Array-based DNA methylation profiling in follicular lymphoma.

Quantitative methylation profiling was performed using the Illumina GoldenGate Assay in untreated follicular lymphoma (FL) (164), paired pre- and post-transformation FL (20), benign haematopoietic (24) samples and purified B and T cells from two FL cases. Methylation values allowed separation of untreated FL samples from controls with one exception, based primarily on tumour-specific gains of methylation typically occurring within CpG islands. Genes that are targets for epigenetic repression in stem cells by Polycomb Repressor Complex 2 were significantly over-represented among hypermethylated genes. Methylation profiles were conserved in sequential FL and t-FL biopsies, suggesting that widespread methylation represents an early event in lymphomagenesis and may not contribute substantially to transformation. A significant (P<0.05) correlation between FL methylation values and reduced gene expression was shown for up to 28% of loci. Methylation changes occurred predominantly in B cells with variability in the amount of non-malignant tissue between samples preventing conclusive correlation with survival. This represents an important caveat in attributing prognostic relevance to methylation and future studies in cancer will optimally require purified tumour populations to address the impact of methylation on clinical outcome.

Stromal gene signatures in large-B-cell lymphomas.

BACKGROUND: The addition of rituximab to combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or R-CHOP, has significantly improved the survival of patients with diffuse large-B-cell lymphoma. Whether gene-expression signatures correlate with survival after treatment of diffuse large-B-cell lymphoma is unclear. METHODS: We profiled gene expression in pretreatment biopsy specimens from 181 patients with diffuse large-B-cell lymphoma who received CHOP and 233 patients with this disease who received R-CHOP. A multivariate gene-expression-based survival-predictor model derived from a training group was tested in a validation group. RESULTS: A multivariate model created from three gene-expression signatures--termed "germinal-center B-cell," "stromal-1," and "stromal-2"--predicted survival both in patients who received CHOP and patients who received R-CHOP. The prognostically favorable stromal-1 signature reflected extracellular-matrix deposition and histiocytic infiltration. By contrast, the prognostically unfavorable stromal-2 signature reflected tumor blood-vessel density. CONCLUSIONS: Survival after treatment of diffuse large-B-cell lymphoma is influenced by differences in immune cells, fibrosis, and angiogenesis in the tumor microenvironment.

Loss of chromosome 11q21-23.1 and 17p and gain of chromosome 6p are independent prognostic indicators in B-cell non-Hodgkin's lymphoma.

Comparative genomic hybridization (CGH) was employed to study chromosomal aberrations in relation to cell proliferation, apoptosis, and patient survival in 94 cases of B-cell non-Hodgkin's lymphoma diagnosed between 1983 and 1993. Eighty cases had aberrations by CGH. Chromosomal regions 1p21-31.1 (10%), 6cen-q24 (12%), 8p (11%), 9p21-ter (14%), 11q21-23.1 (11%), 13q13-21.1 (12%), and 17p (15%) were frequently lost. Gains were found at 3q21-ter (22%), 6p (11%), 7p (12%), 8q23-ter (13%), 12cen-q15 (17%), 17q24-ter (13%), and 18q13.3-21 (20%). A high number of aberrations (> or = 4, 33 cases) was associated (P < or = 0.001) with the mantle cell and diffuse large B-cell lymphoma subtypes, a high fraction of tumour cells in S phase, and short survival (RR (relative risk) = 3.7). Loss of 1p21-31.1, 8p, 9p21-ter, 11q21-23.1, and 13q13-21.1 were associated with mantle cell lymphoma (P < or = 0.03), while gain of 6p and 12cen-q15 were more frequent in diffuse large B-cell and small lymphocytic lymphoma, respectively (P = 0.04). Loss of 8p and 17p, and gain of 3q21-ter, 6p, 7p, and 8q23-ter were associated with a high S phase fraction (P < or = 0.03), but none of the aberrations were associated with tumour apoptotic fraction (P > or = 0.13). The most important prognostic CGH parameters (P < 0.001) were losses of 11q21-23.1 (RR = 3.8) and 17p (RR = 4.4), and gain of 6p (RR = 4.2). The latter parameters and IPI were the only ones with independent prognostic value (RR = 10, 5.0, 6.7, and 3.7, respectively; P < 0.001) when assessed together with lymphoma sub-type, primary versus relapse cases, treatment, B symptoms, S phase fraction, and presence of BCL1 and BCL2 translocations. A combined CGH/IPI binary parameter had high prognostic value for patients receiving different treatments, with various lymphoma sub-types, and for primary as well as relapse cases.

[Gene therapy in cancer]. / Genterapi ved kreft.

BACKGROUND: There has been a substantial increase in our understanding of the pathogenesis of cancer over the past decades, and new treatment modalities are being developed on the basis of this knowledge. MATERIAL AND METHODS: The literature is reviewed, and the status of gene therapy protocols approved in Norway is presented. RESULTS: About 70% of the more than 400 clinical gene therapy studies started are targeted at cancer. Several different principles are used, including gene therapy targeted at tumour suppressor genes, oncogenes, or central signaling molecules, as well as "suicide gene" therapy. In addition, various gene therapy protocols aim at strengthening immune responses. Most studies have been early clinical trials primarily designed to study safety, applicability and toxicity. Several of these phase I and II studies have, however, shown partial remission of tumours and, in rare cases, complete remission, although curation has not yet been shown. In some trials, including TP53 gene therapy trials, effects on tumour size have been observed in up to 50% of the patients. Up until now, only two phase III and one phase II/III studies have been initiated, but results from these studies have not yet been published. The two first gene therapy protocols approved in Norway are also targeted at cancer. So far, six patients in Norway have undergone gene therapy. INTERPRETATION: As of today, the results should be seen as promising for some of the principles which are being tried out; their clinical importance must, however, be documented in larger controlled clinical trials.

[Gene therapy--status and recommendations in Norwegian health care system]. / Genterapi--status og anbefalinger for norsk medisin.

BACKGROUND: This article presents the main conclusions and the recommendations of a multidisciplinary group of experts appointed by the Norwegian Centre for Health Technology Assessment to assess the potential of gene therapy in clinical medicine. MATERIALS AND METHODS: Clinical gene therapy protocols, ongoing or completed with published results, if any, were identified through a systematic survey of descriptive protocols and publications. RESULTS: So far 3,000-4,000 patients have been treated with gene therapy strategies in more than 400 clinical trials. In Norway the six first patients have been treated with gene therapy at The Norwegian Radium Hospital as part of two approved protocols for treatment of cancer. Gene therapy today is dominated by preclinical and clinical research. Most of the gene therapy protocols identified are in early phases (phases I and II) with few patients in each study; only three of the protocols represent phase III studies. Apart from the use of soluble antisense oligonucleotides against cytomegalovirus, gene therapy is not an established treatment modality today. In early clinical studies, however, promising results have been seen in treatment of cancer, in certain forms of cardiovascular diseases and also in a subgroup of inherited severe combined immunodeficiency. INTERPRETATION: The expert group recommends that it is now important to build up national competence in the field in two ways: 1) by building up infrastructure in selected milieus; 2) by starting a national programme for gene therapy research including both preclinical and clinical research. The article also considers ethical and legislative aspects and emphasises that gene therapy should continue to be carefully monitored for side effects.

Identification of nucleolar protein No55 as a tumour-associated autoantigen in patients with prostate cancer.

Four different genes were identified by immunoscreening of a cDNA expression library from the human prostate cancer cell line DU145 with allogeneic sera from four prostate cancer patients. A cDNA encoding the nucleolar protein No55 was further analysed and shown to be expressed at the mRNA level in several normal tissues, including ovaries, pancreas and prostate and in human prostate cancer cell lines PC-3, PC-3m and LNCaP. By reverse transcriptase/polymerase chain reaction, expression of No55 was several-fold higher in two out of nine prostate cancer primary tumours and two out of two metastatic lesions, compared to normal prostate tissue. Antibodies to No55 were detected in sera from seven out of 47 prostate cancer patients but not in sera from 20 healthy male controls. Sequence analysis of the No55 open reading frame from normal and tumour tissues revealed no tumour-specific mutations. The No55 gene was located to chromosome 17q21, a region reported to be partially deleted in prostate cancer. Considering the immunogenicity of the No55 protein in the tumour host, the expression profile and chromosomal localization of the corresponding gene, studies evaluating No55 as a potential antigen for immunological studies in prostate cancer may be warranted.

Oncogenic aberrations in the p53 pathway are associated with a high S phase fraction and poor patient survival in B-cell Non-Hodgkin's lymphoma.

The implications of aberrations in the p53 pathway for induction of apoptosis and regulation of S phase entry, and for patient survival, were investigated in 83 B-cell Non-Hodgkin's lymphomas. Eight cases had missense mutations in exons 5, 7, 8 and 9 as revealed by constant denaturant gel electrophoresis and sequencing. Fifteen cases had lost 1 TP53 allele as revealed by fluorescent in situ hybridization and comparative genomic hybridization. Ten cases expressed high levels of p53 as assessed by immunoblotting and immunohistochemistry. S phase fractions were higher, apoptotic fractions were the same and survival times were shorter in all aberration groups compared with the cases with no TP53/p53 aberrations. Since many tumors had more than one TP53/p53 aberration, the tumors were divided into groups with the following characteristics: no TP53/p53 aberrations; loss of one TP53 allele only (9 cases), TP53 point mutation (8 cases), high-level p53 expression and no TP53 mutation (3 cases). Tumors from the 3 latter groups had higher median S phase fractions (5%, 7.6%, and 5%, respectively, p<0.02) than the cases without any aberrations (1.1%), and survival time for these patients was much shorter (relative risks of 5.9, 8.9, and 6.6, respectively, p<0.003). Apoptotic fractions were similar in all these groups (p=0.09). Multivariate analysis showed that the presence of TP53/p53 aberrations is a strong and independent prognostic parameter in B-cell Non-Hodgkin's lymphoma.

Differential expression of bcl-2 homologs in human CD34(+) hematopoietic progenitor cells induced to differentiate into erythroid or granulocytic cells.

The Bcl-2 family of proteins has been shown to play a central role in the regulation of apoptosis. We have examined the expression of several Bcl-2 homologs upon stimulation of CD34(+) human hematopoietic progenitor cells. CD34(+) cells were induced to differentiate into predominantly erythroid cells in the presence of erythropoietin (Epo) and stem cell factor (SCF), while the addition of G-CSF and SCF led to differentiation predominantly into granulocytic cells, as demonstrated by immunophenotyping and morphological examination of cultured cells. In Epo- and SCF-stimulated cells, we found a marked increase in the level of Bcl-x(L) protein expression and downregulation of Bax expression, apparent from day 4 and more pronounced on days 8 and 21. In contrast, Bcl-x(L) protein expression was downregulated in G-CSF- and SCF-stimulated cells compared with cells cultured in medium alone, whereas there was no sign of change in the level of Bax. Mcl-1 expression showed a biphasic expression pattern in both early erythropoiesis and early granulopoiesis, but with an inverse regulation. Thus, Mcl-1 levels initially decreased in granulocytic progenitor cells and increased in erythroid progenitor cells. Finally, Bcl-2 expression was significantly downregulated in both Epo and SCF and G-CSF- and SCF-stimulated cells. The role of the distinct upregulation of Bcl-x(L) in early erythroid differentiation was further examined by use of specific ribozymes against Bcl-x(L). Addition of Bcl-x(L) ribozymes promoted a clear increase in cell death of Epo- and SCF-stimulated cells, while erythroid differentiation was not affected. In conclusion, we found a distinct regulation of several Bcl-2 family members in CD34(+) cells dependent on the cytokine stimulation given. The use of Bcl-x(L)-specific ribozymes suggested that Bcl-x(L) is important for survival but not for differentiation of erythroid progenitor cells.

cAMP-mediated growth inhibition of lymphoid cells in G1: rapid down-regulation of cyclin D3 at the level of translation.

cAMP is an important physiological mediator of lymphoid growth inhibition. The purpose of the present study was to establish the link between cAMP and the cell cycle machinery leading to inhibition of G1/S transition in human peripheral blood lymphocytes (PBL). To unravel immediate effects of cAMP on this part of the cell cycle machinery, lymphocytes were synchronized in mid to late G1 after stimulation with phytohemaglutenin (PHA) for 32 h. We report that addition of forskolin or cAMP analogues to the cells resulted in dephosphorylation of retinoblastoma protein commencing as early as 30 min. A rapid effect of forskolin was noted on the activity of cyclin-dependent kinase (cdk) 4, which decreased significantly within 30 min of treatment. The decrease in cdk4 activity was concurrent with reduced levels of cyclin D3 protein and a decrease in the fraction of cdk4 associated with cyclin D3. The down-regulation of cyclin D3 was at the level of translation, and this event was preceded by a pronounced inhibition of Akt/protein kinase B phosphorylation at Ser 473. Taken together, our data imply that cyclin D3 is a major effector of cAMP-mediated inhibition of cell cycle progression in PBL, and that cAMP exerts its effect on cyclin D3 expression at the level of translation.

Protein kinase C-alpha isoform is involved in erythropoietin-induced erythroid differentiation of CD34(+) progenitor cells from human bone marrow.

Protein kinase C (PKC) is a family of serine/threonine protein kinases involved in many cellular responses. Although the analysis of PKC activity in many systems has provided crucial insights to its biologic function, the precise role of different isoforms on the differentiation of normal hematopoietic progenitor cells into the various lineages remains to be investigated. The authors have assessed the state of activation and protein expression of PKC isoforms after cytokine stimulation of CD34(+) progenitor cells from human bone marrow. Freshly isolated CD34(+) cells were found to express PKC-alpha, PKC-beta2, and PKC-epsilon, whereas PKC-delta, PKC-gamma, and PKC-zeta were not detected. Treatment with erythropoietin (EPO) or with EPO and stem cell factor (SCF) induced a predominantly erythroid differentiation of CD34(+) cells that was accompanied by the up-regulation of PKC-alpha and PKC-beta2 protein levels (11.8- and 2.5-fold, respectively) compared with cells cultured in medium. Stimulation with EPO also resulted in the nuclear translocation of PKC-alpha and PKC-beta2 isoforms. Notably, none of the PKC isoforms tested were detectable in CD34(+) cells induced to myeloid differentiation by G-CSF and SCF stimulation. The PKC inhibitors staurosporine and calphostin C prevented EPO-induced erythroid differentiation. Down-regulation of the PKC-alpha, PKC-beta2, and PKC-epsilon expression by TPA pretreatment, or the down-regulation of PKC-alpha with a specific ribozyme, also inhibited the EPO-induced erythroid differentiation of CD34(+) cells. No effect was seen with PKC-beta2-specific ribozymes. Taken together, these findings point to a novel role for the PKC-alpha isoform in mediating EPO-induced erythroid differentiation of the CD34(+) progenitor cells from human bone marrow. (Blood. 2000;95:510-518)

Fas ligand promotes cell survival of immature human bone marrow CD34+CD38- hematopoietic progenitor cells by suppressing apoptosis.

Fas (CD95, APO-1) is a member of the TNF receptor family, and engagement of Fas by its ligand, Fas ligand (FasL), can induce apoptotic death of Fas expressing cells. Signaling through Fas has previously been shown to induce apoptosis of CD34+ human hematopoietic progenitor cells after exposure to IFN-gamma or TFN-alpha. In contrast, we found that FasL promoted a significantly increased viability of primitive CD34+CD38- cells. Thus, incubation with FasL for 48 hours reduced cell death from 46 to 29% compared to cells cultured in medium alone as measured by propidium iodide (PI) incorporation (n = 8, p < 0.02). Inhibition of apoptosis was confirmed by morphological analysis and by the Nicoletti technique. Furthermore, by using a delayed addition assay at the single cell level we found that sFasL treatment had a direct viability-promoting effect on CD34(+)CD38(-) cells. The effect of sFasL was completely blocked by NOK-1, a neutralizing mAb against FasL. In agreement with previous reports, FasL alone slightly increased cell death of more mature CD34(-)CD38+ cells, indicating an interesting shift in the responsiveness to FasL during early hematopoiesis.

Prognostic value of lymphoma-specific S-phase fraction compared with that of other cell proliferation markers.

The proliferation-associated antigens Ki67 (immunohistochemistry) and proliferative cell nuclear antigen (PCNA) (immunohistochemistry and immunoblotting) were analysed together with DNA synthesis (3H-thymidine incorporation) and cell-cycle distribution (tumour-specific S-phase fraction determined by flow cytometry) in lymph node suspensions from 63 patients with newly diagnosed B-Cell non-Hodgkin's lymphomas. Details of clinical parameters, treatment and patient outcome were available for all patients, and retrospectively analysed. Of the proliferation-associated parameters, only high S-phase fraction (p < 0.00001) and high PCNA expression by immunoblotting (p = 0.012) were predictive of a poor prognosis. Of the conventional parameters, high-grade malignancy, high International Prognostic Index (IPI) score, bulky disease and presence of B symptoms predicted a patient for poor survival. High S-phase fraction was predictive of a short survival for the low-grade lymphomas analysed separately (p < 0.00001), as well as for patients treated with an Adriamycin- and a non-Adriamycin-containing regimen (p < 0.005 for both groups). In a multivariate analysis, S-phase fraction (p = 0.00006), IPI score (p = 0.015) and B symptoms (p = 0.017) had independent prognostic values, but not histological grade.

Activation of the cAMP signaling pathway increases apoptosis in human B-precursor cells and is associated with downregulation of Mcl-1 expression.

During B- and T-cell ontogeny, extensive apoptosis occurs at distinct stages of development. Agents that increase intracellular levels of cAMP induce apoptosis in thymocytes and mature B cells, prompting us to investigate the role of cAMP signaling in human CD10+ B-precursor cells. We show for the first time that forskolin (which increases intracellular levels of cAMP) increases apoptosis in the CD10- cells in a dose-dependent manner (19%-94% with 0-1,000 microM forskolin after 48 hours incubation, IC50 = 150 microM). High levels of apoptosis were also obtained by exposing the cells to the cAMP analogue 8-chlorophenylthio-cAMP (8-CPT-cAMP). Specific involvement of cAMP-dependent protein kinase (PKA) was demonstrated by the ability of a cAMP antagonist, Rp-isomer of 8-bromo-adenosine- 3', 5'- monophosphorothioate (Rp-8-Br-cAMPS), to reverse the apoptosis increasing effect of the complementary cAMP agonist, Sp-8-Br-cAMPS. Furthermore, we investigated the expression of Bcl-2 family proteins. We found that treatment of the cells with forskolin or 8-CPT-cAMP for 48 hours resulted in a fourfold decline in the expression of Mcl-1 (n = 6, P = 0.002) compared to control cells. The expression of Bcl-2, Bcl-xL, or Bax was largely unaffected. Mature peripheral blood B cells showed a smaller increase in the percentage of apoptotic cells in response to 8-CPT-cAMP (1.3-fold, n = 6, P = 0.045) compared to B-precursor cells, and a smaller decrease in Mcl-1 levels (1.5-fold, n = 4, P = 0.014). Taken together, these findings show that cAMP is important in the regulation of apoptosis in B-progenitor and mature B cells and suggest that cAMP-increased apoptosis could be mediated, at least in part, by a decrease in Mcl-1 levels.

Retinoic acid induces apoptosis of human CD34+ hematopoietic progenitor cells: involvement of retinoic acid receptors and retinoid X receptors depends on lineage commitment of the hematopoietic progenitor cells.

Retinoids are bifunctional regulators of growth and differentiation of hematopoietic cells. In this study we explored the effects of retinoic acid (RA) on apoptosis of human CD34+ hematopoietic progenitor cells isolated from normal bone marrow. RA (100 nM) induced an increase in the percentage of dead cells from 24% to 44% at day 6 (p < 0.05, n = 6) as compared to control cells cultured in medium alone. The effect was dose dependent and appeared relatively late. Significant differences were observed from day 4 onward. Apoptosis, or programmed cell death, was demonstrated as the mode of cell death by using the TUNEL assay, which detects single strand nicks in DNA, or by the Nicoletti technique demonstrating a subdiploid population by DNA staining. RA previously was found to inhibit granulocyte colony-stimulating factor--and not granulocyte-macrophage colony-stimulating factor--stimulated proliferation of CD34+ cells. However, we found that RA opposed anti-apoptotic effects of G-CSF and GM-CSF on CD34+ cells (G-CSF: 8% dead cells at day 6; G-CSF + RA: 20%; GM-CSF: 12%; GM-CSF + RA: 27%). Moreover, RA induced apoptosis of CD34+ cells and CD34+CD71+ cells stimulated with erythropoietin. To explore the receptor signaling pathways involved in RA-induced apoptosis, we used selective ligands for retinoic acid receptors (RARs; RO13-7410) and retinoid X receptors (RXRs; RO 25-6603). We found that RARs were involved in RA-mediated apoptosis of myeloid progenitor cells, whereas RARs as well as RXRs were involved in RA-mediated apoptosis of erythroid progenitor cells.

Evaluation of lymphoid cell populations in cytology specimens using flow cytometry and polymerase chain reaction.

Differential diagnosis between lymphomas and reactive lymphoid proliferations often requires ancillary techniques and morphologic evaluation. Flow cytometry (FCM) and polymerase chain reaction (PCR) can aid the detection of monoclonal B-cell populations. In the present study, the sensitivity and specificity of these two methods in the study of cytology specimens were compared. Eighty-six cytologic specimens from 81 patients (lymph nodes, solid organs, and body cavities) were evaluated. These specimens were taken from three groups of patients: those who underwent an initial evaluation for suspected lymphoma; those who were previously diagnosed with B-cell lymphoma and were now evaluated for possible disease recurrence; and those who were diagnosed with a nonhematologic malignancy. Histologic diagnosis was available for 51 samples. All samples were tested by FCM for the detection of monoclonality using kappa:lambda ratio and for clonal immunoglobulin heavy chain (IgH) gene rearrangements using a single-round PCR after cytologic evaluation. Tissue morphology, FCM and PCR results, and clinical findings in specimens without histologic diagnosis were correlated. Histologic evaluation (N = 51) revealed 44 specimens with B-cell malignancy. Twenty of the 44 lymphoma specimens (45%) were accurately diagnosed in cytologic smears, 18 (41%) were classified as suspicious of lymphoma, and 6 (14%) were diagnosed as reactive. FCM had superior sensitivity compared with PCR (77% vs. 64%). Fifty-six percent of specimens with B-cell malignancy were FCM+/PCR+, 23% were FCM+/PCR-, 14% were FCM-/PCR+, and 7% were FCM-/PCR-. The combined use of FCM and PCR resulted in a diagnosis of B-cell lymphoma in 41 (93%) of 44 B-cell lymphoma specimens and increased the sensitivity of fine needle aspiration by 48%. Both FCM and PCR aid in the diagnosis of lymphoid lesions in cytology specimens, and both can detect monoclonal B-cell populations that may be interpreted in cytology smears as reactive, even by experienced cytologists. Although FCM had higher sensitivity than PCR test in the present study, their combined use should be considered because of a relatively large number of specimens that were detected as monoclonal only with PCR.

[Molecular genetic diagnosis of leukemias and lymphomas]. / Molekylaergenetisk diagnostikk av leukemier og lymfomer.

Leukemias and lymphomas are genetically characterized by pathogenetically important, disease-specific mutations in the malignant cells. Many of the mutations arise through balanced chromosomal rearrangements, often translocations, that fuse oncogenes with other gene loci. In some instances, this leads to the formation of a qualitatively new hybrid gene encoding a leukemogenic or lymphomogenic protein product, whereas on other occasions the result is deregulation of an otherwise normal gene. The demonstration of such gene rearrangements is of both diagnostic and prognostic value and hence serves as an important supplement to the more traditional phenotype-based investigative techniques. All available methods to detect neoplasia-specific, acquired mutations have both advantages and disadvantages, depending partly on the level of resolution at which they operate (chromosomes, nucleic acids, proteins), but also on the specific characteristics of each individual technique. Ideally, the initial genetic diagnosis of a given tumor should be reached by means of an open-framed screening technique, whereas less labour-intensive, specific techniques may be used during the subsequent follow-up of the patient. The most reliable and complete information is always obtained by combining cytogenetic, molecular cytogenetic, and strictly molecular genetic investigations. However, such an approach may be too cumbersome and expensive to be taken routinely.

Proliferation and apoptosis in malignant and normal cells in B-cell non-Hodgkin's lymphomas.

We have examined apoptosis and proliferation in lymph node cell suspensions from patients with B-cell non-Hodgkin's lymphoma using flow cytometry. A method was developed which allowed estimation of the fractions of apoptotic cells and cells in the S-phase of the cell cycle simultaneously with tumour-characteristic light chain expression. Analysis of the tumour S-phase fraction and the tumour apoptotic fraction in lymph node cell suspensions from 95 B-cell non-Hodgkin's lymphoma (NHL) patients revealed a non-normal distribution for both parameters. The median fraction of apoptotic tumour cells was 1.1% (25 percentiles 0.5%, 2.7%). In the same samples, the median fraction of apoptotic normal cells was higher than for the tumour cells (1.9%; 25 percentiles 0.7%, 4.0%; P = 0.03). The median fraction of tumour cells in S-phase was 1.4% (25 percentiles 0.8%, 4.8%), the median fraction of normal cells in S-phase was significantly lower than for the tumour cells (1.0%; 25 percentiles 0.6%, 1.9%; P = 0.004). When the number of cases was plotted against the logarithm of the S-phase fraction of the tumour cells, a distribution with two Gaussian peaks was needed to fit the data. One peak was centred around an S-phase fraction of 0.9%; the other was centred around 7%. These peaks were separated by a valley at approximately 3%, indicating that the S-phase fraction in NHL can be classified as 'low' (< 3%) or 'high' (> 3%), independent of the median S-phase fraction. The apoptotic fractions were log-normally distributed. The median apoptotic fraction was higher (1.5%) in the 'high' S-phase group than in the 'low' S-phase group (0.8%; P = 0.02). However, there was no significant correlation between the two parameters (P > 0.05).

Tumour cell proliferation, but not apoptosis, predicts survival in B-cell non-Hodgkin's lymphomas.

Tumour S-phase fraction, but not the apoptotic fraction, had prognostic value in 92 patients with B-cell non-Hodgkin's lymphoma (P < 0.0001 and P = 0.85 respectively). Multivariate analysis showed that S-phase fraction was the strongest prognostic indicator in all cases (P = 0.0003, relative risk 4.3; age: P = 0.16; grade: P = 0.81), as well as in the 63 primary biopsy cases (P = 0.0006, relative risk = 7.3; international prognostic index: P = 0.015, relative risk = 3.2; B symptoms: P = 0.017, relative risk = 3.3; bulkiness: P = 0.65; grade: P = 0.91).

Combination chemotherapy with mitoguazon, ifosfamide, MTX, etoposide (MIME) and G-CSF can efficiently mobilize PBPC in patients with Hodgkin's and non-Hodgkin's lymphoma.

Many centers use CY and G-CSF to mobilize PBPC. In this study we explored whether a standard chemotherapy regimen consisting of mitoguazon, ifosfamide, MTX and etoposide (MIME) combined with G-CSF was capable of mobilizing PBPC in lymphoma patients. Twelve patients with Hodgkin's disease (HD) and 38 patients with non-Hodgkin's lymphoma (NHL) were mobilized with MIME/G-CSF. Most patients were heavily treated with different chemotherapy regimens receiving a median of 11 cycles (range 3 to 20) of chemotherapy prior to mobilization. It was found that the optimal time of PBPC harvest was at days 12 and 13 after initiating the mobilization regimen. The median number of collected CD34+ cells per kg body weight was 7.1 x 10(6) (range 0.5-26.2). More than 2.0 x 10(6) CD34+ cells/kg were achieved in 69% of the patients after one apheresis. When additional cycles of apheresis were done, only 6% failed to harvest this number of CD34+ cells. There was a statistically significant inverse correlation between the number of prior chemotherapy cycles and CD34+ cell yield (P = 0.003). No such association was found between CD34+ cell yield and prior radiotherapy. When MIME/G-CSF was compared with Dexa-BEAM/G-CSF, it was found that MIME/G-CSF tended to be more efficient in mobilizing PBPC in spite of being less myelotoxic. All patients transplanted with MIME/G-CSF mobilized PBPC had fast and sustained engraftment. These results demonstrate that an ordinary salvage chemotherapy regimen, such as MIME combined with G-CSF can be successfully used to mobilize PBPC.