Novel t(14;19)(q32.2;p13.12) structural variant identified by genome sequencing in a myeloid neoplasm post cytotoxic therapy: a case report

Introduction

Myeloid neoplasms post cytotoxic therapy (MN-pCT) typically arise in patients treated with cytotoxic or radiation therapy for a previous malignancy or non-neoplastic disease (1). A number of cytotoxic drugs have been associated with MN-pCT. The MN-pCTs due to alkylating agents typically have a latency period of 5 to 10 years (2). Since the drugs induce centromeric chromosome breakage, this type of MN-pCTs is usually associated with monosomy or deletions of long arms of chromosomes 5 and 7 (2). On the other hand, MN-pCTs associated with topoisomerase II inhibitors have a shorter latency period (less than 5 years) (3). This type of drug is usually associated with balanced translocations, as the pharmacologic mechanism of the drugs is prevention of the re-ligation step of the topoisomerase reaction (2). The 11q23 (KMT2A gene) locus is frequently involved in MN-pCTs associated with topoisomerase II inhibitors, often rearranging with the ELL gene at 19p13.1 (4). MN-pCTs linked to fluorouracil (5-FU) is relatively rare with a few case reports (5,6). 5-FU is a pyrimidine analog, and inhibits DNA and RNA synthesis by blocking the production of thymidine and incorporating abnormal nucleotides into RNA (7). This disrupts cell division and leads to cell death. We report a case of MN-pCT occurring 12 years after 5-FU-based therapy for primary rectal squamous cell carcinoma that presented as acute myeloid leukemia (AML). We present this article in accordance with the CARE reporting checklist (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-14/rc).

Case presentation

All procedures performed in this case were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki and its subsequent amendments. Publication of this case report and accompanying images was waived from patient consent according to the Washington University institutional review board.

The patient is a 52-year-old woman who presented to her primary care physician with new onset leukocytosis and anemia, and was referred to hematology/oncology clinic. A review of past medical history revealed a history of rectal squamous cell carcinoma with metastasis. The initial diagnosis was made in 2010 and she was treated with 5-FU. The patient stated that she did not tolerate the therapy and it was stopped early. The tumor metastasized to the liver and was treated with radiation. The patient achieved remission after treatment. She presented in 2022 with a complete blood count (CBC) of: 20.5×10³/µL white blood cell, 6.6 g/dL hemoglobin, 15×10³/µL platelet. The peripheral blood and bone marrow aspirate showed marked granulocytic and monocytic proliferation with left-shift, dysgranulopoiesis and increased blasts (Figure 1A). Due to marked dysplasia, it was challenging to distinguish granulocytic and monocytic cells morphologically (Figure 1B). Blast count was best estimated at 25% to 30%. Flow cytometry of the bone marrow aspirate identified an immunophenotypically abnormal blast population that was consistent with myeloid differentiation comprising 28–30% of total cellular events. Antigens expressed were CD34, CD38, CD200, CD56, HLA-DR, CD13, CD14 dim, CD64, CD4 dim, CD123, and CD117. There was also an expanded and immunophenotypically abnormal CD34 negative monocytic population (25% of CD45⁺ events) which expressed CD14, CD64, CD56, and CD11b.

Figure 1 Morphologic and genetic findings of the bone marrow. (A) A Wright-Giemsa stain of the bone marrow aspirate showed increased blasts with fine chromatin and prominent nucleoli, increased monocytes, dysgranulopoiesis and dyserythropoiesis (60×). (B) An H&E stain of the bone marrow biopsy showed diffuse sheets of blasts (40×). (C) Karyotyping showed a complex karyotype with multiple duplications, deletions as well as balanced translocations. The arrows indicate chromosomal breakpoints. (D) Illustration of the balanced translocation identified by genome sequencing. H&E, hematoxylin and eosin.

Karyotyping studies revealed a complex karyotype involving multiple copy number changes as well as apparently balanced translocations (Figure 1C): 53,XX,+4,del(5)(q22q33),+del(5)(q22q33),del(7)(q22),del(8)(q11.2q21),+del(8),add(10)(q24),+11,i(11)(q10), +13,t(14; 19)(q32;p13),+der(14)t(14; 19)(q32;p13),+22[cp20].

Interphase FISH analysis confirmed the duplication of 5p15.31 (D5S630/D5S2064) and deletion of 5q31.2 (EGR1 locus) in 85.5% of nuclei (171/200), the deletion of 7q31 (D7S486) in 96.5% of nuclei (193/200), the duplication of 8q21.3 (RUNX1T1 locus) in 78.5% of nuclei (157/200), and the duplication of 22q11.23 (BCR locus) in 72% of nuclei (144/200). Additional subclones were also observed.

The targeted myeloid next-generation sequencing panel (8) revealed the following variants with known clinical significance: TP53 p.Gly266Glu [42% variant allele frequency (VAF)], TP53 p.Val157Gly (45% VAF), and TET2 p.His1380Tyr (63% VAF). Given the history of carcinoma and subsequent cytotoxic therapies, the diagnosis of MN-pCT was made per the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (9). The patient opted for hospice care after the initial diagnosis was made and further clinical data was not available.

Tumor-only clinical genome sequencing was performed on the bone marrow specimen (10). Genomic library was prepared using the Illumina DNA library preparation kit (Illumina, Inc., San Diego, CA, USA). Sequencing was performed on NovaSeq 6000 sequencing instruments (Illumina) using an S4 flowcell with a target coverage depth of 60X. Data was demultiplexed into FASTQ files and then aligned to GRCh38 using the DRAGEN platform (Illumina), following by variant filtering a custom analysis workflow as previously described (10). The genome sequencing confirmed all sequence variants, copy number alterations, and structural variants previously reported by targeted sequencing panel and conventional karyotype, and further identified the fusion partners of the balanced translocation (Tables 1-3).

Discussion

Genome sequencing demonstrated the capacity to comprehensively characterize copy number alterations, structural variants, and gene-level variants in this individual case of MN-pCT (Figure 1D). In addition, the assay identified, at the base-pair level, the genomic loci involved in the t(14;19) translocation. While karyotyping located the translocation at t(14;19)(q32;p13), the genomic informatics pipeline refined the breakpoints to t(14;19)(q32.2;p13.12). Contigs from two break-end reads, consistent with the presence of two derivative chromosomes, pointed to the reciprocal products resulting from breakpoints on chromosomes 14 and 19. The breakpoint on chromosome 14 occurred between chr14:100893948-100893961, in intron 2 for the RTL1 gene on the reverse strand. A non-coding gene, RP11-909M7.3, is also present on the forward strand. The chr19:14551581-14551589 breakpoint was in intronic regions of two genes, intron 1 of the TECR gene on the forward strand and intron 2 of the DNAJB1 gene on the reverse strand. Translocation of none of the genes has been reported in myeloid neoplasms. The close proximity of the breakpoints from the two break-end reads indicated that this rearrangement is largely balanced at the molecular level, with just 13 and 8 base pairs lost during the formation of the derivatives.

5-FU acts primarily as a thymidylate synthase inhibitor and blocks the synthesis of the pyrimidine thymidylate, a nucleotide required for DNA replication (7). There have been only a few case reports of MN-pCTs associated with 5-FU. Both monosomy 7 and an apparently balanced translocation t(3;21)(q26;q22) have been reported in separate t-AML cases related with 5-FU (5,6). Selective pressures of the chemotherapy lead to the clonal expansion of myeloid progenitor cells carrying TP53 mutations, although TP53 mutations are not associated with increased mutation burden in t-AML (11). Our case presented with bi-allelic TP53 mutations, and a unique combination of copy number changes and structural variation generating a complex karyotype.

Due to the presence of genes on both strands at each breakpoint, the t(14;19)(q32.2;p13.12) translocation disrupts four genes. The RTL1 gene, located in the DLK1–DIO3 genomic region on chromosome 14q32, is a paternally-expressed imprinted gene involved in fetal development (12). The DLK1-DIO3 genomic region contains three paternally expressed protein-coding genes (DLK1, RTL1, and DIO3) and three maternally expressed noncoding genes (MEG3, MEG8, and antisense RTL1). Expression and methylation changes in the DLK1-DIO3 region have been reported in myelodysplastic syndrome (MDS) (13).

The TECR gene, located on the forward strand of chromosome 19p13, encodes a trans-2,3-enoyl-CoA reductase which belongs to the steroid 5-alpha reductase family. Germline variants in the gene have been reported in patients with autosomal recessive non-syndromic mental retardation (14). TECR::PKN1 fusion has been reported in prostate carcinoma (15). Fusion involving TECR gene has not been reported in myeloid neoplasms.

The DNAJB1 gene, located on the reverse strand of the same chromosomal region, encodes a chaperone which is a member of the DnaJ or Hsp40 (heat shock protein 40 kD) family of proteins. DNAJB1::PRKACA fusion kinase has been identified as a driver mutation for fibrolamellar hepatocellular carcinoma through its interaction with β-catenin and role in the liver regenerative response (16). A small number of studies have suggested that DNAJB1 protein interacts with both wild-type and mutant p53 proteins and regulates p53 activity in tumor cells as a tumor suppressor (17). Fusion involving DNAJB1 gene has not been reported in myeloid neoplasms. Its role remains poorly understood.

Conclusions

In conclusion, we report a rare case of MN-pCT after 5-FU and radiation therapy, with bi-allelic TP53 mutations, multiple deletions and a novel structural variant t(14; 19)(q32.2;p13.12). The clinical whole genome sequencing (WGS) assay identified the majority of the copy number alterations, gene-level variants as well as the novel structural variant as a single assay. This assay provided more precise information about the translocation compared to conventional karyotyping method. This case provides an example of how WGS can complement or refine findings from karyotyping when investigating rare or novel structural rearrangements. Although the 19p13.1 genomic locus has been reported to be involved in balanced translocations in MN-pCT, the TECR and DNAJB1 genes at the genomic locus have not been reported in previous studies of MN-pCT. Identification of the novel fusion partners and genomic loci may facilitate individualized therapeutic strategies, and could provide an opportunity for personalized minimal residual disease (MRD) monitoring with RT-PCR in the era of precision medicine.

Acknowledgments

The authors thank the medical staff who participated in this patient’s care.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-14/rc

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-14/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-14/coif). X.Z. serves as an unpaid editorial board member of Journal of Laboratory and Precision Medicine from February 2025 to January 2027. E.J.D. reports receiving royalties or licenses from Caris Diagnostics; as well as consulting fees and payments from Illumina. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this case were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki and its subsequent amendments. Publication of this case report and accompanying images was waived from patient consent according to the Washington University institutional review board.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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