KIR3DL2 (CD158k) is a potential therapeutic target in primary cutaneous anaplastic large-cell lymphoma.

BACKGROUND: KIR3DL2, an inhibitory receptor expressed by natural killer cells and a subset of normal CD8(+) T cells, is aberrantly expressed in neoplastic cells in transformed mycosis fungoides and Sézary syndrome. Anti-KIR3DL2 targeted antibody therapy has shown potent activity in preclinical models for these diseases. OBJECTIVES: To examine the expression of KIR3DL2 and its potential use as a therapeutic target in patients with primary cutaneous anaplastic large-cell lymphoma (pcALCL), the most aggressive cutaneous CD30(+) lymphoproliferative disease. METHODS: Samples from 11 patients with pcALCL and three CD30(+) lymphoproliferative disease cell lines - Mac1, Mac2a and Mac2b - were used in KIR3DL2 expression studies using immunohistochemistry, flow cytometry and reverse-transcriptase quantitative polymerase chain reaction. The effect of IPH4102, a monoclonal humanized IgG1 targeting KIR3DL2, was assessed by in vitro cytotoxicity assays against Mac1, Mac2a and Mac2b using allogeneic peripheral blood mononuclear cells as effectors. RESULTS: KIR3DL2 mRNA and protein were found in all human samples of pcALCL, and in the Mac2a and Mac2b cell lines. KIR3DL2 protein expression was present on 85·8 ± 14·0% of CD30(+) skin-infiltrating tumour cells. In vitro functional studies showed that KIR3DL2(+) Mac2a and Mac2b pcALCL lines are sensitive to antibody-derived cytotoxicity mediated by IPH4102, through activation of natural killer cells, in a concentration-dependent manner. CONCLUSIONS: pcALCL tumour cells express KIR3DL2, and we provide preclinical proof of concept for the use of IPH4102, a humanized anti-KIR3DL2 antibody, to treat patients with primary cutaneous CD30(+) ALCL.

γδ T-cell-rich variants of pityriasis lichenoides and lymphomatoid papulosis: benign cutaneous disorders to be distinguished from aggressive cutaneous γδ T-cell lymphomas.

BACKGROUND: T cells with a γδ phenotype have been associated with aggressive lymphomas. Yet, inflammatory skin disorders and low-grade lymphoproliferative disorders have rarely been described with a predominant γδ T-cell infiltrate. OBJECTIVES: To review our experience and determine the clinical relevance of the γδ T-cell phenotype in lymphomatoid papulosis (LyP) and pityriasis lichenoides (PL). METHODS: A retrospective dermatopathology file review looking for LyP and PL characterized by a γδ T-cell phenotype was performed. Clinical manifestations and course, histological features and molecular data were analyzed. RESULTS: Six of 16 cases of LyP and four of 23 cases diagnosed as PL during a 5-year period (2009-14) were identified. The median follow-up for the whole group was 16 months (range 3-64), showing an indolent clinical course in all cases. CONCLUSIONS: The detection of a predominantly γδ T-cell phenotype in papular lymphoid-rich infiltrates in the absence of other lesions is not associated with a clinically aggressive course. γδ T-cell-rich variants of LyP and PL may reflect a spectrum of related conditions. This is a single academic centre retrospective chart review of a relatively small sample.

[cFLIP diminishes CD95-induced apoptosis of CD30-stimulated cutaneous anaplastic large T cell lymphoma (cALCL) cells]. / Kutanes anaplastisches großzelliges Lymphom. Apoptoseresistenz durch CD30-induzierte cFLIP-Expression.

Stimulation of the TNF receptors CD30 and CD95 exerts opposite effects. Crosstalk of both receptors is unknown. We aimed to reveal regulatory mechanisms of CD30-induced effects on CD95 signaling of cALCL cell lines. "CD30/CD95 crosstalk analysis" was performed in cALCL cell lines by comparison of CD30 or CD95 stimulation and CD30/CD95 costimulation. Receptor expression and induction of apoptosis was investigated by flow cytometry. mRNA expression of CD30-inducible genes (cFLIP, TRAF1, cIAP2, and A20) was compared by semiquantitative reverse transcription (RT-RQ-) PCR in stimulated and unstimulated cells. Protein expression of IκBα, p100/p52, caspase-8, caspase-3, and cFLIP was analyzed by immunoblotting. A lentiviral-based shRNA-mediated approach was used to inhibit cFLIP expression. CD30/CD95 crosstalk experiments revealed that CD30 ligation leads to NFκB-mediated cFLIP upregulation in cALCL cells, which in turn enhanced resistance to CD95-mediated apoptosis. This effect is based on the CD30-induced upregulation of cFLIP. Knockdown of cFLIP restores sensitivity to CD95-mediated apoptosis. We conclude that the anti-apoptotic function of CD30 antibodies should be kept in mind if CD30 antibody-based therapeutic concepts for ALCL lymphoma are considered.

L-428 nodular sclerosing Hodgkin's cell secretes a unique transforming growth factor-beta active at physiologic pH.

Nodular sclerosing Hodgkin's disease is characterized by dense collagen fibrosis. Although transforming growth factor-beta (TGF-beta) is an important bifunctional growth factor for fibroblasts and is stored and released by many cells, it requires acidification to pH 2.0-3.0 before it becomes a biologically active growth factor. We show here that the L-428 Hodgkin's cell releases a high molecular weight TGF that competes for the TGF-beta cell membrane receptor but not the TGF-alpha receptor. This growth factor is most active at physiologic pH and is 97% inactivated by acidification. Hodgkin's TGF is also inactivated by proteases and can be preserved by protease inhibitors. The Hodgkin's TGF can be separated from an autocrine growth factor using either column chromatography or electroelution from gels and is shown to have a molecular weight of approximately 350,000. Incubation of the Hodgkin's TGF in SDS releases a 25,000-D protein with reduced biological activity but which cross-reacts with anti-TGF-beta IgG. We propose that L-428 nodular sclerosing Hodgkin's disease fibrosis is mediated by a potent high molecular weight TGF-beta which, unlike TGF-beta characterized to date, is secreted in a form most active at physiologic pH.

Upregulation of tumor necrosis factor-alpha gene by Epstein-Barr virus and activation of macrophages in Epstein-Barr virus-infected T cells in the pathogenesis of hemophagocytic syndrome.

A potentially fatal hemophagocytic syndrome has been noted in patients with malignant lymphomas, particularly in EBV-infected T cell lymphoma. Cytokines, such as interferon-gamma (IFN-gamma), TNF-alpha, and IL-1alpha, are elevated in patients' sera. To verify whether infection of T cells by EBV will upregulate specific cytokine genes and subsequently activate macrophages leading to hemophagocytic syndrome, we studied the transcripts of TNF-alpha, IFN-gamma, and IL-1alpha in EBV-infected and EBV-negative lymphoma tissues. By reverse transcription PCR analysis, transcripts of TNF-alpha were detected in 8 (57%) of 14 EBV-infected T cell lymphomas, higher than that detected in EBV-negative T cell lymphoma (one of six, 17%), EBV-positive B cell lymphoma (two of five, 40%) and EBV-negative B cell lymphomas (one of seven, 14%). Transcripts of IFN-gamma were consistently detected in T cell lymphoma and occasionally in B cell lymphoma, but were independent of EBV status. IL-1alpha expression was not detectable in any category. Consistent with these in vivo observations, in vitro EBV infection of T cell lymphoma lines caused upregulation of TNF-alpha gene, and increased secretion of TNF-alpha, but not IFN-gamma or IL-1alpha. Expression of TNF-alpha, IFN-gamma, and IL-1alpha was not changed by EBV infection of B cell lymphoma lines. To identify the specific cytokine(s) responsible for macrophage activation, culture supernatants from EBV-infected T cells were cocultured with a monocytic cell line U937 for 24 h. Enhanced phagocytosis and secretion of TNF-alpha, IFN-gamma, and IL-1alpha by U937 cells were observed, and could be inhibited to a large extent by anti-TNF-alpha (70%), less effectively by anti-IFN-gamma (31%), but almost completely by the combination of anti-TNF-alpha and anti-IFN-gamma (85%). Taken together, the in vivo and in vitro observations suggest that infection of T cells by EBV selectively upregulates the TNF-alpha expression which, in combination with IFN-gamma and probably other cytokines, can activate macrophages. This study not only highlights a probable pathogenesis for virus-associated hemophagocytic syndrome, but also suggests that anti-TNF-alpha will have therapeutic potential in the context of their fatal syndrome.

A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T-cell lymphoma.

In many cancers, inactivating mutations in both alleles of the transforming growth factor beta (TGF-beta) type 11 receptor (TbetaRII) gene occur and correlate with loss of sensitivity to TGF-beta. Here we describe a novel mechanism for loss of sensitivity to growth inhibition by TGF-beta in tumor development. Mac-1 cells, isolated from the blood of a patient with an indolent form of cutaneous T-cell lymphoma, express wild-type TbetaRII and are sensitive to TGF-beta. Mac-2A cells, clonally related to Mac-1 and isolated from a skin nodule of the same patient at a later, clinically aggressive stage of lymphoma, are resistant to TGF-beta. They express both the wild-type TbetaRII and a receptor with a single point mutation (Asp-404-Gly [D404G]) in the kinase domain (D404G-->TbetaRII); no TbetaRI or TbetaRII is found on the plasma membrane, suggesting that D404G-TbetaRII dominantly inhibits the function of the wild-type receptor by inhibiting its appearance on the plasma membrane. Indeed, inducible expression, under control of a tetracycline-regulated promoter, of D404G-TbetaRII in TGF-beta- sensitive Mac-1 cells as well as in Hep3B hepatoma cells results in resistance to TGF-beta and disappearance of cell surface TbetaRI and TbetaRII. Overexpression of wild-type TbetaRII in Mac-2A cells restores cell surface TbetaRI and TbetaRH and sensitivity to TGF-beta. The ability of the D404G-TbetaRH to dominantly inhibit function of wild-type TGF-beta receptors represents a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.

Distinctive phorbol ester-induced morphological and surface antigen changes in mycosis fungoides, the Sézary syndrome, and adult T-cell leukemia.

Clinical and pathological studies of cutaneous T-cell lymphomas (CTCL) reveal variations in tumor cell morphology and surface membrane phenotype that are of diagnostic and prognostic importance. Our study investigates blastic transformation and surface antigen change on CTCL cells in vitro under the influence of tumor-promoting phorbol ester (TPA) and phytohemagglutinin. Both agents transformed tumor cells with cerebriform nuclei into blast cells within 5 days; however, Sézary cells were somewhat resistant to transformation with phytohemagglutinin. Multinucleated cells with prominent nucleoli resembled Reed-Sternberg cells of Hodgkin's disease. These morphological changes simulated the appearance of the aggressive tumor stage of mycosis fungoides. During blastic transformation, the erythrocyte rosette receptor was induced by TPA on sheep erythrocyte-rosette-negative Sézary cells from one patient. During the first 24 hr in vitro, Sézary and MF cells stimulated by TPA lost Leu 3a (T4) antigen while maintaining original high levels of Leu 1 antigen. In contrast, leukemia cells from patients with adult T-cell leukemia (ATL) were resistant to modulation of Leu 3a antigen by TPA; 3A1 antigen on CTCL and ATL cells was unaffected by TPA. Blastic transformation of CTCL cells was observed with both TPA and phytohemagglutinin, but helper T-cell antigen Leu 3a (T4) and erythrocyte rosette receptor changes occurred only with TPA. Thus, blastic transformation and surface differentiation were not directly related. These results provide a possible model for the study of blastic transformation and surface antigen/receptor variation in CTCL. They also may provide an independent test for the distinction of CTCL and ATL in vitro. Finally, they illustrate the relative resistance of ATL to surface antigen modulation as previously shown for Tac antigen modulation by anti-Tac antibody.

Clinical presentation and outcome in lymphocyte-predominant Hodgkin's disease.

PURPOSE: The patterns of presentation, histologic pattern (nodular or diffuse), treatment, and long-term outcome were studied in patients with lymphocyte-predominant (LP) Hodgkin's disease (HD) to determine whether these patients should be treated differently than patients with other subtypes of HD. PATIENTS AND METHODS: Pathology was reviewed for 97 patients with an initial diagnosis of LPHD made between 1970 and 1993. Seventy-five patients had LPHD on review: 55 had nodular LPHD, 14 had diffuse LPHD, and six had LP histology without subclassification. There were 60 males (80%) and 15 females (20%). Sixty-six patients (88%), presented with clinical stage (CS) I or II disease. Seventy-one patients were treated at the Joint Center for Radiation Therapy (JCRT) and were considered for analysis of treatment outcome. Sixty-one of these 71 were treated with radiation (RT) alone; 17 received mantle RT alone, 27 mantle and paraaortic RT, and seven total-nodal irradiation (TNI). Ten patients with subdiaphragmatic HD received pelvic and paraaortic RT. Of the 10 remaining patients, four were treated with RT and chemotherapy (CT) and six were treated with CT alone. The median follow-up time was 10.8 years. RESULTS: The 10-year actuarial freedom-from-first-relapse (FFR) and 10-year overall survival rates for the 71 patients with LPHD treated at the JCRT were 80% and 93%, respectively. The 10-year actuarial FFR by nodular (n = 51), diffuse (n = 14), and unspecified (n = 6) histologic pattern was 74%, 100%, and 60%, respectively. Overall, 14 of 71 patients have relapsed: nine of 61 with stage IA, IB, or IIA disease and five of 10 with stage IIB to IVB disease have relapsed. The median time to relapse was 53 months. Nine of 71 patients have died. Only one death has been from HD: five patients died of second cancers, two of cardiac disease, and one of alcoholic liver cirrhosis. Of seven patients with second malignancies, five died. None of the second malignancies were non-Hodgkin's lymphoma (NHL). CONCLUSION: Patients with LPHD have different patterns of presentation, sex and age distribution, and likelihood of occult abdominal disease than patients with nodular-sclerosing (NS) or mixed-cellularity (MC) disease. The median time to relapse for LP patients was later than reported for other histologic subtypes; however, there was no pattern of continuous late relapse. With pathologic staging and standard treatment, mortality from LPHD is low; nearly all deaths have been cardiac- or second tumor-related. This suggests that less aggressive treatment for LPHD might continue to yield excellent results, while perhaps lowering the long-term risk of complications.

Large-cell lymphoma arising in the mediastinum in children and adolescents is associated with an excellent outcome: a Children's Cancer Group report.

PURPOSE: Large-cell lymphoma (LCL) arising in the mediastinum (LCL-M) is a heterogeneous group of non-Hodgkin's lymphoma (NHL) that includes B-cell lymphomas as well as T-cell lymphomas, including anaplastic LCL. LCL-M is well recognized in young adults but is less well characterized and infrequent in children and adolescents. METHODS: A retrospective review of Children's Cancer Group therapeutic studies for nonlymphoblastic lymphomas (CCG-551, CCG-503, CCG-552, and CCG-5911) identified 20 patients with LCL-M, representing 7.2% of all LCLs classified by central pathology review. RESULTS: The patients ranged in age from 4 to 19 years (median, 12.5 years; mean, 12 years); 55% of the patients were male. Although a variety of chemotherapy regimens were used, response was excellent, with all 20 patients (100%) achieving a complete response. Four patients (20%) experienced relapse locally or in distant sites including brain and kidney. One patient died of sepsis during therapy. For the 20 patients with LCL-M, the product-limit estimated 5-year event-free survival (EFS) and 5-year overall survival (OS) rates are 75% +/- 10% and 85% +/- 8%, respectively. For disseminated LCLs (192 cases), the EFS and OS rates were 50% +/- 4% and 63% +/- 4%, respectively, which differ significantly from the those of the LCL-M cases (EFS, P =.025; OS, P =.034). The 5-year EFS and OS rates for patients with localized LCL (67 cases) were 92 +/- 3% and 97 +/- 2%, respectively. CONCLUSION: LCL-M is a heterogeneous group of NHLs that makes up approximately 7.2% of LCL in children and adolescents. Response to therapy and OS in this young age group seems excellent and superior to that of disseminated LCLs but inferior to that of other localized LCL. Future studies of LCL-M will evaluate short intense chemotherapy administered without radiation therapy.

Three-color flow cytometry in the diagnosis of malignant lymphoma based on the comparative cell morphology of lymphoma cells and reactive lymphocytes.

This study examines the identification of unusual cell populations highly associated with lymphoma cells (UCP-L) in diagnostic biopsy specimens using three-color flow cytometry (3-FCM). Patterns of surface antigen expression were used to compare the morphology of distinct lymphoid cell populations present in biopsy specimens and determine the presence or absence of UCP-L. UCP-L were identified by their larger size as compared to admixed reactive lymphocytes, and the method is based on the concept that neoplastic lymphoma cells are larger than reactive lymphocytes. The comparison of relative cell sizes was determined by overlaying forward scatter histograms by multicolor gating using PAINT-A-GATE software. In order for separate gates to be set on UCP-L and reactive cell populations, UCP-L had to fulfill one or more immunophenotypic criteria. These included: (1) belonging to a subset of B cell antigen-positive cells showing restricted expression of kappa or lambda light chains; (2) belonging to a subset of CD4-positive cells having dim or absent expression of CD45RA; (3) showing alterations in antigen expression (loss, dimmer or brighter); or (4) expressing an immunophenotype that is present on only rare cell populations or is absent from reactive lymph nodes. The immunophenotypic profiles of the respective cell populations were demonstrated by cubic representations to assess more easily the co-expression of three antigens. The common morphology of UCP-L as defined by forward and side scatter grams was consistent with a 'lymphoid appearance' except in several cases of HTLV-I-positive T cell lymphoma and gammadelta T cell lymphoma. The immunophenotypic profiles of UCP-L were confirmed to correspond to the presumptive lymphoma cell population by use of a live gating procedure on the large cells, which eliminated interference by reactive cells or necrotic tissue fragments. Using this method, we identified UCP-L in 208 of 293 (71%) consecutive cases of non-Hodgkin's lymphomas, while no UCP-L were seen in 72 cases of non-specific hyperplasia of lymph nodes. Twenty-seven cases could not properly be examined about the existence of UCP-L because of massive necrosis, extensive fibrosis or strong non-specific staining reactions of unknown cause. When those cases were eliminated from the analysis, 80% of non-Hodgkin's lymphoma were found to contain UCP-L. In B cell lymphoma, the incidence of UCP-L in nodal lymphomas (80%) was much higher than in extranodal lymphomas (47%). Only one of 21 cases of Hodgkin's lymphoma was found to have UCP-L. The 3-FCM procedure was validated by the combined use of immunohistochemistry, morphologic examination, cytogenetic and antigen receptor gene rearrangement analysis by Southern blot hybridization. Our findings indicate that detection of UCP-L by 3-FCM is a reliable method to distinguish non-Hodgkin lymphomas from reactive hyperplasias in the majority of cases, even when the reactive cell population predominates over the malignant cell population.

Long-term follow-up of short intensive multiagent chemotherapy without high-dose methotrexate ('Orange') in children with advanced non-lymphoblastic non-Hodgkin's lymphoma: a children's cancer group report.

Despite prolonged therapy (18 months), children with advanced non-lymphoblastic, non-Hodgkin's lymphoma (NHL) treated on previous Children's Cancer Group (CCG) trials achieved less than a 60% 5-year event-free survival (EFS). In this study we piloted a shorter but more intensive protocol ('Orange') to determine the feasibility, safety, and efficacy of this alternative treatment approach. Thirty-nine children received a CHOP-based induction, etoposide/ifosfamide consolidation, DECAL (dexamethasone, etoposide, cisplatin, cytosine arabinoside (Ara-C) and L-asparaginase) intensification, and either one or two similar but less intense maintenance courses. Patients were stratified to standard-risk (5 months) vs high-risk (7 months) treatment. High risk was defined as either bone marrow disease, CNS disease, mediastinal mass > or = one-third thoracic diameter at T5 and/or LDH > or =2 times institutional upper limits of normal. All other patients were considered to be standard risk. Results were compared with the previous CCG NHL study (CCG-503). Sixteen and 23 patients were considered standard- vs. high-risk, respectively. The 5-year EFS and overall survival (OS) were 77 +/- 7% and 80 +/- 7%, respectively. The 5-year EFS and OS were significantly better in the standard- vs. high-risk subgroups (100% vs. 61 +/- 11%) (P < 0.003) and (100% vs. 65 +/- 11%) (P < 0.01), respectively. Lactate dehydrogenase (LDH) > or =2 x normal (NL) was associated with significantly poorer outcomes (LDH > or =2 x NL vs. <2 x NL) (5-year EFS: 55 +/- 12% vs. 100%) (P < 0.0004). This CCG hybrid regimen, 'Orange', of short and more intensive therapy resulted in a significant improvement in outcomes compared with the previous CCG trial of more prolonged but less intense therapy. This regimen that deletes high-dose methotrexate, if confirmed in a larger trial, could be considered as an alternative treatment approach in children without high tumor burdens (LDH <2 x NL) and Murphy stage III disease.

The t(2;5)(p23;q35): a recurring chromosomal abnormality in Ki-1-positive anaplastic large cell lymphoma.

In lymphoid neoplasms, nonrandom cytogenetic abnormalities correlate with clinical, morphologic and immunophenotypic features. A subtype of non-Hodgkin's lymphoma, which expresses the Ki-1 antigen (CD30) and has distinct morphologic and clinical features, has recently been described. We now report the association of a reciprocal translocation involving the short arm of chromosome 2 (band p23) and the long arm of chromosome 5 (band q35), t(2;5)(p23;q35), with Ki-1 positive anaplastic large cell lymphoma. Rearrangement of the genes that are located at the breakpoints on chromosomes 2 and 5 may be a critical step in the pathogenesis of this lymphoma.

Ph-negative non-Hodgkin's lymphoma occurring in chronic phase of Ph-positive chronic myelogenous leukemia is defined as a genetically different neoplasm from extramedullary localized blast crisis: report of two cases and review of the literature.

This report describes two cases of Philadelphia chromosome-negative (Ph(-)) non-Hodgkin's lymphomas (NHLs) recognized in patients with chronic phase Ph-positive (Ph(+)) chronic myelogenous leukemia (CML). Lymph node biopsy of patient 1 was initially diagnosed as diffuse large B cell non-Hodgkin's lymphoma (NHL, T cell rich variant), but at relapse showed immunoblastic features with a marked decrease of admixed lymphocyte components. Patient 2 presented with thickened parietal pleura which revealed a CD30-positive anaplastic large cell lymphoma showing null cell phenotype and genotype with abundant admixed neutrophils and lymphocytes. At the time of lymphoma diagnosis, the patients had CML for 33 and 10 months, respectively. DNA obtained from bone marrow cells at the time of lymphoma diagnosis showed BCR/ABL gene rearrangements by both Southern blot analysis and reverse transcription polymerase chain reaction (RT-PCR), but lacked both immunoglobulin and T cell receptor gene rearrangements. BCR gene rearrangement and BCR/ABL fusion gene were also identified in lymph node and pleural biopsies by Southern blot and RT-PCR analysis, respectively. However, both biopsy specimens also contained reactive lymphocytes and neutrophils, and no fusion signals between BCR and ABL genes were identified in the hyperdiploid lymphoma cells of either case by fluorescence in situ hybridization (FISH). These data suggest the lymphoma cells in both cases were not genetically associated with BCR/ABL. Therefore, these cases were not diagnosed as an extramedullary localized blast crisis in CML, but as Ph(-) NHLs. This represents the first definitive demonstration of peripheral B cell lymphoma occurring by a separate genetic pathway, lacking BCR/ABL, in patients with Ph(+) CML. A review of the literature identified two different subtypes of malignant lymphomas arising in patients with an antecedent or concurrent diagnosis of CML. The most common are T cell lymphomas displaying an immature thymic phenotype, while peripheral B cell lymphomas are more rare. Our study shows, however, that 'Ph(+) NHL' occurring in CML or acute lymphocytic leukemia (ALL) may represent an unrelated neoplasm, even if standard cytogenetic analysis reveals a Ph(+) chromosome, and that FISH is required to confirm whether a localized lymphoid neoplasm is either a true extramedullary localized blast crisis or genetically distinct neoplasm. Leukemia(2000) 14, 169-182.

Tocainide-induced reversible agranulocytosis and anemia.

Tocainide is an effective oral antiarrhythmic agent. We report a 77-year-old man who developed agranulocytosis and anemia while receiving tocainide therapy. These hematologic abnormalities were detected on routine evaluation six weeks after beginning tocainide therapy. The absolute granulocyte count decreased to 50/mm3 (0.05 X 10(9)/L). The anemia was mild; hemoglobin count, 10.9 g/dL (109 g/L). These abnormalities were associated with local and stromal adipocytic bone marrow damage, and decreased production of red blood cells and granulocytes. The platelet count was not affected. The patient had no evidence of infection. Hematologic values were restored to normal two weeks after discontinuation of tocainide therapy, indicating that bone marrow toxicity of tocainide is reversible.

The dominant T cell clone is present in multiple regressing skin lesions and associated T cell lymphomas of patients with lymphomatoid papulosis.

This study was undertaken to determine the clonality of lymphomatoid papulosis (LyP), its clonal relationship to lymphomas, which occur at high frequency in LyP patients, and to define the cell lineage of Reed-Sternberg-like cells in type A lesions of LyP. Punch biopsies of skin of 11 adult patients with LyP were analyzed for morphologic subtype of LyP, surface antigens, and clonal T-cell receptor (TCR) gene rearrangements. Clonal rearrangements were identified by semiquantitative polymerase chain reaction amplification and sequencing of TCR-beta chain genes in nine patients and TCR-gamma chain genes in two patients. A single dominant clone was detected in multiple separate LyP lesions, often of different histologies, in nine patients. The same clone was detected in LyP lesions and the anaplastic large cell lymphoma (ALCL) of 2 patients and the mycosis fungoides (MF) of 2 other patients. No dominant clone could be detected in one patient with LyP uncomplicated by lymphoma or in a second patient with LyP and MF. A T-cell lineage was evident for RS-like cells in cell culture and in type A lesions. These results show that multiple regressing skin lesions and associated T cell lymphomas (MF and ALCL) are clonally related in most LyP patients, which suggest that the disease in these patients was initiated by a non-random genetic event.

Detection of clonal T-cell receptor gamma gene rearrangements in early mycosis fungoides/Sezary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE).

We used a gene amplification strategy to analyze T-cell receptor (TCR) gene rearrangements in 185 specimens, including mycosis fungoides/Sezary syndrome (MF/SS), other cutaneous neoplasms, inflammatory dermatoses, reactive lymphoid tissues, and normal skin. Genomic DNA was extracted from lesional tissues and rearrangements of the TCR-gamma chain gene were amplified using the polymerase chain reaction (PCR) with primers specific for rearrangements involving V gamma 1-8 or V gamma 9 gene segments. The resulting PCR products were then separated according to their nucleotide sequence as well as size by denaturing gradient gel electrophoresis (DGGE). Dominant clonal TCR-gamma gene rearrangements were detected in 61 of 68 MF/SS cases by PCR/DGGE. This sensitivity of 90% compared to a sensitivity of only 59% when dominant clonality was sought in 17 of these same cases by Southern blot analysis of TCR-beta gene rearrangements. This difference in sensitivity was greatest in early, minimally infiltrated skin lesions. PCR/DGGE was also more sensitive than Southern blot analysis for detecting peripheral blood involvement in two cases of early MF. Among 12 additional specimens of suspected MF/SS, nine (75%) showed clonal TCR-gamma gene rearrangements by PCR/DGGE including six of eight cases with a previously confirmed diagnosis of MF/SS and three of four cases without prior known MF/SS. Among 105 non-MF/SS specimens, dominant TCR-gamma gene rearrangements were detected in only six cases (6%). Four were diagnosed as chronic dermatitis and two were diagnosed as cutaneous lymphoid hyperplasia. We conclude that the large majority of MF/SS cases, including patch phase disease, possess dominant clonal TCR-gamma gene rearrangements. PCR/DGGE is more sensitive than Southern blot analysis for detecting dominant clonality and staging disease in patients with a confirmed diagnosis of MF/SS. However, because PCR/DGGE is sensitive enough to detect dominant TCR-gamma gene rearrangements in a subset of patients with chronic dermatitis, it cannot be used as the sole criterion for establishing a diagnosis of T-cell lymphoma. As with other molecular biologic clonality assays, clinicopathologic correlation is essential. Nevertheless, the detection of dominant clonality in some cases of histologically nonspecific dermatitis allows the identification of a previously unrecognized subset of patients, i.e., those with "clonal dermatitis." It will be important to determine the long-term risk of MF/SS among these patients because our study indicated that MF/SS can sometimes present with lesions indistinguishable from clonal dermatitis.

Distinct effects of CD30 and Fas signaling in cutaneous anaplastic lymphomas: a possible mechanism for disease progression.

Lymphomatoid papulosis is part of a spectrum of CD30+ cutaneous lymphoproliferative disorders characterized by spontaneous tumor regression. The mechanism(s) of regression is unknown. In a recent study, a selective increase in CD30 ligand expression in regressing lesions of lymphomatoid papulosis and cutaneous CD30+ anaplastic large cell lymphoma was shown, suggesting that activation of the CD30 signaling pathway may be responsible for tumor regression, whereas no difference in Fas/Fas ligand expression was found between regressing and nonregressing lesions. Therefore we tested the effects of CD30 and Fas activation on three CD30+ cutaneous lymphoma cell lines (Mac-1, Mac-2 A, JK) derived from nonregressing tumors of two patients who had progressed from lymphomatoid papulosis to systemic anaplastic large cell lymphoma. To evaluate the effects of CD30 signaling, the cell lines were incubated with a CD30 agonistic antibody, HeFi-1. Proliferative responses, mitogen-activated protein kinase, and nuclear factor kappa B activities were determined with and without CD30 activation. Mac-1 and Mac-2 A showed increased proliferative responses to incubation with CD30 activating antibody, HeFi-1. Inhibition of the mitogen-activated protein kinase activity caused growth inhibition of the Mac-1, Mac-2 A, and JK cell lines. Activation of the Fas pathway induced apoptosis in all three cell lines. Taken together, these findings suggest that resistance to CD30-mediated growth inhibition provides a possible mechanism for escape of cutaneous anaplastic large cell lymphoma from tumor regression. Mitogen-activated protein kinase inhibitors are potential therapeutic agents for the treatment of advanced cutaneous anaplastic large cell lymphoma. J Invest Dermatol 115:1034-1040, 2000

Epstein-Barr virus in Hodgkin's disease: correlation of risk factors and disease characteristics with molecular evidence of viral infection.

Risk factors suggestive of relatively late exposure to EBV have been consistently associated with Hodgkin's disease (HD) in younger adults. In addition, evidence of EBV infection has been found in the Reed-Sternberg cells themselves in about one-third to one-half of all HD cases. However, no study yet published has correlated these childhood social environment risk factors with the presence of EBV in Hodgkin's tumor cells. We examined whether EBV-positive HD occurs in those patients whose childhood environment would predispose them to relatively late exposure to EBV. The study population consisted of 102 cases of mixed cellularity (MC; n = 25) or nodular sclerosing (n = 77) HD. Samples that tested positive for either EBV-encoded RNA or latent membrane protein or both were considered EBV-positive. Of the 102 cases, 83 completed a questionnaire regarding childhood social environment. The association with EBV-positivity was estimated by the odds ratio (OR) with 95% confidence intervals (CI). Twenty-two percent of the cases were EBV-positive. These cases were more likely to be MC (OR, 6.2; CI, 2.3-16.3) and male (OR, 3.4; CI, 1.3-9.0). History of infectious mononucleosis (IM) was not predictive of EBV-positivity, with only 3 of 14 such patients being EBV-positive (P = 0.82). Contrary to our hypothesis, no association between EBV and childhood environment risk factors was identified. The association of EBV with MC histology and male gender agrees with previous reports. The most intriguing finding was the dissociation between IM history and EBV-positivity, in that almost all of the cases with a history of IM were EBV-negative.

Hepatosplenic and subcutaneous panniculitis-like gamma/delta T cell lymphomas are derived from different Vdelta subsets of gamma/delta T lymphocytes.

Gamma/delta T cell lymphomas (gamma/delta TCL) represent rare, often aggressive types of T cell malignancy that are clinically and pathologically diverse. Most gamma/delta TCL occur as a hepatosplenic or subcutaneous type. To date, analysis of the T cell receptor delta (TCRS) gene repertoire of hepatosplenic gamma/delta TCL (gamma/delta HSTCL) and subcutaneous panniculitis-like gamma/delta TCL (gamma/delta SPTCL) has been reported only in a limited number of cases. In this study we analyzed 11 gamma/delta HSTCL and 4 gamma/delta SPTCL by polymerase chain reaction and immunostaining to determine their usage of the Vdelta subtypes (Vdelta1-6). It is noteworthy that 10 of 11 gamma/delta HSTCL expressed the Vdelta1 gene. The remaining case also expressed T cell receptor delta (TCRS) as determined by flow cytometry and TCRdelta rearrangement in Southern blot. However, the Vdelta gene expressed by this lymphoma could not be determined, which suggests usage of an as yet unidentified Vdelta gene. In striking contrast to the gamma/delta HSTCL, all 4 gamma/delta SPTCL expressed the Vdelta2 gene. Our data demonstrate that gamma/delta HSTCL are preferentially derived from the Vdelta1 subset of gamma/delta T lymphocytes, whereas gamma/delta SPTCL are preferentially derived from the Vdelta2 subset. The pattern of Vdelta gene expression in HSTCL and SPTCL corresponds to the respective, predominant gamma/delta T cell subsets normally found in the spleen and skin. This finding suggests that gamma/delta TCL are derived from normal gamma/delta T lymphocytes which reside in the affected tissues. Furthermore, the selective, lymphoma type-specific Vdelta gene segment usage may provide a molecular tool to distinguish better among various types of gamma/delta TCL lymphoma particularly in the clinically advanced, widely disseminated cases.