Urinary markers in treatment monitoring of lung cancer patients with bone metastasis
Pei Cheng Jin, Bo Gou, Wei QianFirst Published May 21, 2019 Research Article
https://doi.org/10.1177/1724600819848762
Article information
Open Access Creative Commons Attribution, Non Commercial 4.0 License
Abstract
Background:
Bone metastasis remains critical for advanced stage non-small cell lung cancer (NSCLC)—a disease that is challenging to manage. Urinary markers present opportunities for non-invasive testing.
Methods:
Urine specimens were collected from patients prior to treatment. Urinary cell-free DNA was subsequently purified from these samples. To address the specificity of the test, driver mutations in epidermal growth factor receptor L858R and L861Q were analyzed. Clinical specificity was established by comparison with healthy volunteers. Regular monitoring was established during treatment with tyrosine kinase inhibitors. The overall survival of patients was correlated with changes in circulating tumor DNA (ctDNA).
Results:
Baseline clinical correlation of urinary ctDNA and matched tumor specimens achieved 89% concordance. The clinical specificity was 100%. The average background level of urinary ctDNA was 20.7 ng/mL. Comparing patients with and without bone metastasis, the latter had significantly lower baseline levels. During treatment, more pronounced decline in urinary ctDNA was observed in patients without bone metastasis. In our Kaplan–Meier estimator, we observed that patients with a more significant reduction in ctDNA had a better overall survival outcome.
Conclusion:
Our study demonstrates clear benefits and allows better risk profiling for NSCLC patients with bone metastasis. The non-invasive specimen collection is attractive and complements existing cancer management tools.
Keywords Urinary ctDNA, bone metastasis, lung cancer, liquid biopsy, cancer prognosis
Introduction
Cancer is a leading cause of death. Among them, non-small cell lung cancer (NSCLC) is highly prevalent in Asia, mainly due to several associated risk factors.1, 2 Early detection is challenging and a large number of patients are in their advanced stages at first diagnosis.3 In the last decade, the advent of targeted therapies presented more therapeutic options for these patients.4 For instance, numerous clinical studies have shown tyrosine kinase inhibitors (TKIs) are effective for NSCLC patients with several epidermal growth factor receptor (EGFR) mutations.5 The disease is highly dynamic and is the main challenge in clinical management. At the genetic level, it constantly morphs in response to treatment and interventions.6 In addition, patients with bone metastasis tend to have poorer prognosis and these patients requires closer surveillance.7 Current methods of treatment monitoring rely heavily on radiographic imaging to track disease progression.8, 9 This does not enable the detection of molecular profile changes, which is needed for treatment interventions. Secondary mutations, such as T790M in EGFR, confers resistance to first- and second-generation TKIs, and early detection is crucial. The common clinical standard for molecular testing of genetic mutations via tumor tissues is challenging for serial repeated measurements. The need for more tools in disease monitoring cannot be over-emphasized.
Liquid biopsy via cell-free circulating tumor DNA (ctDNA) in the blood stream has been gaining popularity for disease management. In EGFR profiling, numerous studies have highlighted its strong clinical correlation to primary tumors.10, 11 For early disease, a number of studies demonstrated feasibility for screening applications.12, 13 The key advantage for clinical adoption is its ease for sample recovery, and that it does not require invasive surgical procedures. A number of research investigations also revealed the capture of the heterogeneous conditions within a tumor mass or from different metastatic sites.14 Currently, tumor tissue sampling is largely confined to a local region during tumor biopsy, which may miss key driver mutations.15 The liquid biopsy assays are thus suitable for long-term serial measurements of NSCLC patients, especially for those whose tumors are hard to access, such as patients with bone metastasis or with aggressive disease. However, the challenges remain as some patients’ conditions do not allow for the extraction of large volumes of blood. The blood quality is also poor and makes subsequent purification for cell-free DNA difficult.
Alternative body fluid, such as urine specimens, have been explored and early studies have indicated good correlations similar to plasma ctDNA. While extensive data are available on plasma ctDNA, results in urinary ctDNA are incomplete. The current study explores the use of urinary ctDNA for NSCLC patients with bone metastasis as their tumor specimens are difficult to reach and they tend to have a poorer prognosis. Also, we aimed to compare its characteristics with NSCLC patients with other metastatic patterns to better understand its significance. The levels of urinary ctDNA are tracked at regular intervals to investigate the fluctuations during treatment. This study aims to explore the use of urinary cell-free DNA to complement existing methods in cancer management.
Materials and methods
Study design and sample population
All patients and healthy volunteers provided informed consent to participate in the study. Recruitment procedures and sample processing protocols were approved by the institutional review board. As the trial involved human tissues and body fluids, this was done in accordance with the ethical standards laid down in the Declaration of Helsinki. A total of 100 NSCLC patients and 50 healthy volunteers were recruited for the entire study. The patient demographics are provided in Table 1. The average age for NSCLC patients and healthy volunteers were 55 and 54 years old, respectively. The gender ratio (male: female) among the study subjects was 1.70 and 1.63 for NSCLC patients and healthy volunteers, respectively. The majority of patients were detected with stage IV disease. Only patients with EGFR mutations were recruited for the study and were separated by their metastatic patterns. EGFR baseline testing was confirmed via tumor tissue biopsies as part of routine clinical investigations. Patients were placed on TKIs as per treatment guidelines. For the healthy volunteers, subjects had similar smoking profiles and gender ratios, but had negative results on radiographic imaging analysis. These provided control experiments for clinical comparisons with NSCLC patients.
Table
Table 1. NSCLC patient profiles and clinical data.
Table 1. NSCLC patient profiles and clinical data.
View larger version
EGFR: epidermal growth factor receptor; NSCLC: non-small cell lung cancer
Urine specimen collection at different intervals
Urine specimens were collected in EDTA-containing receptacles within 2 weeks of biopsy for baseline comparison. Serial urine collections were performed at monthly intervals thereafter for 9 months. For consistency, the first morning urine midstream sample was collected. Approximately 50 mL of urine volume was taken. The processing of urine specimens had to be performed fresh to prevent degradation of the nucleic acid. All samples were processed within 2 hours of collection to recover urinary ctDNA. The storage condition is highly sensitive, as shown by the study conducted by Hilhorst et al.16 The quality of nuclei acid is also influenced by the time interval from sampling to analysis.17 To remove debris and possible sediments, urine specimens were spun at 3000 xg for 15 minutes at 4°C, and 30 mL of the sample was transferred to a fresh centrifuge tube. For purification of nucleic acid from spun urine specimens, the Qiagen’s QIAamp Circulating nucleic acid kit (Qiagen Inc., USA) was used. Spin columns were utilized for each specimen and centrifuge conditions strictly followed the manufacturer’s recommendations. Urinary ctDNA was eluted in 10 uL nuclease-free water, and 1 µL of purified urinary ctDNA was quantified using the Nanodrop 2000 (ThermoFisher, USA) and the remainder stored at −80°C. For EGFR molecular profiling, samples were batch processed. The same procedures were employed on the urine specimens of healthy volunteers.
Molecular profiling of urinary cell-free DNA using droplet digital polymerase chain reaction
Positive identification of EGFR mutations prior to patient recruitment was performed using tumor tissue biopsies. For urinary cell-free DNA, droplet digital polymerase chain reaction (ddPCR) is employed, which is a sensitive assay to detect low allelic frequencies of EGFR mutants in the sample. The PCR primers and probes were commercially procured from Bio-Rad PrimePCR ddPCR mutational assay (Bio-Rad Lab., USA). All procedures strictly followed the verified manufacturer’s protocols. Briefly, reaction mixes were prepared fresh as recommended, and each reaction contained 20 µL volume. Urinary ctDNA was added into mastermixes together with primers and probes, and was thoroughly mixed via pipetting to homogenize the reaction. Droplet generation was done in the QX200 (Bio Rad Lab., USA) and transferred to the thermocycler for PCR amplification. The cycling conditions were set as follows at 95°C for 10 minutes (1 cycle); 94°C for 30s and 55°C for 1 minute (40 cycles); 98°C for 10 minutes (1 cycle); and finally held at 4°C. For readout, the PCR plate was transferred to the QX200 droplet reader. A sample analysis was performed using the QuantaSoft software (Bio Rad Lab., USA).
Statistical analysis
Quantities of urinary cell-free DNA among healthy and NSCLC subjects were compared using the Student t test. Similarly, comparison among NSCLC patients with and without bone metastases was also performed via the Student t test. Receiver operating characteristics (ROC) analysis was performed to gauge the urinary ctDNA diagnostic accuracy for clinical patients. Analysis of variance (ANOVA) was performed to compare serial measurement results. For risk stratifications, the Kaplan–Meier (KM) analysis was conducted with patient follow-up data. Hazard ratios (HR) were computed using the COX regression model. Statistical analysis was calculated and computed with the aid of the PRISM software (GraphPad Inc., USA).
Results
NSCLC trial design and baseline index comparisons
Bone metastases is one of the main challenges in advanced stage NSCLC and directly impacts disease prognosis as several studies have shown.7 Addressing this patient cohort, we aim to establish the clinical utility for urinary ctDNA, collected from a non-invasive body fluid that can satisfy routine clinical monitoring. Supplementary Figure 1 shows the clinical trial design comparing different groups of NSCLC patients and healthy volunteers. Serial measurements during treatment provide further understanding of the variability in the potential marker, and its application in cancer management. As shown in Table 1, the majority of patients had numerous metastases affecting multiple organs and were positive for EGFR mutations. Healthy volunteers provided a reference comparison with cancer patients, and serial measurements allowed the baseline characterization of inherent urinary cell-free DNA among individuals.
As shown in Figure 1(a), quantifications of urinary ctDNA from pre-treated patients were compiled and compared. Average concentrations derived from patients and healthy subjects were 47.9 ng/mL (95% confidence interval (CI) 44.8, 51.1 ng/mL) and 20.7 ng/mL (95% CI 18.2, 23.3 ng/mL), respectively. The concentration of urinary ctDNA was markedly higher for NSCLC patients, and the Student t test confirmed significant statistical differences (P < 0.001). We performed a ROC analysis using nucleic acid concentrations from urine specimens and compared healthy subjects with NSCLC patients. The area under the ROC (AUROC) was 0.94 and indicated a good separation in urinary cell-free DNA between the two study groups (Figure 1(b)). Addressing NSCLC patients with and without bone metastases, we also observed statistical significance in average concentrations (P = 0.0006). Patients with bone metastases had approximately 1.24-fold higher nucleic acid concentrations compared with patients with other metastatic profiles, as shown in Figure 1(c).
figure
Figure 1. Index measurements of urinary ctDNA prior to therapy commencement. (a) Comparison of recovered concentrations of nucleic acid from healthy and NSCLC subjects. (b) Receiver operating curve (ROC) analysis comparing the results derived from recover cell-free DNA quantities from NSCLC patients and healthy controls. (c) Comparison of urinary ctDNA concentrations among NSCLC patients. (d) Concordance rates among NSCLC patients with matched tumor tissues. ** refers to statistical significance with P value < 0.01.
ctDNA: circulating tumor DNA; NSCLC: non-small cell lung cancer.
Using a sensitive detection method, we examined the agreement of EGFR profiles of matched tumor tissue samples from all 100 NSCLC patients. As a control, healthy volunteers were assayed as well. The clinical specificity of the urinary cell-free DNA assay as determined from healthy controls showed 100% wildtype EGFR signature (data not shown). The results of patient comparisons are shown in Figure 1(d). For NSCLC patients with bone metastases, we observed an overall agreement of 89%. The concordance rates for patients with other metastatic patterns were slightly lower at 86%. Overall, the agreement for the entire study cohort was 93% (Table 2). Using Kappa statistics, we observed that the outcome was significant, with a κ value of 0.84 (95% CI 0.75, 0.93). This may indicate good clinical relevance for urinary ctDNA in NSCLC patients.
Table
Table 2. Agreement with matched tumor tissue at index measurement.
Table 2. Agreement with matched tumor tissue at index measurement.
View larger version
Serial measurements of urinary ctDNA levels for different cohorts
We performed the serial monitoring of cancer patients and healthy volunteers to examine the fluctuations over time. Results from healthy volunteers provided the background levels and variability of urinary cell-free DNA. Figure 2(a) shows the results derived from healthy subjects. The overall mean concentration detected was 21.3 ng/mL. Using ANOVA, no statistical significance was observed among the means at different time points (P > 0.05). Month 4 was the period with highest SD and the corresponding SD value was 9.53 ng/mL. Addressing the clinical specimens from NSCLC patients, we observed several interesting trends. All patients had sensitizing EGFR mutations and were on TKIs. Positive treatment response was expected, and in the preceding months after treatment initiation we observed a corresponding decline in urinary ctDNA concentrations (Figure 3(b) and (c)). For NSCLC patients with bone metastasis, a significant decline in levels was observed in the first 2 months and the trend then moved upwards. The overall average ctDNA concentration was 57.3 ng/mL and was 1.08-fold higher than baseline quantity as shown in Figure 2(b). For NSCLC patients without bone metastasis (Figure 2(c)), we observed a significant decline in urinary ctDNA levels for the first 4 months during monitoring. Concentrations for urinary ctDNA then moved slowly upwards. The average nucleic acid concentration detected was 38.4 ng/mL, and, unlike the results observed in Figure 2(d), this value was significantly lower than the baseline detected concentration for this patient group.
figure
Figure 2. Serial tracking of urinary cell-free DNA profiles among different study subjects. (a) Healthy volunteers showed little mean variations within 8 months. (b) Results showing recovered urinary ctDNA among NSCLC patients with bone metastasis over time. The average detected urinary ctDNA concentration is higher than the index measurement. (c) Results showing recovered urinary ctDNA among NSCLC with other metastatic profiles. The average urinary ctDNA concentrations were lower than the index measurements.
ctDNA: circulating tumor DNA; NSCLC: non-small cell lung cancer.
figure
Figure 3. Kaplan–Meier (KM) estimate to address overall survival of NSCLC patients. (a) Results comparing patients with bone metastasis and patients with other metastatic patterns. (b) Risk stratification of NSCLC patients with bone metastasis by separating the group based on maximum detected urinary ctDNA. The results indicated patients with higher ctDNA had worse outcome. (c) Risk stratification of NSCLC patients with bone metastasis using the maximum drop in urinary ctDNA over the index measurement.
ctDNA: circulating tumor DNA; NSCLC: non-small cell lung cancer.
Survival analysis demonstrates strong links to bone metastases
In further follow-up via phone calls, house visits, or during regular checkups, the overall survival of the cancer patients was collated and analyzed. The KM estimator was used to derive the correlative analysis among different sub-categories of patients as shown in Figure 3. In a direct comparison among NSCLC patients with and without bone metastasis (Figure 3(a)), we observed better survival outcomes for the latter. The HR, as calculated using the COX regression model, was 1.38 (95% CI 0.90, 2.12). Within the patient group with bone metastasis, we postulate that specific trends in urinary ctDNA may aid in better patient stratifications. In Figure 3(b), patients in this group were separated equally into two subgroups by ranking the maximum detected urinary ctDNA during the entire monitoring period. The current analysis clearly highlighted the clinical significance of the parameter and the HR was computed to be 1.64 (95% CI 0.92, 2.94). Median survival was 1 month less for NSCLC patients with concurrent higher nucleic acid counts and bone metastases than patients with lower ctDNA concentrations. Subsequently, we focused on the earlier monitoring period where detected urinary ctDNA showed a declining trend. NSCLC patients with bone metastases were split equally into two subgroups based on the levels of maximum drop in urinary ctDNA (Figure 3(c)). The cohort that had a much higher decline had a better survival outcome. The HR was calculated to be 2.76 (95% CI 1.39, 5.48) and the median survival rates were 2 months less. The results demonstrated the clear benefits of constant monitoring and trend analysis of urinary ctDNA in this patient cohort.
Discussion
In the current study, NSCLC patients with bone metastases were targeted. EGFR mutations are fairly prevalent18 for NSCLC and the current study aims to understand if urinary ctDNA can be employed in the clinical monitoring of these patients. Both L858R and L861Q are fairly common and respond well to first- and second-generation tyrosine kinase inhibitors.19 The clear advantage of the technique is the non-invasive nature of the sample collection, which clearly appeals to patients and clinicians. This also provides direct access to tumor materials for possible regular molecular profiling for real-time tracking of genetic aberrations. In the current study, we aimed to address NSCLC patients with bone metastases as this critical group are not clearly understood. Using urinary ctDNA, we hypothesize this to be useful to track disease changes and to allow better risk stratifications. In the future, this may allow earlier clinical inventions to better target the changes in the disease.
We computed the diagnostic accuracy of the assay at the index measurement prior to treatment and compared it with the matched tumor biopsy tissues. In addition, a comparison of purified nucleic acid concentrations was undertaken on NSCLC patients and healthy populations to address clinical relevance. The observed results clearly highlighted that urinary ctDNA was elevated for NSCLC patients, and the ROC analysis presented a high AUROC value > 0.9. Our results were consistent with other studies that utilized other forms of cell-free circulating nucleic acid mostly from plasma20 for NSCLC. Wei et al.21 noted that the average quantity detected from NSCLC patients was approximately 2-fold higher than healthy controls. In these studies, the elevated nucleic acid quantity was attributed to the metastatic disease and tumors shedding genetic materials into the bloodstream.22 Possibly in the filtration process performed by the kidneys, nucleic acid content is expelled into urine. To affirm that the urinary ctDNA contained tumor-specific material, we performed molecular profiling of the collected sample for EGFR mutations that were present in the bulk cancer tissue. A sensitive detection method employing ddPCR was selected to ensure that the minute presence of mutant DNA could be picked up. The technical challenges associated with plasma ctDNA had been the large amounts of accompanying normal nucleic acid.23 We hypothesized that this will be similarly challenging in urinary ctDNA specimens. Our results highlighted reasonably good concordance among patients’ matched samples of solid and liquid biopsies. Overall concordance was determined at 89%. Among NSCLC patients with bone metastases in this preliminary trial, the agreement with matched tumor tissues was higher than in patients with other metastatic patterns. Interestingly, the levels of urinary ctDNA for NSCLC with bone metastasis was also higher.
In subsequent serial profiling of urinary ctDNA concentrations, we further observed several interesting trends. Targeted therapy for EGFR using TKI is recommended as a first-line treatment option and has shown a good initial response among the majority of patients.24 We observed a dip in concentrations detected in following months after treatment initiation. The decline was less pronounced for NSCLC patients with bone metastases (Figure 2). This trend could be indicative of treatment effects and resulted in less nuclei acid being released. In later parts of the clinical monitoring trial, we observed an upward trend for both patient groups. The issue of drug resistance for TKI-treated patients is well characterized, and the incidence rates detected in several studies are significant.25 The upward trend may indicate therapy resistance development among patient groups, and warrants further investigation. In all, we observed that for NSCLC patients with bone metastases, the average detected urinary ctDNA concentration within 8 months was higher than the index measurements, whereas in patients with other metastatic patterns the concentration increased at a much slower rate. We postulate that this may affect disease outcome in the long term; therefore we performed a KM analysis to assess its relationship with overall survival.
We observed two interesting trends within the NSCLC patient cohort with bone metastasis associated with urinary ctDNA. By comparing the absolute urinary ctDNA concentrations among patients, the subgroup with higher nucleic acid content had a worse outcome. The result provides a straightforward means for patient stratification. This also highlights the importance of serial measurements to understand the variability in each individual patient. More interestingly, when we analyzed the initial response of each patient by taking the ratio of the lowest detected urinary ctDNA concentration over the index measurement, we observed better risk profiling. The HR determined in this case was higher at 2.07. This likely infers that a patient’s initial response to treatment may play a significant role in disease outcome, and this can be reflected in the urinary ctDNA measurements. Our study significantly provided a better understanding of the use of urinary ctDNA in lung cancer management.
Conclusion
Clinical management of cancer is a challenging task and requires better tools to be able to accurately profile the disease. In addressing NSCLC, our study shows the potential opportunities of urinary ctDNA for molecular profiling, treatment monitoring, and prognosis. Patients with bone metastasis benefit from this assay with better risk stratification. The clear advantage of urinary ctDNA over other methodologies is in sample collection, which is straightforward and non-invasive. This provides opportunities for long-term tracking of the disease, and it can be an aid to complement current management routines dominated by radiographic imaging.
Authors’ contributions
Pei Cheng Jin and Bo Gou contributed equally and are designated as co-first authors.
Declaration of conflicting interest
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a research grant provided by Renmin Hospital, Hubei University of Medicine
Ethical approval
All human and animal studies have been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
Supplemental material
Supplemental material for this article is available online.
ORCID iD
Wei Qian https://orcid.org/0000-0002-0645-5247
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View Abstract
Pei Cheng Jin, Bo Gou, Wei QianFirst Published May 21, 2019 Research Article
https://doi.org/10.1177/1724600819848762
Article information
Open Access Creative Commons Attribution, Non Commercial 4.0 License
Abstract
Background:
Bone metastasis remains critical for advanced stage non-small cell lung cancer (NSCLC)—a disease that is challenging to manage. Urinary markers present opportunities for non-invasive testing.
Methods:
Urine specimens were collected from patients prior to treatment. Urinary cell-free DNA was subsequently purified from these samples. To address the specificity of the test, driver mutations in epidermal growth factor receptor L858R and L861Q were analyzed. Clinical specificity was established by comparison with healthy volunteers. Regular monitoring was established during treatment with tyrosine kinase inhibitors. The overall survival of patients was correlated with changes in circulating tumor DNA (ctDNA).
Results:
Baseline clinical correlation of urinary ctDNA and matched tumor specimens achieved 89% concordance. The clinical specificity was 100%. The average background level of urinary ctDNA was 20.7 ng/mL. Comparing patients with and without bone metastasis, the latter had significantly lower baseline levels. During treatment, more pronounced decline in urinary ctDNA was observed in patients without bone metastasis. In our Kaplan–Meier estimator, we observed that patients with a more significant reduction in ctDNA had a better overall survival outcome.
Conclusion:
Our study demonstrates clear benefits and allows better risk profiling for NSCLC patients with bone metastasis. The non-invasive specimen collection is attractive and complements existing cancer management tools.
Keywords Urinary ctDNA, bone metastasis, lung cancer, liquid biopsy, cancer prognosis
Introduction
Cancer is a leading cause of death. Among them, non-small cell lung cancer (NSCLC) is highly prevalent in Asia, mainly due to several associated risk factors.1, 2 Early detection is challenging and a large number of patients are in their advanced stages at first diagnosis.3 In the last decade, the advent of targeted therapies presented more therapeutic options for these patients.4 For instance, numerous clinical studies have shown tyrosine kinase inhibitors (TKIs) are effective for NSCLC patients with several epidermal growth factor receptor (EGFR) mutations.5 The disease is highly dynamic and is the main challenge in clinical management. At the genetic level, it constantly morphs in response to treatment and interventions.6 In addition, patients with bone metastasis tend to have poorer prognosis and these patients requires closer surveillance.7 Current methods of treatment monitoring rely heavily on radiographic imaging to track disease progression.8, 9 This does not enable the detection of molecular profile changes, which is needed for treatment interventions. Secondary mutations, such as T790M in EGFR, confers resistance to first- and second-generation TKIs, and early detection is crucial. The common clinical standard for molecular testing of genetic mutations via tumor tissues is challenging for serial repeated measurements. The need for more tools in disease monitoring cannot be over-emphasized.
Liquid biopsy via cell-free circulating tumor DNA (ctDNA) in the blood stream has been gaining popularity for disease management. In EGFR profiling, numerous studies have highlighted its strong clinical correlation to primary tumors.10, 11 For early disease, a number of studies demonstrated feasibility for screening applications.12, 13 The key advantage for clinical adoption is its ease for sample recovery, and that it does not require invasive surgical procedures. A number of research investigations also revealed the capture of the heterogeneous conditions within a tumor mass or from different metastatic sites.14 Currently, tumor tissue sampling is largely confined to a local region during tumor biopsy, which may miss key driver mutations.15 The liquid biopsy assays are thus suitable for long-term serial measurements of NSCLC patients, especially for those whose tumors are hard to access, such as patients with bone metastasis or with aggressive disease. However, the challenges remain as some patients’ conditions do not allow for the extraction of large volumes of blood. The blood quality is also poor and makes subsequent purification for cell-free DNA difficult.
Alternative body fluid, such as urine specimens, have been explored and early studies have indicated good correlations similar to plasma ctDNA. While extensive data are available on plasma ctDNA, results in urinary ctDNA are incomplete. The current study explores the use of urinary ctDNA for NSCLC patients with bone metastasis as their tumor specimens are difficult to reach and they tend to have a poorer prognosis. Also, we aimed to compare its characteristics with NSCLC patients with other metastatic patterns to better understand its significance. The levels of urinary ctDNA are tracked at regular intervals to investigate the fluctuations during treatment. This study aims to explore the use of urinary cell-free DNA to complement existing methods in cancer management.
Materials and methods
Study design and sample population
All patients and healthy volunteers provided informed consent to participate in the study. Recruitment procedures and sample processing protocols were approved by the institutional review board. As the trial involved human tissues and body fluids, this was done in accordance with the ethical standards laid down in the Declaration of Helsinki. A total of 100 NSCLC patients and 50 healthy volunteers were recruited for the entire study. The patient demographics are provided in Table 1. The average age for NSCLC patients and healthy volunteers were 55 and 54 years old, respectively. The gender ratio (male: female) among the study subjects was 1.70 and 1.63 for NSCLC patients and healthy volunteers, respectively. The majority of patients were detected with stage IV disease. Only patients with EGFR mutations were recruited for the study and were separated by their metastatic patterns. EGFR baseline testing was confirmed via tumor tissue biopsies as part of routine clinical investigations. Patients were placed on TKIs as per treatment guidelines. For the healthy volunteers, subjects had similar smoking profiles and gender ratios, but had negative results on radiographic imaging analysis. These provided control experiments for clinical comparisons with NSCLC patients.
Table
Table 1. NSCLC patient profiles and clinical data.
Table 1. NSCLC patient profiles and clinical data.
View larger version
EGFR: epidermal growth factor receptor; NSCLC: non-small cell lung cancer
Urine specimen collection at different intervals
Urine specimens were collected in EDTA-containing receptacles within 2 weeks of biopsy for baseline comparison. Serial urine collections were performed at monthly intervals thereafter for 9 months. For consistency, the first morning urine midstream sample was collected. Approximately 50 mL of urine volume was taken. The processing of urine specimens had to be performed fresh to prevent degradation of the nucleic acid. All samples were processed within 2 hours of collection to recover urinary ctDNA. The storage condition is highly sensitive, as shown by the study conducted by Hilhorst et al.16 The quality of nuclei acid is also influenced by the time interval from sampling to analysis.17 To remove debris and possible sediments, urine specimens were spun at 3000 xg for 15 minutes at 4°C, and 30 mL of the sample was transferred to a fresh centrifuge tube. For purification of nucleic acid from spun urine specimens, the Qiagen’s QIAamp Circulating nucleic acid kit (Qiagen Inc., USA) was used. Spin columns were utilized for each specimen and centrifuge conditions strictly followed the manufacturer’s recommendations. Urinary ctDNA was eluted in 10 uL nuclease-free water, and 1 µL of purified urinary ctDNA was quantified using the Nanodrop 2000 (ThermoFisher, USA) and the remainder stored at −80°C. For EGFR molecular profiling, samples were batch processed. The same procedures were employed on the urine specimens of healthy volunteers.
Molecular profiling of urinary cell-free DNA using droplet digital polymerase chain reaction
Positive identification of EGFR mutations prior to patient recruitment was performed using tumor tissue biopsies. For urinary cell-free DNA, droplet digital polymerase chain reaction (ddPCR) is employed, which is a sensitive assay to detect low allelic frequencies of EGFR mutants in the sample. The PCR primers and probes were commercially procured from Bio-Rad PrimePCR ddPCR mutational assay (Bio-Rad Lab., USA). All procedures strictly followed the verified manufacturer’s protocols. Briefly, reaction mixes were prepared fresh as recommended, and each reaction contained 20 µL volume. Urinary ctDNA was added into mastermixes together with primers and probes, and was thoroughly mixed via pipetting to homogenize the reaction. Droplet generation was done in the QX200 (Bio Rad Lab., USA) and transferred to the thermocycler for PCR amplification. The cycling conditions were set as follows at 95°C for 10 minutes (1 cycle); 94°C for 30s and 55°C for 1 minute (40 cycles); 98°C for 10 minutes (1 cycle); and finally held at 4°C. For readout, the PCR plate was transferred to the QX200 droplet reader. A sample analysis was performed using the QuantaSoft software (Bio Rad Lab., USA).
Statistical analysis
Quantities of urinary cell-free DNA among healthy and NSCLC subjects were compared using the Student t test. Similarly, comparison among NSCLC patients with and without bone metastases was also performed via the Student t test. Receiver operating characteristics (ROC) analysis was performed to gauge the urinary ctDNA diagnostic accuracy for clinical patients. Analysis of variance (ANOVA) was performed to compare serial measurement results. For risk stratifications, the Kaplan–Meier (KM) analysis was conducted with patient follow-up data. Hazard ratios (HR) were computed using the COX regression model. Statistical analysis was calculated and computed with the aid of the PRISM software (GraphPad Inc., USA).
Results
NSCLC trial design and baseline index comparisons
Bone metastases is one of the main challenges in advanced stage NSCLC and directly impacts disease prognosis as several studies have shown.7 Addressing this patient cohort, we aim to establish the clinical utility for urinary ctDNA, collected from a non-invasive body fluid that can satisfy routine clinical monitoring. Supplementary Figure 1 shows the clinical trial design comparing different groups of NSCLC patients and healthy volunteers. Serial measurements during treatment provide further understanding of the variability in the potential marker, and its application in cancer management. As shown in Table 1, the majority of patients had numerous metastases affecting multiple organs and were positive for EGFR mutations. Healthy volunteers provided a reference comparison with cancer patients, and serial measurements allowed the baseline characterization of inherent urinary cell-free DNA among individuals.
As shown in Figure 1(a), quantifications of urinary ctDNA from pre-treated patients were compiled and compared. Average concentrations derived from patients and healthy subjects were 47.9 ng/mL (95% confidence interval (CI) 44.8, 51.1 ng/mL) and 20.7 ng/mL (95% CI 18.2, 23.3 ng/mL), respectively. The concentration of urinary ctDNA was markedly higher for NSCLC patients, and the Student t test confirmed significant statistical differences (P < 0.001). We performed a ROC analysis using nucleic acid concentrations from urine specimens and compared healthy subjects with NSCLC patients. The area under the ROC (AUROC) was 0.94 and indicated a good separation in urinary cell-free DNA between the two study groups (Figure 1(b)). Addressing NSCLC patients with and without bone metastases, we also observed statistical significance in average concentrations (P = 0.0006). Patients with bone metastases had approximately 1.24-fold higher nucleic acid concentrations compared with patients with other metastatic profiles, as shown in Figure 1(c).
figure
Figure 1. Index measurements of urinary ctDNA prior to therapy commencement. (a) Comparison of recovered concentrations of nucleic acid from healthy and NSCLC subjects. (b) Receiver operating curve (ROC) analysis comparing the results derived from recover cell-free DNA quantities from NSCLC patients and healthy controls. (c) Comparison of urinary ctDNA concentrations among NSCLC patients. (d) Concordance rates among NSCLC patients with matched tumor tissues. ** refers to statistical significance with P value < 0.01.
ctDNA: circulating tumor DNA; NSCLC: non-small cell lung cancer.
Using a sensitive detection method, we examined the agreement of EGFR profiles of matched tumor tissue samples from all 100 NSCLC patients. As a control, healthy volunteers were assayed as well. The clinical specificity of the urinary cell-free DNA assay as determined from healthy controls showed 100% wildtype EGFR signature (data not shown). The results of patient comparisons are shown in Figure 1(d). For NSCLC patients with bone metastases, we observed an overall agreement of 89%. The concordance rates for patients with other metastatic patterns were slightly lower at 86%. Overall, the agreement for the entire study cohort was 93% (Table 2). Using Kappa statistics, we observed that the outcome was significant, with a κ value of 0.84 (95% CI 0.75, 0.93). This may indicate good clinical relevance for urinary ctDNA in NSCLC patients.
Table
Table 2. Agreement with matched tumor tissue at index measurement.
Table 2. Agreement with matched tumor tissue at index measurement.
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Serial measurements of urinary ctDNA levels for different cohorts
We performed the serial monitoring of cancer patients and healthy volunteers to examine the fluctuations over time. Results from healthy volunteers provided the background levels and variability of urinary cell-free DNA. Figure 2(a) shows the results derived from healthy subjects. The overall mean concentration detected was 21.3 ng/mL. Using ANOVA, no statistical significance was observed among the means at different time points (P > 0.05). Month 4 was the period with highest SD and the corresponding SD value was 9.53 ng/mL. Addressing the clinical specimens from NSCLC patients, we observed several interesting trends. All patients had sensitizing EGFR mutations and were on TKIs. Positive treatment response was expected, and in the preceding months after treatment initiation we observed a corresponding decline in urinary ctDNA concentrations (Figure 3(b) and (c)). For NSCLC patients with bone metastasis, a significant decline in levels was observed in the first 2 months and the trend then moved upwards. The overall average ctDNA concentration was 57.3 ng/mL and was 1.08-fold higher than baseline quantity as shown in Figure 2(b). For NSCLC patients without bone metastasis (Figure 2(c)), we observed a significant decline in urinary ctDNA levels for the first 4 months during monitoring. Concentrations for urinary ctDNA then moved slowly upwards. The average nucleic acid concentration detected was 38.4 ng/mL, and, unlike the results observed in Figure 2(d), this value was significantly lower than the baseline detected concentration for this patient group.
figure
Figure 2. Serial tracking of urinary cell-free DNA profiles among different study subjects. (a) Healthy volunteers showed little mean variations within 8 months. (b) Results showing recovered urinary ctDNA among NSCLC patients with bone metastasis over time. The average detected urinary ctDNA concentration is higher than the index measurement. (c) Results showing recovered urinary ctDNA among NSCLC with other metastatic profiles. The average urinary ctDNA concentrations were lower than the index measurements.
ctDNA: circulating tumor DNA; NSCLC: non-small cell lung cancer.
figure
Figure 3. Kaplan–Meier (KM) estimate to address overall survival of NSCLC patients. (a) Results comparing patients with bone metastasis and patients with other metastatic patterns. (b) Risk stratification of NSCLC patients with bone metastasis by separating the group based on maximum detected urinary ctDNA. The results indicated patients with higher ctDNA had worse outcome. (c) Risk stratification of NSCLC patients with bone metastasis using the maximum drop in urinary ctDNA over the index measurement.
ctDNA: circulating tumor DNA; NSCLC: non-small cell lung cancer.
Survival analysis demonstrates strong links to bone metastases
In further follow-up via phone calls, house visits, or during regular checkups, the overall survival of the cancer patients was collated and analyzed. The KM estimator was used to derive the correlative analysis among different sub-categories of patients as shown in Figure 3. In a direct comparison among NSCLC patients with and without bone metastasis (Figure 3(a)), we observed better survival outcomes for the latter. The HR, as calculated using the COX regression model, was 1.38 (95% CI 0.90, 2.12). Within the patient group with bone metastasis, we postulate that specific trends in urinary ctDNA may aid in better patient stratifications. In Figure 3(b), patients in this group were separated equally into two subgroups by ranking the maximum detected urinary ctDNA during the entire monitoring period. The current analysis clearly highlighted the clinical significance of the parameter and the HR was computed to be 1.64 (95% CI 0.92, 2.94). Median survival was 1 month less for NSCLC patients with concurrent higher nucleic acid counts and bone metastases than patients with lower ctDNA concentrations. Subsequently, we focused on the earlier monitoring period where detected urinary ctDNA showed a declining trend. NSCLC patients with bone metastases were split equally into two subgroups based on the levels of maximum drop in urinary ctDNA (Figure 3(c)). The cohort that had a much higher decline had a better survival outcome. The HR was calculated to be 2.76 (95% CI 1.39, 5.48) and the median survival rates were 2 months less. The results demonstrated the clear benefits of constant monitoring and trend analysis of urinary ctDNA in this patient cohort.
Discussion
In the current study, NSCLC patients with bone metastases were targeted. EGFR mutations are fairly prevalent18 for NSCLC and the current study aims to understand if urinary ctDNA can be employed in the clinical monitoring of these patients. Both L858R and L861Q are fairly common and respond well to first- and second-generation tyrosine kinase inhibitors.19 The clear advantage of the technique is the non-invasive nature of the sample collection, which clearly appeals to patients and clinicians. This also provides direct access to tumor materials for possible regular molecular profiling for real-time tracking of genetic aberrations. In the current study, we aimed to address NSCLC patients with bone metastases as this critical group are not clearly understood. Using urinary ctDNA, we hypothesize this to be useful to track disease changes and to allow better risk stratifications. In the future, this may allow earlier clinical inventions to better target the changes in the disease.
We computed the diagnostic accuracy of the assay at the index measurement prior to treatment and compared it with the matched tumor biopsy tissues. In addition, a comparison of purified nucleic acid concentrations was undertaken on NSCLC patients and healthy populations to address clinical relevance. The observed results clearly highlighted that urinary ctDNA was elevated for NSCLC patients, and the ROC analysis presented a high AUROC value > 0.9. Our results were consistent with other studies that utilized other forms of cell-free circulating nucleic acid mostly from plasma20 for NSCLC. Wei et al.21 noted that the average quantity detected from NSCLC patients was approximately 2-fold higher than healthy controls. In these studies, the elevated nucleic acid quantity was attributed to the metastatic disease and tumors shedding genetic materials into the bloodstream.22 Possibly in the filtration process performed by the kidneys, nucleic acid content is expelled into urine. To affirm that the urinary ctDNA contained tumor-specific material, we performed molecular profiling of the collected sample for EGFR mutations that were present in the bulk cancer tissue. A sensitive detection method employing ddPCR was selected to ensure that the minute presence of mutant DNA could be picked up. The technical challenges associated with plasma ctDNA had been the large amounts of accompanying normal nucleic acid.23 We hypothesized that this will be similarly challenging in urinary ctDNA specimens. Our results highlighted reasonably good concordance among patients’ matched samples of solid and liquid biopsies. Overall concordance was determined at 89%. Among NSCLC patients with bone metastases in this preliminary trial, the agreement with matched tumor tissues was higher than in patients with other metastatic patterns. Interestingly, the levels of urinary ctDNA for NSCLC with bone metastasis was also higher.
In subsequent serial profiling of urinary ctDNA concentrations, we further observed several interesting trends. Targeted therapy for EGFR using TKI is recommended as a first-line treatment option and has shown a good initial response among the majority of patients.24 We observed a dip in concentrations detected in following months after treatment initiation. The decline was less pronounced for NSCLC patients with bone metastases (Figure 2). This trend could be indicative of treatment effects and resulted in less nuclei acid being released. In later parts of the clinical monitoring trial, we observed an upward trend for both patient groups. The issue of drug resistance for TKI-treated patients is well characterized, and the incidence rates detected in several studies are significant.25 The upward trend may indicate therapy resistance development among patient groups, and warrants further investigation. In all, we observed that for NSCLC patients with bone metastases, the average detected urinary ctDNA concentration within 8 months was higher than the index measurements, whereas in patients with other metastatic patterns the concentration increased at a much slower rate. We postulate that this may affect disease outcome in the long term; therefore we performed a KM analysis to assess its relationship with overall survival.
We observed two interesting trends within the NSCLC patient cohort with bone metastasis associated with urinary ctDNA. By comparing the absolute urinary ctDNA concentrations among patients, the subgroup with higher nucleic acid content had a worse outcome. The result provides a straightforward means for patient stratification. This also highlights the importance of serial measurements to understand the variability in each individual patient. More interestingly, when we analyzed the initial response of each patient by taking the ratio of the lowest detected urinary ctDNA concentration over the index measurement, we observed better risk profiling. The HR determined in this case was higher at 2.07. This likely infers that a patient’s initial response to treatment may play a significant role in disease outcome, and this can be reflected in the urinary ctDNA measurements. Our study significantly provided a better understanding of the use of urinary ctDNA in lung cancer management.
Conclusion
Clinical management of cancer is a challenging task and requires better tools to be able to accurately profile the disease. In addressing NSCLC, our study shows the potential opportunities of urinary ctDNA for molecular profiling, treatment monitoring, and prognosis. Patients with bone metastasis benefit from this assay with better risk stratification. The clear advantage of urinary ctDNA over other methodologies is in sample collection, which is straightforward and non-invasive. This provides opportunities for long-term tracking of the disease, and it can be an aid to complement current management routines dominated by radiographic imaging.
Authors’ contributions
Pei Cheng Jin and Bo Gou contributed equally and are designated as co-first authors.
Declaration of conflicting interest
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a research grant provided by Renmin Hospital, Hubei University of Medicine
Ethical approval
All human and animal studies have been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
Supplemental material
Supplemental material for this article is available online.
ORCID iD
Wei Qian https://orcid.org/0000-0002-0645-5247
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