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Original Article

Prognostic utility of transrenal DNA for colorectal

cancer patients with bone metastases

Jiguang Jia1*, Zhengling Zhuang1*, Bin Huang1, Jiangtao Liu1, Sen Chen2

1Department of Orthopedics, Xiangyang No.1 People’s Hospital, Hubei University of Medicine, China;2Department

of Orthopedics, Renmin Hospital of Wuhan University, China.*Equal contributors.

Received June 15, 2017; Accepted December 22, 2017; Epub January 15, 2018; Published January 30, 2018

Abstract: Purpose: The current study aims to address disease monitoring and prognosis for metastatic colorectal cancer (CRC) patients with bone metastasis using transrenal DNA. Methods: Transrenal DNA was isolated from urine specimens of CRC patients and matched to the tumor tissue profiles to gauge clinical relevance. KRAS muta -tions were analyzed in these samples. Monitoring of transrenal DNA quantity and KRAS muta-tions were performed at monthly intervals to track its variations between patients with or without bone metastases. For prognosis as-sessment, the Cox proportional hazards model was performed for all CRC patients and subsequently on patient subgroups with and without bone metastases. Results: The agreement in KRAS mutations between transrenal DNA and tumor tissues in CRC patients was more than 90%. Transrenal DNA quantities were significantly higher in CRC patients with bone metastases. In this patient cohort, quantities of transrenal DNA increased 2.58 folds during subsequent monitoring. Prognosis was poor for CRC patients who had bone metastases and higher transrenal DNA quantities. Hazard ratio was determined to be 2.11 (95% CI 1.58 to 4.04). Conclusion: Taken together, this demon-strated the usefulness of transrenal DNA for disease monitoring and prognosis of CRC patients with bone metas-tases. Our results showed that transrenal DNA carries KRAS mutations similar to tumor tissues and its quantity in urine specimen can be potentially useful to identify high-risk patients. This can lead to better disease management or tailored treatment.

Keywords: KRAS, colorectal cancer (CRC), disease monitoring, liquid biopsy, transrenal DNA

Introduction

Colorectal cancer (CRC) is one of the major dis-eases that affect millions worldwide [1]. Metastatic CRC is also a leading cause of death [2], and efforts have been placed on its early detection, at the stage where the disease is localized. Furthermore, investigating the epide-miology of the disease is challenging as CRC morphs quickly [3]. The most critical factors that contribute to disease relapse and survival are the extent of the cancer, lymph nodes involvement and its cellular differentiation. Metastatic spread in CRC has been extensively studied and bone metastasis is one common site accounting for 6-10% of the disease popu-lation [4]. The symptoms can be life threatening and are usually accompanied by considerable pain and may interfere with normal physical activities [5].

More importantly, the clinical outcomes for CRC patients with bone metastases are generally

not encouraging [6]. Studies have shown that these patients have a poor prognosis [6] and metastatic lesions are usually abundant. It also invades the axial skeleton and proximal seg-ments of the limbs [4]. With the progress in can-cer treatments [7], patients have a wider selec-tion of therapeutic opselec-tions. It is imperative that close monitoring is needed to accurately profile the disease. Current methods for monitoring rely heavily on diagnostic imaging. Its primary purpose includes measuring the extent of sec-ondary lesions and to evaluate treatment response [8]. Although these techniques are useful, it does not provide real-time disease updates to the molecular changes that can affect treatment efficacy. The molecular signa-tures are also important as many new treat-ments target unique mutations associated with the disease [9].

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that resides in peripheral blood of cancer patients as a result of necrotic and apoptotic tumor cells [10] show good CRC correlations [11]. This has been performed in longitudinal studies to understand the pathogenesis of the disease [12, 13]. Siravegna et al. for instance tracked ctDNA in patients for use in genotyping and monitoring the molecular evolution of CRC. Patients involved in the trial were undergoing treatment with cetuximab and panitumumab that targets the epidermal growth factor recep-tor (EGFR). The dynamics of the disease was revealed and key genetic aberrations like KRAS mutations were highly correlative of treatment response [13]. The major drawback of the tech-nique is the continual need for blood sampling of considerable volume, which limits the extent and frequency for measuring these events. Interestingly, ctDNA has been found within urine specimens as well [14]. Within the urinary system, ctDNA ends up in urine by diffusing

[image:2.612.89.341.81.469.2]

People’s Hospital or were patients who were transferred from other primary or secondary healthcare institutions. Participants of the trial provided informed consent, which was taken by their respective consulting doctor. The institu-tional review board (IRB) approved all patient recruitment procedures and sample recovery processes. Among the 150 CRC patients, 100 had confirmed bone metastases, established using magnetic resonance imaging (MRI) analy-sis. Details of the CRC patients are summarized in Table 1. The remaining 50 patients without bone metastases were recruited as the experi-mental control and reference. Tissue biopsies used for profiling were mainly extracted by colo-noscopy. The median age of the study cohort was 54 years old. The gender ratio was 1.08 (male/female). The baseline reference mea-surements were taken before treatment com-mencement. Serial urine samplings for transre-nal DNA atransre-nalysis were taken monthly for six

Table 1. CRC patients and their characteristics

Description N=150

Age

CRC Patients with Bone Metastases

Mean 53 (45-68)

CRC Patients without Bone Metastases

Mean 56 (46-69)

Gender

CRC Patients with Bone Metastases

Male 53 (53%)

Female 47 (47%)

CRC Patients without Bone Metastases

Male 25 (50%)

Female 25 (50%)

PS at inclusion

0 63 (42%)

1 75 (50%)

2 9 (6%)

ND 3 (2%)

Locus of primary

Colon 97 (65%)

Rectum 49 (33%)

ND 4 (3%)

Molecular Profile (Tissue)

CRC Patients with Bone Metastases

Wildtype 37 (25%)

KRAS 63 (42%)

CRC Patients without Bone Metastases

Wildtype 25 (17%)

KRAS 25 (17%)

through the kidney barrier during the waste removal process [15]. We hypothesize that the information that can be gathered will have practical clinical applications. Performing dis-ease related testing in urine samples is appealing, as sample collection is non-invasive. It also exists in abun-dance to allow frequent collections and measurements.

In this current work, we aim to estab-lish the clinical relevance of transre-nal DNA from urine. Specifically, CRC patients that suffer from bone metas-tases were examined. We hypothesize that this will provide a sensitive cap-ture of the disease dynamics that may aid in CRC management and progno-sis. Serial specimen extractions at monthly intervals were conducted to gauge the variability in transrenal DNA. This is correlated directly to the overall survival of different patient groups, and measures the clinical sig-nificance of transrenal DNA in CRC.

Materials and methods

Colorectal patients characteristics and data collection

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months consecutively. Comparisons were made with matched tumor profiles for KRAS mutation and transrenal DNA quantity varia-tions were compared during treatment.

Specimen retrieval and transrenal DNA purifi-cation

Urine specimens were collected from each patient using the following criteria to ensure consistency. Participants collected approxi-mately 50 ml of the midstream morning urine into a container with 0.5 M EDTA, pH 8.0. To ensure the integrity of the transrenal DNA, each sample was processed within 2 hours of collec-tion. Retrieval of cell-free nucleic acids was done by centrifugation to remove all contami-nating debris. A two-step centrifugation pro-cess was undertaken. The first centrifugation step was performed at 10,000 g for 15 minutes at 4°C and the supernatant carefully trans-ferred to a new centrifuge tube. The second centrifugation step was performed at 10,000 g for 10 minutes at 4°C to ensure remaining contaminants were removed. For DNA purifica-tion, Qiagen’s QIAmp Circulating Nuclei Acid Kit (Qiagen Inc, USA) was used in accordance with the instructions by the manufacturer. 16 ml of each urine sample was processed and eluate of 20 μl in Tris-EDTA, pH 8.0 was recovered. These samples are then quantified using the Nanodrop 1000 (ThermoScientific, USA) and stored at -20°C before molecular analysis.

Molecular profiling of transrenal DNA using digital droplet PCR

Digital droplet PCR (ddPCR) was used to per-form the KRAS molecular testing in the CRC patients. This technique is capable of detecting low-frequency mutations and has demonstrat-ed its usefulness in various ctDNA detections [16]. Purified DNA from each urine specimen was processed through the QX100 ddPCR sys-tem (Bio-Rad Lab Inc., USA) using the ddPCR KRAS G12/G13 Screening kit (Bio-Rad Inc. USA). The laboratory procedures strictly fol-lowed manufacturer’s recommendations. Brief- ly, reaction master mixes and DNA templates were prepared in 20 μl reactions. PCR settings were as follows:

1. 95°C, 10 minutes; 2. 94°C, 30 s and 55°C, 1 minute (40 cycles); 3. 98°C, 10 minutes. Sam- ple plate was held at 4°C thereafter.

For data analysis, each sample batch was eval-uated using the QuantaSoft software (Version 1.6, Bio-Rad Lab Inc., USA).

Statistical analysis

[image:3.612.90.285.71.243.2]

The concordance between tumor tissue and transrenal DNA for KRAS mutations was tabu-lated to determine the clinical significance of transrenal DNA analysis. The Cohen’s Kappa that measures inter rater agreement was also computed. We compared the quantity of tran-srenal DNA among CRC patients using an unpaired Student t test. Patients in different groups were closely matched for demographic and other clinical parameters (such as age, gender and ratio of KRAS positive patients) as shown in Table 1. To ascertain the variability of transrenal DNA content in different patient groups, we computed the coefficient of varia-tion (CV) among them. A repeated measure analysis of variance (ANOVA) was performed on the serial measurements of transrenal DNA quantities. To ascertain clinical relevance, Ka- plan-Meier (KM) analysis was done by following up with patients and hazard ratios (HR) was achieved by the Cox proportional hazards regression. For all statistical computations, Microsoft Excel (Microsoft Inc, USA) or the Prism statistical software (GraphPad, USA) was used. In all tests, a p-value less than 0.05 was considered statistically significant.

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Results

Experimental trial and assay validation

The prevalence of bone metastases in CRC is significant. Its incidence rate ranges from 6-10%, with median detection time approxi-mately 11-21 months after primary tumor resection [4]. More importantly, bone metasta-ses patients were also linked to lung and/or liver metastases [17]. This was consistent in the current trial, with more than 95% of the patients showing these secondary metastases. Studies have suggested that these patients have poor survival, and better clinical monitor-ing of this patient group can aid in their treat-ment selection to improve outcome. Transrenal DNA offers a relatively pain-free method for tumor material sampling and we hypothesize it can aid in the disease management of this group of advance stage CRC patients. We aim to measure the variations within transrenal DNA and ascertain its clinical relevance. Besides quantifying the nucleic acid content in each urine specimen, we profiled the samples for KRAS mutations as well. This provided direct evidence of the clinical relevance of transrenal DNA in CRC. Using ddPCR, we included spiked conditions of 1000, 100 and 10 copies of mutant KRAS plasmids. It is important that the detection assay is sensitive to pick up low copy numbers of mutant DNA, as it is expected that clinical samples will be of low frequencies as

well [18]. Figure 1 shows the result of the detec-tion assay. Each data point was independently repeated six times and the data is shown in Supplementary Table 1. The results demon-strated good linearity within these input condi-tions and r2 for the assay performance test was 0.978.

Transrenal DNA at baseline and agreement between tumor tissue for CRC

[image:4.612.94.521.73.269.2]

Baseline transrenal DNA content was mea-sured in each CRC patient prior to treatment and the results are shown in Figure 2A. This is to compare if patients with bone metastases had different transrenal DNA quantity. At base-line, it was observed that the mean transrenal DNA recovered from CRC patients with bone metastases was 2.00 folds higher than patients without bone metastases. An unpaired Student t test for one tail showed that this was statisti-cally significant (p-value < 0.05). Measurements of transrenal DNA content clearly showed a good separation of the patients, which might have further clinical implications. Comparisons were also made for the KRAS gene profile among CRC patients as illustrated in Figure 2B. This provided direct associative links of transre-nal DNA to CRC, and the results were matched with the corresponding tumor biopsy KRAS pro-files. Overall concordance between transrenal DNA and tumor tissue profiling was 89.3% (κ = 0.788; 95% CI 0.689 to 0.886) for all patients. A further breakdown of the different patient

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subgroups yielded an agreement of 87% and 94% for CRC patients with and without bone metastases respectively. It is interesting to note that for all patients with wildtype KRAS, the concordance rate was 100%. To gauge the

[image:5.612.90.375.71.541.2]

itoring was unaffected. This also ascertained the validity of the assay for use in CRC patients with bone metastases. We noted that the increasing trend could indicate changes to the disease and performed a case study using

Figure 3. Variations in transrenal DNA measurements with different patients during serial monitoring. A. A monotonic increasing trend was observed with CRC patients with bone metastases. CVs remains relatively constant throughout. B. Case report of one CRC patient with bone metastases at base-line and after 6 months. Variations in transrenal DNA was more than dou-bled comparing with baseline results. C. Transrenal DNA trend within CRC patients without bone metastases. CVs across different time points were relatively constant for the patients as well. D. Discordant CRC patient group with KRAS+ detected using tissue profiling during serial monitoring. Intermit -tent positive signatures were detected using ddPCR. Overall concordance inclusive of this group increases to over 90%.

inter-patient variability for dif-ferent groups, the CVs were computed. The overall CV for CRC patients with bone me- tastases was 13.4%. In con-trast, the CV for patients with-out bone metastases was 32.0%. A lower dispersion was observed with the former, indicating that transrenal DNA may be a good measurement parameter for this patient group. Taken together, the ba- seline transrenal analysis sh- owed good agreement to the tissue profiles, which indicat-ed a strong disease associa-tion. Variability measuremen- ts showed promising out-comes among different CRC patient groups to further use transrenal DNA for prognosis and disease monitoring.

Serial sampling importance to capture the dynamic changes in CRC

The attractiveness of transre-nal DNA atransre-nalysis is the ease of specimen collection. In subsequent serial measure-ments, the stability and vari-ability of the assay were assessed. Patients were mon-itored for a total of six months where possible. Several inter-esting trends were observed. For CRC patients with bone meta- stases, we detected a clear monotonic increasing trend for mean transrenal DNA qu- antity over time as shown in

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mon-imaging modalities as depicted in Figure 3B. This MRI scan showed a worsening condition and correspondingly a higher measured tran-srenal DNA quantity of 2.58 folds was regis-tered compared with the baseline measure-ment. For the other patient group without bone metastases, the mean transrenal DNA quanti-ties showed a less pronounced increase over time (Figure 3C). Comparing with baseline mea-surements, the maximum increase in mean transrenal DNA quantities was 1.23 folds. The repeated measures ANOVA analysis showed significant differences within the serial mea-surements but mean DNA quantity showed alternating trends. In the discordant cases detected at baseline, we noted several patients with positive detection during the six months monitoring period as shown in Figure 3D. A common trend that coexisted among them is the low concentration of detection mutant KRAS DNA using ddPCR. The positive

identifica-determined to be 1.17 (95% CI 0.83 to 1.74) as shown in Figure 4B. Next, we subdivided the analysis for CRC patients with and without bone metastases. Figure 4C shows the KM analysis using a median spilt of patients with bone metastases. The median survival for the patient cohort with higher DNA quantity was 4 months lesser. Using Cox regression, the hazard ratio was determined to be 2.12 (95% CI 1.58 to 4.04) with a p-value less than 0.05. With the same criteria for patient stratification, we ana-lyzed the results of CRC patients without bone metastases. The hazard ratio was 1.90 (95% CI 1.18 to 4.94) with a p-value of 0.03 as shown in

[image:6.612.92.377.73.321.2]

Figure 4D. The median survival of the patients’ cohort with higher transrenal DNA content was 4 months lesser. The results demonstrated the usefulness of transrenal DNA profiling within CRC patients and the ability to identify patients with a potentially worse outcome.

Figure 4. KM analysis of the CRC patient cohort using transrenal DNA se -rial measurements. Patients were ranked in accordance to their maximum detected transrenal DNA quantity and a split of the cohort using the median value into two equal groups was used to compare their survival status. A. Maximum transrenal DNA quantity detected for the six-month monitoring pe-riod for each of the patients and ranked in descending order. B. Comparison of all CRC patients using a median split for maximal transrenal DNA quan-tity detected within a six month period. C. Comparison of CRC patients with bone metastases using a median split for maximal transrenal DNA quantity detected within a six-month period. D. Comparison of CRC patients without bone metastases using a median split for maximal transrenal DNA quantity detected within a six month period.

tions were also not stable throughout the entire moni-toring period. This critically showed the importance of continual monitoring of pa- tients.

Based on the monitoring re- sults of nucleic acid quantity and KRAS profiling, we hypoth-esize that transrenal DNA is useful for clinical prognostica-tion. KM analyses were per-formed on different subgro- ups of clinical patients. As dis-ease severity could be directly related to the quantity of tran-srenal DNA, we rank the patients by the maximum measured transrenal DNA obtained during monitoring.

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Discussion

The need to address clinical monitoring of can-cer is critical to enhance the survival of patients and offer timely therapeutic changes. Transrenal DNA provides alternative methods for cancer monitoring. The uses of urine speci-mens in various medical and forensic evidence gathering are common [19-21]. Urine sampling has proven to be valuable for extracting various markers, and in our study for disease profiling of CRC patients. The major benefits associated with sampling urine are that it has relatively abundant volume and is patient friendly for sample extraction. In the present study, we evaluated the clinical relevance in CRC patients with and without bone metastases. Patients with bone metastases are a critical group that will benefit from enhanced supervision. This can better complement current methods used in diagnostic imaging to profile the disease.

Our study was effective to show the strong links between CRC and transrenal DNA. In our base-line results, good agreements with transrenal DNA and matched tissue profiles had been made between patients with KRAS mutations and those of wildtype characteristics. Fur- thermore, we noted that serial monitoring of patient samples aided to reduce false negative results. Including the cases that were subse-quently confirmed to be KRAS positive, this brought the overall concordance between tran-srenal DNA and tumor tissue to over 90%. Our results are consistent with several studies that had used ctDNA from plasma to demonstrate the close associations to cancer [16, 22-24]. The consolidated results from our serial moni-toring data showed better agreement in KRAS mutations than studies on single time point measurements performed on plasma conduct-ed by Guibert et al. [16]. It was registerconduct-ed that 78% of their patient population was positive for the KRAS mutations. An important deduction from their study was the close correlation of ctDNA to treatment response. In our case study, we noted a strong associative link with disease progression. Potentially, this suggests that transrenal DNA quantity could be a good treatment response marker. Our work provided the basis for future analysis to explore the use of transrenal DNA to monitor drug response and disease progression. CRC patients with bone metastases in our study registered higher quantities of transrenal DNA compared with

the control wildtype group. Supposedly, this could also be used as a gauge of disease sever-ity. In our analysis, we noted the transrenal DNA assay was specific, which is needed to reduce false positive results. For our tests of patients with wildtype KRAS characteristics, the profiles showed 100% agreement using transrenal DNA compared with tumor tissues.

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for lung cancer patients at different treatment cycles correlated to disease outcome. We observed that by considering the maximum transrenal DNA quantity during serial measure-ments, this was a good parameter for patient stratifications. Our results are one of the first to address CRC patients using transrenal DNA, which may enable a faster turn-around for molecular diagnosis.

Future key questions that can be addressed with this assay include treatment response and clinical interventions. Transrenal DNA recovery and profiling open up new opportunities to bet-ter probe the disease. The limitations of the current study are insufficiently statistical power for tumor burden associations and this can be further expanded in future work. Concurrently, possibilities of clinical interventions based on transrenal DNA measurements can be investi-gated. We also observed that the sensitivity of the detection assay may be limiting as a num-ber of cases had intermittent positive identifi-cation for KRAS mutations. This is due to low frequencies of mutant DNA within these urine specimens. The implications are possible false negative cases and this can be mitigated by having DNA pre-enrichment strategies [30].

Conclusions

Our study investigated the use of transrenal DNA for the assessment of CRC and its benefits for patients with bone metastases. The good agreement between tumor tissue samples and transrenal DNA highlighted the strong clinical associations for this urine based test. Results of the current serial monitoring suggest that this can be a useful clinical parameter to probe the disease. This can also be especially useful in cases where tumor tissue samples cannot be obtained. The technique is attractive as it is non-invasive and specimens are easy to obtain. This will ensure higher patient compliance and may enable faster turnover time for molecular diagnosis alongside conventional testing meth-ods. Measuring transrenal DNA in our patients demonstrated strong prognostic abilities to identify high-risk patients and will help lead to better predictions for survival.

Acknowledgements

This study was supported by a research grant provided by the Xiangyang No.1 People’s Hospital, Hubei University of Medicine.

Disclosure of conflict of interest

None.

Address correspondence to: Jiguang Jia, Depart- ment of Orthopedics, Xiangyang No.1 People’s Hospital, Hubei University of Medicine, China. Tel: +867103420098; E-mail: [email protected]

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Supplementary Table 1. Target inputs and recovered quantities using ddPCR performed on six independent runs

Target Input (Copies) Run 1 Run 2 Run 3 Run 4 Run 5 Run 6

10. 8.0 8.2 6.05 7.8 3.4 7.3

100. 75.4 70.7 74.3 68.3 85.6 88.5

Figure

Table 1. CRC patients and their characteristics
Figure 1. Simple linear regression was performed for spiked controls, which demonstrated good sensitivity and linearity in using ddPCR to detect low quantities of mutant KRAS DNA
Figure 2. Association of transrenal DNA to clinical utility. A. Comparison of transrenal DNA quantity in CRC patients with and without bone metastases
Figure 3A. Using repeated
+2

References

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