Comparative genomic hybridization study of nasal-type NK/T-cell lymphoma

BACKGROUND: Nasal-type NK/T-cell lymphoma is a rare type of non-Hodgkin's lymphoma. The genetic changes associated with pathogenesis have not been well defined. This study investigates the nonrandom genetic alteration of nasal-type NK/T-cell lymphoma. METHODS: Nine cases were studied. Comparative genomic hybridization (CGH) was carried out using fresh tumor tissues of seven nasal-type NK/T-cell lymphomas. To complement the data by CGH, loss of heterozygosity (LOH) of chromosomes 6q, 1p, and 17p using polymorphic markers and p53 gene mutation by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) were analyzed. RESULTS: The DNA copy number changes of seven nasal-type NK/T-cell lymphomas were gains on chromosomes 2q(5), 13q(4), 10q(3), 21q(2), 3q(2), 5q(2), and 17q(2), and losses involving chromosomes 1p(4), 17p(4), 12q(3), 13q(2), and 6q(1). One of six cases informative for at least two markers for chromosome 6q showed LOH at D6S300, D6S1639, D6S261, D6S407, and D6S292. Two cases showing loss of 1p and 17q by CGH revealed LOH at D1S214, D1S503, and D17S559. P53 mutation was detected in exon 8 in one of nine cases. CONCLUSIONS: Frequent DNA losses at 1p, 17p, and 12q and gains at 2q, 13q, and 10q suggested that these regions could be targets for further molecular genetic analysis to investigate tumor suppressor genes or oncogenes associated with tumorigenesis of NK/T-cell lymphoma. Infrequent alteration of 6q contrary to previous studies raises doubt about an implication of 6q loss in the pathogenesis of early-stage NK/T-cell lymphoma. Further studies on more defined cases are required to verify their association.     

NK/T-cell lymphoma of bilateral adrenal glands in a patient with pyothorax

ABSTRACT: Primary lymphoma of adrenal glands is rare, and non-B-cell lymphoma associated with pyothorax is also very rare. Here we report the first autopsy case of non-B-cell lymphoma in bilateral adrenal glands of a 79-year-old woman with pyothorax who had an aggressive clinical course. Immunohistochemically, tumor cells showed CD3+, CD45RO+, CD5-, CD7-, CD4-, CD8-, CD10-, CD20-, CD30-, CD79a-, CD138-, CD56-, granzyme B-, TIA-1+ and ALK-. In addition, tumor cells were strongly EBER1-positive by in situ hybridization. In genomic DNA of tumor cells, T-cell receptor rearrangements were not detected by southern blotting. We finally diagnosed this case as extranodal NK/T-cell lymphoma (nasal type).Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/8050621197741854.     

NK cell lymphoma, nasal type, with massive lung involvement: a case report

Extranodal NK/T cell lymphoma, nasal type, is an Epstein–Barr virus-associated lymphoma that most commonly involves the nasal cavity and upper respiratory tract. Lung involvement by NK/T cell lymphoma is rare and seldom reported in the literature. We describe the unusual case of a 41-year-old male with NK cell lymphoma, nasal type, who presented with massive secondary lung involvement 2.5 years after the detection of a retroperitoneal mass. The diagnosis was made by open lung biopsy. Despite aggressive treatment, the patient died shortly after the initiation of therapy. Lung involvement by NK/T cell lymphoma occurs most commonly as part of widely disseminated disease and carries a poor prognosis for the patient. Novel agents and innovative therapies need to be developed for this aggressive lymphoma.     

Nodal presentation of nasal-type NK/T-cell lymphoma. Report of two cases with fine needle aspiration cytology findings

BACKGROUND: Epstein-Barr virus (EBV)-associated NK/T-cell lymphoma typically occurs in extranodal sites, such as nasal cavity, nasopharynx, gastrointestinal tract, skin, testis and salivary gland. Secondary lymph node involvement is rarely encountered until late in the disease course. The fine needle aspiration cytology of NK/T-cell lymphoma with a nodal presentation has not been described before. CASES: Two cases of nasal-type (extranasal) NK/T-cell lymphoma with a nodal presentation were seen at Pamela Youde Nethersole Eastern Hospital, Hong Kong. Both patients presented with submandibular lymph node enlargement but unremarkable peripheral blood and bone marrow findings. Fine needle aspiration cytology was available in both cases, showing a heterogeneous population of small to medium-sized lymphoid cells, follicular center cells, plasma cells, eosinophils and some histiocytes. The medium-sized lymphoid cells showed readily discernible nuclear atypia with an irregular nuclear outline. Cell block sections revealed occasional lymphoid cells with pleomorphic nuclei. Immunocytochemical study confirmed the presence of CD56-positive lymphoma cells. In situ hybridization for EBV-encoded RNA also revealed positive nuclear signals. Histologic examination of the surgical biopsies showed interfollicular expansion by malignant lymphoid cells. Immunoglobulin heavy chain gene and T-cell receptor gene rearrangement studies demonstrated a germline pattern, confirming the putative NK (natural killer cell), non-B and non-T lineage of the lymphoma cells. CONCLUSION: Nodal presentation of NK/T-cell lymphoma, though rare, is diagnosable on the basis of fine needle aspiration biopsy alone, especially in view of its distinctive immunophenotype and EBV association. Recognition of the subtle but definite cytologic atypia of malignant lymphoid cells and presence of an appropriate background (including more eosinophils than usual), together with proper application of ancillary techniques, is crucial to arriving at a correct diagnosis.     

Biologic,Immunocytochemical, and Cytogenetic Characterization of Two New Human Melanoma Cell Lines: IIB-MEL-LES and IIB-MEL-IAN

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD₃ and GD₂ gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10,20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.     

Collecting Duct Carcinomas Represent a Unique Tumor Entity Based on Genetic Alterations

Collecting duct carcinoma (CDC) is a rare renal neoplasm that is associated with poor prognosis due to its highly aggressive course and limited response to immuno- or chemotherapy. Histologically, CDC is defined as a subtype of renal cell carcinomas, but in some cases, it is difficult to differentiate from urothelial carcinomas (UC). Therefore the aim of this study was to determine genetic alterations of CDC in comparison to that of urothelial carcinomas of the upper urinary tract (UUT-UC) to clarify the histological origin of this rare tumor entity. Twenty-nine CDC samples were obtained from seven different German centers and compared with twenty-six urothelial carcinomas of the upper urinary tract. Comparative genomic hybridization (CGH) was used to investigate the genetic composition of patients’ tumors and allowed the detection of losses and gains of DNA copy numbers throughout the entire genome. The clinical data were correlated with CGH results. CGH analysis of CDC revealed DNA aberrations in many chromosomes. DNA losses were more frequently observed than gains, while high-level amplifications were not detected. The mean frequency of CDC chromosomal aberrations (4.9/case) was slightly lower than that in UUT-UC (5.4/case). Recurrent CDC DNA losses occurred at 8p (n=9/29), 16p (9/29), 1p (n=7/29) and 9p (n=7/29), and gains occurred in 13q (n=9/29). In contrast to CDC, the most frequently detected UUT-UC DNA aberration was a loss at 9q (n=13/26). DNA losses at 9q, 13q and 8q as well as gains at 8p showed significant variations in UUT-UC compared to CDC. There was no correlation between the patients’ clinical course and the presence or absence of these recurrent genetic alterations. CDCs are characterized by a different genetic pattern compared to UUT-UC. Regarding the published data on renal cell carcinoma, we conclude that CDC appears to be a unique entity among kidney carcinomas.     

Specific sites for EBV association in the Namalwa Burkitt lymphoma cell line and in a lymphoblastoid line transformed in vitro with EBV

Localization of Epstein-Barr virus (EBV) DNA was studied by in situ hybridization on chromosomes from the Namalwa Burkitt lymphoma cell line and from a lymphoblastoid cell line transformed in vitro (ATL9/g). The five chromosome bands 1p32, 1q31, 5q21, 13q21, and 16p13 showed the presence of EBV DNA in both of the lines. Grain deposition at the site on chromosome 1q of the Burkitt line was particularly intense. It was also found that EBV DNA in the lymphoblastoid cell line co-localized with a stable achromatic gap at 1p32 whose presence seems to confer a proliferative advantage on the cells.     

CD16+ NK lymphoproliferative disorders: cellular and molecular characterization

We studied the mononuclear cells obtained from 2 patients with CD16+ lymphoproliferative disorders. In both subjects, over 80% of the circulating peripheral blood mononuclear cells were CD16+, CD2+, CD7+, CD3-, CD4-, and CD8-. In 1 patient, greater than 60% of the cells expressed HLA-DR and HLA-DQ gene products. Functional analysis of the natural killer (NK) cell activity of cells from this patient demonstrated 76% killing of K562 targets at ratios as low as 1:1 effector:targets. Karyotype analysis demonstrated a deletion on the long arm of chromosome 6, supporting the contention that the lymphocytosis in this patient was due to a clonally expanded population of cells. In additional studies, Southern blot analysis of DNA extracted from cells of this patient revealed that the beta-chain of the T cell receptor was of germ line configuration. This information supports the hypothesis that the clonally expanded NK population in this patient is of a lineage distinct from T cells and represents a true NK leukemia.     

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein expression pattern distinguishes different lymphoid neoplasms

To identify proteins associated with the histological subtypes of lymphoid neoplasms, we studied the proteomes of 42 cell lines from human lymphoid neoplasms including Hodgkin's lymphoma (HL; four cell lines), B cell malignancies (19 cell lines), T cell malignancies (16 cell lines), and natural killer (NK) cell lymphoma (three cell lines). The protein spots were sequentially selected by (i) Wilcoxon or Kruskal-Wallis tests to find the spots whose intensity was significantly (p <0.05) different among the cell line groups, (ii) by statistical-learning methods to prioritize the spots according to their contribution to the classification, and (iii) by unsupervised classification methods to validate the classification robustness by the selected spots. The selected spots discriminated (i) between HL cells and other cells, (ii) between the cells from B cell malignancies, T cell malignancies, and NK cell lymphoma cells, and (iii) between HL cells and anaplastic large cell lymphoma cells. Among the 31 informative protein spots, MS identified 24 proteins corresponding to 23 spots. Previous reports did not correlate these proteins to lymphocyte differentiation, suggesting that a proteomic study would identify the novel mechanisms responsible for the histogenesis of lymphoid neoplasms. These proteins may have potential as differential diagnostic markers for lymphoid neoplasms.     

Characterization of a subset of human B lymphocytes interacting with natural killer cells

Tonsil B cells were analyzed for their capacity to interact directly with NK cells in vitro. A specific, direct interaction between NK cells and B cells could be detected by direct conjugation and by cold target inhibition using the B lymphoblastoid cell line BJA.B as a labeled target. The data further suggest that the B cell interaction with NK cells specifically activates the NK effectors and induces their production of IFN-gamma. The NK-interactive population of tonsil B cells were characterized as low-buoyant density cells (by Percoll gradient fractionation) that stained more brightly with Hoechst 33342, both characteristics of activated B cells. Immunofluorescent staining of NK cell-B cell conjugates allowed determination of the cell-surface antigenic phenotype of conjugate-forming B cells. B cell targets were ICAM-1bri, 4F2+, TfR+, CD32+, BB1+, and CD77-. They tended to be CD38-, but overlapped the CD38+ population. No correlation was seen with CD37, CD44, CD75, CD76, HC2, or Ig kappa. This phenotype is most consistent with a late activation stage of differentiation, just before and overlapping the expression of CD38. These B cells do not appear significantly sensitive to NK-mediated cytolysis, suggesting that NK cell cytokine synthesis and secretion (e.g., IFN-gamma) may be more important in the NK cell regulation of the humoral response.     

Molecular pathogenesis of lymphomas of mucosa-associated lymphoid tissue—from(auto)antigen driven selection to the activation of NF-?B signaling

Lymphomas of mucosa-associated lymphoid tissue(MALT) are typically present at sites such as the stomach, lung or urinary tract, where lymphoid tissues scatter in mucosa lamina propria, intra- or sub-epithelial cells. The infection of certain pathogens, such as Helicobacter pylori, Chlamydophila psittaci, Borrelia burgdorferi, hepatitis C virus, or certain autoantigens cause these sites to generate a germinal center called the "acquired lymphoid tissue". The molecular pathogenesis of MALT lymphoma is a multi-step process. Receptor signaling, such as the contact stimulation of B cell receptors and CD4 positive T cells mediated by CD40/CD40-ligand and T helper cell type 2 cytokines like interleukin-4, contributes to tumor cell proliferation. A number of genetic alterations have been identified in MALT lymphoma, and among them are important translocations, such as t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21) and t(3;14)(p13;q32). Fusion proteins generated by these translocations share the same NF-?B signaling pathway, which is activated by the caspase activation and recruitment domain containing molecules of the membrane associated guanylate kinase family, B cell lymphoma-10 and MALT1(CBM) protein complex. They act downstream of cell surface receptors, such as B cell receptors, T cell receptors, B cell activating factors and Toll-like receptors, and participate in the biological process of MALT lymphoma. The discovery of therapeutic drugs that exclusively inhibit the antigen receptor signaling pathway will be beneficial for the treatment of B cell lymphomas in the future.     

鼻NK／T细胞淋巴瘤间质清道夫受体A的表达及意义

目的 观察清道夫受体A（scavenger receprorA,SR-A）在鼻NK／T细胞淋巴瘤间质中的表达,探讨其意义。方法 对鼻NK／T细胞淋巴瘤,鼻B细胞淋巴瘤以及鼻部炎症的石蜡标本进行HE染色和SP免疫组织化学染色检测SR-A的表达。结果 SR-A蛋白在鼻NK／T细胞淋巴瘤,鼻B细胞淋巴瘤和鼻部炎症中阳性率分别为92．7％,29．2％和6．25%。统计学分析发现,鼻NK／T细胞淋巴瘤中SR-A的表达与B细胞淋巴瘤和炎症中SR-A的表达均有显著差异（P〈0．001）。结论 SR-A可能在鼻NK／T细胞淋巴瘤的诊断及鉴别诊断中有一定的参考价值。     

Characterization of the immunophenotype and the metastatic properties of a murine T-lymphoma cell line. Unexpected expression of cytoplasmatic CD4

We report the first characterization of a mouse T-lymphoma cell line that surprisingly expresses cytoplasmatic (cy) yCD4. Phenotypically, LBC cells are CD5+, CD8+, CD16+, CD24+, CD25+, CD2-/dim, CD3-/dim, TCRbeta-/dim, TCRgammadelta, CD154 , CD40-, and CD45R. Coexpress cyTCRbeta, cyCD3, cyCD4, and yet lack surface CD4 expression. Transplantation of LBC cells into mice resulted in an aggressive T-lymphoblastic lymphoma that infiltrated lymph nodes, thymus, spleen, liver, ovary, and uterus but not peripheral blood or bone marrow. LBC cells display a modal chromosome number of 39 and a near-diploid karyotype. Based on the characterization data, we demonstrated that the LBC cell line was derived from an early T-cell lymphocyte precursor. We propose that the malignant cell transformation of LBC cells could coincide with the transition stage from late double-negative, DN3 (CD4- CD8 CD44-/low, CD25+) or DN4 (CD4-low, CD8-/low, CD44-, CD25-) to double-positive (DP: CD4+CD8+) stage of T-cell development. LBC cells provide a T-lymphoblastic lymphoma model derived from a malignant early T-lymphocyte that can be potentially useful as a model to study both cellular regulation and differentiation of T-cells. In addition, LBC tumor provides a short latency neoplasm to study cellular regulation and to perform preclinical trials of lymphoma-relatel clisorders.     

Immunologic monitoring studies in advanced cancer patients treated with recombinant human gamma interferon (IFN-gamma 4A)

Thirty-nine patients with a variety of advanced malignancies were treated with recombinant IFN-gamma 4A (AMGen, specific activity 1 to 5 x 10(7) U/mg protein). IFN-gamma 4A was administered at a dose of 10-2,000 micrograms/m2/d. Following a 2-week rest, a maintenance phase was continued with injections 3 d/wk. Immunologic monitoring studies were performed on patients' peripheral blood cells before administration of IFN-gamma 4A, then on Days 15 and 90. Flow cytometric analysis was used to determine the absolute number of CD 3+, CD 4+, CD 8+, CD 19+, and CD 16+ cells using a panel of monoclonal antibodies. Natural killer (NK) cell function was assayed by monitoring lysis of the K562 cell line in the Cr51 release assay. Changes from baseline were observed on Days 15 and 90 in all parameters studied, although the ratio of helper to suppressor cells seemed to remain within the normal range. Whereas there were no substantial changes in CD 3+ and CD 4+ cells on Day 15, IFN-gamma 4A had an enhancing effect on CD 8+, CD 19+, and CD 16+ cells. This trend continued at Day 90 only for CD 19+ and CD 16+ cells at the higher dose levels. An increase in functional NK cell activity at Day 15 was less noted on Day 90. Comparison of intravenous (IV) to intramuscular-subcutaneous (IM-SC) administration showed differences in the effect on lymphocyte subpopulations at 450 and 1,000 micrograms. The effect of IFN-gamma 4A on the equilibrium among lymphocyte subpopulations and the possibility of its role in combination therapy with other biologic response modifiers are discussed.     

Natural killer cell clones can efficiently process and present protein antigens.

NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 3-13 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.     

A t(4;22) in a meningioma points to the localization of a putative tumor-suppressor gene.

Cytogenetic analysis of meningioma cells from one particular patient (MN32) displayed the stem-line karyo-type 45, XY, -1, 4p+, 22q-, 22q+, which thus had rearrangements of both chromosomes 22. The 22q+ marker appeared as a dicentric: 22 pter----q11::1p11----qter. The reciprocal product of this translocation has presumably been lost because it lacked a centromere. The 22q- chromosome also appeared to have lost sequences distal to band q11. We assumed that this marker could have been the result of a reciprocal translocation between chromosomes 4 and 22. To investigate the 4p+ and 22q- chromosomes in more detail, human-hamster somatic cell hybrids were constructed that segregated the 22q- and 4p+ chromosomes. Southern blot analysis with DNA from these hybrids showed that sequences from 22q were indeed translocated to 4p+ and that reciprocally sequences from 4p were translocated to 22q-, demonstrating a balanced t(4;22)(p16;q11). On the basis of these results we presume that in this tumor a tumor-suppressor gene is deleted in the case of the 22q+ marker and that the t(4;22) disrupts the second allele of this gene. The latter translocation was mapped between D22S1 and D22S15, a distance of 1 cM on the linkage map of this chromosome. The area in which we have located the translocation is within the region where the gene predisposing to neurofibromatosis 2 has been mapped.     

Amplification at 9p in cervical carcinoma by comparative genomic hybridization.

DNA copy number changes were studied by comparative genomic hybridization on 10 tumor specimens of squamous cell carcinoma of cervix obtained from Korean patients. DNA was extracted from paraffin-embedded sections after removal of non-malignant cells by microdissection technique. Copy number changes were found in 8/10 tumors. The most frequent changes were chromosome 19 gains (n=6) and losses on chromosomes 4 (n=4), 5 (n=3), and 3p (n=3). A novel finding was amplification in chromosome arm 9p21-pter in 2 cases. Gains in 1, 3q, 5p, 6p, 8q, 16p, 17, and 20q and losses at 2q, 6q, 8p, 9q, 10p, 11, 13, 16q, and 18q were observed in at least one of the cases.