described herein is the evolution in the diagnosis and classification of tumors that are variously known as blastic or blastoid natural killer cell lymphoma, CD4+/CD56+ hematodermic neoplasm, or tumors of plasmacytoid dendritic cell lineage. As illustrated by cases submitted to the 2005 Society for Hematopathology/European Association for Haematopathology Workshop, these tumors display a range of clinical manifestations and various morphologic and immunophenotypic features. Recent evidence suggests a close histogenetic relationship for most cases to plasmacytoid dendritic cells. We outline the typical presenting features and the clinical course of CD4+/CD56+ hematodermic tumors, including leukemic relapses with shifts to myelomonocytic differentiation. The currently unresolved issues are largely related to the limits of this entity, particularly the relationship to myeloid leukemia and undifferentiated blastic neoplasms, with which there is diagnostic overlap. Although questions remain, delineation of the CD4+/CD56+ hematodermic tumor and its improved recognition has already served as a catalyst toward better understanding of the multilineage potential of hematopoietic malignancies.

Sequence analysis proves clonal identity in five patients with typical and blastoid mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is typically composed of small irregular lymphoid cells. Blastoid variants, composed of lymphoblast-like (classic type) or large (pleomorphic type) cells, arise de novo or in patients with typical MCL. Although it has been assumed that blastoid variant represents histologic transformation of typical MCL, the clonal relationship between the two tumors has rarely been assessed at the molecular level. We identified five patients with typical MCL who subsequently developed the blastoid variant. There were two men and three women with a median age of 65 years (range, 34–70) at diagnosis of typical MCL involving lymph nodes. The median interval between typical and blastoid MCL was 36 months (range, 11–103). Subsequent blastoid variant MCL involved soft tissue (two), lymph node (one), ileum (one), or rectum (one). All typical and blastoid neoplasms were positive for CD20, cyclin D1, and monotypic surface immunoglobulin light chain, and all typical cases were positive for CD5. Two blastoid neoplasms lost CD5 expression, one of which aberrantly expressed CD10. Immunostaining for Ki-67 showed a median proliferative fraction of 20% in typical and 70% in blastoid neoplasms. Sequence analysis of the VDJ regions of the rearranged IgH allele proved clonal identity in each set of paired samples in all five patients. These results support the concept that blastoid MCL arising in patients with typical MCL represents histologic transformation of the original neoplastic clone.