Altered profile of immune regulatory cells in the peripheral blood of lymphoma patients.

BACKGROUND: Regulatory immune cells may modulate the lymphoma microenvironment and are of great interest due to the increasing prevalence of treatment with immunotherapies in lymphoma patients. The aim was to explore the composition of different immune regulatory cell subsets in the peripheral blood of newly diagnosed lymphoma patients in relation to treatment outcome. METHODS: Forty-three newly diagnosed patients with lymphoma were included in the study; 24 with high-grade B-cell lymphoma (HGBCL) and 19 with classical Hodgkin lymphoma (cHL). Peripheral blood was prospectively collected and immune regulatory cells were identified by multi-color flow cytometry and analyzed in relation to healthy blood donors and clinical characteristics and outcome. RESULTS: The percentage of CD3-positive T-cells was lower (p = 0.03) in the peripheral blood of lymphoma patients at diagnosis compared to healthy blood donors regardless of lymphoma subtype, although statistically, neither the percentage of monocytes (p = 0.2) nor the T-cell/monocyte ratio (p = 0.055) differed significantly. A significant decrease in the percentage of a subset of regulatory NK cells (CD7+/CD3-/CD56bright/CD16dim/-) was identified in the peripheral blood of lymphoma patients compared to healthy blood donors (p = 0.003). Lymphoma patients also had more granulocytic myeloid-derived suppressor cells (MDSCs) (p = 0.003) compared to healthy blood donors, whereas monocytic MDSCs did not differ significantly (p = 0.07). A superior disease-free survival was observed for cHL patients who had an increase in the percentage of granulocytic MDSCs (p = 0.04). CONCLUSIONS: An altered profile of immune cells in the peripheral blood with a decrease in T-cells and regulatory NK-cells was observed in newly diagnosed lymphoma patients. CHL patients with higher percentages of regulatory NK cells and higher percentages of granulocytic MDSCs might have a better outcome, although the number of patients was low.

Reduced expression of TRIM21/Ro52 predicts poor prognosis in diffuse large B-cell lymphoma patients with and without rheumatic disease.

OBJECTIVE: TRIM21 (also known as Ro52) is an autoantigen in rheumatic disease and is predominantly expressed in leucocytes. Overexpression is associated with decreased proliferation, and the TRIM21 gene maps to a tumour suppressor locus. We therefore investigated the expression of TRIM21 in patients with diffuse large B-cell lymphoma (DLBCL) and its potential usefulness as a prognostic biomarker. MATERIALS AND METHODS: TRIM21 expression levels were assessed by immunohistochemistry in lymphoma biopsies from three cohorts of patients with DLBCL: 42 patients with rheumatic disease treated with a cyclophosphamide, vincristine, doxorubicin and prednisone (CHOP)-like regimen, 76 CHOP-treated and 196 rituximab-CHOP-treated nonrheumatic patients. Expression was correlated with clinical and biomedical parameters. TRIM21 expression was assessed in relation to lymphocyte proliferation by quantitative PCR and correlated with (3) H-thymidine incorporation and propidium iodine staining. RESULTS: TRIM21 expression levels differed in the lymphomas compared to normal lymphoid tissue, with reduced expression correlating with shorter overall survival in all three cohorts. In the two larger cohorts, progression-free survival was assessed and was also found to correlate with TRIM21 expression. The association was independent of commonly used clinical prognostic scores, lymphoma subtype and several previously reported prognostic biomarkers. In agreement with this clinical observation, we noted an inverse correlation between TRIM21 expression and proliferation of leucocytes in vitro. CONCLUSIONS: We show that loss of TRIM21 expression is associated with more aggressive lymphoma and increased proliferation, whereas maintenance of TRIM21 expression is associated with better prognosis in patients with DLBCL. Based on our findings, we suggest that TRIM21 should be considered as a novel biomarker for lymphoma characterization and for predicting patient survival.

Donor or recipient origin of posttransplant lymphoproliferative disorders following solid organ transplantation.

Previous studies of donor or recipient origin of posttransplant lymphoproliferative disorders (PTLDs) following solid organ transplantation (SOT) have either been small or with selected patient groups. We studied tumor origin in a population-based cohort of 93 patients with PTLD following SOT. Tumor origin of PTLD tissue was analyzed by fluorescence in situ hybridization of the sex chromosomes in cases of sex mismatch between donor and recipient (n = 41), or HLA genotyping in cases of identical sex but different HLA type (n = 52). Tumor origin of PTLD could be determined in 67 of the 93 cases. All 67 PTLDs were of recipient origin. They were found in recipients of kidney (n = 38), liver (n = 12), heart (n = 10) and lung (n = 7). The most common recipient-derived lymphomas were monomorphic B-cell PTLDs (n = 45), monomorphic T cell PTLDs (n = 9), indolent lymphomas (n = 6), and polymorphic PTLD (n = 4). Half of the recipient-derived PTLDs were Epstein-Barr virus-positive. Twelve of the recipient-derived PTLDs were located in the grafts: in four cases exclusively and in eight cases in combination with disseminated disease outside the graft. Tumor origin was indeterminable in 26 cases, probably due to low DNA quality. We conclude that the vast majority of PTLDs after SOT was of recipient origin.

Role of microRNAs and microRNA machinery in the pathogenesis of diffuse large B-cell lymphoma.

Deregulation of microRNA (miRNA) expression has been documented in diffuse large B-cell lymphoma (DLBCL). However, the impact of miRNAs and their machinery in DLBCL is not fully determined. Here, we assessed the role of miRNA expression and their processing genes in DLBCL development. Using microarray and RT-qPCR approaches, we quantified global miRNAs and core components of miRNA-processing genes expression in 75 DLBCLs (56 de novo and 19 transformed) and 10 lymph nodes (LN). Differential miRNA signatures were identified between DLBCLs and LNs, or between the de novo and transformed DLBCLs. We also identified subsets of miRNAs associated with germinal center B-cell phenotype, BCL6 and IRF4 expression, and clinical staging. In addition, we showed a significant over-expression of TARBP2 in de novo DLBCLs as compared with LNs, and decreased expression of DROSHA, DICER, TARBP2 and PACT in transformed as compared with de novo cases. Interestingly, cases with high TARBP2 and DROSHA expression had a poorer chemotherapy response. We further showed that TARBP2 can regulate miRNA-processing efficiency in DLBCLs, and its expression inhibition decreases cell growth and increases apoptosis in DLBCL cell lines. Our findings provide new insights for the understanding of miRNAs and its machinery in DLBCL.

Inflammatory cells and immune microenvironment in malignant lymphoma.

It has become clear that the biological and clinical behaviour of malignant lymphoma is not only determined by the properties of the tumour cells themselves but are also largely by the interaction of the tumour cells with their nonmalignant microenvironment. The composition and functional status of the tumour microenvironment is highly variable between different classes of malignant lymphoma and may provide both growth-supportive and growth suppressive signals via components of the adaptive and innate immune response. In this review, the functional interactions and clinical consequences of these insights are discussed in indolent and aggressive B-cell lymphomas and in classical Hodgkin's lymphoma.

Impact of germinal center and non-germinal center phenotypes on overall and failure-free survival after high-dose chemotherapy and auto-SCT in primary diffuse large B-cell lymphoma.

Non-germinal center (non-GC) phenotype is an adverse prognostic factor in chemotherapy (CT)-treated diffuse large B-cell lymphoma (DLBCL) patients. To determine how high-dose therapy (HDT) supported with auto-SCT as first line therapy influences GC-associated outcome in young high-risk DLBCL patients GC and non-GC phenotypes were determined immunohistochemically from 63 patients. Of these, 29 primary high-risk DLBCL patients were treated with auto-SCT, whereas 34 CT-treated patients served as a control group. Consistent with previous studies, non-GC phenotype was associated with adverse outcome in CT-treated high-risk patients. In contrast, immunohistochemical classification by cell of origin did not associate with survival after auto-SCT. When the impact of treatment on the predictive value of cell of origin was analyzed, the non-GC patients, who received HDT, had a better failure-free survival (FFS) and overall survival (OS) than the patients treated with CT alone. In multivariate analyses, both age-adjusted International Prognostic Index (aaIPI) and treatment were independent prognostic factors for FFS and OS. For the patients with GC phenotype, the influence of auto-SCT on survival was not significant. The data imply that auto-SCT can overcome the adverse prognostic impact of the non-GC phenotype in patients with high-risk DLBCL and warrant additional prospective studies.

Family history of cancer as a risk factor for second malignancies after Hodgkin's lymphoma.

This study estimated the risk of second primary malignancies after Hodgkin's lymphoma (HL) in relation to family history of cancer, age at diagnosis and latency, among 6946 patients treated for HL in Sweden in 1965-1995 identified through the Swedish Cancer Register (SCR). First-degree relatives (FDRs) to the HL patients and their malignancies were then ascertained together with their malignancies through the Multi-Generation Registry and SCR. The HL patient cohort was stratified on the number of FDRs with cancer, and standardised incidence ratios (SIRs) of developing SM were analysed. In the HL cohort, 781 SM were observed 1 year or longer after HL diagnosis. The risk for developing SM increased with the number of FDRs with cancer, SIRs being 2.26, 3.01, and 3.45 with 0, 1, or >or=2 FDRs with cancer, respectively. Hodgkin's lymphoma long-term survivors treated at a young age with a family history of cancer carry an increased risk for developing SM and may represent a subgroup where standardised screening for the most common cancer sites could be offered in a stringent surveillance programme.

Treatment of a patient with a nodal peripheral T-cell lymphoma (angioimmunoblastic T-Cell lymphoma) with a human monoclonal antibody against the CD4 antigen (HuMax-CD4).

A patient with a CD4+ refractory peripheral T-cell lymphoma (PTL), subtype angioimmunoblastic T-cell lymphoma (AILD), was treated with a human monoclonal anti-CD4 antibody (HuMax-CD4) iv once weekly for 10 wk. Early during treatment all palpable enlarged lymph nodes disappeared. A decline of normal CD4+ T-cells in the blood mirrored the treatment effect. Shortly after stopping treatment the patient relapsed with new enlarged lymph nodes. This time no antitumor effect was seen when HuMax-CD4 treatment was reinstituted. No severe side effects were observed during the antibody treatment. This case report is the first describing that HuMax-CD4 has antilymphoma activity in PTL and is an interesting drug to study further in patients with CD4+ PTL.

Increased serum levels of interleukin-9 correlate to negative prognostic factors in Hodgkin's lymphoma.

Hodgkin's lymphoma (HL) is characterised by an unbalanced cytokine secretion. Many of these cytokines have been implicated in the regulation of malignant and infiltrating cells. Interleukin-9 (IL-9) has been described to act in an autocrine fashion in HL, stimulating proliferation of the malignant cells. To investigate the potential clinical implication of this observation, a novel ELISA method was used to examine the serum levels of IL-9 in lymphoma patients. High levels of IL-9 were found in the sera from patients with HL (18/44), but not in the sera from non-Hodgkin's lymphoma patients (3/21) or healthy controls. The highest serum IL-9 levels, up to 3350 pg/ml, were observed in the nodular sclerosis subtype, and there was a correlation between IL-9 levels and the negative prognostic factors advanced stage, B-symptoms, low blood Hb and high erythrocyte sedimentation rate. Furthermore, there was no correlation between serum levels of IL-9 and IL-13, a cytokine where serum levels have been speculated to be of clinical importance. This is the first report showing that IL-9 can be measured in serum samples. A novel correlation between increased serum IL-9 levels, HL and clinical features is shown, suggesting that IL-9 is a candidate factor contributing to the development of HL.

The serum levels of eosinophil cationic protein (ECP) are related to the infiltration of eosinophils in the tumours of patients with Hodgkin's disease.

We have previously described a relation between abundance of eosinophilic granulocytes in Hodgkin's disease (HD) tumours and poor prognosis. In order to further explore the importance of the eosinophilic infiltration, we immunohistochemically examined the presence of eosinophils, using the monoclonal antibodies EG 1 and EG 2, in the tumours of 54 newly diagnosed patients with HD and related the degree of infiltration to clinical characteristics and the serum levels of eosinophil cationic protein (S-ECP). S-ECP levels (upper normal value 16 micrograms/l) varied between 2.2 and 71.7 micrograms/l, mean 25.4 micrograms/l. There was an association (p = 0.01) between the number of eosinophils in the tumour tissue and S-ECP. S-ECP levels were also associated to high erythrocyte sedimentation rate (ESR, p < 0.01) and nodular sclerosis (NS) histology (p < 0.05), and there was a tendency of a correlation to bulky disease (p = 0.06). The number of eosinophils stained with EG 2 correlated to high ESR (p < 0.05), and to high leukocyte count (p = 0.02). A follow-up value of S-ECP after treatment was, in most of the cases measured, lower than the initial value. The high values of S-ECP in several patients with HD probably originates from eosinophils infiltrating the tumours. The same patients had a higher ESR and tended to have a more advanced stage and bulky disease. There are no significant correlations with disease-free and overall survival, as the follow-up time is short, and prognosis favourable.

Mast cells express functional CD30 ligand and are the predominant CD30L-positive cells in Hodgkin's disease.

Hodgkin's disease (HD) tumours are characterized by the presence of few tumour cells, the Hodgkin and Reed-Sternberg (HRS) cells, surrounded by a large amount of non-neoplastic cells. The role of this cell infiltrate for the development of HD is not known. CD30, belonging to the tumour necrosis factor receptor superfamily, is highly expressed on HRS cells and believed to be involved in tumourigenesis and tumour progression. Tumour samples from 42 patients were immunohistochemically double-stained for tryptase, a mast cell-specific proteinase and CD30 ligand (CD30L). Tryptase-positive mast cells were present in all tumours. Of these cells, 50% expressed CD30L and 66% of the CD30L-positive cells were mast cells. CD30L mRNA in in vitro developed normal mast cells and malignant human and murine mast cell lines was detected using reverse transcription polymerase chain reaction. CD30L protein expressed on human mast cells was detected using flow cytometry. In a co-culture assay, the human mast cell line HMC-1 stimulated thymidine uptake in HRS cell lines, and the stimulation could be blocked using CD30L-specific monoclonal antibodies. In conclusion, mast cells are present in HD tumours and are the predominant CD30L-expressing cells. CD30L-CD30 interaction is a pathway by which mast cells may stimulate DNA synthesis in HRS cells.

High-dose therapy with autologous stem cell transplantation in patients with peripheral T cell lymphomas.

Peripheral T cell lymphomas (PTCL) have a poorer prognosis after conventional treatment than do high-grade B cell lymphomas. The place for high-dose therapy (HDT) with autologous stem cell support in these patients is still not clear. Forty patients, 10 women and 30 men, median age 41.5 years (range 16-61) with PTCL were treated with HDT and autologous stem cell support at The Norwegian Radium Hospital, Oslo, Norway and The University Hospital, Uppsala, Sweden, between February 1990 and September 1999. The histologic subtypes were: PTCL unspecified, 20 patients; intestinal, two patients; angioimmunoblastic (AILD), two patients; angiocentric, two patients and anaplastic large cell lymphoma (ALCL), 14 patients. All patients had chemosensitive disease and had received anthracycline-containing regimens prior to transplantation. At the time of HDT, 17 patients were in first PR or CR and 23 were in second or third PR or CR. Conditioning regimens were BEAM in 15 patients, BEAC in 14 patients, cyclophosphamide and total body irradiation (TBI) in eight patients, BEAC, without etoposide and TBI in one patient and mitoxantrone and melphalan in two patients. There were three (7.5%) treatment-related deaths. The estimated overall survival (OS) at 3 years was 58%, the event-free survival (EFS) 48% and the relapse-free survival (RFS) 56%, with a median follow-up of 36 months (range 7-100) for surviving patients. The patients with ALCL tended to have a better prognosis compared to those with other PTCL subtypes, OS 79% vs 44%, respectively. In conclusion, patients with chemosensitive PTCL who are failing to achieve CR with first-line chemotherapy or are in relapse can successfully be treated with HDT and autologous stem cell support.

A more favourable clinical course of lymphoma relapsing after high-dose therapy: evidence of tumour heterogeneity?

Patients with Hodgkin's disease (HD) or large cell lymphomas who relapse after conventional chemotherapy have a poor prognosis without high-dose chemotherapy and stem cell transplantation (HDCT). Patients who relapse after HDCT have an extremely poor outcome. In the present study we describe four patients with relapsed HD (n=1) and large cell lymphomas (n=3) after HDCT. All had an aggressive clinical course before HDT. At relapse, however, they all have had prolonged remissions and/or spontaneous regressions on mild or no treatment. Possible explanations could be selection of less malignant clones or a lymphoma controlled by the immune system. The mechanisms are, however, unknown but warrant further studies of the molecular biology in these and similar cases.

Long-term follow-up of autologous stem-cell transplantation for follicular and transformed follicular lymphoma.

Despite the fact that follicular lymphomas are both chemo- and radiosensitive, the disease is generally non-curable. These lymphomas often undergo transformation to a more malignant state. In order to improve the prognosis, high-dose treatment with stem cell support has been tested, but its role in the treatment of this disease is still unclear. Fourteen men and eight women with a median age of 45 yr (34-59) were treated with high-dose therapy with autologous stem cell transplantation between 1987 and 1996. The patients were selected to undergo intensive therapy because of an estimated short survival (median < 3 yr), even though they had chemosensitive disease and adequate performance status. Eleven patients' lymphomas had transformed, and the other eleven patients had one or more unfavourable prognostic signs such as advanced stage, bulky disease, multiple relapses, or short remission duration. The conditioning regimen has varied over the period, but BEAC (Becenum, etoposide, cytarabine, cyclophosphamide) or etoposide/cyclophosphamide with or without total body irradiation (TBI) was used in most patients. Nine patients had their stem cells purged. After a median follow-up time of 74 months overall survival was 81% and disease-free survival 72%. One toxic procedure-related death occured. There was no difference in outcome between patients with a transformed lymphoma compared to those without transformation. The patients treated with TBI had a significantly worse outcome. Toxicity was also much higher in TBI-treated patients, including four cases of secondary malignancy (three myelodysplastic syndrome (MDS) cases and one patient with breast carcinoma). This retrospective study, with the longest follow-up time so far reported, shows a promising 6-yr DFS of 72% in a group of follicular lymphoma patients with a bad prognosis. The outcome of patients with transformed lymphoma compared to historical controls is especially encouraging. The high incidence of MDS is worrying. The role of TBI should be questioned because this and other studies have not shown any advantage of using TBI. In the absence of randomised trials the role of high-dose treatment for patients with follicular lymphoma is still not defined.

Treatment outcome in patients younger than 60 years with advanced stages (IIB-IV) of Hodgkin's disease: the Swedish National Health Care Programme experience.

BACKGROUND: Despite improved treatment results achieved in Hodgkin's disease (HD), only about 70% of patients with advanced stages are cured. The primary aim of this study was to evaluate the outcome of advanced stages (IIB-IVB) of HD in younger patients in an unselected population-based group of patients. The patients were recommended individualized treatment with respect to number of chemotherapy (CT) courses and post-CT radiotherapy (RT) based on pretreatment characteristics and tumour response. Secondly, we investigated if variables of prognostic importance could be detected. PATIENTS AND METHODS: Between 1985-92, 307 patients between 17-59 yr of age (median 36) were diagnosed with HD in stages IIB-IVB in 5/6 health care regions in Sweden. Median follow-up time was 7.8 yr (1.3-13). Retrospectively, laboratory parameters were collected. RESULTS: In total, 267 (87%) patients had a complete response (CR). The overall and disease-free 10-yr survivals in the whole cohort were 76% and 67%, respectively. There was no difference in survival between the groups of patients who received 6 or 8 cycles of CT. Survival was not higher for patients in CR after CT when RT was added. For those in PR after CT, additional RT raised the frequencies of CR. A selected group of pathologically staged patients was successfully treated with a short course (2 cycles) of CT + RT. In univariate analyses survival was affected by age, stage IVB, bone-marrow involvement, B-symptoms, S-LDH, S-Alb and reaching CR or not after 2, 4 and 6 cycles of CT. In a multivariate analysis, age and reaching CR after 6 cycles of CT remained statistically significant. CONCLUSIONS: The lack of difference in survival between the groups of patients who received 6 versus 8 cycles of CT indicates a successful selection of patients for the shorter treatment. Reaching a rapid CR significantly affected outcome. Whether some patients need less CT than the generally recommended 8 courses can properly only be evaluated in a randomised study. Additional RT may play a role in successful outcome, particularly if residual tumours are present, but its precise role can also only be defined in prospectively randomised studies. Reaching CR after CT was the most important variable affecting survival besides age.

Epstein-Barr virus distribution in Hodgkin's disease in an unselected Swedish population.

All patients with Hodgkin's disease (HD) (n = 117) identified in the Uppsala/Orebro region of Sweden between 1985 and 1988 were examined for the presence of Epstein-Barr virus (EBV) in the Hodgkin and Reed-Sternberg (HRS) cells. EBV was detected with LMP-1 immunostaining and in situ hybridization for EBERs. Overall, 32 (27%) tumours were EBV-positive but there were significant differences in EBV-positivity between histopathological subgroups (p = 0.03). In MC, 8/21 (38%) were positive, in NS 20/67 (23%), LD 3/3, LP 1/5, and in unclassified 0/1. Patients with EBV-positive tumours were significantly older, mean 52 vs. 42 years (p = 0.02), and were likely to have significantly more B-symptoms or advanced stage disease. Patients with EBV-positive tumours tended to have a poorer survival rate (p = 0.11). The proportion of EBV-positive tumours, and especially the proportion of EBV-positive MC, was lower than previously reported. This could be explained by selection of patients from previous studies, or by differences in EBV-positivity in different geographical or ethnic populations of HD.