Optical Genome Mapping Helps to Identify BCR::JAK2 Rearrangement Arising from Cryptic Complex Chromosomal Aberrations: A Case Report and Literature Review

We report a case of myeloproliferative neoplasm, not otherwise specified (MPN-NOS)-transformed AML with BCR::JAK2 rearrangement. Chromosomal analysis indicated a simple abnormal karyotype 46,XY,t(7;17)(q21;q24),t(9;22)(p24;q11.2). Fluorescence in situ hybridization (FISH) using a BCR/ABL1/ASS1 probe set suggested a possible BCR rearrangement and a reflex JAK2 breakapart probe indicated JAK2 rearrangement, most likely partnered with BCR. Optical genome mapping (OGM) analysis confirmed BCR::JAK2 derived through an inv(9)(p24p13) after a t(9;22)(p13;q11.2) in this case. Due to the complexity of chromosomal aberrations, disruption and/or rearrangement of other genes such as KIF24::BCR, JAK2::KIF24/UBAP1, and CDK6:SOX9 were also identified by OGM. Although the functionality and clinical importance of these novel rearrangements were unknown, disruption of these genes might be associated with a poorer response to chemotherapy and disease progression. We also reviewed all cases with BCR::JAK2 rearrangement reported in the literature. In conclusion, a suspected t(9;22)/BCR::JAK2 rearrangement warrants further characterization with genomic assays such as OGM, whole chromosome sequencing, and RNA sequencing to explore other gene disruptions and/or rearrangements.

Since both JAK2 and BCR are in an orientation of plus strand on chromosomal loci 9p and 22q, respectively, a simple, reciprocal t(9;22)(p24;q11.2) will generate a head-to-head (5 BCR::5 JAK2) and a tail-to-tail (3 BCR::3 JAK2) recombination that are insufficient to form an in-frame, functional 3 BCR::5 JAK2 or BCR::JAK2 fusion.Therefore, in those cases with morphologically balanced t(9;22) and/or three-way translocations, the BCR::JAK2 rearrangement must have been derived from a complex genomic rearrangement, such as translocation plus inversion, which might not be totally detected by standard chromosomal analysis.Here, we report a new BCR::JAK2 rearranged case that arose from complex aberrations involving chromosomes 9 and 22. Intensive FISH studies and the novel technology of optical genome mapping (OGM) analysis were applied to confirm BCR::JAK2 rearrangement and further characterize genome-wide structural variants (SVs) and copy number variants (CNVs) in this case.OGM analysis confirmed all findings detected by both chromosomal and FISH analyses and revealed several novel rearrangements/fusions which may be associated with disease progression.

Case Report
A 39-year-old man presented with a myeloproliferative/myelodysplastic syndrome (MPN/MDS) diagnosed at an outside institution.According to the submitted reports, the initial bone marrow examination showed 95% hypercellularity with grade I myelofibrosis without any increase in blasts.Conventional cytogenetic analysis of bone marrow (BM) showed 46,XY in six cells and then, in a subsequent specimen, t(7;17)(q22;q25),t(9;22)(p13;q11.2).BCR/ABL Dual Fusion (DF) FISH analysis showed a negative result for BCR::ABL1 rearrangement but did detect an extra BCR signal in 76% of analyzed cells.The patient received chemotherapy and ruxolitinib for 3 months but developed disease progression.He was then referred to our hospital in December 2022.
Table 1.The detailed information of aberrations involving chromosome 9 and 22, including breakpoints, confidence, VAF, putative gene fusion, and ISCN called by using Bionano Access Software (v1.7) (the Genome Reference Consortium Human Reference 38 (GRCh38) has been applied).1 and 2.

SV Type
Table 1.The detailed information of aberrations involving chromosome 9 and 22, including breakpoints, confidence, VAF, putative gene fusion, and ISCN called by using Bionano Access Software (v1.7) (the Genome Reference Consortium Human Reference 38 (GRCh38) has been applied).22) and der( 9) and locations of certain gene rearrangements on them.der: derivative.

Discussion
Although BCR::JAK2 rearrangement is relatively rare in MPNs, detection/confirmation of this mutation may qualify affected patients for targeted therapy, such as ruxolitilib alone [13,14] and/or in a combination with BCL2 inhibitors such as venetoclax [11].To the best of our knowledge, there are 17 cases with confirmed BCR::JAK2 rearrangement in the literature, with an additional 5 cases with t(9;22)(p24;q11.2) and suspicious BCR::JAK2 rearrangement solely based on karyotype only and/or limited FISH studies (Table 3) [20][21][22][23][24]. Interestingly, 4 of the 5 cases suspicious for BCR::JAK2 rearrangement also showed a morphologically balanced t(9;22)(p24;q11.2).In fact, the B-ALL case reported by Tirado et al. [20] had a JAK2 rearrangement due to an additional JAK2 signal located on the der( 22), but the authors could not prove a simultaneous BCR rearrangement or a BCR::JAK2 fusion by intensive FISH studies.Chen et al. [24] also reported a B-ALL case with t(9;22)(p24;q11.2).However, they failed to detect either JAK2::BCR or BCR::JAK2 fusion transcripts in that case.By adding the case with BCR::JAK2 confirmed by intensive FISH and OGM analyses in this study, the database of BCR::JAK2-positive cases has been increased to a total of 18 cases.This is also the second BCR::JAK2-positive AML case transformed from a MPN-NOS [12] (Table 3).
The complexity of chromosomal aberrations usually indicates the possibility of mutations simultaneously involving multiple genes, in addition to BCR and JAK2.Although chromosomal analysis was reported as a simple abnormal karyotype with t(9;22)(p24;q11.2) in a B-ALL case, Chen et al. [16] showed a co-existence of JAK2::PPM1F rearrangement and PRAMENP::BCR rearrangement by whole genome sequencing (WGS).A novel JAK2::PPM1F/E16::E2 fusion was further identified by RT-PCR and Sanger sequencing, although it was postulated as non-functional due to induction of a stop codon in the fusion gene.In a myeloid/lymphoid neoplasm with eosinophilia case reported by Snider et al. [10], as further characterized by mate-pair sequencing, involvement of four derivative chromosomes (der(8), der (9), and two copies of der( 22)) to form the BCR::JAK2 rearrangement and a novel PCM::JAK2/E7::E9E10 rearrangement were identified.In the case we report here, the co-existing KIF24::BCR and JAK2::KIF24/UBAP1 rearrangements were detected by OGM.Although a functional fusion transcript from these two novel rearrangements cannot be determined, KIF24 and/or UBAP1 were certainly disrupted in our case.
As a novel technology, OGM has played an important role in further characterizing cryptic chromosomal aberrations and confirming BCR::JAK2 rearrangement.OGM analysis also revealed various novel rearrangements such as KIF24::BCR, JAK2::KIF24/UBAP1, and CDK6:SOX9 and microdeletion of del(7)(q21.13q21.2) in the case we reported.All these novel findings were not detected by routine chromosomal analysis and targeted FISH, and these abnormalities might be associated with a poor response to chemotherapy and disease progression from MPN-NOS to AML, as occurred in this patient.As suggested by Snider and others, all cases with confirmed and/suspected BCR::JAK2 rearrangement warrant analysis using integrated genomic approaches such as WGS, RNA sequencing, and OGM to fully characterize the underlying complex chromosomal aberrations.In summary, cases with BCR::JAK2 rearrangement are infrequent and chromosomal aberrations producing a functional BCR::JAK2 fusion can be complicated.Very likely, disruption and/or rearrangement of other important gene(s) can be co-existent.Utilizing integrated genomic approaches such as OGM, WGS, and/or RNA sequencing is strongly recommended.

Figure 2 .
Figure 2. The OGM analysis in this case.(Left) OGM Circos plot illustrates the chromosomal aberrations in this case.(Right) The enlarged views of the areas containing chromosomes 7 to 9 indicated two types of t(7;17)s, two types of t(9;22), and intra-chromosomal fusions involving chromosomes 7 and 9, respectively.Please see detailed information of these chromosomal aberrations in Tables1 and 2.

Figure 2 .
Figure 2. The OGM analysis in this case.(Left) OGM Circos plot illustrates the chromosomal aberrations in this case.(Right) The enlarged views of the areas containing chromosomes 7 to 9 indicated two types of t(7;17)s, two types of t(9;22), and intra-chromosomal fusions involving chromosomes 7 and 9, respectively.Please see detailed information of these chromosomal aberrations in Tables1 and 2.

Figure 3 .
Figure 3. Illustration of the aberrations and locations of affected genes on chromosomes 9 (blue color) and 22 (red color).(Left) Normal chromosomes 9 and 22 and locations of certain genes on them.(Right) der(22) and der(9) and locations of certain gene rearrangements on them.der: derivative.

Figure 3 .
Figure 3. Illustration of the aberrations and locations of affected genes on chromosomes 9 (blue color) and 22 (red color).(Left) Normal chromosomes 9 and 22 and locations of certain genes on them.(Right) der(22) and der(9) and locations of certain gene rearrangements on them.der: derivative.

Table 2 .
The detailed information of aberrations involving chromosomes 7 and 17 detected by OGM analysis (the Genome Reference Consortium Human Reference 38 (GRCh38) has been applied).

Table 3 .
Twenty-three cases with confirmed and suspicious BCR::JAK2 rearrangement reported in the literature, including the case reported in this study.
* Transformed from previous MPN-NOS.** Previous history of HL and chemotherapy.