CAPP-Seq

Peripheral blood is collected from patients and ctDNA is isolated from ≥1 mL of plasma. Input DNA can be as low as 4 ng.[1]

There were four main goals in adapting this protocol for ctDNA work:

• 1) to optimize the adapter ligation efficiency
• 2) to reduce the number of PCR cycles needed after ligation
• 3) to preserve the naturally occurring size distribution of ctDNA (median 170 base pairs)
• 4) to minimize the variability in depth of sequence coverage across all the captured regions

These were achieved by allowing adaptor ligation to be carried out at 16°C for 16 hours to increase adaptor ligation efficiency and recovery. The most important adaptation is during enzymatic and clean-up steps; they are performed with-bead, in order to minimize tube transfer steps which increases recovery.

In CAPP-Seq, design of selector is a crucial step that identifies recurrent mutations in a particular cancer type using publicly available next generation sequencing data. For inclusion in CAPP-seq selector, the recurrent mutations that are enriched in a population is described by an index- Recurrence Index (RI). RI is the number of mutations per kilobase of a given genomic locus of a patient carrying particular mutations. RI represents a patient level recurrence frequency estimated for somatic mutations and all mutations. Known and driver recurrent mutations in a population can be ranked based on the RI and therefore RI is used to design a selector. A six phase design strategy is employed to design selector.[1]

• Phase-1: Identifying frequently mutated known driver mutations using the publicly available data.
• Phase-2: Maximum coverage of SNVs among the patients was identified by ranking their exonic RI.
• Phase-3 and 4: Exons with higher RI were selected.
• Phase-5: Addition of previously predicted driver mutations.
• Phase-6: Addition of recurrent gene fusions rearrangement that are specific for particular cancer.

Human cancer is heterogeneous and recurrent cancer mutations are present only a minority of patient. Therefore, a careful and non-redundant design of selector is the vital part in CAPP-Seq and also the size of the selector is related to its downstream costs.

Figure 02: Hybridization capture protocol used in CAPP-Seq.

Hybridization capture with the selector probe set is performed on tumor DNA from a biopsy and sequenced to a depth of ~10,000× coverage. The biotinylated selector probes bind selectively to the regions of the DNA library that were chosen to be where the recurrently mutations occur in the given cancer type. In this way you are left with a smaller library that is enriched for only the regions you want, which can then be sequenced. This allows the determination of patient specific mutations. Hybridization capture with the same selector is then performed on ctDNA from the blood to quantify the previously identified mutations in the patient. CAPP-Seq can be applied to ctDNA from multiple blood samples at different time points in order to follow tumor evolution.

When treating cancer it is useful to have precise measurements of the total body disease burden. It helps with determining prognostic significance and treatment response. This is normally done using computed tomography (CT scans), positron emission tomography (PET scans) or magnetic resonance imaging (MRI).[16] These medical imaging procedures are expensive and are not without their own problems. These imaging techniques are not able to accurately resolve small tumors (≤1 cm in diameter).[14] Imaging can also be affected by radiation-induced inflammation and fibrotic changes, making it hard to determine if there is residual tumor or just effects of treatment.[1]

It has been found that levels of ctDNA in plasma significantly correlate with tumor volume as compared with medical imaging (CT, PET and MRI).,[1][14][17][18] Detection of ctDNA can predict residual tumor or imminent relapse, in some cases even better than medical imaging and current methods.

Detection of ctDNA has been found to be a predictor of relapse in multiple studies thus far. In a study [14] in late-stage NSCLC (non-small cell lung cancer) they found two cases where ctDNA correctly determined the outcome of a patient when medical imaging was wrong. In one case, the imaging predicted relapse based on a suspected residual tumor which turned out to only be radiation-induced inflammation, but ctDNA was not detected and the patient did not relapse. In another case, the imaging showed no tumor but ctDNA was detected and the patient relapsed shortly afterward. In another study [18] on DLBCL (diffuse large B cell lymphoma), ctDNA was also found to be predictive of relapse.

Biopsies are invasive and associated with risks to the patient. Therefore, multiple biopsies to monitor disease progression are rare and diagnostic biopsies are relied on for genetic information. This can be problematic because of tumor heterogeneity and tumor evolution. Firstly, biopsies only sample one portion of the tumor, and because tumors are heterogeneous, this will not cover the full genetic landscape of the tumor. Secondly, after treatment tumors evolve and there may be new mutations not represented in the diagnostic sample.[1][14]

Biopsy-free tumor genotyping, by way of CAPP-Seq and ctDNA, addresses many of these issues. A simple blood test is non-invasive and much safer and easier to subject cancer patients to multiple times through the course of treatment. Using ctDNA gives a better sample of tumor DNA compared to a single area of a tumor collected in a biopsy, allowing a better estimate of tumor heterogeneity. Taking multiple samples of ctDNA at different time points following the course of treatment allows tumor evolution to be uncovered. This can help detect the emergence of mutations that confer resistance to a targeted therapy and allow the course of treatment to be adjusted accordingly. CAPP-Seq specifically allows for the screening of multiple genomic locations which will become important as the list of cancer mutations important for treatment continues to grow.[14] In a study[1] for late stage NSCLC, they performed a version of CAPP-Seq where the tumor biopsy was not sequenced first, and they were able to correctly classify 100% of patient plasma samples with a 0% false positive rate. This shows that even without previous knowledge of tumor mutations, they can be accurately discovered by ctDNA alone.