Gunilla Enblad

Evaluation of immunophenotype in diffuse large B-cell lymphoma and its impact on prognosis.

Diffuse large B-cell lymphoma (DLBCL) has been shown to be comprised of at least two prognostic entities, depending on its resemblance to normal germinal center or activated B cells, using global gene expression profiling. Also, the expression patterns of bcl-6, CD10 and IRF-4 (also known as MUM1) have been suggested as alternative means of identifying the germinal- and nongerminal center (activated B-cell like) groups. In the present study, we evaluated by immunohistochemistry the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 in a large material of 161 DLBCL patients. Using two different approaches, patients with germinal center phenotype displayed a significantly better survival than the nongerminal center group. Positive staining for bcl-6 or CD10 predicted for superior survival, while expression of IRF-4 alone showed no association with prognosis. Furthermore, expression of bcl-2 was associated with worse event-free survival and overall survival. In a multivariate analysis, a high international prognostic index score (3–5), non-GC phenotype and bcl-2 were independent adverse prognostic factors. Here we confirm the prognostic importance of determining the germinal- or nongerminal center phenotype in patients with DLBCL.

Mast cell infiltration correlates with poor prognosis in Hodgkin's lymphoma

Summary. Hodgkins lymphoma (HL) is characterized by a few Hodgkin, Reed–Sternberg cells (HRS) surrounded by benign cells. We recently reported that mast cells were the predominant CD30L‐positive cells in HL tumours, and that they activate HRS in vitro through CD30L–CD30 interaction. Here, we investigated the clinical importance of mast cell infiltration in the tumours of 123 patients. Tumour specimens were stained with a mast‐cell‐specific antibody that detects tryptase. Mast cells were detected in virtually every case and increasing numbers of mast cells correlated to nodular sclerosis histology (P = 0·008). Patients with higher mast cell infiltration had a worse relapse‐free survival (P = 0·01).

Adapted treatment guided by interim PET-CT scan in advanced Hodgkin's lymphoma

BACKGROUND We tested interim positron-emission tomography-computed tomography (PET-CT) as a measure of early response to chemotherapy in order to guide treatment for patients with advanced Hodgkins lymphoma. METHODS Patients with newly diagnosed advanced classic Hodgkins lymphoma underwent a baseline PET-CT scan, received two cycles of ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) chemotherapy, and then underwent an interim PET-CT scan. Images were centrally reviewed with the use of a 5-point scale for PET findings. Patients with negative PET findings after two cycles were randomly assigned to continue ABVD (ABVD group) or omit bleomycin (AVD group) in cycles 3 through 6. Those with positive PET findings after two cycles received BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone). Radiotherapy was not recommended for patients with negative findings on interim scans. The primary outcome was the difference in the 3-year progression-free survival rate between randomized groups, a noninferiority comparison to exclude a difference of 5 or more percentage points. RESULTS A total of 1214 patients were registered; 937 of the 1119 patients (83.7%) who underwent an interim PET-CT scan according to protocol had negative findings. With a median follow-up of 41 months, the 3-year progression-free survival rate and overall survival rate in the ABVD group were 85.7% (95% confidence interval [CI], 82.1 to 88.6) and 97.2% (95% CI, 95.1 to 98.4), respectively; the corresponding rates in the AVD group were 84.4% (95% CI, 80.7 to 87.5) and 97.6% (95% CI, 95.6 to 98.7). The absolute difference in the 3-year progression-free survival rate (ABVD minus AVD) was 1.6 percentage points (95% CI, -3.2 to 5.3). Respiratory adverse events were more severe in the ABVD group than in the AVD group. BEACOPP was given to the 172 patients with positive findings on the interim scan, and 74.4% had negative findings on a third PET-CT scan; the 3-year progression-free survival rate was 67.5% and the overall survival rate 87.8%. A total of 62 patients died during the trial (24 from Hodgkins lymphoma), for a 3-year progression-free survival rate of 82.6% and an overall survival rate of 95.8%. CONCLUSIONS Although the results fall just short of the specified noninferiority margin, the omission of bleomycin from the ABVD regimen after negative findings on interim PET resulted in a lower incidence of pulmonary toxic effects than with continued ABVD but not significantly lower efficacy. (Funded by Cancer Research UK and Others; ClinicalTrials.gov number, NCT00678327.).

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. Bone Marrow Transplantation (2001) 27, 711–716.

Mast cell CD30 ligand is upregulated in cutaneous inflammation and mediates degranulation-independent chemokine secretion

Mast cells are involved in many disorders where the triggering mechanism that leads to degranulation and/or cytokine secretion has not been defined. Several chronic inflammatory diseases are associated with increased mast cell numbers and upregulation of the TNF receptor family member CD30, but the role of elevated CD30 expression is poorly understood. Here we report what we believe to be a novel way to activate mast cells with CD30 that leads to degranulation-independent secretion of chemokines. CD30 induced a de novo synthesis and secretion of the chemokines IL-8, macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta, a process involving the MAPK/ERK pathway. Mast cells were found to be the predominant CD30 ligand-positive (CD30L-positive) cell in the chronic inflammatory skin diseases psoriasis and atopic dermatitis, and both CD30 and CD30L expression were upregulated in lesional skin in these conditions. Furthermore, the number of IL-8-positive mast cells was elevated both in psoriatic and atopic dermatitis lesional skin as well as in ex vivo CD30-treated healthy skin organ cultures. In summary, characterization of CD30 activation of mast cells has uncovered an IgE-independent pathway that is of importance in understanding the entirety of the role of mast cells in diseases associated with mast cells and CD30 expression. These diseases include Hodgkin lymphoma, atopic dermatitis, and psoriasis.

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

Hodgkins 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.

Expression of CCL5/RANTES by Hodgkin and Reed-Sternberg cells and its possible role in the recruitment of mast cells into lymphomatous tissue

HL is a malignant lymphoma characterized by a small number of malignant HRS cells among a major population of infiltrating reactive cells, e.g., lymphocytes and eosinophils. We previously reported that mast cells are present in HL‐affected lymph nodes and therein are the predominant CD30L‐expressing cells. The CD30L expressed on mast cells is functionally active and can provide stimulatory signals to HRS cells. Thus, mast cells constitute an important portion of the infiltrating reactive cells that contribute to tumor progression in HL. Control of the recruitment of this previously unrecognized cell and its interactions with tumor cells are essentially unknown. To elucidate if mast cells might be specifically attracted to the tumor area by chemokines produced by HRS cells, we investigated chemokine expression in HL cell lines and in vivo. By RNase protection assay, mRNA expression of several chemokines could be detected in the cell lines. Despite the heterogeneous expression profile exhibited by the cell lines, 4 of 5 expressed CCL5 (RANTES) mRNA. RT‐PCR and immunohistochemistry confirmed expression of CCL5 in vivo. Furthermore, secreted CCL5 was detected in conditioned media from 3 of the cell lines. In a migration assay, we found that CCL5 present in conditioned medium could induce mast cell migration. Taken together, our results suggest that CCL5 produced by HRS cells is one mechanism by which mast cells can be attracted into the tumor tissue in HL.

Somatic hypermutation and VH gene usage in mantle cell lymphoma

Abstract:  Mantle cell lymphoma (MCL) is considered to derive from naïve, pregerminal center (GC) CD5+ B‐cells. However, the cell of origin has been questioned in recent studies that showed somatic hypermutations in the immunoglobulin (Ig) variable heavy chain (VH) genes in subsets of MCL. To clarify this issue, we analyzed the IgVH genes for the presence of somatic hypermutations in 51 MCL cases. Twenty percent of the MCL cases displayed somatically mutated VH genes (defined as >2% mutated), whereas 80% showed unmutated VH genes. This finding suggests that MCL is a genetically heterogeneous disease, with the majority of cases originating from unmutated pre‐GC B‐cells and a subset deriving from more mature B‐cells which have been exposed to the GC environment and have undergone somatic hypermutation. A biased VH gene usage has been demonstrated in several B‐cell malignancies; however, this has not yet been investigated in MCL, although VH4‐34 overusage has been indicated by small studies. Interestingly, we found a restricted usage of three individual VH genes in our MCL material; VH4‐34 (22%), VH3‐21 (16%) and VH5‐51 (12%). This novel finding of preferential VH gene usage in half of the MCL cases may suggest an antigen driven process occurring in B‐cells expressing specific VH genes, thus implicating that Ig specificity could be involved in mantle cell lymphoma development.

Chromosomal Imbalances in Diffuse Large B-Cell Lymphoma Detected by Comparative Genomic Hybridization

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma. In contrast to many other hematological malignancies, no chromosomal abnormalities with a diagnostic or prognostic value have been identified in DLBCL. Numerical chromosomal imbalances were characterized by comparative genomic hybridization (CGH) performed on 54 DLBCL tumors from a total of 40 patients. The clonal relatedness was demonstrated in 9 of 11 pairs of matched diagnostic tumors and their relapses as determined by IGH gene rearrangement analysis and/or the CGH profiles. Furthermore, immunohistochemical expression analyses of BCL2 and BCL6/LAZ3 were performed on all cases. Copy number changes were detected in 94% of the diagnostic tumor samples and in all of the relapses. Chromosomal losses in diagnostic tumors were preferentially observed at 8p22-pter (29%), 1p34-pter (26%), 6q23-qter (20%), 17p12-pter (17%) and 22q (17%), 9p23-pter (14%), whereas gains were mainly seen in Xq25–26 (43%), 13q22 (26%), 12cen-q14 (20%), 3q24–25 (11%), 7 (11%), and 18q12–21 (11%). Loss of 22q was significantly more commonly seen in the diagnostic tumor samples with more advanced clinical stage in other words, Stage III–IV compared with Stage I–II, and band 18q21 was significantly more often gained in relapses as compared to diagnostic tumors. None of the recurrent alterations were detected as a single abnormality, suggesting that other genetic lesions below the detection level of CGH may be the initiating event in the tumorigenesis of DLBCL. However, the distribution of CGH alterations support the idea of a progression of genetic events where loss of 8p and 9p and gain of 3q, 13q, and 18q would represent relatively early events because they were distributed in tumors with only two abnormalities.

Antibodies to LMP2A/2B in EBV-carrying malignancies

Antibodies to the Epstein-Barr virus (EBV)-encoded membrane proteins, LMP2A and LMP2B, were assayed in 540 individuals, including 154 patients with nasopharyngeal carcinoma, 16 with African Burkitts lymphoma, 113 with Hodgkins disease, 14 with EBV-carrying gastric carcinoma, 14 with oral hairy leucoplakia (HIV+ patients), 37 with non-Hodgkins lymphoma, 49 with tumours of the head/neck, 19 with infectious mononucleosis, 62 with chronic illnesses with EBV titres consistent with re-activations, and 62 healthy controls. A novel assay, mouse monoclonal enhanced indirect immunofluorescence assay (MIFA) was designed and used to test the sera for antibodies to the LMP2A and 2B proteins, expressed in human keratinocytes. Antibody to both LMP2A and LMP2B was strikingly specific to NPC. Virtually all (99 of 101) of the LMP2 antibody positive individuals were NPC patients, 95% of whom had antibodies that reacted both with the LMP2A- and LMP2B-transfected indicator cells, while the remaining 5% reacted only with the LMP2B expressing cells.