[Round robin test for detection of genomic aberrations in non-Hodgkin lymphoma by in situ hybridization]. / Ringversuch zum Nachweis genomischer Veränderungen bei Non-Hodgkin-Lymphomen mittels In-situ-Hybridisierung.

BACKGROUND: The detection of characteristic genomic aberrations by fluorescence in situ hybridization (FISH) has a high diagnostic impact on lymphomas according to the World Health Organization (WHO). To investigate the reproducibility of non-isotopic ISH results a multicenter trial was carried out involving eight institutes for hematopathology. MATERIAL AND METHODS: Analyses were performed on two diffuse large B-cell lymphomas (DLBCL) without known aberrations, on one follicular lymphoma with a IGH/BCL2 translocation and BCL6 split and on two B-cell lymphomas intermediate between DLBCL and Burkitt's lymphoma with c-MYC and BCL2 rearrangements, one with an additional BCL6 split. Break-apart probes for BCL6 and c-MYC, as well as fusion probes for the c-MYC/IGH and the IGH/BCL2 translocations were used. RESULTS: All aberrations were correctly detected by all centres and no false positive or false negative results were obtained. The numbers of positive cells varied from 25% to 94%. Pearson's correlation coefficient between the centres was always > 0.8. CONCLUSIONS: The ISH analysis of recurrent genomic aberrations in formalin-fixed paraffin-embedded (FFPE) tissue is a highly reproducible technique which yields substantial additive help for lymphoma diagnostics.

B-cell receptor signaling and CD40 ligand-independent T cell help cooperate in Helicobacter-induced MALT lymphomagenesis.

Gastric B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) develops in the context of chronic inflammation caused by Helicobacter pylori infection. Most pathophysiological features of the early stages of MALT lymphomagenesis can be reproduced by experimental infection of BALB/c mice with Helicobacter species. We have previously shown that MALT lymphomas are infiltrated by T-helper cell type 2-polarized T cells and that human and murine tumor B cells carry polyreactive surface immunoglobulins. Using the murine model of the disease, in this study we show that explanted tumor B cells proliferate upon stimulation with the same panel of self and foreign antigens that are recognized by their surface antibodies. Tumor cell proliferation is strongly enhanced by the presence of intratumoral CD4(+) T cells in a CD40/CD40L-independent manner. A large proportion of tumor-infiltrating CD4(+) T cells are CD25(+)FoxP3(+) regulatory T cells (Tregs) with highly suppressive activity, which are recruited by the tumor cells through secretion of the Treg-attracting chemokines CCL17 and CCL22. The depletion of CD25(+) cells was as efficient as CD4(+) T cell depletion in blocking tumor growth in vitro and in vivo. In conclusion, our data suggest that B-cell receptor-derived signals cooperate with T-helper cell signals in driving the progression of MALT lymphoma, providing an explanation for the unique antigen dependence of this B-cell malignancy.

A multicentre phase II trial of gemcitabine for the treatment of patients with newly diagnosed, relapsed or chemotherapy resistant mantle cell lymphoma: SAKK 36/03.

Mantle cell lymphoma (MCL) has a poor prognosis with often short and incomplete remissions. We aimed to test the efficacy and tolerability of gemcitabine in treating MCL. Gemcitabine was given in doses of 1000 mg/m(2) as a 30 min infusion on days 1 and 8 of each 3 week cycle for a maximum of nine cycles. Eighteen patients with a median age of 70 years were recruited. MCL was newly diagnosed in half of patients and relapsed in the remainder. Fifteen patients had Ann Arbor stage IV. The best-recorded responses were 1 CR (complete remission), 4 PRs (partial responses), 8 SDs (stable diseases) and 4 PDs (diseases progression). The response rate (RR) (CR + PR) was 5 (28%; 95% confidence interval: 7.1, 48.5). The patient achieving a CR had stage IV disease. Most haematological adverse events occurred during the first chemotherapy cycle. Three patients developed non-haematological serious adverse events: dyspnea, glomerular microangiopathy with haemolytic uremic syndrome (HUS) and hyperglycaemia. The median time-to-progression and treatment response duration (TRD) was 8.0 (95% confidence interval: 5.5, 9.3) and 10.6 (95% confidence interval: 5.5, 10.9) months, respectively. We conclude that Gemcitabine is well tolerated, moderately active and can induce disease stabilization in patients with MCL.

MAGE-C1/CT-7 expression in plasma cell myeloma: sub-cellular localization impacts on clinical outcome.

Plasma cell myelomas (PMs) have a poor prognosis. Cancer-testis (CT) antigens are immunogenic proteins, representing potential targets for tumor vaccination strategies. The expression of the CT antigens GAGE, MAGE-A4, MAGE-C1/CT-7, and NY-ESO-1 was investigated on paraffin-embedded bone marrow biopsies from 219 PM and 8 monoclonal gammopathy of undetermined significance (MGUS) patients. The frequency and prognostic impact of these CT antigens were compared with known morphological prognostic markers (i.e. Mib1 labeling index) and the presence of the translocations t(4;14)(p16.3; q32) and t(11;14)(q13;q32). We show that MAGE-C1/CT-7 is the most prevalent CT antigen, expressed in 57% of PMs in a high percentage of tumor cells. While MAGE-C1/CT-7 was absent in non-malignant plasma cells, plasma cells of patients with MGUS did express MAGE-C1/CT-7, but no other CT antigens. MAGE-C1/CT-7 was more frequently expressed in PMs with an elevated proliferation rate (Mib1 >10%) compared to PMs with a low proliferation rate (Mib1

Sequential high dose chemotherapy as initial treatment for aggressive sub-types of non-Hodgkin lymphoma: results of the international randomized phase III trial (MISTRAL).

INTRODUCTION: Sequential high dose (SHiDo) chemotherapy with stem cell support has been shown to prolong the event-free survival in patients with diffuse large B-cell lymphoma. METHODS: To confirm this result in a multicenter trial, we randomized patients with aggressive NHL, to receive either eight cycles of CHOP or SHiDo. The primary endpoint was overall survival. RESULTS: 129 evaluable patients were randomized to receive either CHOP or SHiDo: median age, 48 years; 62% male; stage III+IV: 73%; age adjusted International Prognostic Index 1/2/3: 21%/52%/27%. Toxicity grades 3+4 were more pronounced in the SHiDo-arm with 13% versus 3% of patients with fever; 34% versus 13% with infections; 13% versus 2% with esophagitis/dysphagia/gastric ulcer. The remission rates were similar in SHiDo and CHOP arms with 34%/37% complete remissions and 31%/31% partial remissions, respectively. After a median observation time of 48 months, there was no difference in overall survival at 3 years, with 46% for SHiDo and 53% for CHOP (P = 0.48). CONCLUSION: In this multicenter trial, early intensification with SHiDo did not confer any survival benefit in previously untreated patients with aggressive NHL and was associated with a higher incidence of grades 3/4 toxicity.

Wotherspoon criteria combined with B cell clonality analysis by advanced polymerase chain reaction technology discriminates covert gastric marginal zone lymphoma from chronic gastritis.

BACKGROUND AND AIMS: Gastric mucosa associated lymphoid tissue lymphoma is a well defined B cell lymphoma yet often impossible to distinguish from severe chronic gastritis on morphological grounds alone. Therefore, it was suggested to use the clonality of the immunoglobulin (Ig) heavy chain (H) genes, as detected by polymerase chain reaction (PCR), as a decisive criterion. However, there is controversy as to whether B cell clonality also exists in chronic gastritis, hence rendering this approach futile at present. METHODS: An expert panel re-examined the histology and immunohistochemistry of a total of 97 cases of gastric biopsies, including clearcut marginal zone lymphoma, chronic gastritis, and ambiguous cases, applying the Wotherspoon criteria on the basis of haematoxylin-eosin and CD20 immunostainings. In addition, a new and advanced PCR system for detection of clonal IgH gene rearrangements was independently applied in two institutions in each case. RESULTS: The overall IgH clonality assessments of both institutions were in total agreement. Overt lymphoma (Wotherspoon score 5) was clonal in 24/26 cases. Chronic gastritis (Wotherspoon scores 1 and 2) was not clonal in 52/53 cases; the clonal case being Wotherspoon score 2. Of 18 cases with ambiguous histology (Wotherspoon scores 3 and 4) four were clonal. CONCLUSIONS: Using advanced PCR technology, clonal gastritis is extremely rare, if it exists at all. Thus B cell clonality in Wotherspoon 3 and 4 cases is regarded as suitable for definitively diagnosing gastric marginal zone lymphoma.

Weekly x 4 induction therapy with the anti-CD20 antibody rituximab: effect on circulating t(14;18)(+) follicular lymphoma cells.

Rituximab 375 mg/m(2) weekly x 4 has been reported to induce a 60% response rate in patients with relapsed follicular lymphomas (FL). Our aim was to examine the effect of this rituximab schedule on circulating FL cells in an ongoing multicenter study. One hundred fifty-four patients with FL were examined by nested polymerase chain reaction (PCR) at baseline for the presence of t(14;18) translocation-carrying lymphoma cells in bone marrow and/or blood. Sixty-four patients (42%) had PCR-detectable t(14;18)(+) FL cells. Pretreatment characteristics of these 64 patients were as follows: one had stage I, nine had stage II, 14 had stage III, and 40 had stage IV disease. Thirty-five patients had bulky disease (> or = 5 cm) and 25 patients had an elevated serum lactate dehydrogenase (LDH) level. Bone marrow was morphologically assessed in 64 patients, and 39 of these patients had an infiltration with FL cells. Blood samples from 51 patients were available for PCR analysis between weeks 8-12 after induction therapy, and 28 of these patients (55%) were PCR negative. Paired blood and bone marrow samples were available for PCR analysis from 39 patients between weeks 8-12 after induction therapy with rituximab. Thirteen of these patients (33%) did not have PCR-detectable cells in blood and bone marrow, while 26 patients (67%) still had circulating t(14;18)(+) cells in either bone marrow (eight patients), blood (one patient), or both (17 patients). PCR negativity in blood and bone marrow in 13 patients was statistically significantly associated with partial or complete response after induction therapy with rituximab (P = 0.006). However, clearance of PCR-detectable t(14;18)(+) cells in bone marrow and/or blood could not be associated with any low tumor burden pretreatment characteristics such as stages I/II, absence of morphological bone marrow infiltration or tumor bulk of > or = 5 cm, and normal serum LDH.

Significantly different bcl-2 expression profiles in gastric and non-gastric primary extranodal high-grade B-cell lymphomas.

Fifty-five cases of primary extranodal high-grade B-cell non-Hodgkin's lymphoma were investigated for bcl-2 and p53 protein expression as well as for t(14;18) translocations and p53 mutations. Phenotypic and genotypic profiles were compared between tumours of gastric (27 cases) and non-gastric (28 cases) origin. bcl-2 protein expression was significantly lower in gastric (11/27) than in non-gastric (28/28) lymphomas (p<0.0001), while nuclear p53 protein expression did not differ significantly between these two groups. In the stomach, there were no significant differences in either bcl-2 or p53 expression profiles between high-grade lymphomas with (n=14) and without (n=13) evidence of a low-grade component of MALT type. However, secondary high-grade lymphomas showed a significant down-regulation of bcl-2 protein (p<0.0001) and, conversely, an up-regulation of p53 protein (p<0.0001) as compared with their low-grade tumour components. In extranodal high-grade B-cell lymphomas, bcl-2 protein expression was not associated with t(14;18) translocation. Only one gastric lymphoma had a p53 point mutation with potential alteration of the amino acid sequence. These findings indicate that primary gastric high-grade B-cell lymphomas are immunohistologically distinct from primary extranodal high-grade B-cell lymphomas of an origin other than in the stomach.

Mild renal dysfunction is sufficient to induce erythropoietin deficiency in patients with unexplained anaemia.

Current guidelines suggest that anaemia due to erythropoietin deficiency almost exclusively occurs with creatinine concentrations of at least 177 micromol/l or above. The aim of this prospective case control pilot study was to evaluate whether borderline renal function or mild renal dysfunction with creatinine concentrations well below 177 micromol/l is sufficient to induce inadequate erythropoietin secretion. Patients referred for work-up of otherwise unexplained anaemia with mildly abnormal creatinine concentrations (104-129 micromol/l; study group: eight patients) and patients referred for work-up or therapy of other diseases who also presented with anaemia but normal creatinine levels (<100 micromol/l; control group: nine patients matched for gender, age and degree of anaemia) were included. All but two patients in the control group had bone marrow biopsies to exclude other pathologies. Mild renal dysfunction (as evidenced by creatinine concentrations between 100 and 140 micromol/l, median concentration 112 micromol/l) was found to be sufficient to induce inadequate erythropoietin secretion. The physiologic hemoglobin-dependent erythropoietin regulation demonstrated in the control group was abolished in the study group. Patients with mild renal dysfunction and unexplained anaemia should be investigated for erythropoietin concentration. If the erythropoietin concentration is found to be inadequate for the degree of anaemia, substitution therapy should be considered.

T-cell-rich B-cell non-Hodgkin's lymphoma mimicking Hodgkin's disease.

We report on a patient with recurrent T-cell-rich B-cell lymphoma (TCRBCL), initially misdiagnosed as a lymphocyte-rich Hodgkin's disease. This case exemplifies the diagnostic problems of TCRBCL and the need for immunophenotypic analysis to differentiate TCRBCL from Hodgkin's disease, nodular paragranuloma and peripheral T-cell lymphoma. A rather unusual aspect is the long disease-free interval between the excision of the node in and the late relapse in 1996. The significance of the abundant T-cell infiltration in this B-cell neoplasm will be discussed and the concepts concerning antitumor response will be reviewed. Based on epidemiological data and the clinical behaviour TCRBCL does not seem to represent a distinctive pathological entity.

Monocytoid/marginal zone B-cell differentiation in follicle centre cell lymphoma.

We report on two cases of low grade follicle centre cell lymphoma with a pronounced parafollicular monocytoid/ marginal zone B-cell component. One patient had a history of preceeding follicular high grade B-cell lymphoma of centroblastic type showing the same light chain restriction and identical immunoglobulin heavy chain gene rearrangement as the low grade lymphoma diagnosed 15 months later. Morphologically, in both cases the two constituents of the low grade tumours were clearly distinguishable. Immunohistochemically, the follicular component strongly expressed bcl-2 protein in contrast to a weak staining of the marginal zone B-cell component. Performing PCR, a rearrangement of the major breakpoint region of bcl-2 was not found. Identical light chain restriction of the follicular and the monocytoid B-cell/marginal zone components strongly indicates a clonal relationship between them. A monocytoid/marginal zone B-cell component in follicular lymphoma probably results from differentiation of the follicle centre cells and does not indicate a composite lymphoma.

Cutaneous follicular lymphoid hyperplasia with monotypic plasma cells. A clinicopathologic study of 18 patients.

Twenty cases of cutaneous follicular lymphoid hyperplasia with monotypic plasma cells are presented in a clinicopathologic study on 18 patients. The plaque-like or nodular lesions were solitary in 10 and multiple in eight patients. Immunohistochemistry showed well-defined B- and T-cell areas. Sheets of monotypic plasma cells occurred either interfollicularly or adjacent to the sclerotic stroma, with expression of IgG/kappa in 14 and IgG/lambda in six cases. In one patient with multiple lesions, one sample contained polyclonal plasma cells, whereas the other specimen showed light chain restriction. In another patient, disease recurred with a polytypic cutaneous plasma cell infiltrate. Polymerase chain reaction (PCR) revealed clonal immunoglobulin heavy chain gene rearrangements in eight of 13 cases, which was confirmed by Southern blot analysis in three samples. Clonal T-cell receptor chain gene rearrangements were not detected. Disease progression to overt malignant lymphoma did not occur within the follow-up period of up to 12 years, but recurrent disease was seen in three patients. Our data indicate that cutaneous lymphoid hyperplasia with monotypic plasma cells is a biologically distinct clinicopathological entity.

[Primary non-Hodgkin lymphoma of the stomach. A review with special reference to the MALT concept]. / Das primäre Non-Hodgkin-Lymphom des Magens. Eine Ubersicht mit spezieller Berücksichtigung des MALT-Konzepts.

Primary extranodal malignant lymphomas are most often localized in the stomach. In contrast to gastric carcinomas, primary gastric non-Hodgkin's lymphomas show an increasing incidence. According to their grade of malignancy they are divided into low-grade and high-grade non-Hodgkin's lymphomas and according to their immunophenotype into B-cell and T-cell non-Hodgkin's lymphomas. In most cases they show a B-cell phenotype while high-grade tumors are more frequent than those of low-grade malignancy. However, primary gastric Hodgkin's disease is still a rarity. A new entity, the so-called low-grade B-cell non-Hodgkin's lymphoma of mucosa-associated lymphoid tissue (MALT) type, is characterized by a diffuse infiltrate of centrocyte-like cells intermingled with immunoblasts of the same clone, plasma cell differentiation of the tumor cells, lymphoepithelial lesions, and reactive intratumoral lymphoid follicles. It may secondarily transform into a high-grade B-cell lymphoma but remains limited to the stomach for a considerable period of time with a favourable prognosis. The most important prognostic factors of primary gastric lymphomas are stage at initial diagnosis, classification and grading according to the histopathological concept of the MALT, and depth of infiltration. Although a considerable number of early stage gastric lymphomas achieve complete remission after surgical therapy only, primary treatment of gastric lymphoma is still controverted, thus underlining the urgency of a multicenter prospective study. Chronic Helicobacter pylori infection may play a major role in the pathogenesis of low-grade B-cell lymphoma of MALT type. Complete remission of some cases of low-grade B-cell lymphoma of MALT type with concomitant Helicobacter pylori gastritis after Helicobacter pylori eradication may lead to a new pathogenetic, therapeutic, and prognostic concept.

Primary B-cell gastric lymphoma: a clinicopathological study of 145 patients.

Resection specimens from 145 patients with primary B-cell gastric lymphoma at stage IE (n = 88) and at stage IIE (n = 57) were investigated. Histologically, low-grade malignant B-cell lymphomas arising from the mucosa-associated lymphoid tissue, including immunocytoma (n = 71), could be distinguished from high-grade malignant B-cell lymphomas with (n = 25) and without (n = 49) evidence of a low-grade component. The very rare low-grade B-cell lymphomas of centroblastic-centrocytic, centrocytic, and plasmacytic type were not considered. All patients had undergone primary gastric resection, and 65 received additional chemotherapy (n = 33), radiotherapy (n = 22), or both (n = 10). Actuarial overall survival rates calculated by the Kaplan-Meier life-table method were 76% after 5 years and 58% after 10 years. According to the Mantel test and a multivariate analysis using the Cox regression method, patients at stage IE had a significantly better survival probability than those at stage IIE (P less than 0.0001); 5-year survival rates were 87% and 61%, respectively. The survival probability for low-grade malignant lymphomas was significantly better than for tumors with secondary high-grade transformation (P less than 0.05) or for primary high-grade lymphomas (P less than 0.0001), whereas the two high-grade groups were not significantly different. Five-year survival rates were 91% for low-grade, 73% for secondary high-grade, and 56% for primary high-grade malignant lymphomas. Retrospectively, no significantly different survival rates were found between patients who had undergone gastric resection alone and patients who had received additional treatment. However, survival analyses showed that classification and grading according to the histopathological concept of mucosa-associated lymphoid tissue-derived gastric lymphomas into low-grade B-cell lymphomas of mucosa-associated lymphoid tissue type and high-grade B-cell lymphomas with or without evidence of a low-grade component has great prognostic relevance.

Monocytoid B-cell lymphoma: morphological variants and relationship to low-grade B-cell lymphoma of the mucosa-associated lymphoid tissue.

Twenty-eight cases of monocytoid B-cell lymphoma of lymph nodes and 16 lymph node metastases of primary gastric lymphomas, mostly low-grade B-cell lymphomas of mucosa-associated lymphoid tissue (MALT) type were investigated morphologically and immunohistochemically. Both groups showed the same morphological and immunohistochemical features: diagnostically important sites of infiltration were the sinuses and the marginal zones. The tumour cells were either medium-sized or small. The cytoplasm stained grey with Giemsa and was sometimes rather pale. In imprints the grey colour of the cytoplasm was a characteristic feature. The medium-sized cell type was more frequent; in one third of the cases it was combined with a prominent lymphoplasmacytic component from the same clone, and it resembled the monocytoid B-cells of the sinuses. The small cell type was less common, was not combined with a lymphoplasmacytic component and more closely resembled marginal zone cells. The difference was underlined by the negative reaction with the monoclonal antibody Ki-B3 in the small cell type, which, conversely, was positive in the medium-sized cell type and in the monocytoid B-cell reaction of the sinuses. Both of these cell types, however, showed a granular reaction with the new monoclonal antibody Ki-Mlp. The morphological and immunohistochemical parallels are arguments in favour of the assumption that monocytoid B-cell lymphoma is the nodal equivalent of low-grade B-cell lymphoma of MALT type. This is further supported by the fact that in nine of our 28 cases of monocytoid B-cell lymphoma, lymphomas were found simultaneously or subsequently in organs of the MALT. Monocytoid B-cell lymphoma must be differentiated from an infiltration that occurs in the form of clusters of monocytoid B-cells in other low-grade B-cell lymphomas, especially in immunocytoma with a high content of epithelioid cells.

Monocytoid B cell lymphoma: clinical and prognostic features of 21 patients.

Twenty one patients between 34 and 83 years of age with monocytoid B cell lymphoma (MBCL) of the lymph node were studied. The histological picture characteristically showed broad strands of medium sized cells with irregularly shaped nuclei and a fairly broad rim of slightly basophilic cytoplasm. One case developed into a centroblastic polymorphic lymphoma. Bone marrow infiltration was documented in three cases and leukaemic conversion in one case of MBCL. Seven patients had enlarged spleens. Eight patients were in stage I, five in stage II/IIE, two in stage IIIs and six in stage IV at the time of diagnosis. Of 18 patients subsequently followed up, eight were in complete remission, two in partial remission, and three were undergoing treatment at completion of the study; five patients had died. Relapse occurred in nine patients and was a common feature of MBCL. The prognosis of MBCL was comparable with that of other low grade malignant lymphomas. Fourteen patients presented with primary nodal lymphoma. In seven patients with nodal MBCL, however, a concomitant low grade B cell lymphoma of the mucosa-associated lymphoid tissue (MALT) was also found in the stomach (n = 4), nasopharynx (n = 1), salivary glands (n = 1) and thyroid gland (n = 1). Two of these cases developed into high grade lymphoma. These extra-nodal manifestations were found simultaneously with MBCL in five patients. In another two patients, however, these symptoms occurred in a later phase of the disease. It is emphasised that adequate staging procedures must be carried out in any case of nodal MBCL to exclude underlying low grade B cell lymphoma of the MALT.

Diplomyelia: caudal duplication of the neural tube in mice.

Diplomyelia (duplication of the neural tube or spinal cord) was studied histologically in nine cases of T/+ mouse embryos at 10-15 days of gestation. Grüneberg investigated T/+ fetuses and interpreted the extra neural tube to be notochord, but a reexamination of this material demonstrated that the interpretation is incorrect. Diplomyelia is produced in the mutations Fused, Kinky, vestigial tail, homozygous Brachyury, and t-haplotype t9 and in the new mutation, NM 529, described here.