Natural Killer Cell Malignancies Research Articles

Recent Advances in the Diagnosis and Treatment of Natural Killer Cell Malignancies.

Simple SummaryNatural killer (NK)/T-cell lymphomas are aggressive extranodal Epstein–Barr virus (EBV)-positive malignancies. They can be divided into three subtypes: nasal (involving the nose and upper aerodigestive tract), non-nasal (involving skin, gastrointestinal tract, testis and other organs) and disseminated (involving multiple organs). Lymphoma cells are positive for CD3ε, CD56, cytotoxic molecules and EBV-encoded small RNA. There is a predilection for Asian and Central/South American populations. Genome-wide association studies have identified lymphoma susceptibility loci in Asians. Positron emission tomography computed tomography and plasma EBV DNA quantification are crucial at diagnosis and follow-up. Stage I/II patients receive non-athracycline asparaginse-containing regimens, together with sequential/concurrent radiotherapy. Anthracycline-containing regimens are ineffective. Stage III/IV patients receive asparaginase-containing regimens, followed by allogeneic haematopoietic stem cell transplantation (HSCT). Autologous HSCT does not improve outcome. In relapsed/refractory patients, novel approaches include PD1/PD-L1 targeting, EBV-specific cytotoxic T-cells, and monoclonal antibodies. Small molecules including histone deacetylase inhibitors may be beneficial.Natural killer (NK)/T-cell lymphomas are aggressive malignancies. Epstein–Barr virus (EBV) infection in lymphoma cells is invariable. NK/T-cell lymphomas are divided into nasal, non-nasal, and disseminated subtypes. Nasal NK/T-cell lymphomas involve the nasal cavity and the upper aerodigestive tract. Non-nasal NK/T-cell lymphomas involve the skin, gastrointestinal tract, testis and other extranodal sites. Disseminated NK/T-cell lymphoma involves multiple organs, rarely presenting with a leukaemic phase. Lymphoma cells are positive for CD3ε (not surface CD3), CD56, cytotoxic molecules and EBV-encoded small RNA. There is a predilection for Asian and Central/South American populations. Genome-wide association studies have identified lymphoma susceptibility loci in Asian patients. Positron emission tomography computed tomography and plasma EBV DNA quantification are crucial evaluations at diagnosis and follow-up. Stage I/II patients typically receive non-athracycline regimens containing asparaginse, together with sequential/concurrent radiotherapy. Anthracycline-containing regimens are ineffective. Stage III/IV patients are treated with asparaginase-containing regimens, followed by allogeneic haematopoietic stem cell transplantation (HSCT) in suitable cases. Autologous HSCT does not improve outcome. In relapsed/refractory patients, novel approaches are needed, involving PD1/PD-L1 targeting, EBV-specific cytotoxic T-cells, and monoclonal antibodies. Small molecules including histone deacetylase inhibitors may be beneficial in selected patients. Future strategies may include targeting of signalling pathways and driver mutations.

PRDM1 Binds an Extensive Network of Genes to Regulate Human Natural Killer Cell Homeostasis

Natural killer (NK) cells are innate lymphocytes responsible for early defense against infections and malignant cells. Specific transcription factors crucial for NK cell development and function include PRDM1, which also regulates T cell homeostasis and is essential for the terminal differentiation of B cells into plasma cells. Importantly, PRDM1 deletion, methylation and loss-of-function mutations were commonly detected in NK cell malignancies. However, the detailed mechanisms through which PRDM1 regulates NK cell homeostasis are still largely undefined. Here, we employed an in vitro culture system of human NK cells isolated from healthy donors, in which the NK cells were cultured in the presence of IL-2 with or without an engineered feeder cell line, K562-Cl9-mb21, expressing membrane-bound IL-21, 4-1BBL and CD86. The NK cells were able to expand for months when co-cultured with feeder cells, whereas IL-2 alone could only maintain NK cell survival with limited proliferation for one week. We performed ChIP-seq to compare the genome-wide binding profiles of PRDM1 in NK cells grown with or without feeder cells. We found that PRDM1 bound much fewer target genes (802) in NK cells with feeder than in NK cells with IL-2 alone (2880), and 98.5% (790/802) of the binding sites in feeder-stimulated NK cells overlapped with those found in NK cells without feeder. The PRDM1 consensus motifs were almost identical in both conditions. MEME analyses also identified motifs of other transcription factors enriched in the PRDM1 binding sites, such as the RUNX and T-Box families. Interestingly, the RUNX and T-Box motifs, among others, were more enriched in the PRDM1 binding sites lacking the PRDM1 motif than in those with the PRDM1 motif, which indicates that PRDM1 may often bind to DNA indirectly through other transcription factors. PRDM1 bound a large number of genes in the NK or T cell receptor signaling pathway, which are employed during NK cell activation. In addition, several genes encoding immune checkpoints that may restrict NK cell activation such as TIGIT, HAVCR2 (TIM3), and IL-1R8 were targeted by PRDM1. Many genes encoding NK cell inhibitory and activating receptors were also bound by PRDM1. Moreover, PRDM1 was found to target transcription factors that are important for NK cell development and homeostasis, such as the RUNX family, TBX21, MAF, and PRDM1 itself. Despite an extensive overlap of PRDM1 binding sites detected in NK cells grown with or without feeder cells, the most enriched pathways were not exactly the same. Importantly, our RNA-seq data on PRDM1-knockout NK cells validated the regulatory role of PRDM1 on a fraction of these PRDM1-bound genes and interestingly, PRDM1 appeared to be an activator for some of the genes, including those encoding immune checkpoint molecules TIGIT, HAVCR2, and IL-1R8. We also utilized ATAC-seq to examine the chromatin accessibility of NK cells grown with feeder cells or with IL-2 alone. We identified differentially enriched pathways for the NK cells cultured under different conditions. When compared with the PRDM1 ChIP-seq, we found that those PRDM1 binding sites that contain a consensus PRDM1 motif were less likely to be accessible to transposase than those without the PRDM1 motif, thereby confirming the transcriptional repressor role of PRDM1. To further understand how PRDM1 regulates its target genes in NK cells, we performed mass spectrometric analysis on the protein complexes associated with PRDM1. We were able to identify the interaction of PRDM1 with the corepressor Groucho (TLE3) and components from the SIN3, NCoR, and NuRD complex, which have been previously reported in mouse plasma cells. Surprisingly, the protein compositions of the PRDM1-associated complexes from the two NK cell populations were very different. Although PRDM1 is generally associated with transcriptional repression, we also detected the association of PRDM1 with transcriptional activators or coactivators, such as the RUNX-binding protein CBFβ and the T-Box family member EOMES, which may thereby upregulate some of the PRDM1 target genes. In summary, we found that PRDM1 binds and regulates an extensive network of genes responsible for NK cell activation and function. Extrinsic stimuli, as provided by the feeder cells, can alter the extent and profile of PRDM1 binding as well as the associated protein complexes and hence alter its regulatory function in NK cells. Disclosures No relevant conflicts of interest to declare.

Statins inhibit proliferation and cytotoxicity of a human leukemic natural killer cell line

BackgroundNatural killer cells comprise the body’s first line of defense against virus-infected cells. As is true of all lymphocytes, natural killer cell malignancies can develop, however natural killer cell leukemias can be very difficult to treat due to their intrinsic resistance to chemotherapeutic agents. With the recent understanding that statin drugs may have anti-cancer properties, our investigations have focused on the ability of statins to inhibit the growth and cytotoxicity of the YT-INDY natural killer cell leukemia cell line.ResultsOur findings indicate that several statin compounds can inhibit YT-INDY proliferation disrupt cell cycle progression and abrogate natural killer cell cytotoxicity. Since natural killer cell leukemia cytotoxicity may play a role in the pulmonary damage seen in these patients, this is an important finding. Cytotoxicity, proliferation and cell cycle progression could be restored by the addition of mevalonate, signifying that the statin effects are brought about through HMG CoA reductase inhibition. The mevalonate pathway intermediate geranylgeranyl pyrophosphate, but not other intermediates in the mevalonate pathway, partially reversed statin-induced inhibition of YT-INDY proliferation and cytotoxicity. These results suggest that blockage of products made in the latter part of the mevalonate pathway may account for the observed inhibitory effects on YT-INDY proliferation and cytotoxicity. However, geranylgeranyl pyrophosphate could not reverse the statin-induced inhibition of the cell cycle.ConclusionsThese results suggest that the statin drugs should be investigated as a potential therapeutic strategy for human natural killer cell leukemias possibly in combination with chemotherapeutic agents.

Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells via JAK2/STAT3 Pathway Inhibition

Natural killer (NK) cell malignancies, particularly aggressive NK cell leukaemias and lymphomas, have poor prognoses. Although recent regimens with L-asparaginase substantially improved outcomes, novel therapeutic approaches are still needed to enhance clinical response. Resveratrol, a naturally occurring polyphenol, has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities. In this study, we investigated the potential anti-tumour activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92 and NK-YS. Resveratrol induced robust G0/G1 cell cycle arrest, significantly suppressed cell proliferation and induced apoptosis in a dose- and time-dependent manner for all four cell lines. In addition, resveratrol suppressed constitutively active STAT3 in all the cell lines and inhibited JAK2 phosphorylation but had no effect on other upstream mediators of STAT3 activation, such as PTEN, TYK2, and JAK1. Resveratrol also induced downregulation of the anti-apoptotic proteins MCL1 and survivin, two downstream effectors of the STAT3 pathway. Finally, resveratrol induced synergistic effect on the apoptotic and antiproliferative activities of L-asparaginase against KHYG-1, NKL and NK-92 cells. These results suggest that resveratrol may have therapeutic potential against NK cell malignancies. Furthermore, our finding that resveratrol is a bonafide JAK2 inhibitor extends its potential benefits to other diseases with dysregulated JAK2 signaling.

Natural killer cell regulation by microRNAs in health and disease.

Natural killer (NK) cells are innate immune lymphocytes that are critical for normal host defense against infections and mediate antitumor immune responses. MicroRNAs (miRNAs) are a family of small, noncoding RNAs that posttranscriptionally regulate the majority of cellular processes and pathways. Our understanding of how miRNAs regulate NK cells biology is limited, but recent studies have provided novel insight into their expression by NK cells, and how they contribute to the regulation of NK cell development, maturation, survival, and effector function. Here, we review the expression of miRNAs by NK cells, their contribution to cell intrinsic and extrinsic control of NK cell development and effector response, and their dysregulation in NK cell malignancies.

Leucemia/linfoma agressivo de células NK/T.

The authors describe an unusual case of a young man presenting with fever, asthenia, anorexia and jaundice, associated to hepatosplenomegaly, evolving rapidly to multiorganic failure. Final diagnosis revealed an aggressive NK cell leukemia/lymphoma associated to the Epstein-Barr virus (EBV). The diagnosis, suggested clinically and after bone marrow immunophenotyping, was confirmed by morphologic and immunohistochemical findings on the post-mortem hepatic and splenic biopsy .The tumor cells were positive for CD3 and cytotoxic molecules, TIA, granzyme B and perforin. The herein reported case is a rare clinical entity, only recently recognized and with a difficult early diagnosis. We emphasize the necessity to exclude a Natural Killer cell malignancy in cases with identical characteristics.

PRDM1 is a tumor suppressor gene in natural killer cell malignancies

Natural killer cell lymphoma (NKCL) constitutes a rare and aggressive form of non-Hodgkin lymphoma, and there is little insight into its pathogenesis. Here we show that PRDM1 is a tumor suppressor gene in NKCLs that is inactivated by a combination of monoallelic deletion and promoter CpG island hypermethylation. We observed monoallelic deletion of PRDM1 loci in 8 of 18 (44%) NKCL cases. The other allele showed significant promoter methylation in 12 of 17 (71%) cases. In support of its role as a tumor suppressor gene, the reconstitution of PRDM1 in PRDM1-null NK cell lines led to G2/M cell cycle arrest, increased apoptosis, and a strong negative selection pressure with progressive elimination of PRDM1-expressing cells, which was enhanced when IL-2 concentration is limiting. We observed a progressive increase in PRDM1 expression--in particular, PRDM1α--in normal NK cells in response to IL-2 and in normal NK cells activated with an engineered NK cell target, K562-Cl9-mb21, suggesting its role in NK cell homeostasis. In support of this role, knockdown of PRDM1 by shRNA in normal NK cells resulted in the positive selection of these cells. We identified MYC and 4-1BBL as targets of PRDM1 in NK cells. Disruption of homeostatic control by PRDM1 may be an important pathogenetic mechanism for NKCL.

State of the art in natural killer cell malignancies

The recently updated World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, published in 2008, has made great advances in revising the disorders previously included in the pool of natural killer (NK) cell tumors. Although NK cell neoplasms represent a relatively rare group of diseases, accounting for <5% of all lymphoid neoplasms, they include very distinctive conditions both clinically and pathologically. This family of diseases includes the most indolent clinical forms, such as the provisional new entry of chronic lymphoproliferative disorder of NK cells (CLPD-NK) in the WHO classification, as well as one of the most fatal diseases recognized in medical oncology, aggressive NK cell leukemia (ANKL), which is characterized by a prognosis of weeks, or even days. In addition, some disorders previously identified as blastic NK cell lymphoma within the NK cell system have been more properly defined and included in the blastic plasmacytoid dentritic cell neoplasms, although rare cases of bona fide immature NK lymphoid tumors (now classified as NK cell lymphoblastic leukemia/lymphoma) have been reported in the literature. This paper focuses on recent concepts and progress in morphology, pathogenesis, clinicopathological features, treatment approaches, and outcomes of NK cell malignancies.

Diagnosis and management of natural killer-cell malignancies

Natural killer (NK)-cell malignancies are uncommon neoplasms, which have been referred to as polymorphic reticulosis or angiocentric T-cell lymphomas in the past. In the current WHO classification, they are categorized as extranodal NK/T-cell lymphoma, nasal type and aggressive NK-cell leukemia. NK-cell malignancies show a geographical predilection for Asian and South American populations and are rare in the west. Pathologically, NK-cell lymphomas show a polymorphic neoplastic infiltrate with angioinvasion and angiodestruction. The lymphoma cells are CD2+, cytoplasmic CD3ε+ and CD56+, with germline T-cell receptor gene. There is an almost invariable clonal episomal infection with Epstein–Barr virus. Clinically, NK-cell lymphomas can be classified into nasal, non-nasal and aggressive lymphoma/leukemia subtypes. Most nasal NK-cell lymphomas present with stage I/II disease. The early use of radiotherapy, either alone or concomitantly/sequentially with chemotherapy, is the most important factor in achieving successful treatment. Many stage I/II patients receiving radiotherapy alone fail systemically, so the use of chemotherapy is also considered necessary. Chemotherapy is indicated for stage III/IV nasal NK-cell lymphoma, and the non-nasal and aggressive subtypes. Recent regimens that incorporate the use of L-asparaginase have resulted in substantial improvements in outcome in high-risk, refractory or relapsed patients. High-dose chemotherapy and hematopoietic stem-cell transplantation with autologous or allogeneic hematopoietic stem cells may be beneficial to selected patients. Prognostication of patients with clinical prognostic models and presentation circulating Epstein–Barr DNA load may be useful in the stratification of patients for various treatment modalities.

High-dose chemotherapy and hematopoietic SCT in the management of natural killer-cell malignancies

Natural killer (NK)-cell lymphomas are aggressive. Patients with early (stage I/II) diseases respond favorably to radiotherapy and chemotherapy. Patients with relapses and advanced (stage III/IV) diseases have poor outcome. To improve treatment results, high-dose chemotherapy with hematopoietic SCT (HSCT) has been performed. A review of 57 published cases of autologous HSCT showed the status pre-HSCT as the only significant prognostic factor. HSCT at CR had the best survival. As patients achieving CR with chemotherapy and radiotherapy also have favorable outcome, a definite advantage of autologous HSCT cannot be established. Patients with advanced, relapsed or refractory diseases had dismal survivals after autologous HSCT. Allogeneic HSCT had been reported in about 30 patients, with a 2-year OS of 40%. To evaluate the efficacy of allogeneic HSCT, optimal conditioning regimens and a clear graft-versus-lymphoma effect should be defined. Furthermore, clinicopathological characteristics predicting benefits from allogeneic HSCT need to be determined. HSCT is a potential option in NK-cell lymphoma. However, autologous HSCT may not be necessary for good-risk patients in CR. Whether poor risk patients will have improved outcome with autologous HSCT remains to be defined. The role of allogeneic HSCT requires more rigorous future studies.

Genomic analyses reveal global functional alterations that promote tumor growth and novel tumor suppressor genes in natural killer-cell malignancies

Natural killer (NK)-cell malignancies are among the most aggressive lymphoid neoplasms with very poor prognosis. We performed array comparative genomic hybridization analysis on a number of NK cell lines and primary tumors to gain better understanding of the pathogenesis and tumor biology of these malignancies. We also obtained transcriptional profiles of genes residing in these regions and compared them with normal and activated NK cells. Only 30-50% of the genes residing in the gained or deleted regions showed corresponding increased or decreased expression. However, many of the upregulated genes in regions of gain are functionally important for the proliferation and growth of the neoplastic population. Genes downregulated in regions of loss included many transcription factors or repressors, tumor suppressors or negative regulators of the cell cycle. The minimal common region of deletion in 6q21 included three known genes (PRDM1, ATG5 and AIM1) showing generally low expression. Mutations resulting in truncated PRDM1 and changes in conserved amino-acid sequences of AIM1 were detected. Highly methylated CpG islands 5' of PRDM1 and AIM1 correlated with low expression of the transcripts. Reversal of methylation by Decitabine induced expression of PRDM1 and cell death. In conclusion, we have shown a general tumor-promoting effect of genetic alterations and have identified PRDM1 as the most likely target gene in del6q21. ATG5, an essential gene for autophagy and AIM1, a gene implicated in melanoma, may also participate in the functional abnormalities.

Genomic Analyses Reveal Global Functional Alterations That Promote Tumor Growth and Novel Tumor Suppressor Genes in Natural Killer-Cell Malignancies

Background: Natural Killer (NK)-cell lymphomas/leukemias (NKL) account for 1–2 % of all non-Hodgkin lymphomas. Although the incidence of NKL is relatively low, the clinical course of these lymphomas is highly aggressive. To elucidate the recurrent genomic abnormalities and the associated changes in the gene expression profile, we performed GEP and array-CGH studies on seven clinically well defined cases and eight well characterized cell lines derived from NKL patients.Methods: Array-CGH was performed on a tiling BAC array and GEP on an Affymetrix 133 plus2 array. The two data sets were correlated to identify functional alterations associated with the genetic abnormalities. Candidate genes on del 6q21 were identified and further studied for mutations and promoter methylation.Results: Our aCGH study identified frequent recurrent gains (> 25 %) in 1q, 2p, 7q, 13q, 17q and 20pter-qter. Regions of loss with a frequency (> 25%) included 1p, 6q, 7p, 9p, 9q, 10q, 11q, 12q, 13q, 15q, 16p, 17p, 18p, and 18q consistent with other studies. Only 30%-50% of the genes residing in the gained or deleted regions showed corresponding increased or decreased expression. Many of the genes with increased expression were involved in cell proliferation, growth and energy metabolic processes important for the neoplastic cells. In deleted regions, genes showing decreased expression included transcription factors or repressors (e.g. SP4, PRDM1, NCOR1 and ZNF10), tumor suppressors or negative regulators of the cell cycle (e.g. CDKN2C, CDKN2A, CDKN2B, CHFR and FOXO1A). 6q21 is a frequent deleted region and we found a minimal region of involvement including only three known genes (PRDM1, ATG5 and AIM1), that had consistent low expression. Sequence analysis of these genes revealed mutations in PRDM1 and AIM1 in two out of eight cell lines resulting in truncated proteins of PRDM1 and changes in conserved amino acid sequences of AIM1. Only one cell line with a point mutation in a non-conserved region in ATG5 was detected. DNA methylation analysis showed that the promoter of PRDM1 was highly methylated in NK cell lines having low PRDM1 expression. Reversal of promoter methylation with decitabine treatment resulted in increased PRDM1 transcription consistent with down-regulation of PRDM1 expression on promoter methylation. Finally, we observed progressive up-regulation of PRDM1 expression in primary NK cells in response to IL2 treatment suggestive of a role of PRDM1 on the regulation of NK-cell activation.Conclusion: Combination of high resolution genomic and transcriptional profiling in NK-cell malignancies has provided evidence of a general tumor promoting effect of genomic copy number alterations as well as the identification of candidate genes in a commonly deleted region in 6q. Specifically, we propose PRDM1 as a tumor suppressor gene in NK cell malignancies and loss-offunction of PRDM1 in combination with the haplo-insufficiency of ATG5 might have significant roles in the pathogenesis of NK-cell malignancies.

Fluorine-18 fluorodeoxyglucose positron emission tomography in mature T-cell and natural killer cell malignancies

Fluorine-18 fluorodeoxyglucose (FDG)-positron emission tomography (PET) is useful in Hodgkin and B-cell lymphomas. Few data exist on T-cell and natural killer (NK)-cell lymphomas. Thirty consecutive T-cell and NK-cell lymphomas were investigated with PET-computerized tomography (CT). In 12 NK-cell lymphomas, all nasal/extranasal lesions were FDG-avid. In nasal/maxillary areas, FDG-avid tumours were consistently more localised than on CT, suggesting that soft tissue masses on CT were partly due to inflammation. These findings have important implications in radiotherapy planning. In two NK-cell lymphomas, PET did not detect morphologically occult marrow infiltration uncovered by in situ hybridisation for Epstein-Barr-virus-encoded small RNA. In angioimmunoblastic lymphoma (n = 7), peripheral T-cell lymphoma, unspecified (PTCL-U, n = 4) and anaplastic large cell lymphoma (ALCL, n = 3), involved nodal/extranodal sites shown on CT and/or biopsy were concordantly PET-positive. In one PTCL-U, PET detected FDG-avid marrow infiltrations not shown on biopsies. In contrast, cutaneous ALCL (n = 1) and mycosis fungoides (n = 2) showed minimal FDG uptake. In one case of T-cell large granular lymphocyte leukaemia, marrow, nodal and bowel infiltrations were not FDG-avid. PET maximum standardised uptake value did not correlate with clinicopathological features and prognosis. These observations defined the pre-treatment value of PET-CT in T-cell and NK-cell lymphomas. The post-treatment role requires further studies.

The Spectrum of Epstein-Barr Virus-Associated Lymphoproliferative Disease in Korea: Incidence of Disease Entities by Age Groups

This study is to identify the spectrum of Epstein-Barr virus (EBV)-positive lymphoproliferative diseases (LPD) and relationships between these diseases in Korea. The EBV status and clinicopathology of 764 patients, including acute EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV (CAEBV) infections, B-LPD arising in chronic latent EBV infection, T & natural killer (NK) cell non-Hodgkin's lymphomas (NHL), B-NHLs, and Hodgkin's lymphomas (HD), were analyzed. T or NK cell NHLs were the most common forms of EBV-positive NHLs (107/167, 64%); among these, nasal-type NK/T cell lymphomas were the most common (89/107, 83%). According to the age, Burkitt's lymphoma was the most common in early childhood; in teenagers, chronic (active) EBV infection-associated LPD was the most common type. The incidence of NK/T cell lymphoma began to increase from the twenties and formed the major type of EBV-associated tumor throughout life. Diffuse large B cell lymphoma formed the major type in the sixties and seventies. In conclusion, primary infections in early childhood are complicated by the development of CAEBV infections that are main predisposing factors for EBV-associated T or NK cell malignancies in young adults. In old patients, decreased immunity associated with old age and environmental cofactors may provoke the development of peripheral T cell lymphoma, unspecified, and diffuse large B cell lymphoma.

Analysis of Gene Expression Patterns and Gene Copy Number Changes in Human NK Cell Malignancies.

Natural killer (NK)-cell lymphomas/leukemias are a group of aggressive malignancies distinct from the indolent large granular lymphocyte (LGL) proliferations. In the World Health Organization (WHO) classification, NK-cell malignancies are divided into two groups (1) extranodal NK-cell lymphoma, nasal type (ENKL), and (2) aggressive NK-cell leukemia (ANKL). Only three studies of genome wide alteration have been published in these malignancies. We studied eight NK lymphoma cell lines (NK-92, KHYG1, YT, HANK-1, NK-YS, SNK-6, SNK-3 and KAI3) and seven NK lymphomas/leukemias specimens to:determine DNA gains and losses in comparative genomic hybridizations (CGH) and compare these alterations with those reported literature to define recurring chromosomal abnormalities,obtain transcription profiles of NK malignancies and compare them with normal NK cells(resting or IL-2 stimulated), andcorrelate the genomic profiles with transcriptional profiles to identify possible candidate genes involved in lymphoma development.Whole genome tiling arrays, containing 26,819 bacterial artificial chromosomes (BAC) were employed for CGH study.Our CGH study identified frequent recurrent abnormalities that have been reported in all previous studies. These include gains in 17q11.2–q21 (frequency: 40%); 1q22–q23.1 (frequency: 50 %); and 7q21.13–q22.1(frequency: 50%) and all these regions showed at least a frequency of 17% in other reports. A number of regions were also described in at least one other published reports (e.g. 1q32.2–q41, 6p25.5 and 17q 21.2). Regions of loss with a frequency ≥ 20% including 11q23.1–23.2(20%); 7p22.1–p22.2 (40%); 6q21(50%); 6q25.1–25.3(50%); and 17p13–13.1(50%) showed consistent results with previous reports. 6q21 loss was detected in two ANKL cell lines (NK92 and KHYG1) but not in ENKL cell lines . Gains of 8p12–q21.12, 8q22.1–q24.13, and 8q24.21 were also unique to ANKL cell lines.Transcriptional profiling using affymetrix gene chips on these cell lines and tumor specimen were compared with normal resting and IL-2 activated NK cells. We observed significant down-regulation (6–9 folds) of killer cell Ig-like receptor (KIR) (KIR2DL3, KIR2DS1, KIR3DL1, KIR3DL2) in NK-cell lymphoma as compared to normal NK cells. This may be related to the restricted clonal expression of the KIRs in neoplasms. High expression of pro-proliferative genes (e.g. Cyclin B1, A2, BUB1, CDC2, CDC20, CDCA1, CDCA3, and CDC7) was observed in NK lymphoma. On examining gene signatures/pathways (using BRB-array Tools), we observed significant increased expression of genes (p<0.001) involved in oxidative-phosphorylation /electron transport chain and MYC target signature in NK-cell lymphoma. A number of genes involved in TGF-beta signaling (p<0.0001) were down-regulated in lymphoma samples as compared to normal cells. This may correlate with the deletion of two crucial regulators (TGFBR3 and SMAD7) of this pathway, located at 1p32–33 and 18q21.1 regions respectively. In summary, combination of the genomic and transcriptional profiling by microarrays may lead to the identification of candidate genes in genomic alterations and provide clues to the pathogenesis of NK-cell malignancy.

Natural killer-cell malignancies: diagnosis and treatment

Natural killer (NK)-cell malignancies are uncommon diseases. Previously known as polymorphic reticulosis or angiocentric T-cell lymphomas, they are classified by the World Health Organization as NK/T-cell lymphoma, nasal type and aggressive NK-cell leukemia. They are prevalent in Asia and South America, but exceptionally rare in western countries. Pathologically, NK-cell lymphomas show a polymorphic neoplastic infiltrate with an angioinvasive and angiodestructive pattern. Lymphoma cells are characteristically CD2+, CD56+ and cytoplasmic CD3epsilon+. T-cell receptor gene is germline, and clonal Epstein-Barr virus (EBV) infection is almost invariably. Clinically, they can be divided into nasal, non-nasal, and aggressive lymphoma/leukemia subtypes. Most nasal NK-cell lymphomas present with stage I/II disease, and frontline radiotherapy is the most important key to successful treatment. Many stage I/II patients treated with radiotherapy fail systemically, implying that concomitant chemotherapy may be needed. Chemotherapy is indicated for advanced nasal NK-cell lymphoma, and the non-nasal and aggressive subtypes. However, treatment results are unsatisfactory. High-dose chemotherapy with hematopoietic stem cell transplantation may be beneficial to selected patients. The International Prognostic Index and presentation EBV DNA load is of prognostic significance and may be useful in the stratification of patients for various treatment modalities.

Detection of the CD56+/CD45- immunophenotype by flow cytometry in neuroendocrine malignancies.

Antibodies against CD56 are primarily used in flow cytometric studies to detect natural killer cells. However, they may be useful in the identification of neuroendocrine malignancies, especially if the cells do not express CD45, indicating a non-leucocyte origin. A retrospective review was conducted on all solid tissue flow cytometric studies performed between January 1997 and September 2001, to identify all cases with a CD56+/CD45- immunophenotype. Twelve neuroendocrine malignancies (five metastatic small cell carcinomas, three Merkel cell carcinomas, two metastatic undifferentiated neuroendocrine carcinomas, one metastatic pancreatic neuroendocrine carcinoma, and one neuroblastoma) were identified. CD56+/CD45- neuroendocrine malignancies are only rarely detected in the flow cytometric analysis of solid tissue samples. However, the recognition of this immunophenotype is important to avoid their misclassification as natural killer cell malignancies. Furthermore, flow cytometry assists in the rapid identification of such cases, so that appropriate immunohistochemical studies can be performed to facilitate their correct diagnosis.

Natural killer cell malignancies: clinicopathologic and molecular features.

Malignancies of natural killer (NK) cells have increasingly been recognized as distinct clinicopathological entities. The tumor cells are characterized by an immunophenotype of CD2+, surface CD3-, cytoplasmic CD3epsilon+, and CD56+. The T cell receptor gene is in germline configuration, and a consistent association with Epstein-Barr virus is demonstrable. Pathologically, the tumor cells show variable cytological appearances, with frequent angioinvasion and angiocentricity associated with zonal necrosis. Clinically, most cases affect the nasal cavity or other parts of the upper aerodigestive tract, and are referred to as nasal NK cell lymphoma. A minority involve extranasal sites such as the skin, gastrointestinal tract and testis, and are often referred to as extranasal NK cell lymphoma. A particularly aggressive form presents fulminantly as disseminated disease, sometimes with a leukemic phase, and is referred to as aggressive NK cell lymphoma/leukemia. Cytogenetic and molecular analysis have shown DNA losses at chromosomes 6q, 11q, 13q and 17p to be recurrent aberrations in NK cell malignancies. Frequent DNA gains are also found in chromosomes 1p, 6p, 11q, 12q, 17q, 19p, 20q, and Xp. These regions of DNA losses and gains should be targets for further investigation in order to understand the molecular pathogenesis of this lymphoma. Finally, optimal treatment modalities need to be determined, as all subtypes of NK cell malignancies are associated with a poor prognosis.

Specific Patterns of Gene Methylation in Natural Killer Cell Lymphomas: p73 Is Consistently Involved

Aberrant methylation of promoter CpG regions is a putative mechanism whereby tumor suppressor genes are inactivated. We used a candidate gene approach to investigate the patterns and significance of this epigenetic change in natural killer (NK) cell malignancies. Thirty-three patients were studied for promoter methylation in five putative tumor suppressor genes by methylation-specific polymerase chain reaction (MSP), which has a sensitivity of 10(-3). The p73 gene was methylated in 94% of cases, a frequency that is the highest known for any human malignancy. In the NK cell lymphoma line NK92, p73 was also completely methylated, and the p73 transcript was correspondingly not detectable by quantitative polymerase chain reaction. Treatment of the cell line with 5-azacytidine, a demethylation reagent, led to demethylation of the p73 promoter and reinduction of p73 gene expression. These results suggested that promoter CpG methylation might be an important mechanism in suppressing p73 gene expression in NK cells. Other methylated genes included hMLH1 (63%), p16 (63%), p15 (48%), and RAR beta (47%). Methylation of two or more genes occurred in 88% of cases. With promoter methylation as a molecular marker, MSP identified two cases of occult marrow metastasis. Interestingly, the primary tumor and metastasis showed different methylation patterns, implying that separate clonal evolutions might have occurred at these sites. Furthermore, MSP also identified tumor infiltration in random oropharyngeal biopsies in a case where histological examination could not show evidence of tumor involvement. We conclude that NK cell malignancies show a specific pattern of promoter methylation, with p73 being consistently involved. These results suggest that p73 may be an important target in the neoplastic transformation of NK cells, and the demonstration of its methylation may serve as a potential molecular tool for NK cell lymphoma detection.

Consistent Patterns of Allelic Loss in Natural Killer Cell Lymphoma

Natural killer (NK) cell lymphomas are a group of rare but highly aggressive malignancies. Clinically, they can be divided into nasal NK cell lymphomas, nonnasal NK cell lymphomas, and aggressive NK cell lymphoma/leukemia. To determine the patterns of genetic deletions in these tumors, we performed loss of heterozygosity (LOH) analysis on 15 cases (11 nasal and four nonnasal), and fluorescence in situ hybridization on three cases of aggressive lymphoma/leukemia. A panel of 41 microsatellite loci on chromosomes 6q, 11q, 13q, and 17p were investigated. LOH at chromosomes 6q and 13q was frequently detected in NK cell lymphomas, being found in 80 and 66.7% of cases, respectively. LOH at chromosomes 11q and 17p was less common, being found in 28.6 and 30.8% of cases, respectively. Most tumors showed multiple loci deletions at different chromosomal regions, but several patterns of LOH could be defined. LOH at chromosome 6q was found in 90.9% of nasal NK cell lymphomas, but only in 50% of nonnasal NK cell lymphomas. LOH at chromosome 13q was found in 63.6% of nasal NK cell lymphomas and 75% of nonnasal NK cell lymphomas. For nasal NK cell lymphomas, LOH at 13q was found in 33.3% of cases at presentation, but 100% of cases at relapse. Five tumors showed LOH in only one chromosomal region, involving 6q in three cases (two nasal and one nonnasal), and 13q in two cases (both nonnasal). For the three cases of aggressive NK cell lymphoma/leukemia studied by fluorescence in situ hybridization, DNA loss at 13q14 and 17p13 regions were demonstrated. 17p13 seemed to be more commonly involved in aggressive than nasal and nonnasal NK cell lymphomas. Our results suggested that consistent patterns of LOH could be defined in NK cell malignancies. These deleted loci may contain genes important in the initiation and progression of this lymphoma.