Extranodal natural killer/T-cell lymphoma from skin or soft tissue: suggestion of treatment from multinational retrospective analysis.

BACKGROUND: Clinical features and outcomes of extranodal natural killer/T-cell lymphoma (ENKL) arising from extranasal sites are not fully understood. The purpose of this study was to study the prognosis and treatment outcome of skin/soft tissue primary ENKL. PATIENTS AND METHODS: This multicenter retrospective study included 48 patients with skin/soft tissue primary ENKL diagnosed from 1993 to 2010. RESULTS: Patients with Ann Arbor stage I, T1-2N0M0 by International Society for Cutaneous Lymphomas-European Organization of Research and Treatment of Cancer TNM (tumour-node-metastasis) stage, International prognostic index score of 0-1, and a Korean prognostic index (KPI) score of 0-1 were associated with better survival. Four of five patients with T1-2N0M0 disease achieved complete response with radiation alone. In disseminated disease, only 6 of 13 patients responded to anthracycline-containing chemotherapy, and all the two patients receiving SMILE showed response. CONCLUSION: In conclusion, we identified the prognostic value of KPI, and we suggest a treatment recommendation according to the TNM (tumour-node-metastasis) stage. Radiotherapy with/without chemotherapy seemed to be optimal in localized disease. In advanced stages, a more aggressive treatment regimen with newer agents should be sought.

Natural killer-mediated lysis of normal and malignant target cells, and its regulation by monocytes.

After depletion of monocytes, natural killer (NK) cells were partially purified from peripheral blood by Percoll density gradient sedimentation. The NK cells were then cultured for 1 d and assayed for their cytotoxicity against various types of normal and malignant target cells. All types of target cells tested were found to be susceptible to NK cells. The susceptible targets were autologous T and B lymphocytes, mitogen-induced T and B blasts, monocytes, large granular lymphocytes, autologous or allogeneic lymphoma and leukemia cells isolated from patients, and cultured cell lines, including those resistant to interferon-activated lymphocytes. Such a broad spectrum of cytotoxicity was demonstrated in 1 d of culture, and freshly prepared NK cells were not cytotoxic, or, if anything, were less cytotoxic. Monocytes and their supernatants, added throughout the course of culture, markedly inhibited the development of their cytotoxicity. These results may suggest that, although NK cells having ability to lyse autologous normal and malignant target cells are present in vivo, their lytic activity is regulated by coexisting monocytes.

Prognostic factors for mature natural killer (NK) cell neoplasms: aggressive NK cell leukemia and extranodal NK cell lymphoma, nasal type.

BACKGROUND: Patients with natural killer (NK) cell neoplasms, aggressive NK cell leukemia (ANKL) and extranodal NK cell lymphoma, nasal type (ENKL), have poor outcome. Both diseases show a spectrum and the boundary of them remains unclear. The purpose of this study is to draw a prognostic model of total NK cell neoplasms. PATIENTS AND METHODS: We retrospectively analyzed 172 patients (22 with ANKL and 150 with ENKL). The ENKLs consisted of 123 nasal and 27 extranasal (16 cutaneous, 9 hepatosplenic, 1 intestinal and 1 nodal) lymphomas. RESULTS: Complete remission rate for ENKL was 73% in stage I, but 15% in stage IV, which was consistent with that for ANKL (18%). The prognosis of ENKL was better than that of ANKL (median survival 10 versus 1.9 months, P < 0.0001) but was comparable when restricted to stage IV cases (4.0 months, P = 0.16). Multivariate analysis showed that four factors (non-nasal type, stage, performance status and numbers of extranodal involvement) were significant prognostic factors. Using these four variables, an NK prognostic index was successfully constructed. Four-year overall survival of patients with zero, one, two and three or four adverse factors were 55%, 33%, 15% and 6%, respectively. CONCLUSION: The current prognostic model successfully stratified patients with NK cell neoplasms with different outcomes.

Central nervous system is a sanctuary site for chronic myelogenous leukaemia treated with imatinib mesylate.

Imatinib mesylate (IM) is currently used as the first therapeutic choice against chronic myelogenous leukaemia (CML). Because IM poorly penetrates the blood-brain barrier, IM-treated CML patients may have a potential risk of central nervous system (CNS) involvement. Here we report a case with lymphoid blast crisis isolated only in CNS after bacterial meningitis, although the patient achieved and maintained complete cytogenetic response by IM therapy. It is important to consider isolated CNS blast crisis as a possible event in IM-treated CML patients.

Epstein-Barr virus renders the infected natural killer cell line, NKL resistant to doxorubicin-induced apoptosis.

We established two Epstein-Barr virus (EBV)-infected NKL sublines, which acquired stress resistant phenotype against DNA damage and starvation compared with EBV-negative NKL. EBV-rendered doxorubicin resistance at least partially through NF-kappaB activation and the resultant sustenance of antiapoptotic proteins including Bcl-X(L) and FLIP(L/S).

Spindle checkpoint protein Bub1 corrects mitotic aberrancy induced by human T-cell leukemia virus type I Tax.

Bub1 is a component of the mitotic spindle checkpoint apparatus. Abnormality of this apparatus is known to cause multinuclei formation, a hallmark of chromosomal instability (CIN). A549, aneuploid cell line, aberrantly passed through the mitotic phase and became multinuclei morphology in the presence of nocodazole. Time-lapse videomicroscopy showed unreported bizarre morphology, which we named 'mitotic lobulation' in A549 cells just before the exit from mitosis and multinuclei formation. External expression of wild-type Bub1-EGFP clearly suppressed the multinuclei formation by retaining A549 cells at the mitotic phase during 48 h of time-lapse observation. This suppressive effect on mitotic aberrancy should not be mere restoration of normal Bub1 function, because A549 cells express proper amount of Bub1, which distributed cytoplasm during interphase and concentrated at kinetochore in metaphase. Furthermore, external expression of wild-type Bub1-EGFP suppressed multinuclei formation induced by Tax both in A549 and HeLa cells. Tax is known to induce mitotic abnormality by binding and inactivating Mad1. These observations, therefore, suggest functional redundancy between Bub1 and other mitotic checkpoint protein(s) and a possibility of correction of mitotic aberrancy by external Bub1 expression.

Medullasin enhances human natural killer cell activity.

Medullasin, a new serine protease found in bone marrow cells, increased markedly human natural killer cell activity. Whereas the natural killer cell activity measured immediately after the treatment with medullasin remained almost on the same level as the control, an incubation at 37 degrees C for several hours increased markedly the natural killer cell activity of the lymphocytes treated with medullasin. Enhancement of the natural cytotoxicity was caused by the treatment with physiologic concentrations of the protease (5-20 micrograms/ml). Inhibitors of medullasin such as phenylmethylsulfonyl fluoride and elastatinal prevented the activation of natural cytotoxicity. Depletion of lymphocytes bearing Fc receptors for IgG abolished the enhancement of natural killer cell activity by medullasin. Interferon activity was not detected in the supernatant of lymphocyte cultures stimulated with medullasin. The medullasin enhanced further the natural killer cell activity of lymphocytes stimulated with interferon. Medullasin activity was detected neither in unstimulated nor stimulated (by concanavalin A or phytohemagglutinin) human lymphocytes. The protease was released easily from human mature granulocytes into culture medium. It is considered from these results that the level of human natural killer cell activity is regulated by medullasin released by mature granulocytes.

Telomeric DNA in normal and leukemic blood cells.

We studied telomeric DNA in leukemic cells as well as in normal T cells, B cells, monocytes, polymorphonuclear leukocytes, and bone marrow hematopoietic progenitor cells. No marked differences were observed in the sizes of the telomeric repeats in the various populations of normal blood cells obtained from donors in their twenties to sixties, and the telomere length ranged between 8.5 and 9.0 kb. The leukemic cells of 12 patients with acute leukemia (seven with myeloid and five with lymphoid leukemia) showed a variable reduction in the length of telomeric DNA, ranging from 2.7 to 6.4 kb. The average telomere length was 4.8 and 4.7 kb in myeloid and lymphoid leukemia, respectively, while the telomere length in peripheral blood mononuclear cells obtained from the same patients during complete remission was 8.5 and 7.9 kb, respectively. When the same Southern blots were hybridized with Alu or alphoid sequences, no marked changes in the sizes of the repetitive DNA sequences were observed, indicating that the DNA abnormality in the leukemic cells was specific to the telomere region. Investigation of telomeric DNA changes may be helpful in determining the biological properties of leukemic cells.

Hematopoietic stem cell transplantation for natural killer-cell lineage neoplasms.

Neoplasms of natural killer (NK)-lineage are rare. Their prognosis is generally poor except for cases of solitary nasal NK-cell lymphoma. The NK-cell Tumor Study Group performed a survey in Japan on patients diagnosed between 1994 and 1998. Of 228 patients selected for analysis, 40 underwent HSCT (15 allografts and 25 autografts). The underlying diseases were myeloid/NK cell precursor acute leukemia (n = 4), blastic NK-cell lymphoma (n = 11), aggressive NK-cell leukemia (n = 3), and nasal-type extranodal NK-cell lymphoma (n = 22). At the time of HSCT, 22 patients were in complete remission (CR), 11 were in relapse, and seven were primary refractory. All patients received myeloablative conditioning regimens including total-body irradiation. Sixteen died of disease progression, and six of treatment-related causes. Overall, 4-year survival was 39% with a median follow-up of 50 months; this was significantly better than that of patients who did not undergo HSCT (21%, P = 0.0003). For patients transplanted in CR, the 4-year overall survival was 68%, which was significantly better than that of patients who went into CR but did not undergo HSCT (P = 0.03). These findings suggest that the HSCT is a promising treatment strategy for NK-cell lineage.

Dlk1 in normal and abnormal hematopoiesis.

Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth factor-like family, homologous to Notch/Delta/Serrate. We have found by real-time RT-PCR that Dlk1 mRNA levels were high in CD34(+) cells in 10 of 12 MDS samples compared with CD34(+) cells from 11 normals. Also, Dlk1 mRNA was elevated in mononuclear, low density bone marrow cells from 11/38 MDS patients, 5/11 AML M6 and 2/4 AML M7 samples. Furthermore, 5/6 erythroleukemia and 2/2 megakaryocytic leukemia cell lines highly expressed Dlk1 mRNA. Levels of Dlk1 mRNA markedly increased during megakaryocytic differentiation of both CMK megakaryoblasts as well as normal CD34(+) hematopoietic stem cells. High serum levels of Dlk1 occurred in RA (4/10) and essential thrombocythemia (2/10) patients. Functional studies showed that forced expression of Dlk1 enhanced proliferation of K562 cells growing in 1% fetal bovine serum. Analysis of hematopoiesis of Dlk1 knockout mice suggested that Dlk1 contributed to granulocyte, megakaryocyte and B-cell clonogenic growth and was needed for generation of splenic B-cells. In summary, Dlk1 is overexpressed in selected samples of MDS (especially RA and RAEB) and AML (particularly M6, M7), and it appears to be associated with normal development of megakaryocytes and B cells.

Augmentation of mouse natural killer cell activity by a streptococcal preparation, OK-432.

Streptococcal immunopotentiator OK-432 (NSC-B116209) augmented the natural killer (NK) cell activity of peritoneal exudate cells (PEC) in inbred C57BL/6 mice given ip injections of 0.1 mg OK-432 per mouse. The cytotoxic activity of PEC increased as early as 1 day after inoculation, reached its peak on day 3, and gradually declined thereafter, YAC-1, K562, and MOLT-4 target cells were more sensitive to PEC than were EL 4 and P815 target cells. The elimination of adherent cells by a nylon wool column enriched the proportion of cytotoxic cells among PEC. Nylon wool column-passed PEC were resistant to treatment with anti-Thy 1.2 antibody plus complement and sensitive to anti-asialo GM1 serum plus complement. Because 1:40-diluted rabbit antiserum against glycosphingolipid asialo GM1 is capable of eliminating mouse NK cell activity and is not cytotoxic to killer T-cells, the above results strongly suggest that OK-432 augments the NK cell activity in mice.

Heterogeneity of CD7+ 4- 8- 1- leukemia: usefulness of cytoplasmic antigen detection for subclassification.

We identified CD3 and myeloperoxidase (MPO) antigens in the cytoplasm of leukemic cells from 15 patients with CD7+ 4- 8- 1- leukemia. Seven patients, five of whom had a mediastinal mass, had only cytoplasmic CD3 (cCD3) antigen; these seven were regarded as possibly being of T-lineage type. Six patients, one of whom had a mediastinal mass, showed both cCD3 and MPO; they were considered to have mixed lineage type. Two patients had neither cCD3 nor cytoplasmic myeloid antigens; they were therefore considered to have stem cell type. These two latter types were considered as the subgroups: mixed lineage or stem cell type. Patients with T-lineage type were good responders to L-17M therapy; four out of five who received L-17M therapy achieved complete remission. There were significant differences between the two subgroups in relapse-free survival, patients with T-lineage type exhibiting better prognosis than other types (p < 0.05).

Laboratory findings and clinical courses of 33 patients with granular lymphocyte-proliferative disorders.

The hematological and immunological findings and clinical courses of 33 patients (13 male, 20 female; median age at presentation, 60 years) with granular lymphocyte-proliferative disorders (GLPD) are presented. Based on the surface phenotypes of peripheral blood granular lymphocytes (GL), the GLPD were divided into CD3+ T cell-lineage GLPD (T-GLPD) and CD3- CD16+ natural killer (NK) cell-lineage GLPD (NK-GLPD). Twenty-one patients had T-GLPD, and 12 had NK-GLPD. One patient with T-GLPD and two patients with NK-GLPD had progressive clinical courses and died of the disease despite receiving combination chemotherapy. Twelve patients with T-GLPD were found to have severe anemia at presentation or during the course of the disease; four of them fulfilled the diagnostic criteria of pure red cell aplasia, and the others had closely related conditions. Six of these 12 patients were treated with cyclophosphamide, and all responded to the treatment. In 16 patients, the clinical course was stable, and spontaneous regression was observed in two patients. Since some of the patients with NK-GLPD had stable clinical courses while some had progressive clinical courses, clinical findings in these two groups were compared. We found, taking into consideration our cases and those reviewed in the literature, that age less than 40 years, fever, lymph node swelling, hepatosplenomegaly, and GL with CD16(Leu-11)-CD56+CD57- phenotype and low or absent antibody-dependent cellular cytotoxicity seemed to be predictors of a progressive clinical course.

DNA microarray analysis of natural killer cell-type lymphoproliferative disease of granular lymphocytes with purified CD3-CD56+ fractions.

Natural killer (NK) cell-type lymphoproliferative disease of granular lymphocytes (LDGL) is characterized by the outgrowth of CD3(-)CD16/56(+) NK cells, and can be further subdivided into two distinct categories: aggressive NK cell leukemia (ANKL) and chronic NK lymphocytosis (CNKL). To gain insights into the pathophysiology of NK cell-type LDGL, we here purified CD3(-)CD56(+) fractions from healthy individuals (n=9) and those with CNKL (n=9) or ANKL (n=1), and compared the expression profiles of >12 000 genes. A total of 15 'LDGL-associated genes' were identified, and a correspondence analysis on such genes could clearly indicate that LDGL samples share a 'molecular signature' distinct from that of normal NK cells. With a newly invented class prediction algorithm, 'weighted distance method', all 19 samples received a clinically matched diagnosis, and, furthermore, a detailed cross-validation trial for the prediction of normal or CNKL status could achieve a high accuracy (77.8%). By applying another statistical approach, we could extract other sets of genes, expression of which was specific to either normal or LDGL NK cells. Together with sophisticated statistical methods, gene expression profiling of a background-matched NK cell fraction thus provides us a wealth of information for the LDGL condition.

Aggressive natural killer-cell leukemia revisited: large granular lymphocyte leukemia of cytotoxic NK cells.

Aggressive natural killer-cell leukemia (ANKL) is a rare form of large granular lymphocyte leukemia, which is characterized by a systemic proliferation of NK cells. The clinical features of 22 ANKL cases were analyzed. Hepatomegaly (64%), splenomegaly (55%) and lymphadenopathy (41%) were also frequently observed. Leukemic cells were identified as CD1-, CD2+, surface CD3-, CD4-, CD5-, CD7+, CD8+/-, CD10-, CD11b+/-, CD13-, CD16+, CD19-, CD20-, CD25-, CD33(-), CD34-, CD38+, CD56+, CD122+, HLA-DR+ and TCR-. Two of the 16 cases examined for CD57 were positive and three of the seven cases examined for cytoplasmic CD3. Epstein-Barr virus was detected in the tumor cells of 11 of the 13 cases examined. No common cytogenetic abnormalities were identified and 6q anomaly was detected in only one. Three of 13 patients treated with chemotherapy containing anthracycline/anthraquinone attained complete remission, in contrast to none of the eight who were treated with regimens without anthracycline. Although the overall prognosis was poor with a median survival of 58 days, those who attained remission showed better prognosis (P=0.005). These findings suggest that ANKL is an entity of mature cytotoxic NK-cell neoplasms with distinct phenotype and disease presentations. Intensive treatment for ANKL may result in a better prognosis.

Inhibition of human granulocyte-macrophage colony formation by interleukin 2-treated lymphocytes is mediated by interferon gamma and tumor necrosis factor alpha.

We previously demonstrated that human granulocyte-macrophage colony (granulocyte-macrophage colony-forming units, CFU-GM) formation was inhibited by interleukin 2 (IL-2)-treated lymphocytes and their conditioned medium (CM). In the present study, the mechanism of this suppression was investigated. When anti-interferon (IFN)-gamma antibody or anti-tumor necrosis factor (TNF)-alpha antibody was added to CFU-GM agar culture with IL-2-treated lymphocytes or their CM, the inhibition of CFU-GM colony formation was partially abrogated, whereas anti-TNF-beta antibody did not abolish the inhibitory effects. When anti-IFN-gamma and anti-TNF-alpha antibodies were added simultaneously, full recovery of colony formation was observed. In the CM of IL-2-treated lymphocytes, detectable levels of IFN-gamma (81 +/- 15 U/ml) and TNF (3.1 +/- 1.1 U/ml) were found. Addition of IFN-gamma and TNF-alpha at these concentrations to the agar culture inhibited CFU-GM colony formation. Taken together, these results indicate that inhibition of human CFU-GM colony formation by IL-2-treated lymphocytes and their CM is mediated by IFN-gamma and TNF-alpha generated from IL-2-treated lymphocytes.

Lysis of lymphoma cells by cultured large granular lymphocytes.

Studies were undertaken to determine whether in vitro-propagated large granular lymphocytes (LGL), which are known to mediate natural killer (NK) activity, would lyse autologous and allogeneic lymphoma cells. LGL from ten patients and two healthy donors were propagated in vitro for two to four weeks with interleukin 2-containing medium, and their cytotoxicity was tested in a 5-h 51Cr-release assay. Cultured LGL from all the patients and healthy donors demonstrated strong cytotoxicity against K562 target cells, a standard target in NK assay, while cultured LGL from the patients lysed autologous lymphoma cells from three of the ten patients tested and those of the two healthy donors lysed lymphoma cells from four of the ten patients. These findings indicate that, when LGL propagated in vitro in large numbers show lytic activity against autologous lymphoma cells, these LGL may have potential application in clinical trials.

Effect of recombinant interferons on colony formation of blast progenitors in acute myeloblastic leukemia.

The effect of recombinant interferons (rIFNs) on primary and secondary colony formation by blast progenitors (leukemic colony-forming units, L-CFU) from the peripheral blood of patients with acute myeloblastic leukemia (AML) was examined. rIFN-alpha, rIFN-beta, and rIFN-gamma inhibited L-CFU in a dose-dependent manner. When 3000 U/ml rIFN-alpha, -beta, and -gamma were added, L-CFU were suppressed to 20%, 12%, and 40% of the control cultures, respectively. The concentrations of rIFN-alpha, -beta, and -gamma required for 50% inhibition of colony formation were 63, 29, and 250 U/ml, respectively. The self-renewal capacity of L-CFU was inhibited by rIFNs in a dose-dependent manner, the degree of inhibition being the same for all three rIFNs. These rIFNs also inhibited normal granulocyte-macrophage progenitors to a similar degree as L-CFU. Taken together, these in vitro studies indicate that these rIFNs may be efficacious in the treatment of AML, though development of granulocytopenia may be observed as a complication of IFN therapy.

Inhibition of human granulocyte-macrophage colony formation by interleukin 2-treated lymphocytes.

The effects of interleukin 2 (IL-2)-treated lymphocytes on human myeloid progenitors (CFU-GM) were studied. When peripheral blood mononuclear cells (PBMC) were cultured for 3 days with recombinant IL-2, they developed lymphokine-activated killer (LAK) activity against normal bone marrow cells, and also suppressed colony formation by CFU-GM. Suppression of CFU-GM was found to be mediated mainly by natural killer (NK) cells, and to a lesser degree by T cells according to the results showing that isolated NK cells and T cells exhibited strong and moderate suppressor function, respectively. Since the levels of LAK activity and of CFU-GM inhibitory activity were not parallel in each individual, inhibition of CFU-GM does not seem to be due to a direct lytic action by LAK cells. This possibility was supported by our finding that the supernatant of IL-2-treated PBMC contained factor(s) that inhibited CFU-GM colony formation.

A new serum-free culture system for leukemic colony assay.

A new serum-free assay system for leukemic colony formation (leukemic colony-forming units, L-CFU) was established, and, utilizing this system, the colony-promoting activities of recombinant human colony-stimulating factors (rhCSFs) and the effectiveness of CSFs on cellular self-renewal capacity were investigated. The serum-free assay system included deionized bovine serum albumin (1%), cholesterol (7.8 micrograms/ml), and ASF 101 medium. The plating efficiencies obtained by culturing with phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) ranged from 0.01% to 1.35% in this system. Spontaneous colonies were observed in 9 out of 13 cases studied. Recombinant human granulocyte CSF (rhG-CSF), rh granulocyte-macrophage CSF (rhGM-CSF), rh interleukin 3 (rhIL-3), and rh interleukin 1 (rhIL-1) stimulated colony formation in 10, 9, and 6 out of 13, and 5 out of 11 cases, respectively. The magnitude of stimulation by each CSF ranged from 6% to 145%, 21% to 200%, 0% to 1229%, and 14% to 182% of PHA-LCM, respectively. In eight cases, blast colony assays in serum-free and serum-containing cultures were simultaneously performed. The magnitudes of responsiveness of each CSF differed in the two assay systems; this indicated some effect of fetal calf serum. The value of self-renewal capacity was also examined and compared with that in serum-containing culture. Self-renewal capacity could be maintained with a serum-free culture system. It showed marked variation from case to case, and there was no correlation between the primary colony formation and the self-renewal capacity in both culture systems. Taken together, the development of a completely serum-free culture system was found to be efficient as evaluated by a L-CFU colony assay.