Circulating tumor cell free DNA from plasma and urine in the clinical management of colorectal cancer

2.1 Study design and patient population

A total of 150 patients recruited were examined. All patients provided informed consent to be part of the study. The institutional review board (IRB) approved all procedures and ensured ethical compliance. Blood and urine were collected on the same day to prevent discrepancy. Patients were selected randomly before mutational profiling. The inclusion criteria were patients with advanced stage of colorectal cancer and all had multiple metastases with intact primary tumor in the colon. Details of patients’ characteristics are provided in Table 1. As part of the study, serial samplings were taken at different time points from each participant. A total of 30 healthy volunteers participated to provide healthy reference measurements. All healthy volunteers were of certain disease free and had no history of cancer. Serial sampling was performed on both patients and healthy donors at monthly intervals.

Baseline cell free DNA from plasma and urine specimens was also recovered from these volunteers. Mean age of patients and healthy cohorts were 53 and 52 years old respectively. For all 180 subjects of the study, basic clinicopathological factors are shown in Table 1. Including all serial measurements, a total of 1,080 plasma and urine samples were processed. Molecular profiling was performed for both KRAS and BRAF mutations. Patient survivals were subsequently followed up and correlated to cell free DNA measurements from urine and plasma. At baseline profiles, tumor tissue biopsies indicated 72 KRAS and 38 BRAF positive colorectal cancer patients. The rest were of wildtype characteristics. We performed baseline comparisons with healthy volunteer’s blood and urine specimens, assuming all were of wildtype characteristics.

2.2 Liquid biopsy sample collections and initial purifications

Peripheral blood was collected in a 10 ml K2 EDTA blood tube. For urine, the first morning urine was collected. 40–50 ml of urine specimen was taken and transferred to a 50 ml sterile centrifuge tube. Both specimens were processed within 3 hrs. For blood sample pre-processing, plasma was extracted via direct centrifugation at 4 ∘ C. This was performed using a two-step process. Blood cells were separated from plasma by centrifugation at 1,500 g for 10 mins at 4 ∘ C. A repeat of centrifugation for 15 mins at 2,000 g (4 ∘ C) ensured that platelets were removed. 5 ml of plasma was then transferred to a new sterile tube. For urine processing, samples were centrifuged at 2,000 g for 15 mins at 4 ∘ C twice. The first step separated out possible debris and the second centrifuge step ensured the removal of any possible remaining contaminants. 10 ml of urine was recovered from this process. Cell free DNA was then recovered from the plasma and urine specimen before storing genetic materials at - 20 ∘ C.

2.3 Profiling of plasma and urine specimens

Cell free DNA was immediately purified fromplasma and urine specimens as it may degrade with prolong storage in the media. To purify nucleic acid, the Qiagen’s QIAmp Circulating Nucleic Acid Kit (Qiagen Inc, USA) was used. The processes used spin columns coupled with multiple centrifugation steps and followed manufacturer’s instructions. Sample was eluted in TE buffer (20 ul) and measured on the Nanodrop 2000 (ThermoScientific Inc., USA). All purified DNA specimens were then stored at 20 ∘ C. DNA profiling was batched process using digital droplet PCR (ddPCR) as the technique is sensitive to pick up low frequency mutations. Briefly, reactions were prepared fresh as recommended. The PCR reactions were 20 ul each consisting of the mastermix, primers/probes and DNA template. Internal positive and negative controls were also included in each reaction plate to ensure assay validity. Primers and probes for KRAS and BRAF were procured from PrimePCR™ ddPCR™ Mutation Detection Assay Kit (Bio-Rad Inc, USA). Mutations were addressed for KRAS on 3 specific sites on chromosome 12 (c.38G $>$ A; c.35G $>$ T; c.35G $>$ A; c.35G $>$ C; c.34G $>$ A; c.34G $>$ T) and BRAF for p.V600E. Details of the mutations and assays used are provided in supplementary Table 1. PCR thermocycling conditions were as follows

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95 ∘ C, 10 minutes for one cycle;

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94 ∘ C, 30 s and 55 ∘ C, 1 minute for 40 cycles;

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98 ∘ C, 10 minutes for one cycle. Samples were held at 4 ∘ C.

Post PCR, the reaction plates were analyzed on the QX200 droplet reader (Bio-Rad Inc., USA) using the QuantaSoft software included.

3. Statistical analysis

Normality testing using the Shapiro-Wilk test was performed on recovered DNA concentrations from both urine and plasma specimens. Agreement between matched tumor genetic results were compiled to address assay sensitivity and specificity. Correlations between healthy volunteers at reference measurements were performed using Student t test. All data presented were expressed as mean and standard deviation unless otherwise specified. Serial measurements were tested using analysis of variance (ANOVA). Analyses of survival outcomes were performed using Kaplan Meier (KM) estimate. The hazard ratio (HR) between different group of patients was computed to assess the clinical utility of cell free DNA. All statistical analyses were deemed significant if 0.05. All computations and analyses were made with Prism (GraphPad, USA) or Microsoft Excel (Microsoft Inc, USA).

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5. Discussion

In the current comparative analysis of paired plasma and urine samples, we observed several interesting trends and correlations for colorectal cancer patients. The need for alternative tumor material sampling other than tumor tissue biopsy arises as continual monitoring of cancer patients will better aid in their clinical management. Plasma and urine are attractive as specimen collections are relatively straightforward and more acceptable to patients. In the current study addressing advanced stage colorectal cancer, where access to tumor tissue is challenging, these assays provide alternative strategies to profile the disease.

To ascertain relevance of cell free DNA in plasma and urine to the primary tumor, molecular characterization using KRAS and BRAF mutations was investigated. These mutations typically reflect poor prognosis for colorectal cancer patients [19] and are critical for clinical management. For healthy volunteers, we confirmed all were wildtype for KRAS and BRAF using cell free DNA derived from either plasma or urine. This is similarly true for colorectal cancer patients with wildtype KRAS or BRAF detected by tumor biopsies. These demonstrated good specificities in both urine and plasma tests. Majority of patients with either KRAS or BRAF mutations were correctly identified and no significant differences were observed between the concordance rates in KRAS or BRAF detection using cell free DNA. Collectively, plasma cell free DNA had a marginally higher concordance rates with matched tumor biopsy results than urine cell free DNA as shown in Fig. 1. Our results are largely coherent with other studies that investigated the use of plasma cell free DNA [20, 21, 22]. Our results demonstrate much better concordance than the results shown by Kuo et al. [20], who observed than the agreement rate was 78.8% for KRAS mutations. This is likely due to the differences in patient cohort where in our case all patients were in advanced stages. In newer technologies presented by Powell et al. [22], better clinical sensitivities were shown and with the advent of new assays, we expect the efficacy of the assay to improve. In additional, our results demonstrate that urine is also a viable source for accurate detection of key driver mutations of the disease. In direct comparison of both assays in this study, we detected a good correlation among both assays. Using a Spearman’s correlation, we noted that the levels of recovered nucleic acid were similar in both cases. Our analyses also revealed that this is irrespective of the presence of mutations as the correlation coefficients in the wildtype study group was similar to the patient group that had KRAS or BRAF mutations.

Levels of recovered cell free DNA for different study groups highlighted a distinctive pattern for patients with KRAS or BRAF mutations. Both urine and plasma cell free DNA analyses showed similar trends. Mean recovered concentrations of cell free DNA were much higher than patients with wildtype characteristics or healthy volunteers. Given that patients with KRAS or BRAF mutations tended to have poor prognosis, we postulate that the levels of cell free DNA may be related to clinical outcome as well. Overall, we observed that mean recovered cell free DNA from urine and plasma were much higher in colorectal cancer patients, coherent with prior studies [23, 24] performed on plasma samples. A clear advantage of liquid biopsy via urine and plasma is the ability for serial monitoring of patients as shown in Fig. 3. Normalizing by the baseline reference levels obtained with either the plasma or urine specimen, we observed in a span of 5 months a monotonic increasing level of cell free DNA in the patient cohort. Conversely, healthy volunteers’ measurements showed no significant changes as established using a repeated measure ANOVA test. To address if the changes in cell free DNA significantly impact the prognosis of these patients, we split the patient group equally based on an ascending ranking of recovered cell free DNA and performed a KM analysis. This was tracked over 2 years with regular follow up calls or visits. In both cases using either plasma or urine cell free DNA, the results favored patients with lower cell free DNA during the monitoring period. A direct comparison between urine and plasma cell free DNA however showed that the HR was higher in the case for patient stratifications using urine cell free DNA. Potentially, both plasma and urine can be good candidates to aid in long term monitoring of colorectal cancer patients.

Our study showed highly feasible uses of cell free DNA derived from urine and plasma samples. This has clear advantage over classical tumor biopsy. A major limitation of the assay is the small numbers of false negative results in the patient group with mutations. This is likely due to low concentrations of mutant DNA masked in background normal DNA [25, 26, 27] as observed in other studies. It may not be entirely feasible to replace tumor biopsy at this juncture until more sensitive molecular techniques can be applied but plasma and urine cell free DNA serves well to complement existing disease management strategies. The study by Thomsen et al. [27] also showed insignificant correlative analysis to treatment outcome, which demonstrates that these assays require much more validation studies before clinical adoption. We noted that serial monitoring of patients can be beneficial for better risk stratifications and possibly other forms of cancer detection such as circulating tumor cells (CTCs) can further aid to refine the process. CTCs from peripheral blood may work better as background blood cells can be effectively removed [28]. Future studies may focus on implementing other companion tests to enhance the detection of these key driver mutations for colorectal cancer. In the current study, we addressed common mutations in both KRAS and BRAF that are linked to treatment efficacy. Given the wide spectrum of mutations present in these genes, it is of value for a large scale genomic analysis to address disease heterogeneity. This would be valuable to better utilize cell free DNA in colorectal cancer management. Additionally, in earlier concordance analysis (Fig. 1A), we observed different sensitivities associated with KRAS and BRAF testing. Further studies to compare the sensitivities with larger sample sizes or using both assays in tandem will be useful before the adoption into wider clinical practice.

6. Conclusions

Our data established clear and effective use of cell free DNA from either plasma or urine specimens for colorectal cancer patients. The results showed highly concordant data for key driver mutations found on tumor tissues and cell free DNA form urine and plasma. A direct comparison of urine and plasma cell free DNA showed relatively similar trends in serial monitoring of patients. This potentially has clinical utility to address current shortfalls for sensitive monitoring of colorectal cancer patients during disease progression.