ALBERT

Description: A monoclonal B-cell Lymphocytosis (MBL) is detected in the peripheral blood of around 3% of otherwise healthy adults, the majority of these have a CLL immunophenotype. We have previously demonstrated that the cells in MBL are indistinguishable from good risk CLL, sharing the same immunophenotypic profile, genetic aberrations and IgVH gene usage. MBL exerts an increasing burden on haematology clinics with over 100 new patients diagnosed per annum in our regional haemato-oncology laboratory. This is largely as a result of the increased tendency to investigate patients with a mild lymphocyosis although

Description: Analysis of immunoglobulin heavy chain (IgH) rearrangements in B-CLL differentiates subgroups of patients with significantly different clinical outcomes. Cases can be categorised according to mutational status of the variable (V) segment with unmutated VH regions linked to a worse prognosis. A restricted pattern of use of specific VH, DH and JH gene segments has also been reported in B-CLL. It has been hypothesised that B-CLL originates as a clonal expansion of B-cells that have been selected and activated by contact with self or foreign antigens, leading to those small clones to proliferate, mutate their IGH genes, acquire genetic lesions and eventually become malignant. B-CLL cells normally express low levels of Ig on the surface, normally IgM, although a proportion of patients express IgG or IgA, following the class-switch recombination (CSR) process. We have analysed the pattern of SHM and gene segment usage in this particular subgroup for 44 patients with IgG B-CLL. Successful PCR amplification of recombined Smu-Sgamma switch region DNA was achieved in 40 patients, confirming the presence of IgG class-switching. Mutational analysis of IgH V genes revealed 80% of patients contained more than 2% somatic hypermutation (SHM), with 63% of samples having a greater than 5% SHM rate. For VH gene segment usage, a significant predominance of the VH4 family was seen in 22 cases (50%), followed by VH3 in 17 cases (39%), while VH1 family was found in only 3 of 44 samples, this differs from classical IgM B-CLL where VH3 family usage predominates. Overall, VH4-34 was the most frequently used gene segment (34%), followed by VH3-07 (14%) and VH4-39 (9%). DH6-13 was the most frequently used DH segment (21%), followed by DH6-19 (13%). JH gene segment usage did not differ from normal B-cells, with JH4 being the most frequently used, followed by JH6 and JH5. There was a significant association between VH4-39, DH6-13 and JH5 in three samples all containing unmutated sequence. Together this data reveals a distinct pattern of IGH VDJ rearrangements in IgG B-CLL compared to classical IgM B-CLL. Firstly, the rate of SHM in IgG B-CLL (80%) is significantly higher than the 50% observed in IgM B-CLL. Secondly, VH segment usage pattern differs between the two subgroups with a significant under-representation of VH1 as well as an over-representation of VH4 family members in the IgG subgroup. Finally, there is a striking association between VH4-39 and DH6-13/JH5 in the very few unmutated rearrangements. This could be indicative of a different clonal history of these particular B cells in B-CLL. Together with recent published data, this latter finding suggests that this is a further sub-category exclusive to IgG B-CLL, where selection of a specific antigen receptor may have lead to B-CLL development in such cases. We conclude that class-switched IgG B-CLL contains different molecular features in the IgH genes compared with classical IgM B-CLL, and other B-cell malignancies. The clinical implications of these differences, especially the relationship between the mutational status of the VH genes and outcome in IgG B-CLL, will be further investigated.

Description: We have previously shown that strong, uniform expression of the transcription factor FOXP1 is associated with a poor outcome in a sub-group of activated (non-germinal centre) diffuse large B-cell lymphoma (DLBCL). To further define this poor prognostic group we have compared gene expression profiles of 10 cases which had either FOXP1 uniform, positive expression (n=6) or were FOXP1 negative (n=4). For all cases, a non-Germinal Centre (GC) B cell phenotype, namely CD10 negative, BCL6 negative, MUM1 positive, BCL2 positive, was matched as closely as possible between the two groups, with FOXP1 expression being the solitary differential factor. Gene expression profiling using Affymetrix U133 plus 2.0 chips was performed with RNA extracted from the 10 patient samples and differentially expressed genes were identified between the 2 groups following normalisation of the results. FOXP1 positive lymphoma cells were associated with a distinct expression pattern with activation-induced deaminase (AID) being the gene determined as having the biggest difference in expression, namely an approximately 60-fold increase in expression in FOXP1 positive samples. AID is associated with both the somatic hypermutation and class switch recombination processes of immunoglobulin heavy chain (IgH) genes of normal B-cells, being primarily expressed at the GC stage of B-cell development. An RT-PCR undertaken for AID expression of the 8 cases, where further RNA was still available, confirmed that AID expression was present in FOXP1 positive cases (n=5) but either absent or very weakly expressed in FOXP1 negative cases (n=3). PCR amplification of the IgH variable (V) gene of the sample from RNA, where successful was followed by sequence analysis which showed expected levels of mutation of at least 5% variation from germline IgH V genes. Intraclonal variation of the IgH V region genes was also examined in both FOXP1 positive and negative samples, there was no correlation between the expression of AID and the occurrence of SHM or even evidence of ongoing mutation. Reports have variably suggested that AID expression is confined to GC-type DLBCL, or is heterogeneous between GC and non GC-type (activated) DLBCL. Here we have demonstrated expression of AID in association with over-expression of FOXP1 in cases that are of post-GC origin. This would suggest that AID is deregulated, being expressed beyond the normal GC stage of B-cell differentiation. We hypothesise that FOXP1 positive DLBCL arise from cells at a specific phase of B-cell development. Normal B-cells expressing FOXP1 are found predominately in the mantle zone with a small number of cells in the GC. Ectopic expression of AID has been linked with tumour formation in mice, implying AID has oncogenic potential. Continued (or post-GC) deregulated expression of AID in these DLBCL cells may be a significant pathogenic event associated with these tumours. Normally AID is tightly regulated in B-cells, one vital function of this regulation may be to prevent lymphomagenesis. It is therefore conceivable that overexpression of FOXP1 in this poor prognostic subgroup of DLBCL results in deregulated expression of AID and is therefore a major contributing factor to lymphomagenesis.

Description: To define specific pathways important in the multistep transformation process of normal plasma cells (PCs) to monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM), we have applied microarray analysis to PCs from 5 healthy donors (N), 7 patients with MGUS, and 24 patients with newly diagnosed MM. Unsupervised hierarchical clustering using 125 genes with a large variation across all samples defined 2 groups: N and MGUS/MM. Supervised analysis identified 263 genes differentially expressed between N and MGUS and 380 genes differentially expressed between N and MM, 197 of which were also differentially regulated between N and MGUS. Only 74 genes were differentially expressed between MGUS and MM samples, indicating that the differences between MGUS and MM are smaller than those between N and MM or N and MGUS. Differentially expressed genes included oncogenes/tumor-suppressor genes (LAF4, RB1, and disabled homolog 2), cell-signaling genes (RAS family members, B-cell signaling and NF-κB genes), DNA-binding and transcription-factor genes (XBP1, zinc finger proteins, forkhead box, and ring finger proteins), and developmental genes (WNT and SHH pathways). Understanding the molecular pathogenesis of MM by gene expression profiling has demonstrated sequential genetic changes from N to malignant PCs and highlighted important pathways involved in the transformation of MGUS to MM. (Blood. 2003;102:4504-4511)

Description: The 2007 IWCLL guidelines indicate that a diagnosis of Chronic Lymphocytic Leukemia (CLL) requires a B-cell count above 5,000/μL in the absence of other features; below this level the diagnosis is Monoclonal B-cell Lymphocytosis (MBL). There is little outcome data for MBL patients and it is not clear whether the detection of low levels of CLL cells, seen in 3% of the general population, is of clinical relevance. We have therefore investigated two hospital populations: the first with normal blood counts and no history of cancer; and the second MBL patients referred for investigation of a current or prior lymphocytosis. Blood samples from 1520 outpatients aged 60–80 with a normal blood count were screened: CLL cells were detected in 78/1520 (5.1%) with a median CLL cell count of 140/μL (range 15 – 1,248). Chromosomal abnormalities were frequently detected in purified CLL-phenotype cells (deletion 13q14 in 15/38, trisomy 12 in 4/22) although poor-risk abnormalities (deletion 11q or 17p) were not detected. The median IgVH mutation was 6.6% (range 0.5 – 13.7%) with 85% of cases showing 〉2% mutation from germline. The IgVH gene usage was heavily biased with a similar profile to mutated CLL. Detection of CLL cells in individuals with a normal count was not associated with increased mortality (estimated yearly rate 6.2% vs. 8.9% for matched controls, P=0.76) or risk of developing CLL as subsequent lymphocyte counts remained normal in all cases. A diagnosis of MBL was established in 309 of 2228 referrals for investigation of lymphocytosis between 1995 and 2000. A cohort of 185 MBL patients was monitored for a median 6.7 years (range 0.2 – 11.8): the presenting B-cell count was a median 3,100/μL (range 30 – 5,000), age 73 years (range 42 – 96); IgVH mutation rate was 7.1% (range 1.3 – 9.3%) with 96% of cases showing 〉2% mutation from germline. Progression to a lymphocyte count above 30,000/μL occurred in 15% of cases (28/185) and chemotherapy for progressive CLL was required in 7% (13/185). The absolute B-cell count was the only independent risk factor for an increasing disease levels. Neither IgVH mutation status nor CD38 expression predicted risk of disease progression or requirement for treatment. During follow-up 33% died: age above 70, hemoglobin concentration below 11 g/dL and T-lymphopenia (CD3+ 2,400μL) had significantly longer survival. Development of progressive disease did not predict overall survival: 7/13 patients requiring therapy remain alive at a median 1.9 years (range 0–8.6 years) after initiation of treatment. The total lymphocyte count had no impact on the risk of disease progression, time to treatment or overall survival. CLL-phenotype cells are genetically equivalent to CLL even when detected in the general population but are not associated with increased mortality or risk of progression to CLL when present below 1,500/μL. MBL patients with higher levels of CLL cells show a steady increase in disease levels over time with 1–2% per year requiring chemotherapy for progressive disease. As such, periodic monitoring is indicated but this should have a minimal impact on lifestyle as MBL patients are often elderly with multiple health issues. MBL is a newly described disorder which is related to CLL in a similar way that MGUS is related to myeloma.

Description: Immunoglobulin heavy chain (IgH) sequence analysis has proved a very effective tool for the investigation of B-cell lymphoproliferative disorders. It provides insight into the cell of origin and in the case of CLL provides very powerful prognostic data. Waldenstrom’s Macroglobulinemia (WM) is a B-cell lymphoproliferative disorder characterised by IgM gammopathy and bone marrow infiltration. Immunophenotypic studies in our laboratory have previously suggested that WM is derived from post germinal centre / marginal zone B-cells. Initial sequencing studies have supported this hypothesis as they have demonstrated that the majority of cases are characterised by mutated Ig genes without intraclonal variation (ICV). This suggests that the clonal cell in WM is derived from a B-cell that has encountered antigen and exited the germinal centre environment without undergoing isotype switch recombination. In order to further clarify this we have sequenced IgH rearrangements in 20 cases of WM. We have also assessed 2 cases of IgM MGUS (the putative precursor lesion in WM) in order to determine whether these cases are associated with intraclonal diversity in a manner analagous to IgG / IgA MGUS and multiple myeloma. IgM MGUS and WM samples were analysed by PCR using Biomed2 primers (FR1 and JHcons). Gel-purified PCR products were then directly sequenced using Big Dye terminators on the ABI 377 sequencer. The IgM MGUS sequences were then cloned into plasmids and different clones were compared for sequence variation. WM cases were characterised by high levels of somatic hypermutation, none of the cases had a mutation rate

Description: A monoclonal B-cell lymphocytosis (MBL) is detectable in approximately 3% of the general adult population. In most cases the abnormal cells have a phenotype and genotype that is indistinguishable from indolent CLL. Individuals presenting with an MBL count over 500 cells per microlitre progress to CLL requiring treatment at a rate of approximately 1% per year. The relationship between MBL and CLL therefore appears to be similar to that of MGUS and myeloma. Intraclonal variation (ICV) in the immunoglobulin heavy chain gene (IgVH) gene occurs in approximately half of MGUS patients but is not present in myeloma. The majority MGUS patients that progress to myeloma lack intraclonal variation at the MGUS stage suggesting that clonal selection is a critical pathway for disease progression in myeloma. The aim of this study was to compare the rate of intraclonal variation in MBL and CLL and determine whether low rates of ICV are associated with progressive disease. DNA was extracted from ammonium-chloride lysed blood samples from individuals with CLL-phenotype MBL (n=20) and CLL with progressive disease (n=10). IgVH and BCL6 PCR products were directly sequenced and also cloned and at least 10 clones sequenced. Intraclonal variation was defined as the number of unique sequences as a percentage of total clones sequenced. Unmutated CLL was defined as having