Our patient’s clinical, morphological and immunophenotypic features are consistent with the diagnosis of CLL. Although complex cytogenetic findings including t(8;14) usually confers poor prognosis in CLL, a consistent genotype and phenotype correlation remains an unresolved issue. Our patient’s case exhibits an unreported rearrangement involving IGH@ and MYC loci with absence of MYC expression.
In our patient, the FISH signal patterns detected are unique from those previously reported in CLL cases with atypical rearrangements and an apparently balanced t(8;14). These include a cryptic deletion on 8q24.1 including MYC[6, 8], gain of an extra copy of MYC (+MYC) [4, 5, 29], or deletion of IGH@, usually 5’ [3, 4, 6, 30, 31]. Although a deletion of the MYC-specific signal on der 8q24.1 locus was also observed in our patient using IGH-MYC fusion probe (1Y2G1R), it is not identical to the reported deletion by Reddy et al. in 2006 [6, 8]. The deletion reported on here did not show splitting of signals and no concomitant deletion of a 1.6 Mb segment including the MYC locus. Instead, it showed two unsplit MYC probes (yellow) on the normal chromosome 8 and on der 14q32. We interpreted these findings as an atypical rearrangement never reported elsewhere, with the 5’-MYC-3’ removed from the 8q24.1 locus at a breakpoint at least 400 Kb upstream of its 5’ region. We also showed that this deleted region is relocated to the 14q32 locus and apposed to the 3’-IGH@ locus. Neither gain of MYC nor deletion of the 5’-IGH@ locus was observed by FISH or CMA in our case. We have exhaustively searched the available literature and did not find any cases similar to the signal patterns reported on here.
To the best of our knowledge, expression levels of MYC and its correlation to disease progression have not been established in CLL with t(8;14), with or without MYC translocations [4, 6, 7]. MYC expression is generally at low levels in CLL , and similar in groups with either bad or good prognosis . Increased expression even without MYC rearrangement has also been described in CLL with malignant Richter transformation and other higher risk cases for CLL progression . Although, high levels of MYC are expressed as a result of the t(8;14) and its variant translocations in Burkitt’s lymphoma and in some other B-cell malignancies including DLBCL and plasma cell myeloma, these translocations may not necessarily lead to increased expression of MYC in CLL [4, 6, 7]. These variable findings of MYC expression are most likely dependent on specific disruptions of regulatory regions, or characteristic genomic translocation breakpoints either at the MYC or IGH@ locus.
The typical MYC-IGH fusion at der 14q32 expresses the MYC -deregulatting product, while the reciprocal IGH-MYC fusion at 8q24.1 locus is transcriptionally silent [14, 35]. Despite the typical juxtaposition, overall MYC expression in some CLL cases remains within the normal range , or overexpressed through processes other than translocations . It has been reported that the location of the genomic breakpoint influences MYC expression, with highest level when involving Class I breakpoints [15, 24]. The absence of Myc expression in our patient is most likely due to the atypical MYC-IGH fusion on der 14q32, with a Class III breakpoint (at least 400 Kb upstream of MYC) .
The previously reported “gene desert” region upstream of MYC extends up to about 629 Kb  and includes genes and SNPs. Genome-wide association studies (GWAS) have shown the gene POU5F1B and several genetic variants or SNPs (Regions R1, R2, R3) (Figure 5) that are risk factors for various cancers including CLL exist in this region [26–28, 36–38]. The strongest evidence for risk or genetic susceptibility in CLL or monoclonal B-cell lymphocytosis is rs2456449 (8q24.21) [28, 38]. In our patient, the breakpoint that we suggested (at least 400 Kb) is within this interval and includes POU5F1B, and SNPs R1 and R2. POU5F1B is one of the two RefSeq genes within the breakpoint on 8q24.1 and 5’-MYC, is the most adjacent. Although it is not yet well studied, few reports described it as a pseudogene or a gene that encodes for a weak transcription factor that may play a critical role in stem cell pluripotency, eye development and carcinogenesis [36, 37, 39]. At the present time, there are no reports of a specific fusion involving 5’-POU5F1B and 3’-regulatory region of IGH@. It is possible that the breakpoint in our patient is further upstream, however, the paucity of available cells made it impossible for further characterization. In Figure 5, we extended the suggested breakpoint further upstream, from ~400 Kb to ~600 Kb, to include the farthest reported cancer-associated SNP (Region 2: rs16901979) and CLL SNP (rs2456449). To date, the genotype phenotype correlation underlying these associations remains unclear. However, it has been suggested by reported expression studies that MYC expression is influenced by such SNPs variants by altering its transcription regulation and amplification . Despite such plethora of reports, replication of these findings and elucidation of its physiologic function and clinical significance remain an area of thorough investigation. Further in vivo and in vitro functional studies are needed to show consistent association of risk allele status and MYC expression levels.
On the other hand, transcription at the IGH@ locus is controlled by enhancers elements spread out as wide as 2.5 Mb of the locus , and it contains regulatory elements necessary not only for MYC activation but also the promotion of translocation . CMA detected a 455 Kb copy number loss on chromosome band 14q32.2, not detected by FISH since the probe used was outside of this region. It is still a possibility, that the deletion in our patient may have removed some of the regulatory elements within this interval somehow affecting the regulation of Myc expression. No regulatory elements or high conservation data was seen in the UCSC Genome Browser. This microdeletion has been reported in other CMA studies of CLL patients using BAC-based array CGH, with some of the cases exhibiting the same findings as ours, i.e. with no IGH@ deletion by FISH . It is still unclear whether this microdeletion is a polymorphic feature of this locus and represents a region of frequent mutation and recombination, or it exhibits some susceptibility risks for CLL [3, 24, 31, 41].
About 20% of patients with CLL show ATM deletion, an anomaly also seen in almost all cancer, and is usually associated with an adverse outcome [1, 4, 31]. The collaboration of ATM and MYC in normal cell proliferation via an ATM-dependent pathway is well established. When deleted, ATM loses its protective checkpoint function leading to MYC-induced oncogenesis [4, 42]. This indicates that MYC alone is not capable of transforming lymphoid cells into neoplasia . The ATM deletion and removal and relocation of MYC observed in our patient may explain the lymphomagenesis, but not necessarily the absence of Myc expression.
Given the limitations of this case report, we suggest that comprehensive retrospective studies in CLL patients should be performed to characterize the suggested ~400 Kb breakpoint and the region further upstream by sequential metaphase BAC FISH mapping since CMA does not detect the removal and relocation of an intact MYC locus. It is also possible that the absence of Myc expression is a false negative result given the specificity of immunostaining which is below 100%, and about 17% of cases may be overlooked for MYC rearrangements using this technique . A more accurate quantitative approach such as RT-qPCR is recommended. Since variability in MYC breakpoints could still result in similar MYC expression , possibly due to flexible DNA looping [43, 45], reporter expression studies are needed to better understand the clinical impact and significance of long distance deregulation in in loci with atypical MYC rearrangement.
This paper presents an unreported atypical rearrangement involving the IGH@ and MYC loci detected by FISH, adding to the burgeoning cytogenetic data on CLL patients with atypical t(8;14). It also highlights the Class III translocation breakpoint upstream of MYC, including the cancer and CLL-associated SNPs within the interval. This report also provides important and promising findings for further studies correlating Myc expression with a specific type of genomic translocation breakpoint or copy number variants in CLL and in other B-cell disorders. Lastly, overall findings in our report highlight the utility of karyotype analysis, interphase and sequential metaphase FISH studies, CMA, and other molecular tools in approaching the diagnosis and prognosis of CLL in a more comprehensive manner.