Establishment of the T-cell large granular lymphocyte leukemia cell line MOTN-1 carrying natural killer-cell antigens

A novel interleukin-2 (IL-2) dependent leukemia cell line MOTN-1 was established from the peripheral blood of a 63-year-old woman with T-cell large granular lymphocyte (LGL) leukemia in chronic phase. Primary peripheral blood leukemia cells were CD3+, CD5+, CD7+, CD56+, CD94+, CD161+, TcRalphabeta+, and HLA-DR+. The immunoprofile of the established cell line MOTN-1, however, showed CD3-, CD5-, CD7+, CD56+, CD94+, CD159+, CD161+, TcRalphabeta- and HLA-DR+; the MOTN-1 cells were cytoplasmatically positive for CD3varepsilon and the products of the T-cell receptor (TcR) genes beta and gamma. While the TcRbeta and TcRgamma genes were rearranged, the TcRdelta gene was found to be deleted. DNA fingerprinting and chromosome analysis identifying the t(2;6)(q?23;q?21) and t(12;18)(q13;q?22) alterations demonstrated the authenticity and the malignant nature of the cell line. The scientific significance of MOTN-1 lies in (1) the rarity of this type of leukemia cell lines, (2) the co-expression of various T- and natural killer (NK)-cell-associated markers, and (3) its unique chromosomal aberrations.     

A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation.

We present the establishment of a natural killer (NK) leukemia cell line, designated KHYG-1, from the blood of a patient with aggressive NK leukemia, which both possessed the same p53 point mutation. The immunophenotype of the primary leukemia cells was CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16+, CD56+, CD57+ and HLA-DR+. A new cell line (KHYG-1) was established by culturing peripheral leukemia cells with 100 units of recombinant interleukin (IL)-2. The KHYG-1 cells showed LGL morphology with a large nucleus, coarse chromatin, conspicuous nucleoli, and abundant basophilic cytoplasm with many azurophilic granules. The immunophenotype of KHYG-1 cells was CD1-, CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16-, CD25-, CD33+, CD34-, CD56+, CD57-, CD122+, CD132+, and TdT-. Southern blot analysis of these cells revealed a normal germline configuration for the beta, delta, and gamma chains of the T cell receptor and the immunoglobulin heavy-chain genes. Moreover, the KHYG-1 cells displayed NK cell activity and IL-2-dependent proliferation in vitro, suggesting that they are of NK cell origin. Epstein-Barr virus (EBV) DNA was not detected in KHYG-1 cells by Southern blot analysis with a terminal repeat probe from an EBV genome. A point mutation in exon 7 of the p53 gene was detected in the KHYG-1 cells by PCR/SSCP analysis, and direct sequencing revealed the conversion of C to T at nucleotide 877 in codon 248. The primary leukemia cells also carried the same point mutation. Although the precise role of the p53 point mutation in leukemogenesis remains to be clarified, the establishment of an NK leukemia cell line with a p53 point mutation could be valuable in the study of leukemogenesis.     

A monoclonal antibody 1A6 to T-cell recognizes a common antigen expressed on lymphocytes from normal and leukemic patients, and inhibits NK cell activity

A new monoclonal antibody (MAB), IA61gG2a, has been derived from Balb/c mice after immunization with T-cell chronic lymphocytic leukemia (T-CLL). This monoclonal antibody, 1A6 was tested on normal human peripheral blood cells, leukemia patients' cells and leukemia-lymphoma cell lines. The results demonstrated that this antibody reacts with normal human T and B cells, but not with erythrocytes, granulocytes or platelets. Further examinations showed 10/11 leukemic patient cell types were positive with 1A6. In contrast, many leukemia-lymphoma cell lines were negative (14/17), i.e. T and B leukemia lymphoma cell lines, non-T and non-B leukemia cell lines as examined by complement mediated cytotoxicity test and immunofluorescence test. Other prominent characteristics of 1A6 MAB have been demonstrated, which include inhibition of myeloid colony formation (CFU-C) and suppression of human natural killer cell cytotoxicity (NKCC). These results suggest that 1A6 is a common marker for normal T, B, NK cells and leukemic patients cells having T and B cell origin, but 1A6 antigen is not expressed on many leukemia lymphoma cell lines. Therefore, it could be useful for studying malignant transformation of lymphoid cells.     

Myeloid/NK cell precursor acute leukemia lost both CD13 and CD33 at first diagnosis

It has been reported that malignancies of natural killer (NK) cell precursors, which are present in both myeloid and lymphoid antigens, are characterized by immature lymphoblastoid morphology with CD7+, CD33+ and CD56+ phenotype. Here, we report a 18-year-old man who was diagnosed with CD33- and CD13- NK cell precursor acute leukemia at first diagnosis. Following a 3-year remission state, he had a relapse as a testicular tumor and CD33+ myeloid/NK cell precursor acute leukemia after allogenic BMT. This case suggests that myeloid antigens are not necessary for diagnosis of myeloid/NK cell precursor acute leukemia.     

Human natural killer cell line modified with a chimeric immunoglobulin T-cell receptor gene leads to tumor growth inhibition in vivo

The gene transfer of tumor-specific chimeric immunoglobulin T-cell receptors (cIgTCRs) combining antibody-like specificity with the effector cell function could be an attractive tool in immunotherapy. In this study, we directed the human natural killer (NK) cell line YT to tumor cells by gene transfer of a cIgTCR with specificity against the human carcinoembryonic antigen (CEA). The cIgTCR was constructed of a CEA-specific humanized single-chain Fv antibody fragment fused to the IgG1 Fc domain and the CD3 zeta chain. YT cells were transfected with the cIgTCR gene by electroporation and cIgTCR-expressing cells were enriched by immunoaffinity purification. cIgTCR-expressing YT cells specifically lysed CEA(+) colon carcinoma cell lines, which were resistant to the parental YT cell line. The lysis was not inhibited in the presence of soluble CEA. Receptor gene-modified YT cells retained their CEA-specific cytolytic activity after gamma-irradiation in vitro and inhibited the tumor growth in vivo after adoptive transfer into NOD/SCID mice. This gene-modified NK cell line available in unlimited source might be useful in clinical immunotherapy of CEA(+) cancer.     

Malignancies of natural killer (NK) cell precursor: myeloid/NK cell precursor acute leukemia and blastic NK cell lymphoma/leukemia.

Malignant hematolymphoid disorders arising from natural killer (NK) cells have become widely recognized in the past decade. Recently, we as well as others have drawn attention to some neoplasms of conceivable NK cell precursor origin that might represent two distinct entities, i.e. myeloid/NK cell precursor acute leukemia and blastic NK cell lymphoma/leukemia. Both of these diseases were characterized by remarkable extramedullary involvement and lymphoblastoid morphology, although the sites of involvement differed. Myeloid/NK cell precursor acute leukemia exhibited more frequent involvement of bone marrow (BM) and lymph nodes, whereas blastic NK cell lymphoma/leukemia affected extranodal sites, mainly the skin/subcutis. Tumor cells of these two diseases shared the CD16-, CD56+ and CD57- phenotype, but differed in other phenotypic profiles. Indeed, myeloid/NK cell precursor acute leukemia was immunophenotypically characterized by the expression of CD34 and blastic NK cell lymphoma/leukemia by that of CD4. On the theoretical level in the NK cell differentiation pathway, myeloid/NK cell precursor acute leukemia might be derived from a myeloid antigen-positive precursor preceding a NK cell committed precursor as a conceivable counterpart of blastic NK cell lymphoma/leukemia. Most cases with either disease lacked cytotoxic activities or molecules, a finding which seems to support their precursor origin. Notably, Epstein Barr virus (EBV) was negative in all cases, which contrasted with its high level associated with mature NK cell malignancies. Chemotherapy for acute myeloid leukemia was generally effective for myeloid/NK cell precursor acute leukemia, while the regimen for lymphoid malignancy was effective for blastic NK cell lymphoma/leukemia. These data suggests that each of these two diseases constitutes a distinct entity, which is also different from mature NK cell malignancies.     

Acute leukemia with natural killer cells antigens in Brazilian children

Malignancies arising from Natural Killer cells (NK) were described mainly comprising disorders related to large granular lymphocyte expansion. Clinical, morphological and immunophenotypic characteristics of myeloid/NK cell precursor acute leukemia (M/NK-AL) have been mainly described in adult patients. We identified nine children with M/NK-AL precursor cells in a series of 264 childhood acute leukemia diagnosed in our laboratory from consecutive referrals before treatment. CD34 +/-, CD13/33 +, CD11b +, a-MPO + and CD56 +, characterized these cases with TcR alphabeta and gammadelta negative cells. The differential diagnosis between subtypes of NK leukemia with those of lymphoid or myeloid origin in childhood raises the question regarding the pathogenesis and therapeutic implications of this rare entity in children.     

A novel c-kit positive biphenotypic acute leukemia cell line,TMBL-1, carrying a p53 point mutation

We established and characterized a c-kit positive cell line from the bone marrow of a patient with biphenotypic acute leukemia (BAL). The cell line, designated TMBL-1, carried a His-175 mutant p53. The immunophenotype of the primary leukemia cells at diagnosis was cytoplasmic CD3+, CD7+, CD13+, CD33-, interleukin-7 (IL-7) receptor+ and c-kit -. However, leukemia cells in relapse and TMBL-1 cells were CD33+ and c-kit +. Immunophenotypically, TMBL-1 is a BAL cell line that coexpresses T-lymphoid and myeloid markers which fulfill the criteria of the European Group for the Immunological Characterization of Leukemia. Stem cell factor (SCF), a key regulator of hematopoiesis signaling through c-kit, enhanced the proliferation of TMBL-1 cells. Direct sequencing revealed the conversion at codon 175 of the p53 gene in the TMBL-1 cells. Primary leukemia cells in relapse also carried the same point mutation but not at diagnosis. Moreover, TMBL-1 cells are sensitive to paclitaxel, which could induce p53-independent apoptosis. The biphenotypic features and p53 mutation may be associated with progression to a more malignant type. This cell line may provide new information on the role of SCF in the overlapping area between early T-lymphoid/myeloid cells, and help in the design of new therapies targeted towards p53 mutations.     

EBV-NK Cells Interactions and Lymphoproliferative Disorders

Epstein-Barr virus (EBV) usually infects epithelial cells in the oropharynx and B lymphocytes asymptomatically. Occasionally, however, EBV infects T-cells and natural killer (NK) cells, and infection of these cells has been associated with the development of leukemias and lymphomas. EBV-positive lymphoproliferative disorders of NK cells have been reported with increasing frequency, but the interactions between EBV and NK cells are not fully understood, in part because NK cells are not usually infected with EBV in vitro. The lymphoma-derived EBV-positive NK cell line, YT, has been useful in the study of EBV infection of NK cells. YT cells express the EBV-associated nuclear antigen (EBNA)-1, the latent membrane protein (LMP)-1, and LMP-2A, but not EBNA-2 and LMP-2B genes. This pattern of latent gene expression is compatible with a type II latency program, normally associated with nasopharyngeal carcinoma, Hodgkin's disease, and T-cell lymphoma. In this report, we summarize recent information on EBV-NK cell interactions and EBV-positive lymphoproliferative disorders of NK cells.     

Natural killer cell neoplasms

Natural killer (NK) cell tumors are an uncommon and heterogeneous group of disorders. The World Health Organization (WHO) classified mature NK cell neoplasms into 2 types: 1) extranodal NK cell lymphoma, nasal type and 2) aggressive NK cell leukemia. The mature NK cell tumors are prevalent in Asia and Central and South America. These tumors show polymorphic neoplastic infiltrate with angioinvasion and/or angiodestruction, cytoplasmic azurophilic granules, CD2-positive (CD2+)/CD3-negative (CD3-)/cCD3epsilon+/CD56+ phenotype, and strong association with Epstein-Barr virus (EBV). Loss of chromosomes 6q, 11q, 13q, and 17p are recurrent aberrations. Although blastic NK cell lymphoma, currently referred to as CD4+/CD56+ hematodermic neoplasm, also was included in the NK cell lymphoma category in the WHO classification scheme, existing evidence indicates a plasmacytoid dendritic cell derivation as opposed to an NK cell origin. Recently, rare cases of CD56+ immature lymphoid tumors have been reported in the literature. These tumors are characterized by blastic appearance, CD3-/CD4-/CD56+/CD13-/CD33- phenotype, T-cell receptor and immunoglobulin genes in germline configuration, and no evidence of EBV, suggesting a true immature NK cell derivation. For this article, the authors reviewed the recent concepts and progress in clinicopathologic features, pathogenesis, genetic characteristics, diagnosis, differential diagnosis, treatment approaches, and outcomes of all subtypes of NK cell neoplasms.     

Identification of cell surface molecules characterizing human cutaneous T-cell lymphomas

Primary cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of malignant mature T-cell proliferations most often presenting as mycosis fungoides (MF) or its leukemic variant, Sezary syndrome (SS). No specific cell surface markers are presently available to distinguish the circulating malignant clone from normal lymphocytes. Using the previously established CTCL cell lines, Cou-LS and Pno, we have detected two leucocyte cell surface antigens with aberrant expression on CTCL cells. The NK-receptor (NKR) p140/KIR3DL2 normally expressed by NK and CD8+ T-cells was detected on the surface of CTCL cell lines as well as on freshly isolated CD4+PBL from SS patients. Further on, p140 marked in situ SS cells, distinguishing them from p140-negative tumor cells of patch plaque MF. SC5 is a newly described activation-related intracellular inhibitory receptor expressed on the surface of a minor PBL subset. We found that SC5 expression was significantly increased in SS cells and correlated to p140 expression. Moreover, cross-linking of SC5 molecules inhibited the malignant cell proliferation induced by anti-CD3 mAbs. The identification of these new structures on circulating SS tumor cells seems to be important both for the understanding of CTCL pathophysiology and for the clinical management of SS patients.     

High-dose chemotherapy with peripheral blood stem cell rescue in blastoid natural killer cell lymphoma

A 25-year-old man was referred because of skin rash, lymphadenopathy and anemia. Laboratory examinations revealed severe anemia (Hb, 4.8 g/dl) and elevated levels of GOT, GPT, LDH and soluble interleukin-2 receptor. Work-up studies disclosed the involvement of lymphoma cells in lymph nodes, skin, bilateral kidneys and bone marrow. Lymph node biopsy revealed diffuse proliferation of medium- to large-sized lymphoblastic cells. Bone marrow aspiration showed massive infiltration of large blastic cells with no cytoplasmic granules. The lymphoma cells in bone marrow and lymph node showed surface CD3-, cytoplasmic CD3epsilon+, CD4+, CD8-, CD56+, CD57-, CD16- and CD43 (MT-1)+ phenotype. Analyses of T cell receptor beta and gamma genes showed germ line configurations. EBER-1 was not detectable in the lymphoma cells. He was diagnosed as having blastoid natural killer (NK) cell lymphoma. In spite of several courses of combination chemotherapy, the lymphoma was progressive. He was then treated with high-dose chemotherapy and peripheral blood stem cell rescue, achieving remission which has now lasted for more than 12 months. We consider that blastoid NK cell lymphoma is an extremely aggressive subtype of CD56-positive lymphomas, and high-dose chemotherapy with peripheral blood stem cell rescue should be included for the choice of the treatment.     

Specific targeting of CD33(+) leukemia cells by a natural killer cell line modified with a chimeric receptor

We directed the human natural killer (NK) cell line YT by gene transfer of a humanized chimeric immunoglobulin T cell receptor to CD33, a marker on myeloid leukemias. The chimeric receptor was generated using a CD33 specific single-chain Fv (scFv) fragment based on the humanized antibody HuM195, the human IgG1 Fc domains and the human CD3 zeta signal chain. YT cells transfected by electroporation with the chimeric receptor gene specifically lysed the acute myeloid leukemia (AML) cell line KG1. This gene-modified NK cell line available in unlimited source could be an attractive tool in immunotherapy.     

Cytotoxic/natural killer cell cutaneous lymphomas. Report of EORTC Cutaneous Lymphoma Task Force Workshop

BACKGROUND: Cutaneous lymphomas expressing a cytotoxic or natural killer (NK) cell phenotype represent a group of lymphoproliferative disorders for which there is currently much confusion and little consensus regarding the best nomenclature and classification. METHODS: This study analyzes 48 cases of primary cutaneous lymphoma expressing cytotoxic proteins and/or the NK cell marker, CD56. These cases were collected for a workshop of the European Organization for Research and Treatment of Cancer Cutaneous Lymphoma Task Force, to better clarify the clinical, morphologic, and phenotypic features of these uncommon tumors. RESULTS: Several categories with different clinical and pathologic features were delineated: 1) aggressive, CD8+, epidermotropic, cytotoxic T-cell lymphoma; 2) mycosis fungoides, cytotoxic immunophenotype variant; 3) subcutaneous panniculitis-like T-cell lymphoma; 4) NK/T-cell lymphoma, nasal type; 5) CD4+, NK cell lymphoma; 6) blastoid NK cell lymphoma; (7) intravascular NK-like lymphoma; and 8) cytotoxic, peripheral T-cell lymphoma. CONCLUSIONS: Our data show that primary cutaneous cytotoxic/NK cell lymphomas include distinct groups of diseases, clinically, histologically, and biologically. Because the finding of a cytotoxic phenotype often has prognostic significance, the routine use of cytotoxic markers in the diagnosis and classification of cutaneous lymphomas should be expanded.     

Natural killer cell lymphoma in the duodenum

We present a case of duodenal non-Hodgkin lymphoma in a 71-year-old woman. Immunohistochemistry characterized the lymphoma cells as CD2(+); surface CD3(-) but cytoplasmic CD3(+); CD7(+); and CD56(+) without a rearrangement of the T-cell receptor gene. Cells had a high N/C ratio and irregular nuclear outlines and lacked azurophilic granules and these features indicated that the lymphoma cells arose from natural killer (NK) cells. She was treated with intensive chemotherapy including pirarubicin, cyclophosphamide, vincristine, and prednisolone, but died three weeks after diagnosis. CD56(+) lymphomas originate from NK or cytotoxic T cells and are designated "extranodal NK/T-cell lymphoma, nasal type" in the WHO classification. Nasal NK cell lymphoma is most common in East Asians and CD56(+) lymphomas usually occur in the nasal area. Extranasal forms such as gastrointestinal lymphomas are very rare and usually carry a poor prognosis. Extranodal NK/T-cell lymphoma, nasal type, is characterized by a broad morphologic spectrum and have variable prognosis. These lymphomas constitute an heterogeneous group, and their subclassification has not yet been established.     

Detection of Epstein-Barr virus genome in natural-killer-like cell line, YT.

YT cells, originally reported as a natural-killer-like (NK-like) lymphoid cell line, were investigated for Epstein-Barr virus (EBV) genome, gene rearrangement for T-cell receptor (TCR), phenotype and function. The YT cells of the original batch (YT-0) and two subclones (YT2C2 and YTC3) expressed EBV-associated nuclear antigen, and the BamHI-digested DNA showed the 3.4 kb hybridizing band with the BamHIW probe of EBV DNA in Southern blot analysis. When tested with latent-infection membrane protein probe, an identical hybridizing band was shared, indicating that all three sources of YT cells were of monoclonal derivation in terms of the terminal repeat junctional structure of EBV DNA, and that the original YT cells had been infected with EBV before the isolation of the two subclones. The cell-surface antigen analysis revealed the expression of CD7, CD28, CD30, CD45R0, TLiSA and S6F1 antigen besides the originally recorded CD25, CD56 and HLA-DR antigen. Gene rearrangement analysis showed the germ-line genotype, including TCR gamma and delta as well as beta chain. The Northern blot study using the CD3 epsilon and CD3 delta chain gene probes revealed CD3 epsilon, but not CD3 delta RNA. The YT-0 cells exhibited NK and antibody-dependent cellular cytotoxicity activity, but the YT2C2 and YTC3 cells did not. It was not resolved whether the fresh neoplastic NK-like cells of the YT-cell donor carry EBV genome, but YT cells, the first lymphoid cell line found to have EBV genome and non-B lymphoid properties, are valuable for investigating the relationship between EBV and human non-B lymphoid hematopoietic cells.     

鼻型NK/T细胞淋巴瘤26例临床病理分析

目的:研究鼻型NK/T细胞淋巴瘤(ENKL)的免疫表型、病理特点、临床特点。方法:回顾性分析26例ENKL的临床表现、病理组织学特点,采用免疫组化SP法检测LCA、CD3、UCHL1、CD20、CD79a、CD56、TIA-1、Granzyme B、perforin,原位杂交方法检测EBER在ENKL中的表达情况。结果:NK/T细胞淋巴瘤发生在鼻腔占80.77%(21/26),伴坏死、溃疡、鼻出血者100%(26/26),肿瘤细胞嗜血管现象占46.15%(12/26),伴"鳞状细胞癌样反应"占20.08%(6/26)。CD3、CD56、TIA-1、Granzyme B、perforin及EBER阳性表达率达100%。结论:临床及病理形态复杂多样性是NK/T细胞淋巴瘤的特点。病理诊断中须注意与炎症或高分化鳞状细胞癌鉴别。ENKL根据典型的临床表现、病理形态学改变、免疫表型特点及EBER原位杂交阳性能准确诊断。     

Gastrointestinal T cell lymphoma: predominant cytotoxic phenotypes, including alpha/beta, gamma/delta T cell and natural killer cells

Gastrointestinal T cell lymphoma (TCL) is a rare subset of peripheral TCL, presenting with or without cytotoxic phenotype, a history of coeliac disease (CD) and enteropathy. However, CD is rare in Japan. Here, we describe the clinicopathological features of 18 Japanese cases. Lesions were found in the small intestine (n=13), stomach (n=3) and colon (n=2). Seven patients presented with enteropathy but none had a history of CD. Lymphomas appeared as ulceration (n=11), tumour formation (n=6), or polypoid growth (n=1). Histologically (REAL classification), neoplastic lesions were composed of intestinal type T cell lymphoma (ITCL, n=13, including one case with NK type), anaplastic large cell (ALCL, n=2), adult T cell leukaemia/lymphoma (ATLL, n=2), and lymphoblastic type (n=1). Epstein Barr virus infection was detected by EBER-1 in situ hybridization in 6 of 11 cases with ITCL but not in the other types. ALCL expressed CD30. CD56 was expressed in 3 of 11 cases of ITCL but not in other types. Among the 10 examined cases, 8 were alphabeta T cell type [CD2+, CD3+, T cell receptor (TCR)delta-1-, betaF1+], one was gammadelta T cell type [CD2+, CD3+, TCRdelta-1+, betaF1-], and the remaining case expressed natural killer (NK) cell type [CD2+, CD3-, CD56+, TCRdelta-1-, betaF1-]. Among the 8 examined cases, 3 expressed CD103 molecule, which was associated with extrathymic T cells of intraepithelial lymphocytes. All cases except ATLL expressed the cytotoxicity-associated molecule of TIA-1, and 11 of 14 TIA-1 positive cases expressed activated cytotoxic molecules of perforin, granzyme B, and/or Fas ligand. Despite the morphological, genetic and phenotypic heterogeneity, prognosis was poor, and 11 of 13 patients with small intestinal lesions died albeit appropriate treatment, but 3 of 4 patients with gastric or colonic lesions were still alive. The main cause of death was intestinal perforation. The latter might be due to the site specificity of small intestine and tumour cytotoxicity.     

Continuous hematopoietic cell lines as model systems for leukemia-lymphoma research

Along with other improvements, the advent of continuous human leukemia-lymphoma (LL) cell lines as a rich resource of abundant, accessible and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first LL cell lines, Burkitt's lymphoma-derived lines, were established in 1963. Since then, more than 1000 cell lines have been described, although not all of them in full detail. The major advantages of continuous cell lines is the unlimited supply and worldwide availability of identical cell material, and the infinite viable storability in liquid nitrogen. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro under preservation of most cellular features, and specific genetic alterations. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines must be discerned from Epstein-Barr virus (EBV)-immortalized normal cells, using various distinguishing parameters. However, the picture is not quite so straightforward, as some types of LL cell lines are indeed EBV+, and some EBV+ normal cell lines carry also genetic aberrations and may mimic malignancy-associated features. Apart from EBV and human T-cell leukemia virus in some lines, the majority of wild-type LL cell lines are virus-negative. The efficiency of cell line establishment is rather low and the deliberate establishment of new LL cell lines remains by and large an unpredictable random process. Difficulties in establishing continuous cell lines may be caused by the inappropriate selection of nutrients and growth factors for these cells. Clearly, a generally suitable microenvironment for hematopoietic cells, either malignant or normal, cannot yet be created in vitro. The characterization and publication of new LL cell lines should provide important and informative core data, attesting to their scientific significance. Large percentages of LL cell lines are contaminated with mycoplasma (about 30%) or are cross-contaminated with other cell lines (about 15-20%). Solutions to these problems are sensitive detection, effective elimination and rigorous prevention of mycoplasma infection, and proper, regular authentication of cell lines. The underlying cause, however, appears to be negligent cell culture practice. The willingness of investigators to make their LL cell lines available to others is all too often limited. There is a need in the scientific community for clean and authenticated high-quality LL cell lines to which every scientist has access. These are offered by various institutionalized public cell line banks. It has been argued that LL cell lines are genetically unstable (both cytogenetically and molecular genetically). For instance, cell lines are supposed to acquire numerical and structural chromosomal alterations and various types of mutations (e.g. point mutations) in vitro. We present evidence that while nearly 100% of all LL cell lines indeed carry genetic alterations, these alterations appear to be stable rather than unstable. As an example of the practical utility of LL cell lines, the recent advances in studies of classical and molecular cytogenetics, which in large part were made possible by cell lines, are highlighted. A list of the most useful, robust and publicly available reference cell lines that may be used for a variety of experimental purposes is proposed. Clearly, by opening new avenues for investigation, studies of LL cell lines have provided seminal insights into the biology of hematopoietic neoplasia. Over a period of nearly four decades, these initially rather exotic cell cultures, known only to a few specialists, have become ubiquitous powerful research tools that are available to every investigator.     

Primary mediastinal large cell lymphoma

Purpose: Primary mediastinal large cell lymphoma PMLCL is a recently described entity which has provoked considerable interest lately. The unique immunophenotype of the malignant cell, as well as conflicting reports regarding its response to therapy, prompted us to review the current literature focusing on the pathological and clinical aspects of this disease. Design: We reviewed the current literature that contained details on the clinical presentation, pathological profile and clinical outcome. Results: Most reports agree that PMLCL typically affects a young female population and presents with symptoms related to compression of mediastinal structures. Pathologically, the malignant cells may arise from a distinctive thymic medullary B cell subpopulation which is characterized by the following phenotype: CD19+, CD21 –, CD20+, CD22+. Alterations of c-myc and lack of expression of HLA class 1 antigens on the tumour cell surface have been described. Response to treatment and clinical outcome have varied from one series to another and is possibly explained by the small number of patients in most series and by the heterogeneity of therapy. In general, the outcome does not appear to be worse than that of other large cell lymphomas although, given the young age of these patients, it might be expected to be more favourable. Conclusions: Primary mediastinal large cell lymphoma is a distinct clinico-pathological entity characterized by: (1) a predominance of young adults; (2) female predominance (female/male ratio of 2:1) in contrast to the other large cell lymphomas; (3) CD19+/CD21 – immunophenotype; (4) absence of HLA-class 1 antigen expression; (5) possible involvement of c-myc; (6) frequent dissemination at relapse to unusual anatomic sites in a pattern reminiscent of Burkitt's lymphoma. The role of radiotherapy and the optimal treatment for this disorder are issues that remain unresolved.