Microarray-based classification of a consecutive series of 121 childhood acute leukemias: prediction of leukemic and genetic subtype as well as of minimal residual disease status.

Gene expression analyses were performed on 121 consecutive childhood leukemias (87 B-lineage acute lymphoblastic leukemias (ALLs), 11 T-cell ALLs and 23 acute myeloid leukemias (AMLs)), investigated during an 8-year period at a single center. The supervised learning algorithm k-nearest neighbor was utilized to build gene expression predictors that could classify the ALLs/AMLs according to clinically important subtypes with high accuracy. Validation experiments in an independent data set verified the high prediction accuracies of our classifiers. B-lineage ALLs with uncharacteristic cytogenetic aberrations or with a normal karyotype displayed heterogeneous gene expression profiles, resulting in low prediction accuracies. Minimal residual disease status (MRD) in T-cell ALLs with a high (>0.1%) MRD at day 29 could be classified with 100% accuracy already at the time of diagnosis. In pediatric leukemias with uncharacteristic cytogenetic aberrations or with a normal karyotype, unsupervised analysis identified two novel subgroups: one consisting mainly of cases remaining in complete remission (CR) and one containing a few patients in CR and all but one of the patients who relapsed. This study of a consecutive series of childhood leukemias confirms and extends further previous reports demonstrating that global gene expression profiling provides a valuable tool for genetic and clinical classification of childhood leukemias.

A population-based cohort study on early-stage Hodgkin lymphoma treated with radiotherapy alone: with special reference to older patients.

BACKGROUND: Combined modality treatment has reduced the risk of relapse among younger early-stage Hodgkin lymphoma (HL) patients. Older HL patients may not tolerate chemotherapy and their prognosis is less favorable. We conducted a population-based study to evaluate long-term follow-up outcome in older early-stage HL patients initially treated with radiotherapy (RT) alone. PATIENTS AND METHODS: We included 308 consecutive patients (22% were >or=60 years) diagnosed 1972-1999 (median follow-up 20 years; range 1-28). Using Cox regression models we defined risk of relapse and survival in relation to clinical factors. RESULTS: 272/308 (88%) patients obtained complete remission following first-line RT alone. Among these, 42% relapsed within a median of 21 months. The relapse rate was independent of gender and age at diagnosis (median age 32 years, range 14-85); however, lymphocyte-predominant HL was associated with borderline (P=0.049) 56% decreased risk of relapse. Among patients<60 years and >or=60 years, we observed 29 (median latency 10 years, range 2-25) and 11 (median latency 3 years, range 1-10) second tumors, respectively. CONCLUSIONS: Older age (>or=60 years) was not associated with an increased risk of relapse following RT alone. Given the risks of iatrogenic morbidity/mortality of chemotherapy in older patients, RT alone could be an alternative first-line therapy in early-stage older HL patients.

Children and adults with acute lymphoblastic leukaemia have similar gene expression profiles.

OBJECTIVES: To compare the gene expression pattern in children and adults with acute lymphoblastic leukaemia (ALL) in order to improve our understanding of the difference in disease biology and prognosis. METHODS: The gene expression profiles in diagnostic samples from 29 children and 15 adults with ALL were analysed using the oligonucleotide chip Hu95ver2a, produced by Affymetrix. RESULTS: Unsupervised hierarchical cluster analysis revealed that, in spite of differences in outcome, patients clustered irrespective of age, first by T-cell or B-precursor immunophenotype, and second by cytogenetic changes within the B-precursor group. The expression pattern analysis allowed the reclassification of some samples into the proper cytogenetic group. We also showed that separate clustering of samples with the BCR/ABL translocation could be explained by different breakpoint regions in the BCR. No significant difference in gene expression was observed between samples with and without CDKN2A deletion within the B-precursor group. Analysis of different age groups revealed a similarity in expression profiles when infants with the MLL translocation and adults over 40 yr of age were compared irrespective of karyotype. CONCLUSIONS: In spite of the difference in clinical outcome, the gene expression pattern in children and adults with ALL is very similar and is primarily dependent on immunophenotype and cytogenetic aberrations. However, when age groups are compared, the expression patterns of infants and adults over 40 show a remarkable similarity.

Analysis of minimal residual disease in childhood acute lymphoblastic leukemia: comparison between RQ-PCR analysis of Ig/TcR gene rearrangements and multicolor flow cytometric immunophenotyping.

Detection of minimal residual disease (MRD) in follow-up samples from patients with ALL is essential for evaluation of treatment response. We applied multicolor flow cytometry and real-time quantitative PCR (RQ-PCR) to compare MRD results in 71 follow-up samples from 22 children treated for ALL. When results obtained by flow cytometry and RQ-PCR were grouped into positive-negative categories, a significant level of agreement was found in 72% of samples (P<0.001). However, if a cutoff level of 0.01% was applied, the concordance was 89%. MRD could be quantified in 19 samples by both methods, showing a strong correlation (P<0.01). Nevertheless, MRD levels differed more than five-fold between both methods in 4/19 samples. In 20 (28%) samples, the two techniques showed discordant results. Most discordant results (17/20) were due to the limited sensitivity of flow cytometry analysis within the range 0.01-0.001%; remaining discordant results were due to the instable or subclonal IG/TCR gene rearrangements or a limited quantitative range of the applied RQ-PCR targets. Although concordant results could be obtained by flow cytometry and RQ-PCR analysis, MRD levels may differ. Therefore, MRD data obtained by these two techniques are not yet easily exchangeable.

Cellular immune reconstitution after subcutaneous alemtuzumab (anti-CD52 monoclonal antibody, CAMPATH-1H) treatment as first-line therapy for B-cell chronic lymphocytic leukaemia.

Little information is available on long-term immune reconstitution after therapy with alemtuzumab in B-CLL patients. We present long-term follow-up data for blood lymphocyte subsets analysed by flow cytometry in previously untreated B-CLL patients who received alemtuzumab subcutaneously as first-line therapy. All lymphoid subsets were significantly (P<0.001) and profoundly reduced; the median end-of-treatment counts for CD4(+), CD8(+), CD3(-)56(+) (natural killer (NK)), CD3(+)56(+) (NK-T) and CD19(+)5(-) (normal B) cells were 43, 20, 4, 1 and 8 cells/microl, respectively. The median cell count of all subsets remained at <25% of the baseline values for >9 months post-treatment. CD4(+) and CD8(+) levels in blood had reached >100 cells/microl in >50% of the patients at 4 months after the end of treatment. One patient had a cytomegalovirus reactivation and one patient developed Pneumocystis carinii pneumonia during therapy. No opportunistic or other major infections were recorded during unmaintained, long-term follow-up. There was no correlation between the cumulative dose of alemtuzumab and the severity or length of immunosuppression. CD52(-) T-cell subsets occurred during the treatment and comprised >80% of all CD4(+) and CD8(+) cells in the blood at the end of therapy. These subpopulations declined gradually during unmaintained follow-up. The relationship between these observations and the safety/antitumour effects of alemtuzumab is discussed.

Flow cytometric follow-up of minimal residual disease in bone marrow gives prognostic information in children with acute lymphoblastic leukemia.

Using flow cytometry (FC) and live gate (LG) analysis we have followed levels of minimal residual disease (MRD) in the bone marrow (BM) of 70 consecutive patients with childhood acute lymphoblastic leukemia (59 B precursor ALL and 11 T-ALL) treated according to the Nordic (NOPHO-92) protocols. Thorough studies of B and T cell antigen expression patterns in normal BM performed during BIOMED 1 Concerted Action on MRD, made it possible to tailor individual protocols of marker combinations for follow-up in 97% of patients. In 12% of LG analyses, the numbers of cells exceeded 10(6) and in 82% exceeded 10(5), giving the sensitivity level of MRD detection 10(-5) and 10(-4), respectively. The median follow-up time was 53 months. Patients with MRD levels > or = 0.01% at follow-up time-points during and after first induction, and at the end of treatment had significantly lower disease-free survival by comparison to patients with MRD values <0.01%. Seven of nine patient with recurrence in the BM showed under treatment persisting MRD levels > or = 0.01% of BM cells. This was also observed in another two patients with infant leukemia who relapsed. In conclusion, the investigation of levels and the dynamics of MRD by sensitive and quantitative FC can provide a basis for further clinical studies for at least upgrading of therapy.

Antigen expression patterns reflecting genotype of acute leukemias.

Multi-parameter flow cytometry, molecular genetics, and cytogenetic studies have all contributed to new classification of leukemia. In this review we discuss immunophenotypic characteristics of major genotypic leukemia categories. We describe immunophenotype of: B-lineage ALL with MLL rearrangements, TEL/AML1, BCR/ABL, E2A/PBX1 translocations, hyperdiploidy, and myc fusion genes; T-ALL with SCL gene aberrations and t(5;14) translocation; and AML with AML1/ETO, PML/RARalpha, OTT/MAL and CBFbeta/MYH11 translocations, trisomies 8 or 11 and aberrations of chromosomes 7 and 5. Whereas some genotypes associate with certain immunophenotypic features, others can present with variable immunophenotype. Single molecules (as NG2, CBFbeta/SMMHC and PML/RARalpha proteins) associated with or derived from specific translocations have been described. More often, complex immunophenotype patterns have been related to the genotype categories. Most known associations between immunophenotype and genotype have been defined empirically. Therefore, these associations should be validated in independent patient cohorts before they can be widely used for prescreening of leukemia. Progress in our knowledge on leukemia will show how the molecular-genetic changes modulate the immunophenotype as well as how the expressed protein molecules further modulate cell behavior.

Tissue eosinophilia in relation to immunopathological and clinical characteristics in Hodgkin's disease.

Eosinophils frequently infiltrate tissues involved by Hodgkin's disease (HD), and blood eosinophilia is frequently observed. However, the clinical significance and the mechanisms underlying eosinophilia need further elucidation. In this study the grade of eosinophilic infiltration (EoI) was evaluated in biopsies from 259 HD-patients. In a selected group (n=32), the numbers of Hodgkin-Reed-Sternberg (HRS)-cells were counted, and the phenotype of small lymphocytes, the expression of cytotoxic lymphocyte-associated proteins, CD3-zeta-chain, HLA-DR, proliferation markers, latent membrane protein 1 (LMP-1) and blood lymphocyte function were evaluated. Samples from 88 HD patients (34%) showed high EoI. Significantly higher EoI was seen in nodular sclerosis 2 (NS2; p<0.001), bulky disease (p<0.05) and in patients <50 years (p<0.05). Patients with high EoI did not differ from the remainder with regard to distribution of sex, stage, B-symptoms, blood lymphocyte function and outcome. HRS-cells were significantly more frequent in NS HD as compared to mixed cellularity (MC) (p<0.001) irrespective of EoI. LMP-1-expression, proliferative fraction and phenotypes of small lymphocytes did not differ between the cases with low and high EoI, respectively. MC HD samples had significantly higher numbers of small cells positive for CD8 (p<0.01), T-cell intracellular antigen-1 (p<0.01) and Granzyme B (p<0.05) than NS. LMP-1-positive cases had significantly higher frequency of CD8-positive cells than LMP-1-negative. In conclusion, high EoI remains a feature of certain clinical subgroups of HD. However, there was no association between the degree of EoI and numbers of HRS-cells, phenotypes of small lymphocytes, EBV status and clinical outcome. Determination of EoI is of limited diagnostic and prognostic clinical value in HD. However, the differences in small cell distribution of CD8, TIA-1, GrB and CD57 between the histopathological groups and between LMP-1-expressing/non-expressing cases may contribute to our understanding of the biology of the disease.

BIOMED-I concerted action report: flow cytometric immunophenotyping of precursor B-ALL with standardized triple-stainings. BIOMED-1 Concerted Action Investigation of Minimal Residual Disease in Acute Leukemia: International Standardization and Clinical Evaluation.

The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%). Parallel flow cytometric studies in different laboratories finally resulted in highly concordant results (>90%) for all five antibody combinations, indicating the high reproducibility of our approach. In conclusion, the technique presented here with triple-labelings forms an excellent basis for standardized flow cytometric MRD studies in multicenter international treatment protocols for precursor-B-ALL patients.

Flow cytometry and allele-specific oligonucleotide PCR are equally effective in detection of minimal residual disease in ALL.

The analysis of minimal residual disease (MRD) has assumed a growing role in the follow-up of patients with acute lymphoblastic leukemia (ALL). We have applied multiparameter flow cytometry (FC) with 'live-gate' analysis and allele-specific oligonucleotide (ASO)-PCR detecting leukemia-specific T cell receptor gamma and delta gene rearrangements for MRD follow-up in 30 ALL patients. The comparison of results obtained in 89 follow-up samples from 23 patients showed significantly consistent results in 70 samples (78%); (P < 0.001). Bone marrow samples taken during the first phase of treatment (during or immediately after induction) showed a lower level of consistency when compared to samples taken during later phases of treatment (69% vs 85% consistent results, respectively). Some of the discrepant results were due to low cellularity of the samples obtained for FC and some due to the presence of PCR inhibitors. Of 29 patients evaluated at the end of the induction treatment, 18 (62%) had detectable levels of MRD and six of these patients suffered relapse. In all these patients MRD levels by FC increased preceding relapse. Our results suggest that FC offers a MRD detection tool that can be easily applied in clinical practice and is as informative as molecular methods.

Telomerase activity in relation to pro- and anti-apoptotic protein expression in high grade non-Hodgkin's lymphomas.

BACKGROUND AND OBJECTIVES: Telomerase activity (TA) is determined by the catalytic unit telomerase reverse transcriptase (hTERT). In vitro studies show that hTERT is downregulated by wild type p53 and TA is upregulated by BCL-2 expression. The aim of this study was to investigate the relationship of TA and mRNA expression of hTERT, telomerase RNA (hTER) and Tankyrase in 31 samples from patients with high-grade non-Hodgkin's lymphoma (HG-NHL). The results were then related to apoptosis and proliferation and the expression of p53 and BCL-2 family member proteins. DESIGN AND METHODS: The telomeric repeat amplification protocol (TRAP) assay and reverse transcription-polymerase chain reaction (RT-PCR) were used to quantify TA, and hTERT, hTER and Tankyrase mRNA expression. Proliferation (Ki67), p53, BCL-2, MCL-1, BAX and BAK protein expression were evaluated by immunohistochemistry. Apoptosis was evaluated by TUNEL staining. RESULTS: TA was detected in 93% of HG-NHL and tended to be higher in p53+ lymphomas. A positive correlation existed between mRNA expression of hTERT, hTER and Tankyrase. hTERT mRNA expression tended to be higher with increasing levels of apoptosis and proliferation, in HG-NHL samples lacking BAX expression and in samples from patients with survival shorter than 3.5 years. hTER mRNA expression was significantly higher in BAX and BAK negative samples. INTERPRETATION AND CONCLUSIONS: Telomerase is activated or upregulated in the majority of HG-NHL. Enhanced TA combined with deregulation of the factors responsible for cell survival and proliferation may contribute to the development and progression of lymphomas. Observation that high hTERT mRNA expression may be related to shorter survival should prompt further investigation of the clinical significance of TA and its components in HG-NHL.

BIOMED-1 concerted action report: flow cytometric characterization of CD7+ cell subsets in normal bone marrow as a basis for the diagnosis and follow-up of T cell acute lymphoblastic leukemia (T-ALL).

The European BIOMED-1 Concerted Action was initiated in 1994 to improve and standardize the flow cytometric detection of minimal residual disease (MRD) in acute leukemia (AL). Three different protocols were defined to identify the normal subsets of B, T and myeloid cells in bone marrow (BM), and were applied to the different types of AL in order to study aberrant immunophenotypes. Using sensitive acquisition methods ('live gate') T cell subsets in normal BM could be identified with five triple-stains: CD7/CD5/CD3, CD7/CD4/CD8, CD7/CD2/CD3, CD7/CD38/CD34 and TdT/CD7/surface or cytoplasmic (cy)CD3 (antibodies conjugated with FITC/PE/PECy5 or PerCP, respectively). The identification of T cell subsets in BM allowed definition of 'empty spaces' (ie areas of flow cytometric plots where normally no cells are found). All studied T-ALL cases (n = 65) were located in 'empty spaces' and could be discriminated from normal T cells. The most informative triple staining was TdT/CD7/cyCD3, which was aberrant in 91% of T-ALL cases. In most cases, two or more aberrant patterns were found. Apparently the immunophenotypes of T-ALL differ significantly from normal BM T cells. This is mostly caused by their thymocytic origin, but also the neoplastic transformation might have affected antigen expression patterns. Application of the five proposed marker combinations in T-ALL contributes to standardized detection of MRD, since cells persistent or reappearing in the 'empty spaces' can be easily identified in follow-up BM samples during and after treatment.

Alteration of cellular mediated cytotoxicity, T cell receptor zeta (TcR zeta) and apoptosis related gene expression in nasopharyngeal carcinoma (NPC) patients: possible clinical relevance.

We have investigated apoptosis related gene expression in tumour cells, phenotype and function of blood mononuclear cells at diagnosis in relation to clinical response in three patients with nasopharyngeal carcinoma (NPC). We have focused our study on the Epstein Barr virus latent membrane protein-1 (LMP-1) and Bcl-2 expression in the tumour cells, the essential signal-transducing zeta molecule of T cell receptor (TcR zeta) and cellular mediated cytolysis of the blood mononuclear cells. The carcinoma cells of the patients were Bcl-2 negative. They were heterogeneous with regard to the expression of LMP-1 and the number of proliferating or apoptotic cells. Decrease in the expression of mature T cells (CD3, CD4, and CD8), TcR zeta and cellular mediated cytotoxicity was detected in blood mononuclear cells of the patients. IL-2 up-regulated these phenotypes and the cytolytic capacity of the blood mononuclear cells. The patient with LMP-1 negative carcinoma cells, down-regulated TcR zeta expression and impaired IL-2 mediated cytolysis, had the worst clinical outcome. Another patient with low apoptotic, highly proliferating and LMP-1 positive carcinoma cells had recurrent disease only in the irradiated area. Interestingly, NPC with high apoptotic and few LMP-1 expressing cells was detected in the patient with a normal level of TcR zeta expression and cytolytic functions in blood mononuclear cells at the time of diagnosis. After combination treatment with chemotherapy followed by radiotherapy, this patient is still alive with complete remission and disease-free at 36 months. Suppression of the immunological functions may occur in NPC patients. Our study suggests that the immunological functions and apoptosis related gene expression in the carcinoma cells may be used as prognostic factors and help in the decision of therapy of patients with nasopharyngeal cancer.

Heterogeneity of isolated mononuclear cells from patients with acute myeloid leukemia affects cellular accumulation and efflux of daunorubicin.

BACKGROUND AND OBJECTIVE: Pharmacologic studies on blasts from patients with leukemia are generally performed on density gradient isolated blood or bone marrow cells. Thereby, cellular drug accumulation and efflux are determined as mean values of the entire cell population. The objective of the present study was to characterize the heterogeneity in the accumulation and efflux of daunorubicin in various subpopulations of mononuclear cells isolated from patients with acute myeloid leukemia (AML). DESIGN AND METHODS: Mononuclear cells from 33 patients with AML were isolated from peripheral blood by density gradient centrifugation on Lymphoprep (1. 077 g/mL). Cellular accumulation of fluorescent daunorubicin was determined by flow cytometry after incubation of the cells at +37C for 1 hour. Thereafter, the cells were washed and reincubated in drug-free medium. Kinetics of drug efflux were determined by frequent determination of cellular fluorescence during 30 min. Daunorubicin accumulation and efflux were compared in the total isolated mononuclear cell population and in the various blast cell populations gated on FSC/SSC according to the results of immunophenotyping. RESULTS: In 8 of these 33 (24%) patient samples, two distinct blast cell populations could be identified. In 7 out of 8 these cases the more immature blasts had a lower drug accumulation and in 6 out of the 8 cases also a higher efflux rate than the differentiating cell population. Cyclosporin A increased daunorubicin accumulation and reduced efflux in the immature blast population. In the differentiating cell population cyclosporin A increased both the accumulation and the efflux. In patients with a single blast cell population, the gated blast cells had a significantly lower drug accumulation but also a lower drug efflux rate than the total cell population. INTERPRETATION AND CONCLUSIONS: The results imply that drug transport studies on cells isolated from patients with AML give somewhat different results depending on the cell population studied. Some, but not all, of these differences in daunorubicin accumulation and efflux as well as in the effect of cyclo-sporin A can be explained by a heterogenous expression of the mdr1-gene. The observed heterogeneity may be of special relevance with regard to drug resistance. The presence of even a small resistant cell clone may jeopardize the effect of the chemotherapy due to expansion resulting in relapse of disease.

Biological markers may add to prediction of outcome achieved by the International Prognostic Score in Hodgkin's disease.

BACKGROUND: The International Prognostic Score (IPS) identifies seven independent factors predicting progression-free and overall survival in advanced stage Hodgkin's disease (HD). The IPS is also applicable in limited disease. However, the IPS does not identify patients with a very poor prognosis. The aim of this study was to define biological markers which may add to the IPS in predicting outcome. PATIENTS AND METHODS: One hundred forty-five patients (> 15 years) with HD of all stages and histopathology subgroups were included. In addition to factors included in the IPS, serum levels of CRP, sCD4, sCD8, sCD25, sCD30, sCD54, interleukin (IL)-10, beta2-microglobulin and thymidine kinase were analysed. RESULTS: The strongest predictors of a poor cause-specific survival (CSS) in univariate analyses were: increased serum levels of IL-10, sCD30 and CRP, anaemia, low levels of albumin (P < 0.001); stage IV (P = 0.003), age > or = 45 years (P = 0.006), increased serum levels of sCD25 (P = 0.010), low lymphocyte counts (P = 0.020). Serum IL-10 added prognostic information to that achieved by the IPS: patients with a high score and increased serum IL-10 had a very poor outcome with a five-year CSS of 38%. Patients with increased serum levels of sCD30 and a high score also had a poor outcome with a five-year CSS of 54%. CONCLUSION: Serum levels of IL-10 and sCD30 may add to IPS in prediction of outcome in HD, and should be validated in large, prospective studies.

Detailed characterization of a complex karyotype in a patient with primary plasma cell leukaemia using multicolour spectral karyotyping and micro-FISH.

INTRODUCTION: Plasma cell leukaemia is a rare disorder that usually carries an aggressive course with a rapidly fatal outcome. A variety of chromosomal abnormalities have been reported in plasma cell leukaemia but the clinical significance of an abnormal karyotype is still unclear. MATERIALS AND METHODS: We have applied the molecular cytogenetic techniques multicolour spectral karyotyping and microdissection in combination with fluorescence in situ hybridization on metaphases from a patient with primary plasma cell leukaemia and a fatal outcome. RESULTS AND CONCLUSION: The chromosome analysis showed severe hypodiploidy and 12 marker chromosomes. Identification of the structural rearrangements was not possible using routine cytogenetic methods. Utilizing the methods above, all marker chromosomes could be identified in detail and the karyotype was shown to be very complex. Forty-three breakpoints were found, and 25 could be identified at the band level, among others 14q32 where the immunoglobulin heavy chain locus is situated. Thus, these techniques provide the opportunity to resolve very complex chromosomal changes in a way that has not been previously possible and will consequently be of great importance in the search for hot spots that may harbour new cancer genes.

Epstein-Barr virus expression in Hodgkin's disease in relation to patient characteristics, serum factors and blood lymphocyte function.

Epstein-Barr virus (EBV) expression was investigated by immunohistochemistry (latent membrane protein 1 [LMP-1]) and in situ hybridization (EBV encoded RNA [EBER]) in biopsies from 95 patients with untreated Hodgkin's disease (HD). Tumour EBV status was related to EBV antibody titres, spontaneous and concanavalin A induced blood lymphocyte DNA synthesis, serum levels of soluble (s) CD4, sCD8, sCD25, sCD30, sCD54, beta2-microglobulin, thymidine-kinase, routine chemistry, patient characteristics, complete remission and survival. The median follow-up time was 145 months (range 60-257). Tumour EBV-positive (n = 30; 33%) and negative (n = 62; 67%) patients did not differ with regard to sex, age, stage, presence of bulky disease or B-symptoms, remission rate or survival. The proportion of EBV+ cases was significantly higher among patients with mixed cellularity histopathology (58%) as compared to the nodular sclerosis subtype (18%; P < 0.001). The total white blood cell (WBC) counts were significantly lower in EBV+ patients (P < 0.01), who also had significantly higher levels of sCD54 (P < 0.02) and a tendency towards lower levels of sCD30 (P = 0.056). Patients in the tumour EBV+ group had significantly higher IgG antibody titres to restricted early antigen (EA-R) (P < 0.02). Hence, clinical features and outcome were not related to tumour EBV status. However, HD patients with EBV+ tumours had elevated sCD54 levels, higher antibody titres to EA-R and decreased total WBC counts. A potential causal relationship between EBV tumour status and these findings needs to be further explored.