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Two patients presented with post-transplant lymphoproliferative disorder (PTLD). PTLD encompasses a broad range ofoften malignant proliferations of lymphoid tissue arising in the immunocompromised host after transplantation. The first patient, a 62-year-old woman, received a bilateral lung transplant due to end-stage emphysema and was diagnosed with PTLD 27 days after transplantation. Treatment consisted of reduction in immunosuppression and administration of rituximab. The PTLD regressed. The second patient, a 57-year-old woman, presented with a massively disseminated PTLD 12 years after kidney transplantation. Immunosuppression was reduced and rituximab was administered, but no response was observed. Despite salvage chemotherapy, the patient died due to progressive disease. These two cases illustrate the heterogeneous presentation of PTLD. The condition is caused by the proliferation of B lymphocytes infected with Epstein-Barr virus (EBV) that are no longer controlled by EBV-specific cytotoxic T lymphocytes, due to the immunosuppressive medication given to prevent transplant rejection. Regression of the lymphoma may be achieved by reducing the immunosuppression or treating with rituximab, which attacks B lymphocytes.

Background: Treatment of early-stage Hodgkin's disease is usually tailored in line with prognostic factors that allow for reductions in the amount of chemotherapy and extent of radiotherapy required for a possible cure., Methods: From 1993 to 1999, we identified 1538 patients (age, 15 to 70 years) who had untreated stage I or II supradiaphragmatic Hodgkin's disease with favorable prognostic features (the H8-F trial) or unfavorable features (the H8-U trial). In the H8-F trial, we compared three cycles of mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) combined with doxorubicin, bleomycin, and vinblastine (ABV) plus involved-field radiotherapy with subtotal nodal radiotherapy alone (reference group). In the H8-U trial, we compared three regimens: six cycles of MOPP-ABV plus involved-field radiotherapy (reference group), four cycles of MOPP-ABV plus involved-field radiotherapy, and four cycles of MOPP-ABV plus subtotal nodal radiotherapy., Results: The median follow-up was 92 months. In the H8-F trial, the estimated 5-year event-free survival rate was significantly higher after three cycles of MOPP-ABV plus involved-field radiotherapy than after subtotal nodal radiotherapy alone (98% vs. 74%, P<0.001). The 10-year overall survival estimates were 97% and 92%, respectively (P=0.001). In the H8-U trial, the estimated 5-year event-free survival rates were similar in the three treatment groups: 84% after six cycles of MOPP-ABV plus involved-field radiotherapy, 88% after four cycles of MOPP-ABV plus involved-field radiotherapy, and 87% after four cycles of MOPP-ABV plus subtotal nodal radiotherapy. The 10-year overall survival estimates were 88%, 85%, and 84%, respectively., Conclusions: Chemotherapy plus involved-field radiotherapy should be the standard treatment for Hodgkin's disease with favorable prognostic features. In patients with unfavorable features, four courses of chemotherapy plus involved-field radiotherapy should be the standard treatment. (ClinicalTrials.gov number, NCT00379041 [ClinicalTrials.gov].)., (Copyright 2007 Massachusetts Medical Society.)

Intrinsic resistance of lymphoma cells to apoptosis is a probable mechanism causing chemotherapy resistance and eventual fatal outcome in patients with diffuse large B cell lymphomas (DLBCL). We investigated whether microarray expression profiling of apoptosis related genes predicts clinical outcome in 46 patients with primary nodal DLBCL. Unsupervised cluster analysis using genes involved in apoptosis (n = 246) resulted in three separate DLBCL groups partly overlapping with germinal centre B-lymphocytes versus activated B-cells like phenotype. One group with poor clinical outcome was characterised by high expression levels of pro-and anti-apoptotic genes involved in the intrinsic apoptosis pathway. A second group, also with poor clinical outcome, was characterised by high levels of apoptosis inducing cytotoxic effector genes, possibly reflecting a cellular cytotoxic immune response. The third group showing a favourable outcome was characterised by low expression levels of genes characteristic for both other groups. Our results suggest that chemotherapy refractory DLBCL are characterised either by an intense cellular cytotoxic immune response or by constitutive activation of the intrinsic mediated apoptosis pathway with concomitant downstream inhibition of this apoptosis pathway. Consequently, strategies neutralising the function of apoptosis-inhibiting proteins might be effective as alternative treatment modality in part of chemotherapy refractory DLBCL.

Purpose: In early-stage Hodgkin's lymphoma (HL), subtotal nodal irradiation (STNI) and combined chemotherapy/radiotherapy produce high disease control rates but also considerable late toxicity. The aim of this study was to reduce this toxicity using a combination of low-intensity chemotherapy and involved-field radiotherapy (IF-RT) without jeopardizing disease control., Patients and Methods: Patients with stage I or II HL were stratified into two groups, favorable and unfavorable, based on the following four prognostic factors: age, symptoms, number of involved areas, and mediastinal-thoracic ratio. The experimental therapy consisted of six cycles of epirubicin, bleomycin, vinblastine, and prednisone (EBVP) followed by IF-RT. It was randomly compared, in favorable patients, to STNI and, in unfavorable patients, to six cycles of mechlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin, and vinblastine (MOPP/ABV hybrid) and IF-RT., Results: Median follow-up time of the 722 patients included was 9 years. In 333 favorable patients, the 10-year event-free survival rates (EFS) were 88% in the EBVP arm and 78% in the STNI arm (P = .0113), with similar 10-year overall survival (OS) rates (92% v 92%, respectively; P = .79). In 389 unfavorable patients, the 10-year EFS rate was 88% in the MOPP/ABV arm compared with 68% in the EBVP arm (P < .001), leading to 10-year OS rates of 87% and 79%, respectively (P = .0175)., Conclusion: A treatment strategy for early-stage HL based on prognostic factors leads to high OS rates in both favorable and unfavorable patients. In favorable patients, the combination of EBVP and IF-RT can replace STNI as standard treatment. In unfavorable patients, EBVP is significantly less efficient than MOPP/ABV.

Background: It has been suggested that normal concentrations of biogenic amines and 'histamine-releasing foods' may exacerbate symptoms in mastocytosis. The purpose of this study was to look for scientific evidence in the literature on diets restricted in biogenic amines and histamine-releasing foods in the treatment of mastocytosis., Methods: Medline (1966 to 2004), Cinahl (1982 to 2004) and the Cochraine Library were searched for double-blind placebo-controlled food challenge (DBPCFC) studies with biogenic amines and/or histamine-releasing foods in mastocytosis., Results: No studies employing DBPCFC with dietary biogenic amines or histamine-releasing foods in mastocytosis were found. Only a few in vitro studies in other diseases, animal studies and studies in humans in which histamine-releasing agents were incubated directly with duodenal tissues were found. One case was reported of severe adverse reactions to alcohol in mastocytosis, objectified by an open challenge., Conclusion: Despite the widespread belief that biogenic amines and histamine-releasing foods may cause allergy-like, non-IgE-mediated symptoms in certain patients, the role of diets restricted in biogenic amines and histamine-releasing foods in the treatment of mastosytosis remains hypothetical but worthy of further investigation. There is some evidence for adverse reactions to alcohol in mastocytosis.

Although Burkitt's lymphoma (BL) is classified as one entity in the World Health Organisation (WHO) classification, we wondered whether BL should not be considered as a different disease in children compared with adults. Netherlands Cancer Registry (NCR) data were obtained from 1994 to 1998 (n=203). Detailed clinical data from two treatment protocols were compared: one for adults up to the age of 65 years (n=27) and one for children (n=80). All slides of the two clinical studies were centrally reviewed which included immunophenotyping and when necessary breakpoint analysis of MYC/8q24. Only cases with an unambiguous diagnosis of BL (classical and atypical BL) were accepted. The age distribution of BL-patients showed a bimodal distribution with a peak at the paediatric age and a steady increase after approximately 60 years of age. Most of the patients were males (89% for children and 78% for adults) and only male patients showed this bimodality. Children more often had extranodal disease (81% vs. 59%), whereas adults more often had nodal disease (89% vs. 53%). Based on epidemiology and clinical presentation, the concept that BL is one disease should be re-challenged.

Forty years ago, van Putten described in the European Journal of Cancer (see this issue) quantitative studies on the optimal storage techniques of mouse and monkey bone marrow suspensions. Survival of the animals after irradiation following injection with stored bone marrow cell suspensions was the endpoint. He observed some species differences, but based on the data obtained considered a careful trial of the glycerol-polyvinylpyrrolide (PVP) combination for storage of marrow in man was indicated. In spite of this, dimethyl sulphoxide has become the 'standard' cryopreservant for human marrow stem cells. Over the last 40 years, there has been a tremendous increase in knowledge about haematopoietic stem cells and their use in the clinic. Haematopoietic stem cells are now known to travel between the bone marrow and peripheral blood and are the best-characterised adult stem cells. These cells are currently widely used for transplantations in the clinic and are obtained from a wide variety of sources. These include the bone marrow, peripheral blood, cord blood, autologous as well as allogeneic stem cells from related or unrelated donors. Increasingly, data has become available that adult haematopoietic stem cells can generate differentiated cells belonging to other cell types, a process called "developmental plasticity". Thus, they may contribute to non-haematopoietic tissue repair in multiple organ systems. This has created a whole new potential therapeutic armamentarium for the application of haematopoietic stem cells outside of the area of malignancies and haematopoietic disorders.

A higher prevalence of solid tumours in patients with M(onoclonal) proteinaemia without a co-existing haematological malignancy has been reported. We investigated this association by linking a population-based registry of patients with newly diagnosed M-proteinaemia (n = 1464) with the Regional Cancer Registry. Patients were followed for a median of 7.4 years for those still alive. In total 167 (11%) patients with 173 solid tumours were compared with 861 patients with 'M-proteinaemia only' (without a haematological malignancy). The M-protein isotype or level or clinical parameters did not differ between the groups. M-protein isotype was not associated with a specific tumour type. Standardised Morbidity Ratios (SMR) for nearly all solid tumours were elevated in the year of the M-protein discovery, but the excess risk disappeared during follow-up suggesting selection through diagnostic investigations rather than a causal role. In this large series of patients with both newly diagnosed M-proteinaemia and a solid tumour no relationship could be established.

Whether the response to chemotherapy is a prognosticator in multiple myeloma (MM) is still not known. Therefore, the relationship between survival and the rate of monoclonal protein (M-protein) decrement during the first cycles of therapy was prospectively assessed in 262 patients with newly diagnosed MM that were included in a phase III trial (HOVON-16). M-proteins were collected monthly during melphalan-prednisone therapy (MP: melphalan 0.25 mg/kg, prednisone 1.0 mg/kg orally for 5 d every 4 weeks). Patients with light chain disease (n = 18), immunoglobulin M (IgM)-MM (n = 1) and no immunotyping (n = 1) were excluded. Of the 242 patients studied, 75% had IgG M-protein and 25% IgA; MM stages: I: 1%, II: 35% and III: 64%. The median M-protein decrease after the first cycle of MP was 21% for IgG and 27% for IgA, and declined to < 5% after four cycles. An obvious survival advantage was seen for patients who had an M-protein decrease of at least 30% after the first MP cycle, which became significant when an M-protein decrease of 40% or more was reached. As established prognostic parameters (Salmon & Durie stage, serum creatinine, and haemoglobin) also remained prognostically significant, we concluded that early response to MP predicts for survival in MM.

We analysed data from 936 newly-diagnosed patients with advanced, aggressive non-Hodgkin's lymphoma (NHL) treated in three randomised European Organisation for Research and Treatment of Cancer (EORTC) trials performed between 1980 and 1999 (median follow-up of 8.7 (0.2-20.4) years). The CHOP-like regimen CHVmP/BV (cyclophosphamide, doxorubicin, teniposide and prednisone with bleomycin and vincristine at mid-interval), was compared with CHVmP (CHVmP/BV without bleomycin and vincristine), ProMACE-MOPP (methotrexate, doxorubicin, cyclophosphamide, etoposide, mechlorethamide, vincristine, procarbazine and prednisone) and CHVmp/BV with additional, autologous stem-cell transplantation, respectively. Overall, treatment with CHVmP/BV resulted in a better long-term outcome with 63% complete responses being observed and an overall survival (OS) of 59 and 43% at 5 and 10 years, respectively. Remarkably, OS after CHVmP/BV improved across the trials, even after stratifying for the International Prognostic Index (IPI). This finding could not be directly related to better salvage treatments during the last decade. Selection bias appears to be responsible: stepwise corrections for small differences in inclusion criteria eliminated the difference in OS, especially when histological subgroups were studied. This systemic review underlines the difficulties encountered in retrospective sub-set analyses and the biases that can be introduced when recent studies are compared with older ones.

Hairy cell leukemia (HCL) is a chronic mature B-cell leukemia characterized by malignant B cells that have typical hairy protrusions. To characterize possible HCL-associated tumor antigens, we generated an HCL-specific and HLA class II (DPw4)-restricted proliferative CD4+ T-cell clone. To identify the target antigen of these T cells, we constructed a synthetic peptide library dedicated to bind HLA DPw4, and identified a mimicry epitope recognized by the T-cell clone. With this epitope, the recognition motif of the T-cell clone was deduced and a peptide of human synaptojanin 2 (Syn 2) was identified that stimulated the HCL-reactive T-cell clone. Both Northern and Western blot analyses showed that Syn 2 expression was increased in HCL samples compared to other B cells. Besides, the Syn 2-expressing cell line AML193, with the introduced restrictive HLA-DPw4 molecules, was recognized by the HCL-specific T-cell clone. These results indicate that Syn 2 is a target of autoreactive HCL-specific T cells. Since Syn 2 is a phosphatidylinositol 4,5-biphosphatase involved in cell growth and rearrangement of actin filaments, the increased Syn 2 expression may correlate with the disease etiology or the characteristic morphologic alterations caused by the disease.

Breast lymphomas are rare and consensus about their treatment is lacking. A population-based study of 38 breast lymphomas, registered in the databases of two Comprehensive Dutch Cancer Centers from 1981 to 1999, was performed. The median age of all female patients was 65 years (20-92): 25 patients had localized and 13 patients had disseminated lymphoma. The most common type was diffuse large B-cell lymphoma (DLBCL), which accounted for 17 of the localized and 4 of the disseminated cases. Burkitt's lymphoma (BL), three being disseminated, was found in four patients. There were six extranodal marginal zone lymphomas (ENMZL), three being localized. Seven DLBCL and one BL showed additional histological features of mucosa-associated lymphoid tissue (MALT) lymphoma. Localized aggressive lymphomas treated with surgery and/or radiation therapy had relapse rates of 100% and 67%, respectively. Cyclophosphamide, hydroxydaunomycin, vincristine, and prednisone (CHOP)-like chemotherapy with or without local irradiation led to 17% relapses in patients with localized aggressive lymphoma. Median follow-up time was 32 months (0.6-218); 37% of the patients relapsed and 24% had progressive disease. Response to salvage regimens, given to 91% of the patients with recurrent disease, was poor. The 2-year overall survival rate was 63%, 72% for patients with localized disease, and 46% for patients with disseminated lymphoma. The majority of breast lymphomas are localized aggressive lymphomas that should be treated initially with CHOP-like chemotherapy with or without irradiation. The initial choice of treatment is very important because response to salvage regimens is poor.

Background: The use of involved-field radiotherapy after chemotherapy for advanced Hodgkin's lymphoma is controversial., Methods: We randomly assigned patients with previously untreated stage III or IV Hodgkin's lymphoma who were in complete remission after hybrid chemotherapy with mechlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin, and vinblastine (MOPP-ABV) to receive either no further treatment or involved-field radiotherapy. Radiotherapy consisted of 24 Gy to all initially involved nodal areas and 16 to 24 Gy to all initially involved extranodal sites. Patients in partial remission were treated with 30 Gy to nodal areas and 18 to 24 Gy to extranodal sites., Results: Of 739 patients, 421 had a complete remission; 161 of these patients were assigned to no further treatment, and 172 to involved-field radiotherapy. The median follow-up was 79 months. The five-year event-free survival rate was 84 percent in the group that did not receive radiotherapy and 79 percent in the group that received involved-field radiotherapy (P=0.35). The five-year overall survival rates were 91 and 85 percent, respectively (P=0.07). Among the 250 patients in partial remission after chemotherapy, the five-year event-free and overall survival rates were 79 and 87 percent, respectively., Conclusions: Involved-field radiotherapy did not improve the outcome in patients with advanced-stage Hodgkin's lymphoma who had a complete remission after MOPP-ABV chemotherapy. Radiotherapy may benefit patients with a partial response after chemotherapy., (Copyright 2003 Massachusetts Medical Society)

The Comprehensive Cancer Centre West (CCCW) population based non-Hodgkin's lymphoma (NHL) registry contains information on all newly diagnosed NHL patients living in the region covered by the CCCW. Patients were entered from June 1st 1981 to December 31st 1989. Follow-up is still ongoing, median follow-up is 113 months (1-191 months) for patients alive. In this study, patient and tumor characteristics, data on patterns of care, response and (relative) survival are described. As follicular lymphomas and diffuse large B-cell lymphomas are the most frequently occurring NHL subtypes in the database, a separate analysis is performed to characterize the clinical picture of these disease entities in the CCCW population. Our data illustrate that NHL patients in the general population are substantially older than patients included in trials and hospital based series. Due to older age, treatment is withheld or adapted for a substantial number of patients. The resulting survival and relative survival rates are a reflection of these choices.

Background: The definition of primary extranodal non-Hodgkin's lymphoma (NHL) is a controversial issue, especially in patients where both nodal and extranodal sites are involved., Patients and Methods: The impact of different definitions of primary extranodal NHL on incidence and prognosis is explored using data from a population-based NHL registry., Results: Using liberal criteria, 389 (34%) cases were classified as primary extranodal NHL. Overall survival (OS) rates of nodal and extranodal NHL patients defined this way were comparable; however, extranodal NHL patients had a better disease-free survival (DFS). When strict criteria were applied, 231 cases (20%) were classified as primary extranodal NHL. OS and DFS rates of extranodal NHL patients defined this way were superior to nodal NHL patients; however, the difference in OS was reversed after correction for differences in International Prognostic Index and malignancy grade., Conclusion: This study illustrates the selection bias that is introduced when a strict definition of primary extranodal NHL, that excludes cases with disseminated disease, is used. Patients with primary extranodal NHL were found to have a superior DFS, irrespective of which definition of primary extranodal NHL was used.

Many cost analyses of stem-cell transplantations are available, which is in sharp contrast to the level of cost analyses on first-line chemotherapy for aggressive non-Hodgkin's lymphoma (NHL). Given the scarcity of cost analyses of first-line chemotherapy for NHL, it is difficult to assess the economic impact of upcoming new treatment modalities. Therefore we performed an analysis on costs of diagnosis and treatment of patients with newly diagnosed NHL who were treated with standard CHOP (-like) chemotherapy. As many NHL patients are treated in trials and the economic effects of the trial participation are unknown, our analysis included both patients treated according to trial protocols and patients treated according to standard local practice (SLP). The cost analysis was based on the total medical consumption of the patients. It was found that costs of the trial and SLP groups are within comparable ranges, although costs of diagnostic tests were somewhat higher within the trials. In elderly patients, SLP chemotherapy was discontinued more frequently in case of leucocytopenia or thrombocytopenia. This analysis provides basic information about the costs of first-line standard chemotherapy for patients with newly diagnosed aggressive NHL and the plausible ranges in which these costs may vary. Given the results, we will initiate larger studies to investigate whether trial treatments (showing more or less similar costs as SLP treatments) are more cost-effective for patients with aggressive NHL.

A 64-year-old male was treated for acute myeloid leukemia with idarubicin and cytarabin. He developed pulmonary mycosis (radiologically consistent with aspergillosis), which responded to intravenous amphotericin B.

It is debated whether non-Hodgkin's lymphomas originating in Waldeyer's ring (WR NHL) behave as NHL originating in lymph nodes or share common features with extranodal lymphomas originating in mucosa associated lymphatic tissue (MALT). We analyzed data from a population based NHL registry on patterns of dissemination at diagnosis, response to treatment, patterns of failure and survival of 77 primary Waldeyer's ring Non-Hodgkin's lymphomas (WR NHL) patents. Data of completely staged patients with diffuse large cell lymphomas (DLCL) originating in WR (n=44) were compared with those of patients retrieved from the same registry with DLCL originating in lymph nodes or stomach (the latter as prototype of a lymphoma originating in MALT). Primary WR NHL had favorable risk scores according to the International Prognostic Index (IPI), and responded well to therapy: a complete response (CR) rate of 74% was observed. Disease free survival (DFS) and overall survival (OS) were poor, however (47% and 31% at 10 years, respectively). The comparison of DLCL originating in WR, lymph nodes and stomach revealed that WR and gastric NHL patients shared a restricted pattern of dissemination at diagnosis, in contrast to patients with DLCL originating in lymph nodes. Although not all patients were completely restaged at relapse, analysis of patterns of failure suggested that the gastro-intestinal tract is a preferential site for recurrences, both for WR and gastric DLCL patients. CR rates of WR, nodal and gastric DLCL patients were 77%, 55% and 55% respectively (P=0.03), OS of the three patient subgroups did not differ (33%, 27% and 37% at 10 years). DFS of WR DLCL patients was similar to nodal DLCL patients but inferior to gastric DLCL patients (47%, 48% and 73% at 10 years respectively, P=0.006). After Cox regression analysis the relative relapse risk for patients with WR DLCL when compared to patients with DLCL originating in lymph nodes was 2.01 (C.I. 0.99-4.01, P=0.05), and 3.46 (C.I. 1.32-9.00, P=0.01) when compared to patients with gastric DLCL. The clinical picture of primary WR NHL emerging from this population based study is in agreement with data form hospital based studies. In the comparison of WR DLCL, nodal DLCL and gastric DLCL, the observed patterns of dissemination suggest similarities between WR DLCL and gastric DLCL. The frequent relapses after CR observed for WR DLCL patients, however, indicate that these lymphomas clinically behave as nodal DLCL, and should be treated accordingly.

Background: The EORTC clinical trial 20901, activated in 1990, was designed to treat non-Hodgkin's lymphomas (NHL) of intermediate/high-grade malignancy according to the Working Formulation. Established in 1994, the R.E.A.L. Classification on NHL has now replaced all former classifications., Patients and Methods: We reanalysed all cases (n = 273) documented by material available for review according to the R.E.A.L. Classification. In addition, we subdivided cases recognised as diffuse large B-cell lymphoma (DLBCL) into three morphologically distinct categories, namely, large cleaved DLBCL (LC-DLBCL), T-cell-rich/histiocyte-rich B-cell lymphoma (T-cell-rich/histiocyte-rich BCL) and CD30+ DLBCL with anaplastic cell features (CD30+ DLBCL). Finally, T/NULL anaplastic large-cell lymphoma (ALCL) cases were subdivided into ALK+ and ALK- lymphomas. Review was performed independently by two pathologists from two different centres., Results: DLBCL (61%), T/NULL ALCL (15%) and mantle-cell lymphoma (MCL, 50%) were the main NHL categories represented in the study. Fifty-seven of one hundred sixty DLBCL cases were further subclassified as LC-DLBCL (33 cases), T-cell-rich/histiocyte-rich BCL (13 cases) or CD30+ DLBCL (11 cases). The remaining cases were indicated as unspecified DLBCL. A clinico-pathological correlation confirmed the findings of previous studies suggesting that MCL, DLBCL and ALCL represent distinct entities with MCL being characterised by a short survival, in contrast with the longer survival and less frequent progression typical of ALK+ compared to ALK- ALCL. Within DLBCL, T-cell-rich/histiocyte-rich BCL showed distinctive features at presentation whereas CD30+ DLBCL showed a trend towards a more favourable prognosis, that might be comparable to that of ALK+ ALCL., Conclusions: Our data further support the usefulness of the R.E.A.L. Classification and illustrate the feasibility of DLBCL subtyping. Moreover, our results demonstrate the distinct clinical characteristics of T-cell-rich/histiocyte-rich BCL and CD30+ DLBCL with anaplastic cell features suggesting that they may represent clinico-pathologic entities.

Circulating hematopoietic progenitor cells (HPCs) are routinely measured by flow cytometry using CD34 expression. As an alternative, the "immature information" (IMI) channel measurement of the automated hematology analyzer Sysmex SE machines was developed. We tested the IMI channel HPC method with umbilical cord blood specimens. The IMI-HPCs were compared with CD34 counts and numbers of colony-forming units (CFUs). The IMI-HPC data were reproducible and dilution experiments yielded a log-linear relationship. The mean percentage of CD34(+) cells in 50 umbilical cords was 0.43 versus 0.11 of HPCs in the IMI channel (correlation coefficient r = 0.67). Absolute numbers yielded 96.79 x 10(6)/L CD34(+), 33.17 x 10(6)/L IMI-HPC, and 35.04 x 10(6)/L CFU-HPC. Receiver operating characteristics curves were made at various cutoff levels for CD34(+) cells to visualize sensitivity and specificity profiles. With median values of 13.56 x 10(6)/L for IMI-HPC and 20 x 10(6)/L for CD34(+) as cutoff points (the levels used in the laboratory to start stem cell pheresis), the percentage of false-negative observations was 70.4%. To exclude the influence of storage time, tests were repeated until 72 h after umbilical cord collection. Total white blood cell count decreased in most cases, whereas absolute number of IMI-HPC tended to increase in time. In conclusion, if HPC measurements in the IMI channel are used to monitor circulating stem cells during mobilization, one has to be aware of a very low correlation between these results and those of other methods such as CD34(+) analysis and colony growth. False-negative results do occur, but if events are seen in the IMI channel, this simple monitoring technique is useful to predict the presence of circulating stem cells.

T cells from patients with active hairy cell leukemia (HCL), a chronic B cell malignancy, show poor proliferation in response to allogeneic peripheral blood mononuclear cells (PBMC). In order to study the T cell dysfunction, the expression of several adhesion and costimulatory molecules was analyzed by flow cytometry. Circulating T cells from HCL patients showed increased percentages of CD28(-) in all T cell subsets. In some patients the percentage of CD28(-) T cells within the CD4(+) subset was increased up to 80%. These CD4(+)CD28(-) T cells did not proliferate in a mixed lymphocyte culture (MLC) against allogeneic PBMC. After enrichment for CD4(+)CD28(+) T cells, the proliferative response in the MLC was recovered, but this response was still lower than the proliferative response from control T cells. In conclusion, lack of CD28 on T cells and a restricted T cell repertoire may contribute to immune deficiency in patients with HCL., (Copyright 1999 Academic Press.)

We determined interleukin-6 (IL-6) levels in the serum of 212 well-defined patients with newly diagnosed paraproteinaemia and evaluated its discriminatory value and prognostic role in multiple myeloma (MM). Results were compared with serum neural cell adhesion molecule and beta-2-microglobulin, both established prognostic MM markers. Paraproteinaemia-related diagnoses were: MM (60), other haematological diseases (46), solid tumours (35), autoimmune diseases (17) and monoclonal gammopathy of unknown significance (MGUS) (54). The range of IL-6 levels in all diagnostic groups overlapped widely and did not serve as a discriminatory marker in newly diagnosed paraproteinaemia even when patients with infection or fever (42) were excluded. In MM high IL-6 levels (>/= 50 pg/ml) were not associated with a shorter survival (P = 0.24). We compared our results with 20 published studies on serum IL-6 in paraproteinaemia and/or MM. IL-6 data have to be related to the assay used (bio- or immunoassay) and to the status of MM (newly diagnosed, during therapy, progressive disease). We conclude that serum IL-6 is not specific for paraproteinaemia-related diseases and will not serve as a reliable discriminatory or prognostic marker in paraproteinaemia and MM.

A 34-year-old man with chronic myeloid leukaemia (CML) firstly developed a lymphoid blast crisis of B-cell type. After a second chronic phase which lasted for > 4 years with maintenance chemotherapy of hydroxyurea, 6-mercaptopurine and methotrexate, he developed a T-cell acute lymphoblastic leukaemia of TcR-gammadelta+ type. Cytogenetic analysis revealed disappearance of the t(9;22) translocation and appearance of new abnormalities consistent with the diagnosis secondary acute leukaemia. To our knowledge, secondary leukaemia in CML has not previously been reported.

The relationship between aplastic anaemia (AA) and pregnancy remains uncertain. To assess whether an association between pregnancy and severe aplastic anaemia (SAA) exists, we compared the frequency of pregnancy in 35 young women with newly diagnosed SAA with the expected frequency in the general population. The observed pregnancy rate in the SAA group was 3-6%. This percentage approximates the expected pregnancy rate of 4.4% in the general population and is not compatible with a strong association between pregnancy and SAA.

Background: To compare characteristics, treatment and outcome of patients > or = 70 years with patients < 70 years in a population-based non-Hodgkin's lymphoma (NHL) registry., Patients and Methods: All new patients with NHL (n = 1168) in a geographically defined region in the western part of The Netherlands were registered during a nearly 10-year period. Patient, tumour and treatment characteristics, response to therapy and survival were analysed for both age groups. An age-adjusted prognostic index was determined for elderly patients with aggressive lymphoma., Results: The elderly comprised 41% of the registered patients. There were significantly more females, a preponderance of intermediate-grade histology (diffuse large B-cell lymphoma) and a lower performance status. Incomplete staging in the elderly was mostly due to the omission of a bone marrow biopsy. With respect to WF grading the complete remission rate (except for patients with low-grade/stage I NHL, patients with extranodal NHL and for patients with intermediate grade/extensive NHL) and overall survival at five years (except for patients with low-grade/stage I NHL and for patients with intermediate-grade/extensive NHL) were significantly inferior in the elderly. With respect to the R.E.A.L. Classification the exceptions were in patients with high grade MALT lymphomas (elderly good) and patients with mantle-cell and peripheral T-cell lymphomas (younger group bad too). However, once complete remission was reached, the disease-free survival did not differ significantly between the two age groups, emphasising the importance of achieving complete remission. Although 65% of the classified elderly patients presented with intermediate-grade NHL, only 26% of the elderly patients treated with chemotherapy received anthracycline-based chemotherapy. In the elderly, lymphoma (treatment-related toxicity included) contributed to death in 70% and concomitant disease (other malignancy included) in 30%, versus 78% and 22%, respectively, for the younger group (P = 0.04). The age-adjusted prognostic index, made up of the factors serum LDH, stage and Karnofsky index, showed a clear distinction between the four risk categories low, low/intermediate, intermediate/high and high, with a median survival time of 43, 20, seven and four months, respectively. For the younger group the respective numbers were 144, 45, 19 and 11 months., Conclusions: In a population-based NHL registry the elderly, predominately female patients, formed a larger proportion of the patient group than the one usually reported in the literature. In this population-based cohort inferior remission and overall survival rates were seen in the elderly. However, obtaining complete remission was beneficial for the prognosis of this disease in the elderly. By the application of the R.E.A.L. Classification important subgroups emerge.

Diagnoses in patients with paraproteinaemia are diverse; few (mostly single centre based) studies are known that describe incidence, diagnoses and follow-up in patients with paraproteinaemia. In the region of the Comprehensive Cancer Centre West in the Netherlands (population 1.6 million, 1992) a population-based registry was set up in the period 1991-1993. Patients (n = 1464; median age: 72 years; range: 16-102) were entered by clinical chemists, internists, haematologists, and pathologists. Multiple myeloma and plasmacytoma were diagnosed in 261 patients (18%), paraprotein-related haematological diseases in 159 patients (11%) and paraprotein-related internal diseases in 210 patients (14%). After bone marrow examination monoclonal gammopathy of unknown significance (MGUS) was diagnosed in 207 (14%) patients. No further diagnosis could be made in 627 (43%) patients mostly for lack of supplementary bone marrow and (or) X-ray examinations. Consequently, more than two-thirds of all patients with a newly found paraprotein did not show any sign of a haematological malignancy. Using these data a 'myeloma risk score' was developed to predict the presence of a multiple myeloma based on paraprotein type and concentration, aiding the physician in determining which patients should undergo further bone marrow and skeletal examinations.

Background: Interferon alfa is a conservative and widely used alternative to bone marrow transplantation in treatment of patients with early chronic myeloid leukemia (CML). A meta-analysis was conducted to develop a reliable prognostic scoring system for estimation of survival of patients with CML treated with interferon alfa., Methods: Patients treated in prospective studies, including major randomized trials, were separated into learning and validation samples. Cox regression analysis and the minimum P-value approach were used to identify prognostic factors for patient survival and to discover groups in the learning sample with the greatest differences in survival. These findings were then validated by applying the new scoring system to patients in the validation sample., Results: We collected data on 1573 patients who were participants in 14 studies involving 12 institutions; 1303 patients (learning sample, n = 981; validation sample, n = 322) were eligible for inclusion in this analysis, and their median survival time was 69 months (range, 1-117 months). Because two previously described prognostic scoring systems failed to discriminate risk groups satisfactorily, we developed a new scoring system that utilizes the following covariates: age, spleen size, blast count, platelet count, eosinophil count, and basophil count. Among 908 patients with complete data in the learning sample, three distinct risk groups were identified (median survival times of 98 months [n = 369; 40.6%], 65 months [n = 406; 44.7%], or 42 months [n = 133;14.6%]; two-sided logrank test, P< or =.0001). The ability of the new scoring system to discriminate these risk groups was confirmed by analysis of 285 patients with complete data in the validation sample (two-sided logrank test, P = .0002)., Conclusions: A new prognostic scoring system for estimating survival of patients with CML treated with interferon alfa has been developed and validated through use of a large dataset.

Hairy cell leukemia (HCL) is clinically associated with severe T-cell dysfunction. Several new observations have given more insight into the abnormal T-cell responses seen in this disease. T-lymphocytes in the spleen of patients with HCL seem to be abnormally activated. On the other hand, they are non-responsive, possibly as a result of monocytopenia which may lead to inadequate antigen presentation. This, together with the lack of CD28 on T-cells, may cause T-cell dysfunction. Furthermore, there is a very restricted repertoire of the T-cell receptor-beta family, which may also result in non-responsiveness. Otherwise, T-cell clonal excess may be indicative for activated, possibly autoreactive T-cells.

The function of the simultaneous expression of CD5 and its ligand CD72 on B cell malignancies like chronic lymphocytic leukemia and mantle cell lymphoma was assessed. It is unknown if reciprocal interactions between CD72 and CD5 exert an autocrine growth-promoting or -inhibiting effect. CD5+ (n = 13) and CD5- (n = 9) B cell malignancies were cultured with the anti-CD72 mAb WL225. For comparison, five other anti-CD72 mAbs were tested. Only CD5+ B cell malignancies proliferated upon CD72 activation (9 out of 13 cases). A strong suppressive effect of IL-4 on the anti-CD72-induced [3H]thymidine incorporation, partially caused by downmodulation of the CD72 expression, was seen. Stimulation of the CD5 antigen by L cells transfected with human CD72 (LhCD72) and the anti-CD5 mAb 1C12 exerted no (n = 9) or a minor effect (2 out of 8 cases), respectively. Finally, the results of CD72 stimulation were compared with CD40 stimulation, as this "CD40 system" is an effective method for stimulating B cell malignancies. In 4 of the 7 anti-CD72 responsive cases a costimulatory effect was seen.

Purpose: We report the results of a randomized study of the European Organization for Research and Treatment of Cancer (EORTC) Lymphoma Group, which compared a chemotherapy regimen specifically devised for elderly patients, ie, etoposide, mitoxantrone, and prednimustine (VMP), versus the standard regimen of cyclophosphamide, doxorobucin, vincristine, and prednisone (CHOP) in patients older than 70 years of age with intermediate- and high-grade non-Hodgkin's lymphoma (NHL)., Patients and Methods: Patients older than 70 years of age with stage II, III, or IV intermediate- and high-grade NHL, with an Eastern Cooperative Oncology Group (ECOG) performance status less than 4 and acceptable cardiac, renal, and liver function were randomized to receive six courses of VMP or six courses of CHOP. Between February 1989 and June 1994, 130 patients aged 70 to 93 years (median, 75) were enrolled and 120 were assessable for response, 60 patients in each arm., Results: Overall objective response rates were 50% and 77% in VMP- and CHOP-treated patients, respectively (P = .01), while complete response (CR) rates were borderline significant (27% v 45%; P = .06). At 2 years, the progression-free survival (PFS) rate was 25% with VMP versus 55% with CHOP (P = .002) and the overall survival (OS) rate was 30% with VMP versus 65% with CHOP (P = .004). Statistically significant more alopecia and neurologic and gastrointestinal toxicities were reported with CHOP., Conclusion: CHOP is the standard regimen for patients > or = 70 years of age with stage II to IV intermediate- and high-grade NHL.

Patients with monoclonal gammopathies comprise a heterogenous group. The few studies on incidence and follow-up are single-centre-based and may reflect referral bias. To avoid this, all patients (n=1275) in midwestern Netherlands with a newly discovered paraproteinaemia in 1991, 1992 and 1993 were included in a population-based registry and divided into four major diagnostic groups: multiple myeloma and plasmacytoma (n=230, 18%), other haematological diseases (n=141, 11%), paraprotein-related internal diseases (n=191, 15%) and monoclonal gammopathy of undetermined significance (MGUS, n=713, 56%). To avoid a possibly erroneous diagnosis, patients who were classified as having MGUS but who did not undergo confirmatory bone marrow examination were included in a separate group 'provisional MGUS' (n=524, 41%), whereas patients who did were classified as having 'definite MGUS' (n=189, 15%). The 'provisional MGUS' patients were relatively older and had more often a poor performance status, but differences between this and the 'definite MGUS' group were otherwise small. Patients complaining of general malaise more often had a full work-up of their paraproteinaemia. Bone pain, hypercalcaemia, high total protein, and high ESR occurred predominantly in the myeloma group, whereas fever or infection was less often seen in these patients. This registry of patients with paraproteinaemias provided valuable data related to all different diseases associated with paraproteinaemia.

Diagnostic systems for monoclonal gammopathies use bone marrow and X-ray examinations to exclude multiple myeloma (MM). Data from a population-based registry of unselected patients with paraproteinemia indicate that these tests are often done only when MM is suspected. We used 441 randomly selected patients to develop a simple four point "Myeloma Risk Score" based on two readily available laboratory tests. One point was given for paraprotein concentration > or = 10 g/l, one point for IgG and IgA, and two points for IgD and light chains only. A score of 0 or 1 indicated a low risk for MM, with scores of 2 and 3 signifying high risks. Sensitivity, specificity, positive and negative predictive value (PV) for the Myeloma Risk Score in the training sample were 92%, 88%, 79%, and 96% respectively. Extrapolating these results to a larger cohort showed that 90% of patients with a monoclonal gammopathy could be classified correctly as having MM or a non-myeloma condition. The Myeloma Risk Score can identify patients with a paraproteinemia at risk for MM, and who are therefore candidates for bone marrow and X-ray examination.

The development of a population-based registry on paraproteinemia and multiple myeloma is described. A unique feature of this registry is the multidisciplinary approach to obtain and collect new cases. Clinical chemists, internists, hematologists, and pathologists could all enter patients. All patients newly diagnosed in the mid-western part of The Netherlands (1.7 million inhabitants in 1992) with a paraproteinemia or multiple myeloma in 1991, 1992, and 1993 were included. The project was composed of a registry of clinical and laboratory data extracted from the patient's records, storage of 1 ml serum at diagnosis, and a yearly follow-up. A total of 1832 entries was received, of which 83% met the inclusion criteria. Comparison of this database with the Regional Cancer Registry showed that the paraprotein registry was successful as far as registration of myeloma patients was concerned. We conclude that the multidisciplinary approach used in this paraprotein registry is feasible and has resulted in a unique collection of patients for studying potential pre-malignant conditions such as paraproteinemia.

Objectives: To define the cumulative risk of central nervous system (CNS) relapse in systemic non-Hodgkin lymphoma (NHL); to assess the risk factors of age, sex, malignancy grade, stage, localization, and response to initial therapy; and to evaluate the effect of CNS prophylaxis., Patients and Methods: An unselected group of 532 patients with systemic NHL. A retrospective analysis., Results: Eleven patients presented with systemic as well as CNS localization, whereas in 55 patients, CNS relapse occurred later. The cumulative risk of CNS relapse at 4 years for all 532 patients was 19%. High-grade NHL carried a 39% risk of CNS relapse, with the vast majority of relapses occurring in the first 14 months after the initial diagnosis. The cumulative risk in patients with intermediate-grade NHL was considerable (22%) and dispersed throughout a much longer period (6 years). Patients with low-grade NHL still carried a 7% risk of CNS relapse; in all these patients, low malignancy grade was transformed into a higher malignancy grade at that time. In a multivariate analysis, high- and intermediate-grade NHL and advanced stage were independent risk factors for CNS relapse. There was not any strong evidence for a beneficial role of CNS prophylaxis in patients with intermediate- and high-grade NHL, but a retrospective analysis cannot be conclusive with regard to the effect of therapy. Systemic relapse occurred rapidly after CNS relapse, resulting in a median survival time after CNS relapse of only 2 months., Conclusions: Patients with high- and intermediate-grade NHL carry a considerable risk of CNS relapse. Advanced stage is an additional independent risk factor. The role of CNS prophylaxis seems to be disappointing, but a retrospective analysis cannot be conclusive. Prognosis after CNS relapse is poor.

Background: Induced sputum potentially allows monitoring of airway inflammation in patients with asthma in a non-invasive way. However, the relationship between the cellular content in sputum and airway tissue has not been fully clarified., Objective: We compared the cellular compositions of hypertonic saline-induced sputum, bronchoalveolar lavage fluid (BAL) and bronchial biopsies in 18 clinically stable patients with mild to moderate atopic asthma (baseline FEV1: range 61-114%pred, PC20 methacholine: 0.04-4.7 mg/mL). They were treated with inhaled short-acting bronchodilators on demand, with (n = 8) or without (n = 10) regular inhaled steroids., Methods: Each patient underwent sputum induction and fiberoptic bronchoscopy on separate days in random order. Differential cell counts of induced sputum, bronchoalveolar lavage and bronchial wash were determined on May-Grünwald-Giemsa stained cytospins. Flow cytometry was performed on sputum and BAL samples. Immunohistochemical techniques were used to stain inflammatory cells in 6 microm cryostat sections of bronchial biopsies., Results: Sputum cell differentials were not different between the patients with and without inhaled steroids, and showed a median value of 19.4% squamous cells, with 1.0% eosinophils, 3.3% lymphocytes, 28.7% neutrophils, 49.4% macrophages and 6.9% cylindric epithelial cells (in percentage non-squamous cells). The percentage eosinophils in sputum was significantly correlated with their percentage in bronchial wash (Rs = 0.52, P = 0.03) and in BAL (Rs = 0.55, P= 0.02), whilst there was a trend towards such a correlation between the number of eosinophils/mL sputum and the number of EG2+ eosinophils/mm2 lamina propria in bronchial biopsies (Rs = 0.44, P = 0.07). In addition, the percentage of CD4+ lymphocytes correlated between sputum and BAL (Rs = 0.55, P = 0.03)., Conclusion: We conclude that the eosinophil counts in hypertonic saline-induced sputum from patients with asthma are related to those in bronchial wash and BAL and, to a lesser extent, with the counts in bronchial biopsies. This suggests that induced sputum can be used to monitor the presence and severity of airway inflammation in asthma.

Purpose: T-prolymphocytic leukemia (T-PLL) is an aggressive malignancy of mature T cells refractory to conventional chemotherapy, with a median survival duration of 7.5 months. We report here promising results with the use of a genetically reshaped human CD52 antibody, CAMPATH-1H., Patients and Methods: Fifteen patients with T-PLL, most of whom had received the purine analog deoxycoformycin (DCF), were treated with CAMPATH-1H. Results were compared with those of 25 patients treated with DCF., Results: Major responses occurred in 11 patients (73%) treated with CAMPATH-1H compared with 40% with DCF. Complete remissions (CRs) were documented in nine (60%) of the CAMPATH-1H cases and only three (12%) were obtained with DCF. CRs with CAMPATH-1H were durable, and re-treatment with the antibody resulted in second CRs in three relapsed patients. Two of them were successfully autografted with peripheral-blood and bone marrow stem cells collected during the first CR. Apart from first-dose reactions, infusions of CAMPATH-1H were well tolerated. However, two responding patients developed severe bone marrow aplasia that was fatal in one; the second remained moderately pancytopenic 21 weeks after stopping CAMPATH-1H therapy. The cause of this adverse effect is unknown., Conclusion: CAMPATH-1H is an effective agent in T-PLL and represents a significant improvement over other types of therapy. However, CAMPATH-1H alone is not sufficient for long-term remissions, and the role of autologous stem-cell transplantation needs further investigation.

In Hodgkin's disease stage I or II, excellent survival rates have been reached by radiotherapy alone as well as by the combination of radiotherapy and chemotherapy. Unfortunately, patients surviving for longer than 10 to 15 years are at risk for late complications due to the irradiation, such as myocardial infarction and lung and breast cancer. The current treatment strategy is aimed at minimising these late effects without jeopardizing the good survival rates. Randomized trials, such as the Hodgkin trials of the European Organization for Research and Treatment of Cancer (EORTC), are the only way to ultimately determine the relative values of radiotherapy and chemotherapy. Every Hodgkin patient deserves being included in such a trial, because the survival results are optimal. In future clinical trials, radiotherapy alone will probably be replaced by chemotherapy combined with radiotherapy as the principal treatment modality.

Although the results of treatment of Hodgkin's disease (HD) have improved considerably in the last decades, the disease remains fatal in a minority of patients. We have recently shown that numbers of activated cytotoxic T cells (CTLs), present in tumor biopsy specimens, differ considerably among individual HD patients. Because CTLs are the major effector cells in elimination of neoplastic cells, we investigated whether the number of activated CTLs is related to the clinical outcome of the individual patient with HD. Activated CTLs present in tumor biopsy specimens of patients with nodular sclerosis or mixed cellularity HD were identified by immunohistochemistry using an antibody directed against granzyme B (GrB), a major constituent of the cytotoxic granules of activated CTLs and natural killer cells, and an antibody directed against CD8. The presence of a high percentage of GrB+ lymphocytes was found to be an unfavorable prognostic marker. The large majority of GrB+ cells were also CD8+, indicating that these cells are activated CTLs. Prognosis was found to decrease with increasing percentages of GrB+ lymphocytes. Optimal discrimination between patients with good and poor prognosis was obtained when the threshold was set at 15% GrB+ cells; 6 of 10 patients with > or = 15% GrB+ lymphocytes died as a result of the disease, as compared with 6 of 70 patients with less than 15% GrB+ lymphocytes (P < .0001). In stage-2 patients, the percentage of GrB+ lymphocytes retained its predictive value in a multivariate analysis including histology, sex, age, erythrocyte sedimentation rate, and the presence of B symptoms as covariables. In addition, patients with > or = 15% GrB+ lymphocytes had a shortened progression-free survival time (P = .002). We conclude that a high percentage of activated CTLs present in biopsy material of HD patients is a strong indicator for an unfavorable clinical outcome.

CHOP has been and still is regarded by many as the 'standard' treatment of advanced non-Hodgkin's lymphoma. In 1980 the EORTC Lymphoma Cooperative Group started a study to evaluate the addition of vincristine and bleomycin to its standard four-drug combination chemotherapy, CHVmP (cyclophosphamide, hydroxorubicin, Vm-26, prednisone). Eligible patients were stage III or IV, intermediate- to high-grade non-Hodgkin's lymphoma (Working Formulation E-I). One-hundred-eighty-nine patients were entered, of whom 140 were eligible and evaluable. A previous report showed an improved response rate and failure-free survival (FFS) and overall survival for the combination CHVmP-VB. At ten years, the outcome still favors the addition of vincristine and bleomycin. The FFS was 34% vs. 23% and the overall survival 34% vs 22%. This difference was mainly due to a difference in CR rate (74% vs. 49%), Relapse-free survival for patients reaching a CR was the same in both arms. When the patients were grouped according to the International Prognostic Factor Index, no statistically significant difference could be observed in favor of one treatment within either group. This trial clearly demonstrates the benefit gained by the addition of vincristine and bleomycin to 'standard' chemotherapy for intermediate and high-grade non-Hodgkin's lymphoma.

DNA fibers can be released from cell suspensions and frozen tissue and can be used as a template for hybridization with multiple differently labeled probes. Simultaneous hybridization with 5-10 adjacent or partly overlapping probes generates a highly specific "color barcode" for individual DNA segments. Rearrangements in this barcode can be easily detected and mapped. The resolution of DNA fiber FISH is between 2 and 500kb. In mixing experiments of cell lines with different structural abnormalities, we found a sensitivity of approximately 10%. We applied DNA fiber FISH for detection of t(11;14) in mantle cell lymphoma (MCL) and immunoglobulin (Ig) class switching in hairy cell leukemia (HCL). Using a barcode for the Ig and BCL-1 loci at 14q32 and 11q13, we detected and mapped a t(11;14) breakpoint in 35 of 36 MCL. In 5 cases complex mono-allelic rearrangements at both sides of the cyclin D1 gene were identified. In 13 HCL with phenotypic evidence of Ig class switching, including 2 cases with solely IgD, fiber FISH revealed concordant Ig class switch deletions. In most cases both alleles were affected. These results indicate that DNA fiber FISH is a very powerful method to detect and map structural DNA alterations.

During the last decade, biannual quality controls were performed in the Netherlands focusing on the immunophenotyping of leukaemic haematological malignancies. All results on 48 specimens obtained by 18-34 laboratories were analysed. The interlaboratory variability and percentages of discordant results from 30 markers were measured by assessing false positive or negative (cut-off 10%) results in comparison with median results of the group. The quality of the immunophenotypic diagnoses obtained from the interpretation of these markers in relation to clinical data was evaluated by scoring them as 'correct', 'minor fault', 'major fault', 'not based upon the markers used', and 'no diagnosis', CD3, CD8, CD19, CD61 and Sm lambda had the lowest percentage discordancy (sum of total negative and positive discordant values 5-7.5% of assays): CD13, CD15, cyCD22, CD33 and TdT scored worst with 14-20% cumulative discordancy. The analysis of each diagnosis yielded 78% acceptable immunophenotypic conclusions (correct 54% and minor fault 24%). It appeared that the major faults in immunophenotyping were caused by suboptimal antibody selection and erroneous interpretation of the results obtained, rather than by technical errors. Large differences per diagnostic category were observed, with the best scores for mature B-cell leukaemias, AMLs and common-ALL, and the poorest scores for T-cell malignancies which were correctly diagnosed in only 24-60% of specimens. Mature T-NHL and T-PLL were mistakenly diagnosed as T-ALL by 40% of the centres. Misinterpretation of TdT immunofluorescence or omitting this marker contributed significantly to these wrong diagnoses. A median of 4% of immunophenotypic diagnoses were not based on a correct panel of antibodies, but upon the morphology of the accompanying blood smear, and was often flawed by overinterpretation. In conclusion, both the technical performance of immunophenotyping of haematological malignancies in The Netherlands and the procedure by which a final diagnosis is obtained needs improvement, especially for T-cell malignancies.

No reliable culture system exists for B-lineage acute lymphoblastic leukaemia (ALL). Recently we found that many different mature B-cell malignancies proliferate upon stimulation via the CD40 antigen, and this led us to investigate whether a similar CD40 activation on ALL cells could also induce proliferation. First, we measured CD40 expression in 21 ALL cases; all were CD40+, although mostly weak. Next, we triggered the CD40 antigen by anti-CD40 antibodies and by a CD40 ligand-expressing cell line. In addition, we measured the influence of IL-3, IL-4 and IL-7 with and without these stimuli. In 8/10 cases proliferation, measured by 3H-thymidine incorporation, could be induced after CD40 crosslinking, especially in the presence of IL-3. Stimulation via the CD40 ligand was more successful than using crosslinked anti-CD40 antibodies. IL-4 inhibited the spontaneous proliferation found in three cases, but stimulated proliferation after CD40 crosslinking. IL-7 did not contribute to proliferation. Morphology, immunophenotyping and surface marker analysis, combined with DNA flow cytometry confirmed that the proliferation found could be ascribed to the ALL cells. In conclusion, B-lineage ALL cases are CD40+, and many can be cultured using CD40 stimulation and IL-3.

Purpose: We studied the prognostic significance of bcl2 and p53 protein expression in relation to clinical and pathologic characteristics in patients with diffuse large B-cell lymphoma (LCL)., Patients and Methods: Three hundred seventy-two patients with LCL were retrieved from a population-based registry for non-Hodgkin's lymphoma (NHL). bcl2 and p53 protein expression was studied on paraffin-embedded tumor tissue by immunohistochemistry in relation to clinical factors. Response to therapy and survival were analyzed in 165 patients who were uniformly staged and treated and for whom all prognostic data were available according to the International Prognostic Index (IPI)., Results: Forty-five percent of tumors showed strong expression of the bcl2 protein (bcl2++), with a higher frequency in patients with primary nodal involvement. Disease-free survival (DFS) was significantly better in bcl2-negative/intermediate (bcl2-/+) cases as compared with bcl2++ cases (P = .0011). At 5 years, bcl2-/+ patients showed a DFS rate of 74%, in contrast to bcl2++ patients with a DFS rate of 41% (P = .002). Bcl2 was the strongest independent prognostic value in a multivariate analysis, with a relative risk (RR) of 3.0 in comparison to p53 expression and the clinical factors of the IPI. Overall survival (OS) was not significantly influenced by bcl2 protein expression. p53 protein expression was found in 13% of cases, with a higher frequency in patients with extensive disease. p53 expression did not influence the chance to achieve complete remission (CR) and survival., Conclusion: bcl2 protein is frequently expressed in LCL and is a strong independent prognostic factor for DFS. p53 expression is related with high tumor burden, but is not an independent risk factor for CR and survival.

An increasing amount of literature has been published concerning the interaction of the CD40 antigen and its ligand with regard to normal B cell ontogeny. In this review, an overview of the CD40 antigen and the CD40 ligand is given, focussing on their possible role in B cell malignancies. Data on the expression of the CD40 antigen on various B cell malignancies (acute and chronic leukemias, non-Hodgkin's lymphoma and multiple myeloma) are presented. The recently developed novel culture "CD40 system" is described. This system is a powerful tool used to culture normal B cells, but also most malignant B cells. We demonstrate in addition a more prominent role of the human Fc receptor presenting murine fibroblasts in the "CD40 system", especially in relation to cultured plasma cells. Finally, some important applications of the "CD40 system" are also summarized.

Concurrent activation of BCL2 and MYC usually occurs in B cell non-Hodgkin lymphoma (B-NHL) by translocation of both oncogenes to both immunoglobulin heavy chain (IGH) alleles: this abrogates immunoglobulin synthesis. We have studied three B-NHL cell lines (DoHH2, VAL and ROS 50) and show that concurrent activation of BCL2 and MYC may follow translocation of both oncogenes to the same IGH allele. Conventional cytogenetics of DoHH2 suggested the presence of a t(14;18)(q32;q21) translocation. However, fluorescent in situ hybridization (FISH) studies using whole chromosome paints, alpha satellite probes and flow-sorted chromosomes as probes revealed an unexpected complexity of rearrangements involving chromosomes 8, 14 and 18, namely t(8;14;18)(q24;q32;q21). DNA blot and previous PCR analysis confirmed the juxtaposition of BCL2 major breakpoint region (mbr) with IGJH6, but also demonstrated a rearrangement within the first exon of MYC. The centromeric (5') MYC rearranged fragment comigrated with the BCL2-JH6 rearranged fragment in BamHI, EcoRI and Bg/II restriction digests. The der(8)t(8;14;18) therefore comprised 5' MYC (exon I)-Sgamma4-JH6-BCL2 mbr. Similar rearrangements were observed in both ROS 50 and VAL cell lines which contained two and three copies of the der(8)t(8;14;18) respectively. Quantitative flow cytometry for BCL2 and MYC expression showed abundant expression of both proteins in all three lines. These data indicate the der(14)t(14;18)(q32;q21) may itself be the target for any second translocation. The presence of the intact BCL2-JH fusion gene on the der(8)t(8;14;18) allowed concurrent activation of both BCL2 and MYC with no loss of immunoglobulin expression.