Alicja M. Gruszka-Westwood

Prognostic features of splenic lymphoma with villous lymphocytes: A report on 129 patients

Summary. Splenic lymphoma with villous lymphocytes (SLVL) is a low‐grade B‐cell lymphoma defined in the World Health Organization classification as the leukaemic form of splenic marginal zone lymphoma. Presenting features and response to therapy have been described, but information on prognostic factors is scanty. Clinical, laboratory and follow‐up data were collected on 129 patients with SLVL to determine features predicting disease behaviour and survival. Diagnosis was made on clinical, morphological and immunophenotypic features and, where available, bone marrow and spleen histology. Median age was 69 years (range 39–90 years) and male:female ratio, 0·9. The majority had splenomegaly, but lymphadenopathy and hepatomegaly were rare. Median Hb was 11·8 g/dl, white blood cell count was 16 × 109/l and platelet count was 145 × 109/l; 27% of patients had monoclonal protein in serum and/or urine. While 27% of patients remained untreated, 10% transformed to high‐grade lymphoma. Median follow‐up was 61 months and median survival was 13 years, with 72% of patients alive at 5 years. Cox regression analysis showed that increasing age, anaemia, thrombocytopenia and lymphocytosis > 16 × 109/l were independent adverse predictors of overall survival. However, only anaemia and lymphocytosis > 16 × 109/l remained highly significant independent prognostic factors when only deaths due to lymphoma were analysed. Splenectomized patients fared better than those receiving chemotherapy only (P = 0·001 for SLVL deaths). We conclude that SLVL is mainly a disease of the elderly with a relatively benign course but, when treatment is required, splenectomy is beneficial.

Delineation of the minimal region of loss at 13q14 in multiple myeloma

Previous studies have focused on the incidence and prognostic implications of 13q14 deletions in multiple myeloma (MM), but none has sought to delineate the minimal common deleted region (CDR). In an effort to do so, dual‐color interphase fluorescence in situ hybridization (FISH) was applied on 82 myeloma cases, initially by use of three probes for 13q14 (RB1, D13S319, and D13S25). Deletions were detected in 29/82 (35.4%) cases, and all except one were monoallelic. Subsequently, contiguous YACs, PACs, and a BAC spanning the 13q14–q21 region were employed for deletion mapping in addition to a 13q telomere probe. Large deletions extending to the 13q34 region were found in 55% of the deleted cases, whereas an additional 13.8% showed loss of both 13q34 and 13q14 regions with retention of 13q21. A CDR of approximately 350 kb was identified at 13q14 with the proximal border approximately 120 kb centromeric from D13S319, encompassing an area rich in expressed sequence tagged sites and containing DLEU1, DLEU2, and RFP2 genes. Direct sequencing of the RFP2 gene revealed no mutations in six patients and four MM cell lines harboring deletions of the CDR. However, a role for RFP2 in the pathogenesis of MM cannot yet be excluded, given that alternative mechanisms such as haploinsufficiency remain possible.

Splenic marginal zone lymphoma with villous lymphocytes shows on-going immunoglobulin gene mutations.

Splenic marginal zone lymphoma (also splenic lymphoma with villous lymphocytes) is a B-cell non-Hodgkins lymphoma with a characteristic morphology and phenotype. We studied the pattern of somatic hypermutation of the rearranged immunoglobulin heavy chain genes on 23 cases and have correlated these data with survival as well as immunophenotypic and genetic characteristics of the cases. Two-thirds of the cases show immunoglobulin gene mutations, half of which show evidence of antigen selection, whereas one-third of the cases show no significant mutations. On-going mutation, a feature characteristic of follicular lymphoma, was demonstrated in all six cases randomly selected for this analysis, including one case with a low number of mutations (<2%). No statistical significant correlation was found between immunoglobulin mutation status and clinical, immunophenotypic, or genetic characteristics. Our results demonstrate that on-going somatic hypermutation is a prominent feature of splenic marginal zone lymphoma with circulating villous lymphocytes. On-going somatic hypermutation has previously been demonstrated in extra-nodal and nodal marginal zone lymphoma. Our results indicate that marginal zone lymphomas at different anatomical localizations may derive from a similar B-cell subset.

The incidence of trisomy 3 in splenic lymphoma with villous lymphocytes: a study by FISH

Splenic lymphoma with villous lymphocytes (SLVL) is a low‐grade B‐cell lymphoproliferative disorder characterized by splenomegaly and circulating villous lymphocytes. The relationship between SLVL and splenic marginal zone lymphoma (SMZL), a disorder with identical splenic histology to SLVL, is not clear. Previous studies have failed to show a consistent karyotypic abnormality in SLVL whereas trisomy 3 has been reported in patients with SMZL. The presence of trisomy 3 in SMZL and its absence in SLVL has been viewed as evidence that these are different diseases. However, it is possible that the frequency of trisomy 3 in SLVL has been underestimated because previous studies have relied on conventional cytogenetics. We have therefore used interphase fluorescence in situ hybridization (FISH) to re‐assess the frequency of trisomy 3 in SLVL. We studied 70 patients, who were stratified into four groups according to the percentage of circulating villous lymphocytes. Trisomy 3 was found overall in 17% of patients. In particular, trisomy 3 was detected in 13% of cases with >50% of villous lymphocytes and which were considered typical of SLVL. In conclusion, we have demonstrated that some patients with SLVL have circulating cells with trisomy 3, which does not support the view that SLVL and SMZL are different diseases on the basis of the incidence of trisomy 3.

Deletion mapping on the long arm of chromosome 7 in splenic lymphoma with villous lymphocytes.

Splenic lymphoma with villous lymphocytes (SLVL) is a low‐grade lymphoproliferative disorder characterized by splenomegaly and circulating villous lymphocytes in the peripheral blood. It is considered to be the leukemic form of splenic marginal zone lymphoma (SMZL). The genetic basis of this lymphoma type remains unknown. Conventional cytogenetic studies have identified frequent structural abnormalities of chromosome 7, in the form of translocations, mainly unbalanced, and 7q deletions. In this current study, we undertook deletion mapping of the long arm of chromosome 7 in a series of cases with SLVL. Metaphase fluorescence in situ hybridization (FISH) was used in the first instance, followed by a study of loss of heterozygosity (LOH). The common area of deletion identified by FISH spanned from the YAC clone HSC7E1289 (mapping to 7q32.1) to in between YACs HSC7E195 and HSC7E648 (7q32–3). By application of 50 microsatellite markers mapping to the FISH‐CDR and to areas of deletion reported in other studies, four distinct hotspot loci were identified, with abnormalities present in 29–55% cases. In three of them, both LOH and biallelic deletions were found. The LOH in the majority of patients was noncontiguous. The presence of a high incidence of abnormalities in the established hotspot areas and in particular the finding of biallelic deletions is indicative of the existence of genes important for the pathogenesis of SLVL in these areas.

Constitutive Expression of the AP-1 Transcription Factors c-jun, junD, junB, and c-fos and the Marginal Zone B-Cell Transcription Factor Notch2 in Splenic Marginal Zone Lymphoma

Splenic marginal zone lymphoma (SMZL) is a lymphoma type of putative marginal zone B-cell origin. No specific genetic alterations have yet been demonstrated in SMZL. Clinically, SMZL is a low-grade B-cell non-Hodgkin lymphoma. However, the presence of p53 mutation, 7q22-7q32 deletion or the absence of somatic hypermutations of immunoglobulin genes has been correlated with a worse prognosis. In this study, we analyzed genome-wide gene expression of 24 cases of SMZL using the microarray technique. The AP-1 transcription factors c-jun, junD, junB, and c-fos as well as Notch2 were found to be specifically up-regulated. These data were confirmed by real-time PCR and immunohistochemical staining of tissue sections. The absence of concordant high expression of the MAP kinases, the signaling cascade leading to AP-1 up-regulation, suggests autoregulation of the AP-1 transcription factors and an important role in SMZL oncogenesis. High expression of Notch2, a transcription factor that induces marginal zone B-cell differentiation, is highly suggestive for a marginal zone B-cell origin of SMZL. In addition, SMZL with the 7q deletion showed high expression of TGF-beta1 and low expression of the DNA helicase XPB, a crucial part of the nucleotide excision repair complex, possibly explaining the more aggressive clinical course of those cases.

The incidences of trisomy 8, trisomy 9 and D20S108 deletion in polycythaemia vera: an analysis of blood granulocytes using interphase fluorescence in situ hybridization

We have used interphase fluorescence in situ hybridization (IFISH) to detect trisomy 8, trisomy 9 and 20q deletion in circulating granulocytes from patients with polycythaemia vera (PV). Out of 64 PV patients, 15 (23%) exhibited an abnormality. Two patients had trisomy 9, three had trisomy 8 and 10 patients had hemizygous deletion of D20S108 (a locus in the 20q common deleted region). Aberrant nuclei ranged from 10% to 80% in these 15 cases. There was no correlation between the presence of a marker and sex, age, interval between presentation and IFISH analysis, neutrophil or platelet count or therapy. Conventional marrow cytogenetic karyotype results were available in 23 cases and there was concurrence between these and blood IFISH in 16 cases (13 normal and three with 20q/D20S108 deletion by both methods). Three patients with D20S108 deletion by IFISH were normal by previous marrow cytogenetic testing and four cases with 20q deletion by previous marrow cytogenetics had normal blood granulocytes according to IFISH. Thus, we confirm that trisomies 8 and 9 and deletion of 20q are diagnostically useful markers of PV. IFISH analysis of blood granulocytes is a practical method for detecting these markers, but as an adjunct to, not as a substitute for, conventional marrow cytogenetics.

A narrow deletion of 7q is common to HCL, and SMZL, but not CLL

To further characterise the genetic background of the two closely related B‐lymphocytic malignancies hairy cell leukaemia (HCL), and splenic marginal zone lymphoma (SMZL) we have identified characteristic copy number imbalances by comparative genomic hybridisation (CGH). Based on these findings, areas of special interest were fine mapped, and relevant probes constructed for use in interphase‐fluorescence in situ hybridisation (FISH) investigations. Thus, using the CGH data from 52 HCL and 61 SMZL patients, we identified the characteristic profiles of copy number imbalances for both diseases. These were a gain of 5q13‐31 (19%) and loss of 7q22‐q35 (6%) for HCL, and gain of 3q25 (28%), loss of 7q31 (16%), and gain of 12q15 (16%) for SMZL. A partial loss of 7q unsual for low‐malignant B‐cell diseases was found to be common to the two diseases. This loss was therefore fine mapped with BAC/PAC clones. Fine mapping revealed that in SMZL the minimal lost region covers 11.4 Mb spanning from 7q31.33 to 7q33 located between sequence tagged site (STS)‐markers SHGC‐3275 and D7S725. This area was distinct from the commonly deleted 7q region of myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML). A FISH probe specific for the 7q region was constructed. Using this probe in an interphase‐FISH investigation we showed the minimal lost 7q‐region of HCL and SMZL to be one and the same. In one HCL case, this investigation furthermore showed the extent of the deleted region to be below the detection limit of CGH, whereas interphase‐FISH screening of 36 chronic lymphocytic leukaemia (CLL) cases showed no deletion of the 7q area. In conclusion, we have identified characteristic profiles of copy number imbalances in HCL and SMZL and fine mapped the minimal extent of a commonly lost 7q area of special interest. We hypothesise that this region may contain (a) gene(s) important for the pathology of HCL and SMZL.

Comparative expressed sequence hybridization studies of high-hyperdiploid childhood acute lymphoblastic leukemia

The functional consequences of a high‐hyperdiploid karyotype, found in up to one‐third of cases of acute lymphoblastic leukemia (ALL), are unknown. Using the technique of comparative expressed sequence hybridization (CESH), we sought to address the question of whether increased chromosome copies in hyperdiploid ALL lead to increased gene expression. Relative expression of hyperdiploid ALL blasts versus peripheral blood mononuclear cells was analyzed in 18 patients. Common regions of overexpression corresponding to the presence of tri‐/tetrasomies included: Xp22.1–22.2, 4q28, 6q14–15, 6q24, 10p13, 14q23–24, 17q21, 18q12, and 21q21, identified in 28–89% of cases. However, increased expression without underlying trisomy occurred at 3p21.3, 7q11.2, 8p21, and 8q24.1 in 39–90% of cases. High expression at 7q11.2, the most consistent change detected, was confirmed by quantitative PCR. Poor correlation between the presence of tri‐/tetrasomy and overexpression was observed for chromosomes 14 and 17. Two cases were reanalyzed versus (i) B cells, (ii) transformed B cells, and (iii) CD34+19+ cells (the putative counterpart of the leukemic cell). A reduction in the number of relatively overexpressed regions was observed with CD34+19+ cells. In particular, the peak at 7q11.2 disappeared, suggesting up‐regulation of genes from this region in the early ontology of normal B‐cell development. In conclusion, we have shown that tri‐/tetrasomies in hyperdiploid ALL lead to an increase in the expression of associated sequences. The choice of a biologically relevant reference is crucial for data interpretation.

Isolated Bone Marrow Involvement in Diffuse Large B Cell Lymphoma: A Report of Three Cases with Review of Morphological, Immunophenotypic and Cytogenetic Findings

Diffuse large B cell lymphoma (DLBL) comprises a heterogenous entity characterized by the presence of large cells, exhibiting a mature B cell phenotype. The high proliferation rate and aggressive disease remain a therapeutic challenge, but the apparent biological diversity permits a risk-stratification model for prognostic grouping through the International Prognostic Index (IPI). Empirical to this approach is the consideration of cytogenetic data, offering an insight into the pathogenetic events which may underlie neoplastic clonal evolution and disease progression. We describe three cases of DLBL presenting with isolated marrow disease, a rare primary finding in this lymphoma. All three cases showed involvement of blood and bone marrow without evidence of splenic or lymph node involvement on imaging studies. Histological and immunophenotypic findings were similar in all three cases, outlining the phenotypic maturity of this disease. Cytogenetic analysis revealed complex karyotypes in the two cases examined. M-FISH (multicolour fluorescent in situ hybridization) performed on bone marrow from case 1 showed several cryptic translocations not evident on G-banding, including a novel translocation between 2p and 9p, and an unbalanced translocation between 14q and 11q. Cytogenetic analysis in case 2 showed abnormalities involving 7q, 9p at the site of the INK4a gene, and the bcl-2 locus, findings confirmed by M-FISH. These cases serve to highlight the biological and cytogenetic heterogenity of DLBL and emphasize the need for complementary investigations in the characterization of this entity.