ALBERT

Description: EGR1 is an early growth response zinc finger transcription factor with broad actions, including in differentiation, mitogenesis, tumor suppression, and neuronal plasticity. Here we demonstrate that Egr1−/− mice on the C57BL/6 background have normal eyelid development, but back-crossing to BALB/c background for four or five generations resulted in defective eyelid development by day E15.5, at which time EGR1 was expressed in eyelids of WT mice. Defective eyelid formation correlated with profound ocular anomalies evident by postnatal days 1–4, including severe cryptophthalmos, microphthalmia or anophthalmia, retinal dysplasia, keratitis, corneal neovascularization, cataracts, and calcification. The BALB/c albino phenotype-associated Tyrc tyrosinase mutation appeared to contribute to the phenotype, because crossing the independent Tyrc-2J allele to Egr1−/− C57BL/6 mice also produced ocular abnormalities, albeit less severe than those in Egr1−/− BALB/c mice. Thus EGR1, in a genetic background-dependent manner, plays a critical role in mammalian eyelid development and closure, with subsequent impact on ocular integrity.

Description: Multiple myeloma (MM) plasma cells (PCs) express receptor for hyaluronan-mediated motility (RHAMM), a hyaluronan-binding, cytoskeleton and centrosome protein. The most abundant RHAMM isoforms in MM are full-length RHAMM (RHAMMFL) and the splice variant RHAMM-exon4. We separately examined the significance of RHAMM expression, and isoform balance, in 2 groups of MM patients. In oligonucleotide microarray experiments (n = 210, Arkansas), increasing RHAMM mRNA expression in MM PCs is strongly associated with osteolytic bone lesions (P 〈 .001), and event-free (P = .05) and overall (P = .04) survival. Semiquantitative determination of RHAMM isoform expression (Alberta, Canada) used capillary electrophoretic detection and measurement of RHAMM-exon4/RHAMMFL reverse-transcriptase-polymerase chain reaction (RT-PCR) products. RHAMM isoforms are rarely expressed concurrently in single MM PCs; the pattern of isoform expression, at the single-cell level, is approximated in larger numbers of cells by the RHAMM-exon4/RHAMMFL ratio. Absolute RHAMM expression and the RHAMM-exon4/RHAMMFL ratio are only partially correlated in MM PCs; in cell lines, absolute RHAMM expression is elevated in mitosis, while RHAMM ratios remain stable. Temporal examination of MM patients' peripheral blood reveals that the RHAMM-exon4/RHAMMFL ratio increases with disease burden. The RHAMM-exon4/RHAMMFL ratio in diagnostic bone marrow samples (n = 101, Alberta) is an independent prognostic factor. Thus, expression and splicing of RHAMM are important molecular determinants of disease severity in MM. (Blood. 2004;104:1151-1158)

Description: Analysis of immunoglobulin V genes, which undergo stepwise changes during B cell differentiation such as VDJ rearrangement, somatic hypermutation, and class switch recombination, provides insight into the point of transformation of B cell tumors. In Multiple Myeloma (MM), clonotypic VDJ sequences of malignant plasma cells are mutated, homogeneous, and associated with post-switch constant regions (either IgG or IgA, called the clinical isotype), suggesting the malignant arm of the MM clone arises from transformation events in the late stages of the germinal centre reaction. By contrast, the existence of clonotypic VDJ associated with pre-switch IgM is well established, and we have shown persistent clonotypic IgM is associated with advanced disease at diagnosis and poor survival in MM. Whether clonotypic IgM cells represent a malignant progenitor or a non-malignant population that parallels disease severity is unclear. To address these possibilities, we focused our analysis of clonotypic VDJ mutation profiles on IgM+ cells sorted by immunomagnetic separation from MM patient peripheral blood cells (PBMC). IgM clonotypic transcripts were amplified by hemi-nested RT-PCR targeting the CDR2-C mu constant region in IgM+ cells from 4/7 patients. These products were cloned, and 122, 28, 27, and 25 IgM clonotypic colonies were identified by specific CDR2/CDR3 PCR for patients 1–4 respectively. Each of these clones was sequenced, and mutations were identified by comparison with the closest germline V gene and tumor derived plasma cell VDJ sequences. An average mutation frequency of 0.005, significantly greater than the Taq error rate, was obtained for the 250–280 bp fragment downstream of CDR2, including the D-J-C mu region. Typically, MM clones were observed with 1–2 mutations in this region, many localizing to the D-J-C mu region. Small deletions that preserve reading frame were also observed in the D region of single clones of patients 1 and 4 respectively. The detection of intraclonal heterogeneity amongst clonotypic IgM cells may reflect a normal arm of the myeloma clone that co-exists with the post-switch malignant arm. In previous work examining bulk PBMC populations we had detected diversified clonotypic cells in the non-clinical isotype compartment of one patient, but, in accordance with studies performed by several other groups, were unable to detect diversified pre-switch counterparts. In this work we have focused on IgM+ MM B cells, a compartment of the MM clone that may remain driven by antigenic selection and undergo persistent clonal expansion. Our analysis gives insight into the nature of this proposed normal arm of the myeloma clone, revealing two coexisting subsets of pre-switch clonotypic IgM cells: a major set exhibiting homogeneity, identity with post-switch tumor VDJ, and questionable transformation status, and a minor clonally heterogeneous set which may represent the pre-malignant clone from which myeloma arose.

Description: Clonotypic B cells of Waldenstrom’s macroglobulinemia (WM) are characterized as CD20+IgM+IgD+ cells that are usually somatically mutated in IgH VDJ but for some patients, the clonotypic IgH VDJ is germline (unmutated).For both mutated and unmutated clones, WM lack ongoing somatic hypermutation (SHM) and class switch recombination (CSR). This may be due to abnormalities in switching and/or mutator genes. To understand the nature of unswitched tumor B cells, uracil DNA glycosylase (UDG) and activation-induced cytidine deaminase (AID), the two essential elements for CSR, were analysed in WM. Analysis of 12 WM clones characterized by somatic hypermutation showed that the mutation profile of VH genes had normal transition/transversion ratios at C or G, and thus did not suggest UDG abnormalities. Expression of AID was determined by single stage RT-PCR. Out of 14 patients studied (2 unmutated and 12 mutated VH clones), two of them (WM1-01 and WM1-08,with mutation rates of 0% and 6.2% respectively) gave positive bands. In WM1-01, despite having a germline IgH VDJ, AID is consistently expressed in two bone marrow samples collected three years apart and from which the identical unmutated clonotypic VDJ sequence was isolated. Full-length (FL) AID transcripts of WM have a conserved sequence, thus ruling out the possibility of functional defects due to point mutation. In addition, detection of AID in an unmutated VH clone suggested that lack of SHM does not result from an inability to produce AID. In addition to FL transcripts, three other splice variants were identified in both patients. Single cell analysis indicated that only a small compartment (10% or less), not all, of clonotypic B cells expressed AID, and multiple isoforms may be detectable in individual cells. Whether these splice variants that contain truncated C-terminal ends play a role in the regulation of CSR in WM remains to be investigated. Splice variants, nevertheless, may not characterize tumor B cells since up to 10% of AID-expressing normal activated B cells (n=3) also carried them. In vitro activation of clonotypic WM B cells by CD40L and IL4, using conditions that induced CSR in normal B cells, did not yield detectable class switching in WM B cells. In cultures of B cells from WM, the number of non-clonal B cells increased but the clonotypic B cells did not appear to expand, as indicated by the reduction of clonotypic IgM transcript at 5-days of culture. Thus, as well as failing to undergo somatic mutation or class switching, WM tumor B cells appear unresponsive to CD40L+IL4. They may be fundamentally unresponsive to signals for class switching and their clonal expansion may depend upon alternate signaling pathways.

Description: Translocations involving the IgH locus are one of the most common genetic abnormalities observed in multiple myeloma (MM). Unlike several hematological malignancies, MM IgH translocations involve multiple partner chromosomes. Although IgH translocations are not unique to MM, the molecular anatomy of the translocations appears to be different from that observed in most B-cell malignancies. In general, the breakpoints occur within the switch regions of the IgH locus and the translocations appear to result from illegitimate class switch recombination (CSR) events. Previous analysis of the breakpoint junctions from t(4;14) samples suggested that the majority of these translocations result from illegitimate CSR events. These events were characterized by der(4) breakpoints containing Smu-chromosome 4 junctions and der(14) breakpoints with chromosome 4-downstream switch region junctions. However, not all t(4;14) breakpoints fit this “classical” model, as some derivative chromosomes were observed with hybrid switch regions. Unfortunately, the mechanism that generates these hybrid switch regions has been unclear. In general, only a single derivative was cloned from each patient or cell line. In the one case reported elsewhere in which both derivatives were cloned, the mechanism did not appear to be linked to the CSR process and thus represented a “non-classical” translocation. The poor prognostic impact of t(4;14) myeloma has been well established by several groups, including our own. In an attempt to identify recurrent breakpoint sites and to identify the potential mechanism(s) leading to t(4;14) translocations, we cloned the breakpoint junctions of both derivative chromosomes from 4 cell lines and 5 patients with MB4-2 and MB4-3 breakpoints. Furthermore, we cloned der(4) breakpoints from 4 additional patients, three of which are FGFR3 non-expressers for which we could not detect a der(14) breakpoint using our PCR based strategy. We defined the t(4;14) breakpoint region as encompassing 64.5 kb of chromosome 4, flanked by LETM1 exon 3 and MMSET exon 5, based on combining the previously published breakpoints with our newly cloned and sequenced breakpoints. Current dogma suggests that t(4;14) translocation events are randomly distributed throughout the defined breakpoint region, but this idea is not supported by our sequencing data. We identified two hotspots, which contain breakpoints from 9 of the 27 patients or cell lines with at least one cloned derivative. Interestingly, these regions only represent 1 kb of the entire breakpoint region. Therefore 33% of the cloned breakpoints exist within only 1.5% of the total breakpoint region. Moreover, for the 13 MM samples for which both derivatives are cloned, although 6/13 (46%) fit the classical model of CSR mediated switch translocations, surprisingly, 7/13 (54%) appear to be non-classical translocations. The non-classical translocations are defined by little to no loss of sequence from the involved switch region and the presence of a hybrid switch region on one of the two derivative chromosomes. Importantly, the non-classical translocations may not involve B-cell specific mechanisms and could potentially occur before or after a successful CSR event. Therefore, the classical illegitimate CSR event model can explain only half of the t (4; 14) breakpoints cloned to date.

Description: Analysis of clonotypic isotype class switching (CSR) in Waldenström macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (MGUS) reveals a normal initial phase of B-cell activation as determined by constitutive and inducible expression of activation-induced cytidine deaminase (AID). Switch μ (Sμ) analysis shows that large deletions are not common in WM or IgM MGUS. In CD40L/IL-4-stimulated WM cultures from 2 patients, we observed clonotypic IgG exhibiting intraclonal homogeneity associated with multiple hybrid Sμ/Sγ junctions. This suggests CSR had occurred within WM cells. Nevertheless, the estimated IgG/IgM-cell frequency was relatively low (1/1600 cells). Thus, for the majority of WM B cells, CSR does not occur even when stimulated in vitro, suggesting that the WM cell is constitutively unable to or being prevented from carrying out CSR. In contrast to WM, the majority of IgM MGUS clones exhibit intraclonal heterogeneity of IgH VDJ. Furthermore, most IgM MGUS accumulate more mutations in the upstream Sμ region than do WM, making them unlikely WM progenitors. These observations suggest that switch sequence analysis may identify the subset of patients with IgM MGUS who are at risk of progression to WM.

Description: Studies in B cell malignancies such as chronic lymphocytic leukemia have revealed associations between immunoglobulin V gene characteristics and clinical outcomes. Few studies of this kind have been conducted in multiple myeloma (MM), a B lineage cancer characterized by malignant plasma cells that display somatically hypermutated VDJ, clonal homogeneity, and post switch (IgG or IgA) clinical isotypes. In this study we analyzed the clonotypic VDJ sequences for a large cohort of 115 MM patients to define associations with clinical parameters such as staging at diagnosis (e.g. clinical isotype, percent PC, M protein, beta-2-microglobulin) and overall survival. A key feature of this analysis is the rigorous validation of the experimentally derived clonotypic VDJ sequence by confirming its presence in the majority of single MM PC, to exclude any VDJ sequences arising from expanded but unrelated clones of normal PC that are also present in MM bone marrow (BM) samples. This process involves the amplification of patient specific clonotypic VDJ using sets of VH family and constant region primers, direct sequencing of VDJ fragments, and CDR2 / CDR3 patient specific primer design. The proposed VDJ is sequence is confirmed by one of several single cell assays, preferably RT-PCR on CD138+ sorted PC, but also including in-situ RT-PCR and limiting dilution analysis. The set of confirmed clonotypic VDJ sequences were analyzed for several parameters including: VH, DH, and JH gene usage, VH and CDR3 length, percent mutation in the CDR1-CDR3 region, and CDR3 amino acid sequence. We found that V gene usage amongst MM patients was not significantly different from expected values based on the frequency of functional germline alleles: VH3 (52.2%), VH4 (19.1%), VH1 (17.4%), VH2 (7.8%), VH5(3.5%). Frequently used VH alleles included VH3-30 (10.4%), VH1-69 (9.6%), VH3-23 (7.8%). Amongst VH5 alleles, VH5-51 was observed exclusively. VH4-34, which has been associated with autoimmune diseases and is not present in normal plasma cells was observed in a single case. DH and JH usage from most to least frequent was as follows: JH4, JH6, JH3, JH5, JH1, and JH2; DH3, DH2, DH1, DH6, DH5, and DH4. Sequence analysis of the CDR1-CDR3 region revealed 1.8–18.8% mutation with a mean of 9.4%. Using mutation frequency as a guideline, we identified three groups, high (12–18.8%), medium (6–11.9%) and low (1.8–5.9%) frequencies of mutation, and compared their clinical characteristics. In a similar fashion, CDR3 length, selected VH, DH, and JH alleles were also evaluated in the context of clinical parameters. In a preliminary analysis, there were no detectable correlations between any of these V gene characteristics and overall survival. Future studies with this database will include CDR3 sequence analysis to determine if subsets of recurrent amino acid patterns, suggestive of specific antigen involvement are detectable. Unlike other B lineage malignancies, such as B-CLL or Waldenstrom’s macroglobulinemia, the V gene usage of MM appears to be a largely random representation of the normal VH gene repertoire, with no selection for particular VH gene families or individual VH genes, and a broad range of mutation frequencies. This type of analysis has meaning only if the IgH VDJ sequences analyzed are derived from the MM clone itself, making validation of experimentally derived clonotypic sequences a critical priority. Overall, this is the largest analysis to date of rigorously validated IgH VDJ gene sequences for MM.

Description: Clonotypic B cells of Waldenstrom Macroglobulinemia (WM) are characterized as CD20+138− sIgM+sIgD+ cells that are not restricted to CD27 memory marker expression. Clonotypic VDJ sequences show VH3/JH4 gene bias, exhibit intraclonal homogeneity and do not undergo class switch recombination (CSR). To better understand the failure of CSR in WM, switch (S) region analysis, activation induced cytidine deaminase (AID) expression and CD40L/IL-4 activated clonal CSR were studied in comparison to IgM monogammopathy of undetermined significance (MGUS). Large internal deletions of Sμ that could hinder CSR were determined by analysis of CDR2/Sμ fragments amplified from genomic DNA of clonotypic WM or MGUS B cells. Deletions were found in only 1/12 WM and 0/4 IgM MGUS, suggesting that large deletions of Sμ are not common in WM or IgM MGUS. Sequence analysis of Sμ upstream regions revealed striking differences between WM and IgM MGUS. By comparing the 1.6 kb sequences upstream of Sμ tandem repeats, we found that WM clones exhibit less mutation (0–2 bp/1.6 kb or 0.57x10− 3 bp, n=11) than IgM MGUS (0–7 bp/1.6 kb or 2.5x10− 3 bp, n=4, p=0.001). The MGUS clones containing frequently mutated switch region also exhibited intraclonal diversity of IgM VDJ. These differences exclude most IgM MGUS from a precursor relationship with WM and may help to identify those less frequent IgM MGUS that are at risk of progression to WM. Only 2/17 WM and 0/5 IgM MGUS constitutively expressed AID but it can be induced by CD40L/IL-4 activation as shown in 4/4 WM and 3/3 IgM MGUS. Single cell analysis showed that both clonotypic and non-clonotypic B cells accounted for AID induction. Splice variants are colocalized with full-length transcripts in some individual cells from WM, IgM MGUS and normal donors. Thus, AID splice variants are generally present in activated B cells. Using enhanced PCR amplification protocols, clonotypic CDR2/Cγ transcripts were detectable post-stimulation in 2/4 WM and 2/3 IgM MGUS, suggesting that WM and IgM MGUS B cells are capable of CSR. For 2 WM activated in vitro, we detected clonal IgG transcripts, having variable-sized CDR2/Sγ 1 fragments and diversity of clonotypic Sμ/Sγ junctions. This indicates that clonotypic CSR occurred multiple times within the WM tumor population. Although this implies that persistent CSR occurs and although clonotypic IgM B cells were frequent (16%) in cultures, clonotypic IgG producing WM B cells were very rare, with a frequency of only 0.01%. Clonotypic IgG exhibited intraclonal homogeneity of the clonotypic VDJ, with a sequence identical to that of the clonotypic IgM VDJ, suggesting that clonotypic CSR is a post-transformation event. Clonotypic IgG may arise through CSR occurring in vitro, through clonal expansion of pre-existing post-switch WM B cells or both. Overall, our studies show that initial B cell activation as determined by AID induction is normal and that intact CSR machinery is functional in WM, although clonotypic CSR occurs at very low frequency. For the majority of WM B cells, CSR does not occur even when stimulated in vitro, suggesting that the WM cell is constitutively unable to undergo CSR or being prevented from CSR. Further, since mutations in upstream Sμ are found in most IgM MGUS, making them ineligible as WM progenitors, switch sequence analysis may help to identify those IgM MGUS at risk of progression.