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Whole genome sequencing (WGS) is becoming increasingly available for research purposes, but it has not yet been routinely used for clinical diagnosis.
To determine whether whole genome sequencing can identify cryptic, actionable mutations in a clinically relevant time frame.
We were referred a difficult diagnostic case of acute promyelocytic leukemia with no pathogenic X-RARA fusion identified by routine metaphase cytogenetics or interphase FISH. The patient was enrolled in an IRB approved protocol, with consent specifically tailored to the implications of whole genome sequencing. The protocol employs a ‘movable firewall,’ which maintains patient anonymity within the entire research team, but allows the research team to communicate medically relevant information to the treating physician.
Clinical relevance of whole genome sequencing and time to communicate validated results to the treating physician.
Massively parallel paired-end sequencing allowed us to identify a cytogenetically cryptic event: 77 kilobases 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. RT-PCR subsequently validated the expression of the fusion transcript. Novel FISH probes identified two additional cases of t(15;17)-negative acute promyelocytic leukemia that had cytogenetically invisible insertions. Whole genome sequencing and validation were completed in seven weeks, and changed the treatment plan for the patient.
Whole genome sequencing can identify cytogenetically invisible oncogenes in a clinically relevant timeframe.
Acute promyelocytic leukemia (APL) is commonly (>90%) associated with PML-RARA fusion transcripts resulting from pathogenic t(15;17) translocations.1, 2 Unusual cytogenetic rearrangements (e.g. insertions and three, four, or even eight-way translocations)2–4 can also lead to PML-RARA formation. Alternative PML-RARA fusions and splice variants exist, which are not detected by standard RT-PCR,5–7 as well as alternative X-RARA fusions, which may be responsive to all-trans retinoic acid (ATRA) (e.g. NuMA1-RARA, NPM1-RARA, STAT5B-RARA, PRKAR1A-RARA, FIP1L1-RARA, BCOR-RARA, and the non-RARA translocation NUP98-RARG)1, 8–12 or ATRA resistant (PLZF-RARA)1. Timely and accurate diagnosis of APL is essential because the addition of ATRA to chemotherapy leads to significantly improved outcomes (5-year event free survival 69% compared to 29% in patients receiving chemotherapy alone).13
A 39 year-old woman with acute myeloid leukemia (AML) in first remission was referred to our institution for consideration of an allogeneic stem cell transplant. She had initially presented with hypofibrinogenemia, disseminated intravascular coagulopathy (DIC), and pancytopenia (WBC 1.3 K/μl, Hgb 11.6 g/dL, Plts 72 K/μl). Her bone marrow contained 61% atypical promyelocytes with invaginated nuclei (including bilobed forms), and dense primary granules (Figure 1A and B). She started induction chemotherapy with ATRA, cytarabine and idarubicin. However, her metaphase cytogenetics (46, XX, del(9)(q12q32), del(12)(q12q21)/idem, −6, −16, add(16)(p13.2), +2 mar/46, XX, Figure 1C and D) revealed a complex pattern, which is associated with < 15% long-term survival, and is treated with allogeneic transplant in first remission whenever possible.14, 15 Interphase fluorescence in situ hybridization (FISH) suggested a possible fusion between chromosomes 15 and 17 on der(17), but was most consistent with an RARA-PML fusion, not the pathogenic PML-RARA fusion characteristic of M3 AML (Figure 1E and F). RT-PCR to detect a PML-RARA fusion transcript was not performed at the referring institution. These findings lead to a diagnostic conundrum and ATRA was discontinued. Persistent AML was observed on day 14. She entered a complete remission following re-induction with cytarabine, idarubicin and etoposide. She was then referred to our institution for consideration of allogeneic stem cell transplantation. At that time, her bone marrow biopsy revealed no morphologic evidence of AML, and had normal metaphase cytogenetics, normal interphase FISH, and no evidence of PML-RARA by RT-PCR. HLA typing identified one matched sibling. This case posed a diagnostic dilemma with prognostic and therapeutic consequences: does the patient have APL, or does she have AML with unfavorable-risk cytogenetics?
Because her leukemic cytomorphology was consistent with APL, we empirically recommended two cycles of arsenic trioxide consolidation, which she received.16
Little material from her original leukemia remained for subsequent evaluation and no clinical samples were available for FISH or RT-PCR. However, two vials of bone marrow cells had been cryopreserved under a research protocol at her referring institution. DNA and RNA were generated from these respective samples (the RNA sample was severely degraded). We obtained appropriate consent for whole genome sequencing (WGS), and completed this analysis using paired-end reads. Our primary goal was to determine if WGS could identify an actionable mutation (e.g. a cryptic X-RARA rearrangement) in a clinically relevant time-frame (6–8 weeks).
A ‘movable-firewall’ within our research protocol allows for the communication of clinically relevant findings to the patient’s physician and to the patient, while strictly maintaining patient anonymity among all research personnel. De-identified samples are entered into a tissue bank. Clinical information (age, gender, disease, treatment, outcome, etc) is maintained in association with de-identified codes only. A list associating de-identified codes with personal patient information (name, date of birth, treating physician) is maintained in a locked safe. A single protocol administrator has access to this list. The research team can communicate medically relevant information to the administrator. The administrator communicates this information to the treating physician, who is responsible for informing the patient of the results and of their clinical implications.
After obtaining explicit consent for WGS with an IRB approved protocol, DNA libraries were generated from one cryovial of the original bone marrow aspirate and from a skin punch biopsy obtained in remission (matched normal cells). We generated 187.1 and 200.1 billion base pairs of DNA sequence from each of the respective samples, with an average read depth of 43.7x and 46.8x, respectively. Library generation, sequence production, and data analysis were performed as previously described.17–20 Adequate genome-wide coverage (> 99.5% diploid coverage) was assured by assessing the coverage of known single nucleotide polymorphisms (SNPs) in the patient’s genome, as defined with data collected from the Affymetrix Genome-Wide Human SNP Array 6.0. Reagents and methods for PCR validation, RT-PCR, and FISH are described in Supplemental Methods.
Validated WGS results were completed and reported to the patient’s physician seven weeks after obtaining the DNA samples.
This is the timeline of data production, analysis and validation: Day 1: DNA logged in at Washington University Genome Institute; Day 5: libraries completed and sequencing begins; Day 18: sequence completed; Day 22: alignment to reference sequence completed; Day 24: prediction of single nucleotide variants (SNVs) completed; Day 25: structural variants predicted by BreakDancer20; Day 52: insertional fusion completely validated by PCR and results transmitted to treating physician.
Using massively parallel DNA sequencing with paired-end reads, we identified two sets of breakpoints between chromosomes 15 and 17, which occur in the LOXL1/PML locus and RARA locus respectively (schematically described in Figure 2A). PCR amplification across each predicted breakpoint validated the en bloc insertion of a 77 kilobase (Kb) segment of chromosome 15 (containing parts of the LOXL1 and PML genes) into intron 2 of RARA, the invariant site of RARA-associated translocations (Figure 2B).
This insertion generates three novel fusion transcripts: bcr3 PML-RARA; LOXL1-PML; and RARA-LOXL1. Expression of bcr3 PML-RARA was validated by RT-PCR using three different primer pairs (using the degraded RNA from the original banked AML sample), including the CLIA-certified InVivoscribe PML/RARa Mix2b kit (Figure 2C, Supplemental Figure 2A and data not shown). The RARA-LOXL1 fusion was out of frame and is predicted to encode a 67 amino acid protein (Supplemental Figure 2B and data not shown). The LOXL1-PML fusion leads to altered splicing and a premature stop codon prior to the PML junction, and is predicted to encode a 573 amino acid protein (Supplemental Figure 2C).
In addition, we identified and resolved the breakpoints associated with all abnormalities observed with metaphase cytogenetics including del(9), del(12), and add(16)(p32.2); the latter was in fact a translocation t(16;22)(p13.3;q13.31) (Figure 1C, Supplemental Figure 1 and data not shown). Two other large deletions not found by conventional cytogenetics were detected by WGS: del(14) and del(19); the latter was also identified in the skin sample, proving that it is an inherited copy number variant (Figure 2B). The predicted deletions of chromosomes 6 and 16 were not detected by WGS. Instead, we identified a 61 megabase inv(6)(p22.3;q14.1) and a translocation t(6;16)(q22.31;p13.3) (Figure 1C). We further identified and validated 12 SNVs within protein coding sequences (Figure 2D). SNV allele frequency was consistent with the presence of two distinct leukemic clones in the bone marrow, recapitulating the metaphase cytogenetics: 8/12 SNVs had a variant allele frequency of 35–51%, and 4/12 SNVs had a variant allele frequency of 13–21% (Figure 1C, 1D, and and2E).2E). The significance of these somatic mutations for disease pathogenesis is currently unknown.
We designed a new set of fosmid-based FISH probes (each 30–40 Kb in size) for the detection of insertional fusions that target the minimal PML translocation region (the promoter/enhancer and exons 1-3, which is roughly 30 Kb) (Figure 3A and B). We searched the Washington University Department of Pathology database for AML cases diagnosed during the last five years. We identified eleven cases with features suggestive of APL (including any promyelocytic morphology, characteristic CD33+CD34−HLA-DR− immunophenotype, and variable to strong myeloperoxidase staining by enzyme cytochemistry) but that lacked normal dual fusion patterns by FISH. We found that two of these specimens contained PML-RARA fusions resulting from cryptic insertions: one was associated with an insertion of PML into the RARA locus (ins(17;15)), and the other with insertion of RARA into the PML locus (ins(15;17)) (Figure 3C–F). Both cases (as well as the proband) had RT-PCR confirmation of a PML-RARA fusion (bcr1 and bcr3 isoforms), and all had features typical of APL (Table 1).
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.
Funding support: The Washington University Cancer Genome Initiative provided support for this study. JSW is a fellow of the Leukemia Lymphoma Society. This work was also supported by NIH PO1 CA101937, NIH K99 HL103975 (Welch), and the Barnes-Jewish Hospital Foundation 00335-0505-02 (Ley), and by U54 HG003079 (Wilson).
Role of the sponsor: The funding organizations had no role in design and conduct of the study; collection, management, analysis and interpretation of the data; and preparation, review, or approval of the manuscript.
Additional Contributions: We thank the Washington University Cancer Genomics Initiative for their support, and Drs. Charles W. Caldwell and Carl Freter for referring the patient, and for collecting, storing and contributing the cryopreserved bone marrow cells used for this study.
Financial disclosures: Dr. Westervelt has received lecture fees from Celgene and Novartis; Dr. DiPersio has received consulting and lecture fees from Genzyme.
Author Contributions: Drs. Ding and Larson, had full access to all of the sequence data and take responsibility for the integrity of the sequence and the accuracy of the sequence analysis.Study concept and design: Welch, Westervelt, Ding, Larson, Klco, Watson, Tomasson, Link, Graubert, DiPersio, Mardis, Ley, and Wilson.
Analysis and interpretation of data: Welch, Westervelt, Ding, Larson, Klco, Kulkarni, Wallis, Chen, Payton, Fulton, Veizer, Schmidt, Vickery, Mardis, Ley, and Wilson.
Drafting of the manuscript: Welch, Westervelt, and Ley.
Critical revision of the manuscript for important intellectual content: Welch, Westervelt, Ding, Larson, Klco, Heath, Ley, Link, and Wilson.
Statistical and sequence analysis: Ding, Larson, Wallis, Chen, Schmidt, Vickery, Mardis, Ley, and Wilson.
Obtained funding: Welch, Westervelt, Watson, Link, Graubert, Tomasson, DiPersio, Mardis, Ley, and Wilson.
Administrative, technical, or material support: Kulkarni, Payton, Fulton, Heath, Veizer, Schmidt, Vickery, Watson.