Use of whole-genome sequencing to diagnose a cryptic fusion oncogene

Abstract

Context: Whole-genome sequencing is becoming increasingly available for research purposes, but it has not yet been routinely used for clinical diagnosis.

Objective: To determine whether whole-genome sequencing can identify cryptic, actionable mutations in a clinically relevant time frame.

Design, setting, and patient: We were referred a difficult diagnostic case of acute promyelocytic leukemia with no pathogenic X-RARA fusion identified by routine metaphase cytogenetics or interphase fluorescence in situ hybridization (FISH). The case patient was enrolled in an institutional review board-approved protocol, with consent specifically tailored to the implications of whole-genome sequencing. The protocol uses a "movable firewall" that maintains patient anonymity within the entire research team but allows the research team to communicate medically relevant information to the treating physician.

Main outcome measures: Clinical relevance of whole-genome sequencing and time to communicate validated results to the treating physician.

Results: Massively parallel paired-end sequencing allowed identification of a cytogenetically cryptic event: a 77-kilobase segment from chromosome 15 was inserted en bloc into the second intron of the RARA gene on chromosome 17, resulting in a classic bcr3 PML-RARA fusion gene. Reverse transcription polymerase chain reaction sequencing subsequently validated the expression of the fusion transcript. Novel FISH probes identified 2 additional cases of t(15;17)-negative acute promyelocytic leukemia that had cytogenetically invisible insertions. Whole-genome sequencing and validation were completed in 7 weeks and changed the treatment plan for the patient.

Conclusion: Whole-genome sequencing can identify cytogenetically invisible oncogenes in a clinically relevant time frame.

Figure 1. Molecular diagnostics of incident case

A and B. Cytomorphology of initial bone marrow biopsy stained with Wright-Giemsa. A. 10x amplification. B. 60x amplification. C. Metaphase cytogenetics of the dominant clone (46, XX, del(9)(q12q32), del(12)(q12q21), −6, −16, add(16)(p13.2), +2 mar [13 of 20 cells]). D. Metaphase cytogenetics of the minor clone (46, XX, del(9)(q12q32), del(12)(q12q21)[6 of 20 cells]). E. FISH using Abbott/Vysis dual fusion, dual probes. Note one fusion signal, two red signals (chromosome 15) and one green signal (chromosome 17). F. Interphase FISH using Abbott/Vysis dual fusion, dual probes. Note fusion on der(17). Probes were labeled with: Spectrum Orange, PML; Spectrum Green, RARA.

Figure 2. Whole genome sequencing results

A. Schematic representation of ins(17;15) identified by WGS and resulting in PML-RARA fusion. Breakpoints are: chromosome 15: 72027045 and 72104113; chromosome 17: 35742679 and 35742683 (NCBI36/hg18). ATG: CDS start. ZF: zinc finger domain. CC: coiled-coil domain. DBD: DNA-binding domain. Arrows indicate binding sites of primers used in panel C. B. PCR validation of ins(17;15), del(12), del(14) and del(19). PCR of genomic DNA from the patient’s skin (normal: N) and leukemia (tumor: T) using primers that span the junction of RARA-LOXL1 (P1/P2), PML-RARA (P3/P4), del(12), del(14), LOXL1-PML (P5/P6), and del(19). Note amplification across fusion breakpoints in the leukemia sample (T), but not in the skin sample (N) for all but del(19) specific primer pairs. C. RT-PCR validation of PML-RARA expression. RNA prepared from cryopreserved leukemia cells was amplified with a forward primer in PML exon 3 and a reverse primer in RARA exon 3. DNA ladder: 1500, 800, 500, 300, 200, 150, 100, 50 base-pairs. D. Single nucleotide variants that occur in protein coding sequences and the percentage of each SNV allele within the total bone marrow population. Note two distinct clones, consistent with the original metaphase cytogenetics (Figure 1C and D).

Figure 3. Identification of additional patients with PML-RARA insertional fusion events

A. Fosmid selection and position of homology to chromosomes 15 and 17. B. Schematic representation of fosmid homology to the STROML1/PML locus and to the CDC6/RARA locus. C and D. Interphase FISH identifies a fusion event on der(17), consistent with ins(17;15). E and F. Interphase FISH identifies a fusion on der(15), consistent with ins(15;17). Probes were labeled with: Spectrum Orange, STROML1/PML; Spectrum Green, CDC6/RARA.