In order to investigate the incidence of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and unclassified variants in chronic pulmonary disease in children and adults, we studied 20 patients with asthma, 19 with disseminated bronchiectasis (DB) of unknown aetiology, and 12 patients with chronic obstructive pulmonary disease (COPD), and compared the results to 52 subjects from the general Greek population. Analysis of the whole coding region of the CFTR gene and its flanking intronic regions revealed that the proportion of CFTR mutations was 45% in asthma (P<0.05), 26.3% in DB (P>0.05), 16.7% in COPD (P>0.05), compared to 15.4% in the general population. Seventeen different molecular defects involved in disease predisposition were identified in 16 patients. Three potentially disease-causing mutations, T388 M, M1R and V11I, are novel, found so far only in three asthma patients. The hyperactive M470 allele was found more frequently in COPD patients (frequency 70.8%, P<0.01) than in the controls. The study of the TGmTnM470 V polyvariant CFTR allele revealed the presence of CFTR function-modulating haplotypes TG13/T5/M470, TG11/T5/M470, TG12/T5/V470 and TG12/T7, combined with M470 or V470, in six asthma patients, four DB patients (P<0.01), and two COPD patients (P<0.05). These results confirm the involvement of the CFTR gene in asthma, DB and possibly in COPD.

Primary splenic small B-cell lymphomas mostly comprise the distinct entity of splenic marginal-zone lymphoma (SMZL) and the provisional category of splenic lymphoma/leukemia unclassifiable, mainly represented by the hairy cell leukemia variant and splenic diffuse red pulp small B-cell lymphoma (SDRL). Until recently, histopathologic examination of splenectomy specimens was considered mandatory for the diagnosis of SMZL. However, nowadays, mainly because of advances in chemoimmunotherapy, splenectomy is performed much less frequently. We evaluated the diagnostic efficacy of bone marrow biopsy (BMB) histopathology in the diagnostic approach toward SMZL and SDRL and tested whether it may serve as a substitute for spleen histopathology in the differential diagnosis between these 2 entities. To this end, we conducted a paired assessment of BMB and spleen diagnostic samples from 46 cases with a diagnosis of SMZL (n=32) or SDRL (n=14) based on spleen histopathology. We demonstrate that detailed immunohistopathologic BMB evaluation offers adequate evidence for the confirmation of these entities and their differential diagnosis from other small B-cell lymphoma histotypes. Notably, the immunophenotypical profile of SMZL and SDRL was identical in both BMB and spleen specimens for 21 evaluated markers. Paired assessment of BMB and spleen specimens did not identify discriminating patterns of BMB infiltration, cytology, and/or immunohistology between SMZL and SDRL. Accordingly, bone marrow histopathology contributes significantly in confirming the diagnosis of SMZL and SDRL. However, presently it is not possible to distinguish SMZL from SDRL on the basis of BMB evaluation alone; hence, histopathologic examination of the spleen remains the "gold standard" approach.

We analyzed 42 splenic marginal-zone lymphoma (SMZL) cases diagnosed on splenectomy specimens after established World Health Organization criteria. A predominantly nodular growth pattern was observed in 24 cases; the remainder showed predominantly (11/42) or exclusively (7/42) diffuse infiltration. Twenty-one cases showed the "classic" biphasic appearance; 13 cases exhibited marginal-zone morphology; finally, 8 cases were composed predominantly of small cells. CD21 and CD35 were expressed by 12/42 and 17/38 cases, respectively. DBA.44 was detected in 24/42 cases. Seventeen of 37 cases were surface IgD (SIgD)-positive. Twenty-one of 22 analyzed cases were SIgM-positive (12/21 coexpressed SIgD). Five of 37 cases were SIgG-positive. CD27 staining was observed in 21/35 cases; 7/18 CD27-positive cases coexpressed SIgD; 7/14 CD27-negative cases were SIgD-positive. Forty IGHV-D-J rearrangements were amplified in 34/42 cases: the IGHV4-34 gene predominated, followed by IGHV1-2. Using the 98% homology cut-off, 25/40 (62.5%) IGHV sequences were considered as "mutated": 10/11 cases with monomorphous, marginal-zone morphology were IGHV-mutated; in contrast, 4/6 cases with monomorphous, small-cell morphology were IGHV-unmutated. Five of 7 cases expressing IGHV1 subgroup genes had biphasic morphology, whereas 6/9 IGHV3-expressing cases had monomorphous, marginal-zone morphology. Most IGHV-mutated cases (14/20; 70%) were SIgD-negative; in contrast, 8/11 IGHV-unmutated cases expressed SIgD. CD27 was detected in 10/17 IGHV-mutated and 6/10 IGHV-unmutated cases. Seven of 11 CD27-negative cases were IGHV-mutated; 5/7 CD27-negative/IGHV-mutated cases expressed DBA.44. These results confirm the considerable histologic, immunohistochemical, and molecular heterogeneity of SMZL and indicate an origin from the diverse resident B-cell populations of the normal SMZ.

Summary. Follicular lymphoma cells carry surface immunoglobulin whose heavy chain variable (V H ) regions exhibit considerable divergence from the aminoacid sequence predicted by the germline nucleotide sequence as a result of the somatic hypermutation process. The present study examined the extent of somatic hypermutation in follicular lymphoma light chain variable region (V) genes about which the available data is limited. DNA extracted from fresh frozen lymph node tissue of 14 patients with follicular lymphoma at diagnosis was subjected to polymerase chain reaction (PCR) amplification aimed at detecting clonal V H and V L (L; light chain) gene rearrangements. Clonal V gene rearrangements were detected in 10/14 cases. Amplified V H and V genes of these 10 cases were directly sequenced by the dideoxy-chain termination method. In all cases, rearranged V H genes demonstrated numerous mutations clustering in the complementarity determining regions (CDRs), in keeping with previous reports. The degree of divergence of the rearranged V genes from the closest homologous germline V genes varied significantly. Furthermore, two patterns of mutations were observed: (i) in six cases (60%), mutations were most often of the replacement (R) type (changing the aminoacid sequence of the encoded polypeptide) in the CDRs and of the silent (S) type (leaving the aminoacid sequence of the encoded polypeptide unchanged) in the framework regions (FWRs) resulting in R:S ratios significantly greater than would have occurred by chance; (ii) in four cases (40%), very few or no mutations were observed and the distribution of mutations as well as the R:S mutation ratios did not differ significantly from what would have occurred by chance alone. These findings imply that, compared to their partner heavy chains, the light chains of follicular lymphoma neoplastic B-cells' surface immunoglobulin (sIg): (i) are less affected by somatic hypermutation; (ii) play a less significant role in the antigen selection process.Keywords: follicular lymphoma, immunoglobulin genes, somatic hypermutation.The differentiation and growth of normal B cells is critically dependent upon the normal function of their sIgs as testified by previous studies (Cooper, 1987;Kitamura et al, 1991). The three hypervariable regions of the surface immunoglobulin (sIg) heavy and light chains, part of the respective polypeptide's variable region, constitute its antigen-combining sites; for this reason they are also termed complementarity determining regions (CDRs; CDR1, CDR2 and CDR3) (van Noesel & van Lier, 1993). At the DNA level, the gene sequences encoding for the Ig heavy and light chain variable region are formed by an enzymatically driven assembly of distinct variable (V), diversity (D) (for the heavy chains only) and joining ( J) gene segments, a process known as V(D)J recombination. Antigen recognition is effected in the germinal centres of the secondary lymphoid organs. The development of B cells in the germinal centre is affected by two, often interacting, but neverthel...

Lymphomas originating from the lymphatic system comprise about 30 entities classified according to the World Health Organization (WHO). The histopathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in different lymphoma entities, their detection will be increasingly important. Hence, a split-signal fluorescence in situ hybridisation (FISH) procedure would be helpful in discriminating the most difficult classifications. The Euro-FISH programme, a concerted action of nine European laboratories, has validated a robust, standardised protocol to improve the diagnostic approach on lymphoma entities. Therefore, 16 fluorescent probes and 10 WHO entities, supplemented with reactive cases, were selected. The results of the Euro-FISH programme show that all probes were correctly cytogenetically located, that the standardised protocol is robust, resulting in reliable results in approximately 90% of cases, and that the procedure could be implemented in every laboratory, bringing the relatively easy interpretation of split-signal probes within the reach of many pathology laboratories.