Our results show that mutations can be detected at a level of ~1% in a background of wild type BCR-ABL1 making it a useful tool for screening of both high and low level kinase domain mutations. Further, it provides information on the clonal distribution of mutations as well as BCR-ABL1 isoforms in a single assay. This feature is of major clinical relevance as compound mutations show different resistance profiles compared to individual mutants [5]. Standard Sanger sequencing methods routinely used in diagnostic laboratories are unable to distinguish between independent or compound mutations. Until now, this information has only been available through time consuming cloning experiments [5], underlining the potential clinical utility of our assay.
Although recent reports showing MPS-approaches are emerging [8,9], its usefulness in establishing the clonality of mutations has recently been debated. In a recent report, Parker et al. [13] showed that compound mutations detected by MPS technologies might actually be artifacts due to PCR-mediated recombination. However, our assay has a somewhat different setup compared to previous studies. Instead of performing a two round nested PCR, as is required for shorter read technologies, the fusion transcripts were amplified in one single round. This could potentially reduce the rate of PCR recombination. We were able to evaluate the degree of in vitro artifacts in our experiments. For example, in the 49-month sample from patient 3, 91.2% of the reads contained only T315I and 4.2% of the reads contained only F359C (see Figure 4B). In the same sample 0.1% of reads show the presence of both T315I and F359C. These results suggest that the recombination rate in this particular case is very low, well below the frequency of the individual clones. However, since the rate of chimeric reads can be influenced by the experimental conditions and may vary between samples, a more thorough investigation would be required to validate the PCR recombination rate under different circumstances.
On the practical level, the PacBio assay allows for a simple, efficient and streamlined workflow conducive to clinical routine. Our laboratory workflow provides a quick turnaround time of approximately two days, encompassing all steps from RNA isolation to report generation. A simple library preparation procedure, rapid sequencing and straight forward bioinformatics analysis enable this efficient workflow. The library preparation is performed during one day and the sequencing run takes approximately 2–3 hours per sample. Under the current set up, PacBio sequencing is more expensive compared to the more traditional Sanger sequencing and RQ-PCR based assays. However, the cost for sequencing of small target regions such as the BCR-ABL1 transcript is comparable to that of other available MPS technologies. In the present study, we obtained 10,000X coverage of BCR-ABL1 for each of the samples. In light of this rather extensive coverage, it is likely that a similar sensitivity for mutation detection could to be obtained when utilizing a reduced coverage, thus opening up the possibility of barcoding of two or more samples on one SMRT cell. Further, due to the continuous improvements of the PacBio system in terms of quality, read length and throughput, the potential for multiplexing is likely to increase, thus leading to substantial reductions in experimental cost.
This study presents a proof of principle for detection of BCR-ABL1 mutations and our results are based on just a handful of patients, limiting the generality of our conclusions. Nevertheless the analysis of individual patient samples illustrates important aspects and strengths of our approach. One main advantage is the sensitivity of the assay, as illustrated in one of the patients (patient 1) where we could detect the T315I mutation four months earlier than detected by Sanger sequencing. These results indicate that an NGS-screen could be informative when performed at earlier time points, possibly in patients with no or limited responses to TKI therapy already at the three months control. Further studies are needed to specifically address this question.
The sensitivity of the method can also be instrumental in excluding other BCR-ABL1 mutations as responsible factors for the observed TKI resistance. For example, in patient 2, we could only detect the T315I and despite an initial molecular response to ponatinib, the patient remains with a minor molecular response. Thus in this case BCR-ABL1-independent factors might explain the failed therapy. This information is of particular importance when looking for alternative TKI-resistance pathways.
The ability to discern between independent and compound mutations is a major advantage of this assay. For example, patient 3 carried both the T315I and F359C mutations, but present in independent clones. A recent study has shown that the compound mutation F359C and T315I is associated with in vitro profiles implicating mutant pairing of these two positions in moderate and high-level resistance to ponatinib and rebastinib, respectively [5], while the individual mutants are instead sensitive to these substances. Thus, we can speculate that the molecular response observed in this case upon ponatinib treatment, can be explained by the fact that these mutations are located in different molecules in this patient. In contrast, the T315I positive clone (84%) acquired independently two extra mutations (D276G and H396R) upon ponatinib treatment. These low frequency compound sub clones did not seem to impede the molecular response (MR5) attained after 3 years of treatment. The results are somehow conflicting with recent results showing that the compound mutant H396R/T315I has an intermediate to high resistance in vitro profile with an IC50 for ponatinib of 90.8 ± 24.7 nM compared to an IC50 of 20.1 ± 3.5 (H396R) and 29.1 ± 7.8 nM (T315I) for the individual mutants [5]. However, in vitro sensitivity does not necessarily always correlate with the clinical response to treatment.
Similarly, patient 5, harboring the non-compound Y253H/E255V mutations, would clearly benefit today from sensitive examination of the clonal composition of these two mutations prior to therapy decisions because of the high ponatinib-resistance of the Y253H/E255V compound mutant [5]. The fact that in this patient the mutations occur in different molecules would remain indiscernible by routine Sanger sequencing.
Another advantage of our approach is that as we amplify and sequence almost the entire BCR-ABL1 p210 transcript we are able to identify transcript isoforms. We identified elevated levels of transcripts isoforms in two of the patients. Among the variants that we detected, 35INS is best studied in the literature. Some studies have described 35INS as a possible mechanism of imatinib resistance [14,15], while biochemical data from a separate study shows that it does not contribute to TKI resistance in vitro [16]. Although there is at present no basis for taking 35INS into consideration for treatment decisions, these conflicting reports highlight the need for routine screening in CML patients in order to gain more knowledge. Our results suggest that the PacBio assay can be used for screening of 35INS as well as other splice isoforms down to a frequency of at least 5%. Furthermore, it enables simultaneous detection of multiple different alternative isoforms present in a single sample. These results corroborate previous findings that propose alternative splicing as a common mechanism among CML patients undergoing TKI treatment [14,17]. However, to clarify the role of transcript isoforms in drug resistance and response a larger number of samples should have to be analyzed and our approach might simplify this kind of analysis.
Although this study is focused on BCR-ABL1 the same method can be readily applied to the analysis of other cancer-associated fusion transcripts, providing not only information on the clonal distribution of mutations but also on isoform frequencies. Isoform analysis is to this date not performed routinely on CML patient samples and therefore the knowledge on their impact on disease progression and treatment efficiency is very limited.