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.

Deregulated over expression of FOXP1 protein in diffuse large B-cell lymphoma does not occur as a result of gene rearrangement.

Strong uniform expression of FOXP1 protein occurs in a subgroup of non-germinal centre (GC) diffuse large B-cell lymphomas (DLBCL). We have investigated gene rearrangement as a potential mechanism for deregulated expression of FOXP1 however, using FISH FOXP1 translocations were not found in any case with over-expression of the protein.

Immunoglobulin heavy chain sequence analysis in Waldenstrom's macroglobulinemia and immunoglobulin M monoclonal gammopathy of undetermined significance.

In this study, we used IGH sequence analysis to assess the maturational status of Waldenstrom's (WM) macroglobulinemia and its putative precursor immunoglobulin (Ig)-M monoclonal gammopathy of undetermined significance (MGUS). IGH sequence analysis was performed using standard methods in 23 cases (20 WM and 3 IgM MGUS as defined by consensus panel criteria). Waldenstrom's macroglobulinemia cases were characterized by heavily mutated IGH genes (median, 6.3%; range, 3.8%-13.9%) but without intraclonal variation (ICV). IgM MGUS was similarly characterized by somatic hypermutation (median, 7.5%; range, 7%-7.7%), but ICV was evident in 1 of the 3 cases. We would therefore conclude that WM is characterized by somatic hypermutation without ICV, which supports a derivation from postgerminal center/memory B cells. IgM MGUS is also characterized by somatic hypermutation but, in a manner similar to IgA/IgG MGUS, can be associated with ICV, although the significance of this remains unclear.

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.

Complex biallelic IGH rearrangements in IgM-expressing Z-138 cell line: Involvement of downstream immunoglobulin class switch recombination.

Chromosomal translocations involving the immunoglobulin (Ig) receptor loci usually disrupt and silence these loci. On the basis of observations in follicular lymphoma (FL) with downstream Ig heavy chain (IGH) class switch recombination (CSR), we hypothesized that downstream CSR-mediated chromosomal translocations would leave the V(D)J-Cmu transcription unit intact, thereby still allowing IgM expression from the IGH allele involved in the translocation. To test this hypothesis, we analyzed biallelic IGH translocations in the IgM-expressing cell line Z-138 by interphase FISH, DNA fiber-FISH, long-distance vectorette PCR, and DNA sequencing. One IGH allele was involved in a t(11;14), showing a break in the JH region that juxtaposed the Emu enhancer and the 3' Calpha enhancers to the cyclin D1 gene. The other IGH allele contained a t(8;14) breakpoint involving the 3' end of a Sgamma region, whereas the reciprocal breakpoint at 8q24 was approximately 40 kb centromeric of MYC. Molecular analysis showed that this IGH allele harbored a normal V(D)J-Cmu complex, which is responsible for IgM expression. These data show that chromosomal breakpoints such as the t(8;14) can occur in downstream IGH constant regions and do not necessarily interfere with Ig expression.

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.

Positioning of cervical carcinoma and Burkitt lymphoma translocation breakpoints with respect to the human papillomavirus integration cluster in FRA8C at 8q24.13.

Molecular cytogenetic analysis frequently shows human papillomavirus (HPV) integration near translocation breakpoints in cervical cancer cells. We have recently described a cluster of HPV18 integrations in the distal end of the common fragile site FRA8C at 8q24 in primary cervical carcinoma samples. Chromosome band 8q24 contains the MYC gene (alias c-MYC), FRA8C, and FRA8D. The MYC gene is frequently deregulated--usually by translocation or amplification--in various tumor types. In the present study, we performed a molecular cytogenetic analysis of HPV18 integration patterns and the 8q24 translocation in a primary cervical carcinoma and in HeLa cells using combined binary ratio-fluorescence in situ hybridization. Our aim was to determine how the chromosomal breaks involved in these events relate physically to the MYC gene; whether they map to the FRA8C site, the FRA8D site, or both; and how they correlate with the occurrence of DNA flexibility domains. The 8q24 translocation breakpoints mapped between stretches of integrated HPV18 sequences in the distal end of FRA8C. This region contained DNA helix flexibility clusters, several of which mapped in the vicinity of HPV integration sites and translocation breakpoints in cervical carcinomas. DNA helix flexibility clusters were also found near known MYC translocation breakpoints in Burkitt lymphomas (BL), but most BL breakpoints mapped clearly outside FRA8C. Our data revealed that FRA8C is involved in HPV integration and chromosomal translocations in cervical carcinoma; however, this fragile site is not involved in classical MYC translocations in most BLs. In the context of the familial nature of cervical cancer, FRA8C may be considered a candidate susceptibility region for cervical carcinoma.

Strong expression of FOXP1 identifies a distinct subset of diffuse large B-cell lymphoma (DLBCL) patients with poor outcome.

FOXP1 (Forkhead box-P1) is a winged-helix transcription factor that is differentially expressed in resting and activated B cells. FOXP1 expression has been demonstrated in a subset of diffuse large B-cell lymphomas (DLBCLs) and is more common in the nongerminal center (non-GC), activated B-cell type; however, its prognostic significance is uncertain. We analyzed presentation lymph nodes from 126 patients with nodal DLBCL, previously classified according to GC and BCL2 status, for FOXP1 protein expression using standard immunocytochemistry. Uniform high FOXP1 expression was demonstrated in 23 of 126 patients with DLBCL. This high level of expression was almost exclusively confined to patients who lacked the GC phenotype, who expressed MUM-1 and BCL2 in the absence of t(14; 18), and who were identified as a subgroup of patients with particularly poor outcomes in a group with already poor prognoses. The data presented suggest that high FOXP1 expression is an independent prognostic factor in DLBCL.

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 (Smu/Sgamma) 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'Smu 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.

Insights into the multistep transformation of MGUS to myeloma using microarray expression analysis.

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-kappaB 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.

Polymorphic variation in GSTP1 modulates outcome following therapy for multiple myeloma.

Glutathione S-transferase P1 (GSTP1) is a phase 2 drug metabolism enzyme involved in the metabolism and detoxification of a range of chemotherapeutic agents. A single nucleotide polymorphism (Ile105Val) results in a variant enzyme with lower thermal stability and altered catalytic activity. We hypothesized that patients with the less stable variant have a decreased ability to detoxify chemotherapeutic substrates, including melphalan, and have an altered outcome following treatment for multiple myeloma. We have assessed the impact of GSTP1 codon 105 polymorphisms in 222 patients entered into the Medical Research Council (MRC) myeloma VII trial (comparing standard-dose chemotherapy with high-dose therapy). In the standard-dose arm, patients with the variant allele (105Val) had an improved progression-free survival (PFS) (adjusted hazard ratios for PFS were 0.55 for heterozygotes and 0.52 for 105Val homozygotes, compared with 105Ile homozygotes; P for trend =.04); this was supported by a trend to improved overall survival, greater likelihood of entering plateau and shorter time to reach plateau in patients with the 105Val allele. No difference in outcome by genotype was found for patients treated with high-dose therapy. However, the progression-free survival advantage of the high-dose arm was seen only in patients homozygous for 105Ile (P =.008).

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 mu 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.

Monoclonal B lymphocytes with the characteristics of "indolent" chronic lymphocytic leukemia are present in 3.5% of adults with normal blood counts.

Molecular and cellular markers associated with malignant disease are frequently identified in healthy individuals. The relationship between these markers and clinical disease is not clear, except where a neoplastic cell population can be identified as in myeloma/monoclonal gammopathies of undetermined significance (MGUS). We have used the distinctive phenotype of chronic lymphocytic leukemia (CLL) cells to determine whether low levels of these cells can be identified in individuals with normal complete blood counts. CLL cells were identified by 4-color flow cytometric analysis of CD19/CD5/CD79b/CD20 expression in 910 outpatients over 40 years old. These outpatients were age- and sex-matched to the general population with normal hematologic parameters and no evident history of malignant disease. CLL phenotype cells were detectable in 3.5% of individuals at low level (median, 0.013; range, 0.002- 1.458 x 10(9) cells/L), and represented a minority of B lymphocytes (median, 11%; range, 3%-95%). Monoclonality was demonstrated by immunoglobulin light-chain restriction in all cases with CLL phenotype cells present and confirmed in a subset of cases by consensus-primer IgH-polymerase chain reaction. As in clinical disease, CLL phenotype cells were detected with a higher frequency in men (male-to-female ratio, 1.9:1) and elderly individuals (2.1% of 40- to 59-year-olds versus 5.0% of 60- to 89-year-olds, P =.01). The neoplastic cells were identical to good-prognosis CLL, being CD5+23+20(wk)79b(wk)11a(-)22(wk)sIg(wk)CD38-, and where assessed had a high degree (4.8%-6.6%) of IgH somatic hypermutation. The monoclonal CLL phenotype cells present in otherwise healthy individuals may represent a very early stage of indolent CLL and should be useful in elucidating the mechanisms of leukemogenesis.

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

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.

Follicular lymphoma with a novel t(14;18) breakpoint involving the immunoglobulin heavy chain switch mu region indicates an origin from germinal center B cells.

With the use of DNA-fiber fluorescent in situ hybridization, a BCL2 protein positive follicular lymphoma with a novel BCL2 breakpoint involving the immunoglobulin heavy chain (IGH) switch mu (S(mu)) region instead of the J(H) or D(H) gene segments was identified. Sequence analysis showed that the genomic breakpoint is localized between the S(mu) region of the IGH complex and the first intron of BCL2. Reverse-transcriptase polymerase chain reaction showed expression of a unique hybrid IGH-BCL2 transcript involving the transcription initiation site I(mu). Sequence analysis of the V(H) region of the functional nontranslocated IGH allele showed multiple shared somatic mutations but also a high intraclonal variation (53 differences in 15 clones), compatible with the lymphoma cells staying in or re-entering the germinal center. This is the first example of a t(14;18) translocation that results from an illegitimate IGH class-switch recombination during the germinal center B-cell stage.