[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.

Similar clinical features in follicular lymphomas with and without breaks in the BCL2 locus.

Approximately 15% of follicular lymphomas (FLs) lack breaks in the BCL2 locus. The aim of this study was to better define molecular and clinical features of BCL2-breakpoint/t(14;18)-negative FLs. We studied the presence of BCL2, BCL6 and MYC breaks by fluorescence in situ hybridization and the expression of BCL2, MUM1, CD10, P53 and Ki67 in large clinical trial cohorts of 540 advanced-stage FL cases and 116 early-stage disease FL patients treated with chemotherapy regimens and radiation, respectively. A total of 86% and 53% of advanced- and early-stage FLs were BCL2-breakpoint-positive, respectively. BCL2 was expressed in almost all FLs with BCL2 break and also in 86% and 69% of BCL2-breakpoint-negative advanced- and early-stage FLs, respectively. CD10 expression was significantly reduced in BCL2-breakpoint-negative FLs of all stages and MUM1 and Ki67 expression were significantly increased in BCL2-break-negative early-stage FLs. Patient characteristics did not differ between FLs with and without BCL2 breaks and neither did survival times in advanced-stage FLs. These results suggest that the molecular profile differs to some extent between FLs with and without BCL2 breaks and support the notion that FLs with and without BCL2 breaks belong to the same lymphoma entity.

Different biological risk factors in young poor-prognosis and elderly patients with diffuse large B-cell lymphoma.

Prognostically relevant risk factors in patients with diffuse large B-cell lymphoma (DLBCL) have predominantly been evaluated in elderly populations. We tested whether previously described risk factors are also valid in younger, poor-prognosis DLBCL patients. Paraffin-embedded samples from 112 patients with de novo DLBCL, enrolled in the R-MegaCHOEP trial of the German High Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) were investigated using immunohistochemistry (MYC, FOXP1, LMO2, GCET1, CD5, CD10, BCL2, BCL6, IRF4/MUM1) and fluorescence in situ hybridization (MYC, BCL2, BCL6). MYC, BCL2 and BCL6 breaks occurred in 14, 21 and 31%, respectively. In the majority of cases, MYC was simultaneously rearranged with BCL2 and/or BCL6. The adverse impact of MYC rearrangements was confirmed, but the sole presence of BCL2 breaks emerged as a novel prognostic marker associated with inferior overall survival (OS) (P=0.002). Combined overexpression of MYC and BCL2 showed only limited association with inferior OS. All immunohistochemical cell of origin classifiers applied failed to predict survival time. DLBCL tumors with significant proportion of immunoblastic and/or immunoblastic-plasmacytoid cells had inferior OS, independently from from BCL2 break. Younger, poor-prognosis DLBCL patients, therefore, display different biological risk factors compared with an elderly population, with BCL2 translocations emerging as a powerful negative prognostic marker.

Acute myeloid leukaemia with t(8;21) associated with "occult" mastocytosis. Report of an unusual case and review of the literature.

Approximately 20% of patients with systemic mastocytosis (SM) have an associated haematological, clonal, non-mast cell lineage disease, and most exhibit an associated myelogenous neoplasm. This report describes a 48 year old man with acute myeloid leukaemia (AML) and a type t(8;21) cytogenetic abnormality. Associated bone marrow mastocytosis (a defined subtype of SM) was only detected after successful polychemotherapy in the state of bone marrow aplasia, and persisted after complete remission of AML. The diagnosis of mastocytosis was based on the demonstration of a multifocal dense mastocytic infiltrate. The atypical mast cells showed prominent spindling and an aberrant immunophenotype, with coexpression of tryptase, chymase, KIT, and CD25-which is expressed only on neoplastic (not normal) mast cells. In addition, the transforming somatic mutation D816V of the c-kit gene was detected. Re-examination of the pretherapeutic (initial) bone marrow revealed a slight diffuse increase in partially spindle shaped mast cells also exhibiting an abnormal immunophenotype, with CD25 expression, although compact mastocytic infiltrates were not detected. Because the D816V mutation was detected in the initial bone marrow specimen, strict application of three minor diagnostic criteria (spindling, CD25, D816V) enabled a diagnosis of SM-AML to be confirmed retrospectively in the initial bone marrow tissue.

Matrix-metalloproteinases in Hodgkin lymphoma.

BACKGROUND: Classical Hodgkin lymphomas are characterized by relatively few tumour cells and prominent proliferation of plasma cells, histiocytes, lymphocytes and eosinophils. In addition there is a varying degree of sclerosis, which is especially prominent in nodular sclerosis. These morphological peculiarities led to the idea that the interaction between tumour cells and bystander cells as well as the extracellular matrix may be important in Hodgkin lymphomas. MATERIALS AND METHODS: Thirty-four classical Hodgkin lymphomas (CHL) were analysed regarding the expression of EMMPRIN, MMP-2, -7, -9, -10 and-11 using immunohistochemistry. RESULTS: The tumour cells were positive for EMMPRIN in 100% of the cases. In 82% of CHL the Hodgkin and Reed-Sternberg cells (HRS) were negative for MMP-2. In contrast the surrounding non-neoplastic cells were MMP-2-positive in 71% of the cases. The HRS cells stained positive for MMP-7 in 68% of CHL, whereas only a few surrounding cells were positive for this marker. In all but one case (97%) the HRS cells were negative for MMP-9. However, the surrounding cells stained positive in 32%, thus resembling the staining pattern for MMP-2. Only scattered cells of both populations, HRS cells as well as bystander cells, stained for MMP-10 and -11, and no specific staining pattern was observed. CONCLUSION: Our data indicate a complex interaction between tumour cells and bystander cells with regard to metalloproteinases. The expression of EMMPRIN in the tumour cells may induce the expression of MMP-2 in the surrounding non-neoplastic cells. MMP-2 can be activated by MMP-7, which is expressed in the tumour cells. It is tempting to speculate that an interruption of this cycle could be of therapeutic benefit.

Significant high expression of CD23 in follicular lymphoma of the inguinal region.

AIMS: Inguinal lymph nodes are considered to be problematic for the diagnosis of lymphoma due to architectural changes resulting from previous inflammatory processes. The aim was to investigate the morphology and immunophenotype of follicular lymphomas (FL) in order to clarify whether FL presenting in inguinal nodes differs from FL biopsies from other sites. METHODS AND RESULTS: A total of 219 FLs were studied, comprising 78 biopsy specimens of inguinal lymph nodes and 141 from other sites. All samples were assessed for growth pattern, grade, sclerosis and immunophenotype (Bcl-2, CD10, CD23, Mib-1). Cases negative for Bcl-2 were analysed by polymerase chain reaction and fluorescence in situ hybridization. In comparison with the biopsies from other regions, we found a significantly increased number of CD23+ FLs in samples of inguinal lymph nodes (38% versus 21%). Expression of CD23 was more frequently detected in grade 1 FLs than in other grades (grade 1, 37%; grade 2, 18%; grade 3, 23%; transformed, 6%). Other immunohistochemical parameters, however, did not differ between the two groups. CONCLUSION: There is an unexpectedly high frequency of CD23 expression in FL in general, which is even more pronounced in inguinal nodes.

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.