James A. L. Fenton

Monoclonal B-Cell Lymphocytosis and Chronic Lymphocytic Leukemia

BACKGROUND A diagnosis of chronic lymphocytic leukemia (CLL) requires a count of over 5000 circulating CLL-phenotype cells per cubic millimeter. Asymptomatic persons with fewer CLL-phenotype cells have monoclonal B-cell lymphocytosis (MBL). The goal of this study was to investigate the relation between MBL and CLL. METHODS We investigated 1520 subjects who were 62 to 80 years of age with a normal blood count and 2228 subjects with lymphocytosis (>4000 lymphocytes per cubic millimeter) for the presence of MBL, using flow cytometry. Monoclonal B cells were further characterized by means of cytogenetic and molecular analyses. A representative cohort of 185 subjects with CLL-phenotype MBL and lymphocytosis were monitored for a median of 6.7 years (range, 0.2 to 11.8). RESULTS Monoclonal CLL-phenotype B cells were detected in 5.1% of subjects (78 of 1520) with a normal blood count and 13.9% (309 of 2228) with lymphocytosis. CLL-phenotype MBL had a frequency of 13q14 deletion and trisomy 12 similar to that of CLL and showed a skewed repertoire of the immunoglobulin heavy variable group (IGHV) genes. Among 185 subjects presenting with lymphocytosis, progressive lymphocytosis occurred in 51 (28%), progressive CLL developed in 28 (15%), and chemotherapy was required in 13 (7%). The absolute B-cell count was the only independent prognostic factor associated with progressive lymphocytosis. During follow-up over a median of 6.7 years, 34% of subjects (62 of 185) died, but only 4 of these deaths were due to CLL. Age above 68 years and hemoglobin level below 12.5 g per deciliter were the only independent prognostic factors for death. CONCLUSIONS The CLL-phenotype cells found in the general population and in subjects with lymphocytosis have features in common with CLL cells. CLL requiring treatment develops in subjects with CLL-phenotype MBL and with lymphocytosis at the rate of 1.1% per year.

Common variation at 3q26.2, 6p21.33, 17p11.2 and 22q13.1 influences multiple myeloma risk

To identify variants for multiple myeloma risk, we conducted a genome-wide association study with validation in additional series totaling 4,692 individuals with multiple myeloma (cases) and 10,990 controls. We identified four risk loci at 3q26.2 (rs10936599, P = 8.70 × 10−14), 6p21.33 (rs2285803, PSORS1C2, P = 9.67 × 10−11), 17p11.2 (rs4273077, TNFRSF13B, P = 7.67 × 10−9) and 22q13.1 (rs877529, CBX7, P = 7.63 × 10−16). These data provide further evidence for genetic susceptibility to this B-cell hematological malignancy, as well as insight into the biological basis of predisposition.

t(3;14)(p14;q32) results in aberrant expression of FOXP1 in a case of diffuse large B-cell lymphoma

Strong expression of Forkhead box‐P1 (FOXP1), a winged‐helix transcription factor, has been identified as an independent prognostic factor in diffuse large B‐cell lymphoma (DLBCL). However, possible mechanisms of deregulation of this gene, on 3p14.1, have yet to be elucidated. We have identified a breakpoint at the IGA1 gene in the immunoglobulin heavy chain (IGH) locus at 14q32 that was juxtaposed to the FOXP1 gene locus in a gastric DLBCL that showed strong expression of FOXP1. This may be one possible mechanism of deregulating FOXP1 expression by placing it under the control of IGH enhancers.

A global expression-based analysis of the consequences of the t(4;14) translocation in myeloma

Purpose: Our purpose in this report was to define genes and pathways dysregulated as a consequence of the t(4;14) in myeloma, and to gain insight into the downstream functional effects that may explain the different prognosis of this subgroup. Experimental Design: Fibroblast growth factor receptor 3 (FGFR3) overexpression, the presence of immunoglobulin heavy chain-multiple myeloma SET domain (IgH-MMSET) fusion products and the identification of t(4;14) breakpoints were determined in a series of myeloma cases. Differentially expressed genes were identified between cases with (n = 5) and without (n = 24) a t(4;14) by using global gene expression analysis. Results: Cases with a t(4;14) have a distinct expression pattern compared with other cases of myeloma. A total of 127 genes were identified as being differentially expressed including MMSET and cyclin D2, which have been previously reported as being associated with this translocation. Other important functional classes of genes include cell signaling, apoptosis and related genes, oncogenes, chromatin structure, and DNA repair genes. Interestingly, 25% of myeloma cases lacking evidence of this translocation had up-regulation of the MMSET transcript to the same level as cases with a translocation. Conclusions: t(4;14) cases form a distinct subgroup of myeloma cases with a unique gene signature that may account for their poor prognosis. A number of non-t(4;14) cases also express MMSET consistent with this gene playing a role in myeloma pathogenesis.

A molecular study of the t(4;14) in multiple myeloma

Summary. The t(4;14) translocation is found in approximately 10% of myeloma patients and results in the deregulation of at least two genes, MMSET and fibroblast growth factor receptor 3 (FGFR3), with the formation of a fusion product between MMSET and the immunoglobulin heavy chain (IgH) locus and overexpression of FGFR3. We have analysed a series of 80 patient samples, comprising 67 multiple myeloma (MM) cases and 13 monoclonalgammopathy of undetermined significance (MGUS) cases, using RT–PCR to detect IgH–MMSET fusions. The t(4;14) translocation was detected in 7/67 (10%) myeloma cases and all seven expressed FGFR3 which was not seen in t(4;14)‐negative myeloma cases. In the MGUS cases, a similar proportion of t(4;14)‐positive cases was found (2/13; 15%), but none of these expressed FGFR3. All patients with detectable FGFR3 expressed both the FGFR3 IIIb and FGFR3 IIIc isoforms, the result of alternative splicing in the ligand binding domain, and exon‐deleted variants of FGFR3. We also identified a cryptic splice site in MMSET which results in a 277 amino acid deletion downstream of the breakpoint on der(4). FGFR3 mutation analysis revealed no mutations in the presenting myeloma or MGUS samples. However, we also had access to paired presentation and relapse samples which had been taken from a patient 13 months apart. Both samples had the t(4;14) translocation and overexpressed FGFR3, but only the relapse sample possessed the K650E mutation in the kinase domain of FGFR3. This suggests that targeted mutation in the translocated FGFR3 gene when under the control of the immunoglobulin promoters can occur and may provide one mechanism for disease progression.

In multiple myeloma, only a single stage of neoplastic plasma cell differentiation can be identified by VLA‐5 and CD45 expression

The nature of the proliferating fraction in myeloma is still not known and understanding the characteristics of this fraction is central to the development of effective novel therapies. However, myeloma plasma cells typically show a very low rate of proliferation and this complicates accurate analysis. Although the level of CD45 and/or VLA‐5 has been reported to identify proliferating ‘precursor’ plasma cells, there are discrepancies between these studies. We have therefore used a rigorous sequential gating strategy to simultaneously analyse cycle status and immunophenotype with respect to CD45, VLA‐5 and a range of other integrin molecules. In 11 presentation myeloma patients, the proliferative fraction was distributed evenly between CD45+ and CD45− cells, however, cycling plasma cells were consistently VLA‐5‐. There was close correlation between the expression of VLA‐5 and a range of other integrin molecules (CD11a, CD11c, CD103), as well as the immunoglobulin‐associated molecules CD79a/b (Spearman, n = 10, P < 0·0001). In short‐term culture, cells that were initially VLA‐5‐showed increasing VLA‐5 expression with time. However, simultaneous analysis of the DNA‐binding dye 7‐amino‐actinomycin D demonstrated that this was not as a result of differentiation, as VLA‐5+ plasma cells were all non‐viable. This was confirmed in freshly explanted plasma cells from nine patients. Discrete stages of plasma cell differentiation could not be distinguished by the level of CD45 or VLA‐5 expression. The results indicate that there is a single stage of plasma cell differentiation, with the phenotype CD38+CD138+VLA‐5−. These findings support the hypothesis that neoplastic bone marrow plasma cells represent an independent, self‐replenishing population.

Genomic characterization of the chromosomal breakpoints of t(4;14) of multiple myeloma suggests more than one possible aetiological mechanism

Using FISH-based techniques, rearrangements of the immunoglobulin heavy-chain (IgH) locus at 14q32 have been found in the majority of cases of multiple myeloma (MM). Some of these IgH translocations are recurrent and we have characterized the genomic breakpoints of seven t(4;14) translocations from MM patients, using a combination of vectorette and conventional polymerase chain reaction methods, the aim being to understand the molecular mechanism leading to MM. Conventionally, the chromosome 14q32 breakpoints in these reciprocal translocations are believed to be located in the IgH μ switch (S) region and a further downstream S region with deletion of intervening DNA occurring as a result of aberrant class switch recombination (CSR); this was seen in five of the cases analysed. However, in two patients it was possible to demonstrate that the rearranged hybrid switch region sequence was joined to DNA from chromosome 4p16, suggesting that IgH translocations can occur in B cells that have already undergone legitimate CSR. The complex nature of these rearrangements leads us to speculate that primary IgH translocations may occur at different time points in the development in MM plasma cells, either at the time of physiological CSR or at a later stage, possibly involving a different mechanism.

Liposomal daunorubicin : In vitro and in vivo efficacy in multiple myeloma

Liposomal encapsulation of anthracyclines is a potential method of drug targeting, altering both the antitumour activity and side‐effect profile of anthracyclines. Liposomal daunorubicin (daunoxome) shows both altered pharmacokinetics and a potential for reducing dose‐limiting cardiotoxicity compared to conventional daunorubicin. Anthracyclines have a common role in the treatment of multiple myeloma, a prevalent and fatal haematological malignancy. Avoiding cumulative anthracycline toxicity in these patients is important. There is also a need for more effective relapse schedules given that many patients have chemosensitive disease at relapse. We have analysed daunoxome in vitro in myeloma cell lines using a thymidine‐based cytotoxicity assay and show superior efficacy compared to a pegylated liposomal doxorubicin derivative. Subsequently we have treated seven relapsed myeloma patients with a regime consisting of oral CCNU 25–50mg/m2 on day 1, 4 days of oral dexamethasone 10mg/m2 and intravenous daunoxome (liposomal daunorubicin) given for 4 days (total 100mg/m2). The main toxicity was myelosuppression but non‐haematological toxicity was minimal and the regime was well tolerated. Four out of seven of these heavily pretreated patients responded. Together with the in vitro data on its cytotoxicity in myeloma and its favourable pharmacokinetic profile further studies of liposomal daunorubicin in myeloma would be warranted. Copyright

Translocation t(11;14) in multiple myeloma: Analysis of translocation breakpoints on der(11) and der(14) chromosomes suggests complex molecular mechanisms of recombination

We describe the characterization of the genomic DNA breakpoints of two multiple myeloma (MM) patients with t(11;14). IGH translocation events are present in many MM tumors, and it is proposed that they occur early in the pathogenesis, based on the assumption that the translocations are simple reciprocal events mediated by errors in class‐switch recombination (CSR). We provide evidence from two patients that the translocation event can be more complex, with DNA from chromosome band 11q13 joined to apparently already recombined hybrid (Sμ/Sγ) switch region sequences. In one case, there was also evidence that a further rearrangement had occurred at the t(11;14) recombination site, resulting in an inversion of 40 bp of the 5′Sμ flanking sequence. This suggests that primary IGH arrangements in MM may be more complex than previous myeloma models have suggested, but that they essentially occur through illegitimate CSR events.

FICTION-TSA analysis of the B-cell compartment in myeloma shows no significant expansion of myeloma precursor cells

Studies utilizing flow cytometry and PCR have shown that the B‐cell compartment in myeloma contains cells which are clonally related to the myelomatous plasma cells. Current data, however, remains inconclusive regarding the extent of this involvement. By combining fluorescent immunophenotyping, tyramine signal amplification and fluorescence in‐situ hybridization (FICTION‐TSA), we have used the presence of numerical chromosomal abnormalities within plasma cells as a clonal marker to examine the CD20+ B‐cell compartment for the presence of aneuploidy. A series of 54 cases of myeloma were screened for the presence of numerical abnormalities of chromosomes 3 and 11. FICTION‐TSA was performed on 13 cases with either trisomy 3 or 11 and on a control group of six cases known to be disomic for the two chromosomes. B‐cell numbers were reduced in the myeloma cases compared to the normal controls (median 1.8% v 3.0%, P = 0.05). In the cases with a chromosomal marker, three signals were seen in a median of 1.88% of CD20+ B cells compared to 2.58% within the control group. Comparison of the two groups using a Wilcoxon‐Mann‐Whitney U test showed no statistical significant difference. Using this data set, it was possible to exclude a 3.03% expansion of clonally related B cells (95% confidence level). We conclude that the B‐cell compartment in myeloma does not represent the major site of clonal expansion, and if clonally related cells are present then the numbers are few.