This is a case of a 51 year-old female who had received a heart transplant with basiliximab induction therapy 6 years prior to nonischemic cardiomyopathy. She presented with 1-week of exertional chest pain associated with nausea, shortness of breath and dizziness. She had no prior history of cardiac graft rejection and was on stable doses of tacrolimus and mycophenolate mofetil, as well as infectious prophylaxis with acyclovir and trimethoprim/sulfamethoxazole. Initial work-up for a primary cardiopulmonary etiology, including electrocardiogram (ECG), troponins, transthoracic echocardiogram (TTE) and chest radiograph (CXR), was unremarkable.
Complete blood count (CBC) revealed new-onset pancytopenia with a white blood cell count (WBC) of 0.9 x 109/L, absolute neutrophil count (ANC) of 0.2 x 109/L, hemoglobin of 7.4 g/dL, mean corpuscular volume (MCV) of 111.2 fL and platelets of 101 x 109/L (previous CBCs had been entirely within normal limits). Further evaluation for secondary causes of pancytopenia was unrevealing, including infectious and nutritional testing. Peripheral blood smear demonstrated pancytopenia with red blood cell anisopoikilocytosis, hypogranular neutrophils and circulating blasts. Bone marrow biopsy demonstrated findings consistent with AML with myelodysplastic related morphologic changes, including 50% cellularity with 59% blasts (enzyme cytochemistries: alpha naphthyl butyrate esterase negative, myeloperoxidase positive), decreased megakaryocytes, multi-lineage dyspoiesis and increased iron with no abnormal sideroblasts. Flow cytometry identified an immunophenotypically abnormal blast population consistent with myeloid differentiation comprising 19.46% of total cellular events. Blasts expressed CD45 (dim), CD33, CD34, CD13, CD117, CD38, HLA-DR and CD123, but were negative for CD19, CD15, TdT, CD16, CD11b, CD7, CD10, CD14 and CD64.
The bone marrow aspirate was sent for comprehensive genomic analyses. Interphase FISH analyses for MDS (chromosomes 5 and 7) and AML (MECOM, RUNX1T1/RUNX1, PML/RARA, CBFB) probes were negative (Table S1; Fig. S1a-f). However, karyotype analysis (Table 1; Fig. 1a) revealed two abnormal, related clones; 20% with t(8;11) only, and 50% with 13q deletion in addition to t(8;11), with the rest of the metaphase cells being normal. Sequential metaphase FISH studies were performed to determine the possible involvement of FGFR1 (8p11) and NUP98 (11p15) loci. Results showed 23% with 3’FGFR1 on derivative 11p and 36.5% with 5’NUP98 on derivative 8p, indicative of FGFR1 and NUP98 rearrangements (Table 1; Fig. 1b-c). Subsequent clinical genome-wide sequencing was performed via ChromoSeq (Methods S1) [7], a next generation sequencing (NGS)-based assay which detects SNVs and indels (≥73bp) in 40 genes or gene hotspots, and 612 pre-defined structural variants including 624 genes, and genome-wide copy number alterations (≥5Mb). The ChromoSeq assay identified NSD3 (Nuclear Receptor Binding SET Domain Protein 3), a gene 27 kb downstream of FGFR1, as the fusion partner of NUP98 (Table 1; Fig. 1d), specifically between intron 4 of NSD3 (Fig. 1e) and intron 12 of NUP98.
Table 1. Genomic Aberrations Detected by Various Assays
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t(8;11)(p11.2;p15) alone
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t(8;11)(p11.2;p15) and 13q deletion
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13q deletion alone
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12p12.3p13.31 deletion
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WT1, c.C1288T: p.(R430*)
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STAG2, c.G1525T: p.(G509*)
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Karyotype
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20%
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50%
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ND
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ND
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NA
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NA
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FISH
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FGFR1 (23%) NUP98 (36.5%)
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NT
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51%
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NT
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NA
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NA
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ChromoSeq*
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15.4%
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NA
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53.4% (11.5 Mb)
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47.2% (9.5 Mb)
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27.3%
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15.4%
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MyeloSeq**
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NA
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NA
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NA
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NA
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20.0%
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24.0%
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ISCN: 46,XX,t(8;11)(p11.2;p15.4)[4]/46,idem,del(13)(q12q14)[10]/46,XX[6].ish t(8;11)(5'FGFR1+,3'NUP98+;3'FGFR1+,5'NUP98+).nuc ish(FGFR1x2)(3'FGFR1 sep 5'FGFR1x1)[46/200],(NUP98x2)(3'NUP98 sep 5'NUP98x1)[73/200],(D13S319x1,LAMP1x2)[102/200], (MECOM,EGR1,D7S486,RUNX1T1,PML,CBFB,RARA,RUNX1)x2[200]. seq[GRCh38] chr8:g.pter_38335390delins[chr11:g.pter_3741107],chr12:g.7500000_17000000del, chr13:g.42000000_53500000del
Legend:
Karyotype band level: 400
*GRCh38/hg38 genome assembly
**GRCh37/hg19 genome assembly
ND - not detected
NT - not tested
NA - not applicable
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Consistent with karyotyping, subsequent FISH assay confirmed the deletion of the long arm of chromosome 13 (13q14.3) in 51% of the cells (Table 1; Fig. S1g-h). Although deletion of 13q is relatively more common in lymphoid malignancies, it is also found in various types of myeloid neoplasms. The chromosome interval that is deleted in all reported myeloid cases was found to be 13q13-21, which includes RB1 [8]. Studies have shown that the 13q12-q22 deletion interval is more recurrent in MDS, while 13q21 and 13q12-q32 deletions are more common in AML and myeloproliferative neoplasm (MPN), respectively. By ChromoSeq, the deletion in our patient was precisely determined to be 11.5 Mb in size involving a more distal interval, 13q14.11-q14.3 instead of 13q12-q14 (Table 1). Furthermore, ChromoSeq also identified a ~9.5Mb interstitial deletion on the short arm of chromosome 12 (p12.3p13.31) (Table 1) that is not detectable in G-banded chromosomes at a band level of 400. This deletion involves 19 OMIM genes, including ETV6. Deletions and structural alterations involving 12p that includes ETV6 have been described to exhibit tumor-suppressor characteristics and that loss of ETV6 plays a role in leukemogenesis and possibly prognosis [9].
In addition to structural and copy number variations, both MyeloSeq (Methods S2) [10] and ChromoSeq assays (i.e., next generation targeted and whole-genome sequencing, respectively) also identified two premature stop codons in WT1 and STAG2 (Table 1); two genes with known implications in AML [11, 12]. Both were classified as variants of uncertain clinical significance following standard guidelines provided by Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists [13].
The overall combined genomic studies in this case revealed a complex karyotype with aberrations detected by karyotype, FISH, MyeloSeq; and ChromoSeq. Importantly, the use of a single clinical genome-wide sequencing assay along with a streamlined analytical approach (ChromoSeq) not only identified the aberrations initially identified by conventional genomic studies, but further resolved the correct gene fusion partners involved in t(8; 11), and uncovered another large deletion (12p-).
Based on the presence of MDS-related changes in the bone-marrow along with a complex karyotype (>3 abnormalities) and the identification of the previously discussed rearrangements/variants, the patient began induction therapy for high-risk AML with MDS-related changes using liposomal daunorubicin and cytarabine (VYXEOS®). The patient achieved a complete remission with the day-30 post-induction bone marrow biopsy demonstrating a mildly hypocellular marrow with multilineage dysplasia, but no diagnostic features of acute leukemia, and no increase in blasts by flow cytometry. In agreement with these findings, her repeat MyeloSeq assay did not reveal any of the previously identified variants. In addition, the karyotype was normal and there was only a low level NUP98 rearrangement (0.5%) detected by FISH, with no evidence of deletion in the long arm of chromosome 13. The patient was subsequently treated with 2 consolidation cycles of VYXEOS® with a sustained remission and the ultimate plan to pursue consolidation allogeneic hematopoietic cell transplantation, if possible.