The utility of massively parallel DNA sequencing has improved considerably with the introduction of paired-end reads, which allow for better mapping efficiency, and more accurate identification of junctional breakpoints associated with structural variants (translocations, insertions and deletions).
In this report, we describe the use of paired-end read WGS for “real-time” oncologic diagnosis, and describe the genomic details of an oncogenic fusion gene created by an insertional event. Within seven weeks, we completed the process of library generation, massively parallel sequencing, analysis, and validation of a novel insertional fusion that created a classic PML-RARA bcr3 variant. These findings altered the medical management of this patient, who received ATRA consolidation instead of an allogeneic stem cell transplant. The patient remains in first remission 15 months after her presentation.
RT-PCR was not performed at the referring institution, and was negative when evaluated at Washington University when the patient was in remission. We did not initially perform RT-PCR with the RNA generated from the cryopreserved sample because the RNA was severely degraded. Further, because her complex cytogenetics predicted an unfavorable prognosis, it was essential to determine whether the patient had a recognized PML-RARA fusion gene (since t(15;17) supercedes other cytogenetic findings and predicts a favorable outcome in patients treated with ATRA). Fortunately, the sequencing of the patient’s tumor genome resolved the conundrum, allowing the RT-PCR results to serve as confirmatory proof of the fusion event (despite RNA degradation), and providing a novel mechanism for its formation, which assured us that the diagnosis was correct.
Alternative laboratory approaches could have been used to detect potential pathogenic RARA
rearrangements (e.g. nested PCR from a linker-ligated library, long-distance PCR,21
BAC clone screening, targeted 3730 sequencing of the 48.5 Kb RARA
locus, etc). However, such techniques are labor intensive, and can require large amounts of starting material, personalized design, and iterative troubleshooting. Further, many of these techniques have success rates that are not adequate for clinical practice. In contrast, WGS with paired-end libraries can be accomplished with as little as 10 ng of starting DNA, is amenable to an automated ‘pipeline’ strategy, and can consistently de-convolute single nucleotide variants (SNVs), small insertions and deletions, structural variants, and clonality. Further, this approach requires no custom reagents, and no foreknowledge of genomic regions that must be assessed for diagnostic accuracy.
This study also confirms that reciprocal RARA-PML
fusions are not required for the development of APL. RARA-PML
has been proposed to participate in APL pathogenesis, although it is identified in only ~67% of APL cases by RT-PCR.22
Complex rearrangements and deletions can lead to unusual RARA-PML
transcripts of uncertain significance.22–25
Furthermore, bcr3 RARA-PML
does not independently lead to leukemia in a murine leukemia model.26, 27
In this patient, the reciprocal fusion RARA-PML
was absent, and the alternative RARA-LOXL1
was fused out of frame.
WGS resolved not only the PML-RARA insertion event, but also all other abnormalities observed during routine cytogenetics. Loss of chromosome 6 and 16 were not detected with WGS; rather, we identified an additional inversion and translocation, inv(6)(p22.3;q14.1) and t(6;16)(q22.31;p13.3). This suggests that genetic information on chromosomes 6 and 16 was actually present within the two marker chromosomes, but that chromosomal banding patterns were disrupted by a three-way translocation.
FISH has been used to suggest that insertional translocations may occur in t(15;17)-negative promyelocytic leukemia.2, 22, 28–30
The commercially available Abbott/Vysis dual fusion dual probe strategy employs large probes (between 239 and 417 Kb, schematically described in Supplemental Figure 3A and B
). However, the minimal required PML
insertion region (the promoter/enhancer and exons 1-3) is nearly one tenth the size of these probes. The large probes (239+ Kb) improve sensitivity for conventional t(15;17) detection, but they make accurate diagnosis of small insertional events difficult or even impossible.2, 31
Alternative cosmid-based strategies improve the ability to detect small insertions,2
but the reagents are not widely available. Because of these issues, we designed a new set of fosmid based probes, which are publically available. Using these probes, we identified further cases of PML-RARA
insertional fusions that were missed by conventional cytogenetics and FISH.
Insertional translocations are likely to be under-diagnosed due to the technical difficulties associated with FISH using conventional probes (as highlighted by this case). This may be especially true of tumor types interrogated with FISH break-apart strategies, and tumors that lack an alternative molecular diagnostic assay (e.g. Burkitt lymphoma).32
Diagnostic WGS remains cost-prohibitive for universal application in cancer patients (~$40,000 for each tumor/normal pair at the current time). This price has been decreasing rapidly over the last several years, while the expansion of AML-associated genes and mutations (e.g. PML-RARA, ETO-AML1, CBFB-MYH11, BCR-ABL, FLT3, NPM1, KRAS, DNMT3A, TET2, IDH1/2, RUNX1, CEBPA, etc) is increasing the cumulative cost of molecular tests that may be relevant for diagnosis and risk-prediction.
Acute promyelocytic leukemia is likely to be an indicator of the variety of mutations that can present with similar morphology and clinical course. APL may be associated with diverse PML-RARA
splice variants, unusual translocations/insertions, multiple RARA
fusion partners, and even an RARG
fusion partner.1, 8–12
The diagnostic strength of WGS is that it is a generic and stable platform for mutation detection, and special approaches are not required for specific diagnostic settings. All classes of mutations are detected in a totally unbiased fashion, allowing for confirmation of a suspected diagnosis even if it is caused by a rare or unusual mutation; this data can be obtained and interpreted in a clinically relevant timeframe.
The time required to complete WGS is rapidly decreasing. The sequencing timeline for this patient involved 25 days to generate and analyze WGS and an additional 27 days for orthogonal validation; this makes validation a significant, but necessary, bottleneck in the overall time to complete clinical grade sequencing (Supplemental Figure 4
). As costs continue to fall, increasingly deep coverage will become possible (e.g. 60x coverage instead of 30x), allowing for improved variant detection that may reduce the need for validation. With these improvements, “clinical grade” WGS should soon be possible within 4 weeks of sample collection.
Meaningful diagnostic timeframes are dependent on the cancer type being assessed; some cancers have long diagnostic windows before patients need therapy (e.g. follicular lymphoma and myelodysplastic syndrome), others have moderately long diagnostic windows (e.g. breast, lung, colon, etc, where definitive chemoradiotherapy often is not considered until after surgical resection and appropriate healing), and a few require urgent chemotherapy (e.g. AML, ALL, Burkitt lymphoma, blast phase CML, small cell lung cancer). The critical decision in the treatment in AML and ALL is not which induction therapy to use (since uniform approaches to remission-induction are currently employed for both diseases), but whether patients should receive consolidation therapy with chemotherapeutic approaches, or should receive allogeneic transplantation. This decision is generally made within 6–8 weeks of initial presentation; for AML and ALL, a six-week timeframe for WGS is therefore clinically relevant. However, to fully utilize this potentially transformative technology to make informed clinical decisions, standards will have to be developed that allow for CLIA-CAP certification of WGS, and direct reporting of relevant results to treating physicians.