Clinical application of urinary NTx in patients with bone metastases from lung cancer

Abstract

Objective

To investigate the clinical value of urinary N-telopeptide (NTx) of type I collagen in diagnosing bone metastasis (BM) from lung cancer and to optimize a multi-indicator combined detection strategy.

Methods

The study enrolled patients from a healthy control group, a lung cancer without bone metastases (LC-nonBM) group, and a lung cancer with bone metastases (LC-BM) group. The diagnostic performance of NTx-to-creatinine ratio (NTx/Cr) was evaluated by comparing biochemical indicators, staging characteristics, and receiver operating characteristic (ROC) curve analysis.

Results

NTx/Cr showed statistically significant differences across various stages within the LC-nonBM group (P = 0.004). In the LC-BM group, NTx/Cr levels were significantly elevated (P < 0.001) and positively correlated with the number of bone metastatic lesions. ROC curve analysis revealed that NTx/Cr alone achieved an areas under the curve (AUC) of 0.78 for diagnosing bone metastasis, while the three-indicator combination of the urinary NTx/Cr, serum alkaline phosphatase (ALP), and calcium (Ca) reached an AUC of 0.882, indicating a marked improvement in diagnostic performance.

Conclusion

Urinary NTx/Cr is an effective biomarker for diagnosing bone metastasis in lung cancer, and its level is closely associated with tumor stage and metastatic burden. A multi-indicator diagnostic model combining NTx/Cr, ALP, and Ca significantly enhances diagnostic accuracy.

Highlights

• •Urinary NTx levels are significantly elevated in lung cancer patients with bone metastases.
• •NTx/Cr combined with ALP and calcium greatly improves diagnostic accuracy for bone metastasis.
• •A simple urine test combined with routine blood markers offers a noninvasive screening strategy.

1. Introduction

Bone Metastasis (BM) refers to the process in which malignant tumor cells originating from other parts of the body migrate via the bloodstream or lymphatic system, colonize and proliferate within bone tissue, and form secondary tumor lesions. Bone is one of the most common sites of metastasis, following the lungs and liver [1]. BM is a frequent complication in patients with various advanced solid tumors, particularly lung cancer, breast cancer, and prostate cancer, significantly impacting patients' quality of life and prognosis [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. It is estimated that the incidence of BM exceeds 70% in patients with advanced breast and prostate cancers, and reaches 30%–40% in those with advanced lung cancer [13], [14]. The occurrence of BM not only indicates that the tumor has entered an advanced stage but, more critically, triggers a series of severe skeletal-related events (SREs). These include intense and refractory bone pain, hypercalcemia, pathological fractures, spinal cord compression, and resulting neurological dysfunction. These complications severely diminish patients' quality of life, increase the complexity of care and medical costs, and ultimately become key factors leading to poor prognosis and shortened survival [15].

Imaging remains the primary clinical method for diagnosing BM; however, it has multiple limitations: high cost and radiation exposure make it difficult to popularize and use frequently in primary care settings; it lacks sensitivity for early, minute lesions, potentially delaying diagnosis; and its assessment of treatment efficacy is delayed, unable to reflect dynamic changes in bone metabolism in real time, which hampers timely adjustment of therapy [16], [17], [18]. This poses a challenge for clinicians in promptly modifying treatment plans. Consequently, there is a pressing clinical need to identify non-invasive, sensitive, cost-effective biomarkers that can reflect dynamic bone metabolic changes.

The N-telopeptide of type I collagen (NTx) has garnered significant attention due to its high specificity for bone tissue and its direct reflection of osteoclast activity [19]. NTx is a specific peptide fragment containing cross-links from the N-terminus of type I collagen, released during osteoclast-mediated bone resorption. When BM occurs, tumor cells stimulate a sharp increase in osteoclast activity, leading to massive degradation of type I collagen. Consequently, NTx is released in large quantities into the bloodstream, filtered by the kidneys, and eventually appears in urine [20]. Therefore, detecting urinary NTx levels may serve as an effective means to assess the pathological state of heightened bone resorption induced by BM [4], [21]. Although urine concentration is easily influenced by fluid intake, measuring urinary creatinine (Cr) simultaneously and calculating the NTx/Cr ratio can effectively correct for variations in urine dilution, providing standardized and comparable results [22]. Additionally, urinary NTx offers the advantages of convenient sample collection and a simple operational workflow.

This study investigates urinary NTx and other bone metabolism biomarkers during the disease progression of patients with lung cancer and bone metastases. It aims to elucidate the relationship between bone metabolic indicators and the mechanisms underlying BM in lung cancer, thereby further guiding the diagnosis of bone metastases and providing a basis for establishing an economical and effective early-warning system.

2. Materials and methods

2.1. Study population

A total of sixty-eight (68) patients diagnosed with lung cancer and new bone metastases who were admitted to the First Affiliated Hospital of Xinjiang Medical University from December 2023 to December 2024, and ninety-eight (98) patients who were diagnosed with lung cancer with no bone metastases during the same period were selected as the case groups. Eighty-seven (87) individuals without a history of malignant tumors, bone metabolic diseases, or major systemic diseases were selected from the healthy population as the control group, using a population matching method for age (±3 years) and sex. All patients with lung cancer underwent at least one of the following imaging examinations at the time of enrollment: contrast-enhanced CT, fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT), or whole-body bone scintigraphy (ECT). Bone metastasis was diagnosed when any of these modalities clearly demonstrated osteolytic, osteoblastic, or mixed metastatic lesions. Patients in the lung cancer without bone metastasis group had no evidence of bone lesions on all available imaging examinations at enrollment. If a patient underwent multiple imaging modalities, the most sensitive modality (PET/CT > ECT > contrast-enhanced CT) was used as the reference standard. This study was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Ethics Approval Number: K202404–26).

2.2. Inclusion and exclusion criteria

2.2.1. Inclusion criteria

• (1)Primary tumor diagnosis of all lung cancer patients could be determined.
• (2)The imaging diagnosis of bone metastasis in lung cancer patients is clear.
• (3)The patient's age is ≥18 years old.
• (4)Blood calcium concentration ≥ 2 mmol/L.
• (5)Serum creatinine ≤1.5 times the upper limit of normal.

2.2.2. Exclusion criteria

• (1)The functional status score of lung cancer patients is greater than 2 points.
• (2)Two or more primary malignant tumors occur simultaneously.
• (3)Women who are pregnant, breastfeeding or planning to become pregnant.
• (4)Suffering from severe heart disease or systemic diseases.
• (5)Patients who have received anti-bone resorption treatment such as denosumabor have undergone radiotherapy to sites of bone metastases within the past month.
• (6)Suffering from endocrine diseases that affect bone metabolism, such as osteoporosis, primary hyperparathyroidism, Paget's disease, poorly controlled diabetes, rheumatic or osteoarthropathy; Traumatic fractures have occurred within the past six months

2.3. General clinical data

Baseline demographic and clinical characteristics—including age, sex, systemic anti-cancer therapy (chemotherapy, targeted therapy, or immunotherapy), radiotherapy, and laboratory indicators (ALP, Ca, phosphorus [PHOS], magnesium [Mg], and CEA)—were retrospectively extracted from the Jiahe Electronic Medical Record System. Prior systemic therapy was defined as any history of chemotherapy, targeted therapy, immunotherapy, or their combinations administered before enrollment.

2.4. Sample collection and testing

All patients with lung cancer who met the inclusion and exclusion criteria were included in this study. Urine and blood samples were collected at the time of hospital admission, strictly prior to the administration of any bone-modifying agents. Second-voided morning urine samples were collected to minimize circadian variation. Clean midstream urine was obtained according to standard procedures. All collected specimens were strictly tested by qualified technicians in the laboratory department in accordance with the standard operating procedures. The specific detection methods are as follows: Urinary NTx and creatinine levels were measured using the VITROS™ 5600 Integrated System (QuidelOrtho, NJ, US) with the corresponding NTx Reagent Pack (Cat# 6800030) and CREA slides (Cat# 6802584). The NTx/Cr ratio was subsequently calculated in nmol bone collagen equivalents (BCE)/mmol Cr in accordance with the manufacturer's instructions.

2.5. Statistical analysis

Data analysis was conducted using SPSS 27.0 (IBM Corp., NY, USA). The number of cases were expressed as percentage [n (%)], and the testing/measurement data were initially evaluated for normality by the Shapiro-Wilk test. Data conforming to the normal distribution were expressed as mean ± standard deviation ($\overline{x}\pm s$), and independent sample t-tests were used for comparison between groups. The non-normally distributed data were described as the median (interquartile range) [M(P₂₅,P₇₅)]. The differences between groups were analyzed using the Wilcoxon rank sum test or the Mann-Whitney test, and the significance threshold was set at bilateral P < 0.05. ROC were generated, including urine NTx/Cr, with calculation of the area under the curves. The diagnostic efficacy of individual biomarkers and their combinations was evaluated based on the area under the curve (AUC). An AUC > 0.7 was considered to indicate favorable diagnostic performance.

3. Results

3.1. Analysis of differences in the levels of various indicators among the three groups of subjects

The study compared the differences in age, gender, cancer type, stage and multiple biochemical indicators between three groups of patients: the healthy control group, the lung cancer without bone metastasis group and the lung cancer with bone metastasis group (Table 1). The results showed that there was no statistically significant difference in age and sex among the three groups (P > 0.05). In the lung cancer without bone metastasis group, the cancer stages were predominantly stage I-III, while in the lung cancer with bone metastasis group, the proportion of stage IV was relatively high (82.4%). Among the biochemical indicators, NTx/Cr, CEA, ALP, Ca, PHOS, and Mg showed statistically significant differences between the three groups (P < 0.001). Among them, NTx/Cr, CEA, and ALP were significantly increased in the bone metastasis group. Ca was significantly decreased in the bone metastasis group. PHOS and Mg also showed differences between groups.

Table 1.

Analysis of the differences in the levels of various indicators among the three groups of subjects.

Variable	Overall n = 253	Healthy control group n = 87	LC-nonBM group n = 98	LC-BM group n = 68	χ2	P
Age (year)	62.00(54.00,70.00)	59.00(54.00,70.00)	62.00(55.00,69.00)	66.00(54.00,73.00)	3.65	0.161
Male, n (%)	181(71.5%)	64(73.6%)	73(74.4%)	44(64.7%)	2.15	0.342
Cancer staging, n (%)	/	/	/	/	/	/
I	7(4.3%)	/	7(7.2%)	/	/	/
II	12(7.2%)	/	12(12.2%)	/	/	/
III	46(27.7%)	/	34(34.7%)	12(17.6%)	/	/
IV	101(60.8%)	/	45(45.9%)	56(82.4%)	/	/
NTx/Cr (nM BCE/nM Cr)	49.23 (35.38, 77.37)	36.18 (27.59, 47.51)	47.58 (37.51, 72.67)	88.06 (65.28, 126.49)	87.88	<0.001
CEA (ng/mL)	2.99 (1.83, 6.50)	1.99 (1.10, 2.89)	3.59 (2.37, 6.69)	6.67 (3.76, 14.22)	84.90	<0.001
ALP (U/L)	84.52 (70.00, 104.53)	72.00 (65.00, 81.10)	85.48 (69.20, 101.63)	109.54 (91.56, 144.27)	62.28	<0.001
Ca (mmol/L)	2.28 (2.19, 2.37)	2.35 (2.27, 2.38)	2.28 (2.24, 2.39)	2.21 (2.13, 2.28)	34.08	<0.001
PHOS (mmol/L)	1.13(1.00, 1.25)	1.06(0.96,1.14)	1.20(1.07,1.31)	1.14(1.01,1.28)	27.13	<0.001
Mg (mmol/L)	0.86(0.80, 0.91)	0.88(0.84,0.94)	0.82(0.78,0.88)	0.85(0.81,0.91)	22.66	<0.001

Note: Measurement data are expressed as median (interquartile range). The urinary level of NTx in BCE is expressed as a ratio to urine Cr excretion.

Abbreviations: ALP, alkaline phosphatase; BCE, bone collagen equivalents; Ca, calcium; CEA, carcinoembryonic antigen; LC-BM, lung cancer with bone metastases; LC-nonBM, lung cancer without bone metastases; Mg, magnesium. NTx/Cr, N-telopeptide of type I collagen corrected by creatinine; PHOS, phosphorus.

3.2. The differences in indicators among patients at different stages in the non-bone metastasis group

The study further analyzed the levels of various indicators in 98 patients with lung cancer without bone metastases, including those in the early stage (stage I- II), stage III and stage IV. NTx/Cr showed statistically significant differences among different stages (P = 0.004) and showed an upward trend with the progression of stages (Table 2).

Table 2.

Analysis of the differences in the levels of various indicators among 98 patients with lung cancer without bone metastases at different stages.

Variable	Overall n = 98	Stage I-II n = 19	Stage III n = 34	Stage IV n = 45	χ2	P
NTx/Cr (nM BCE/nM Cr)	47.74(37.62,72.92)	38.73(32.45,46.36)	54.22(41.95,74.31)	52.10(40.04,78.39)	11.01	0.004
CEA (ng/mL)	3.59(2.58,6.05)	2.85(2.11,3.96)	3.59(2.47,4.61)	4.11(2.75,7.53)	5.27	0.072
ALP (U/L)	85.48(70.15,100.77)	75.74(63.98,95.96)	85.48(71.17,101.21)	86.96(75.60,104.31)	2.55	0.280
Ca (mmol/L)	2.28(2.24,2.39)	2.27(2.24,2.42)	2.28(2.24,2.38)	2.27(2.24,2.36)	0.48	0.788
PHOS (mmol/L)	1.20(1.07,1.31)	1.20(1.10,1.22)	1.16(1.08,1.36)	1.24(1.05,1.32)	0.89	0.642
Mg (mmol/L)	0.82(0.78,0.88)	0.81 (0.77,0.86)	0.82 (0.78,0.89)	0.83 (0.79,0.88)	0.47	0.790

Note: Measurement data are expressed as median (interquartile range). The urinary level of NTx in BCE is expressed as a ratio to urine Cr excretion.

Abbreviations: ALP, alkaline phosphatase; BCE, bone collagen equivalents; Ca, calcium; CEA, carcinoembryonic antigen; Mg, magnesium. NTx/Cr, N-telopeptide of type I collagen corrected by creatinine; PHOS, phosphorus.

3.3. The index differences in the number of different metastatic sites among patients with bone metastases

The index levels of patients with single-site, two-site and three or more site metastases among 68 patients with lung cancer and bone metastases were compared (Table 3). There were differences in the levels of NTx/Cr and ALP between patients with different numbers of metastatic bone lesions/sites (P < 0.001), and the indicators increased significantly with increased number of metastatic sites. There were no statistically significant differences in CEA, Ca, P and Mg in relation to the number of metastatic sites (P > 0.05).

Table 3.

Analysis of the differences in the levels of various indicators among the number of different bone metastasis sites in 68 patients with lung cancer and bone metastases.

Variable	Overall n = 68	One site n = 34	Two sites n = 15	Three sites n = 19	χ2	P
NTx/Cr (nM BCE/nM Cr)	88.06(65.28, 126.49)	64.71(50.52,75.09)	95.05(86.80,129.59)	151.05(119.56,280.05)	38.89	<0.001
CEA (ng/mL)	6.67(3.76, 14.22)	6.02(3.10,12.19)	5.46(3.63,11.77)	12.55(6.30,18.28)	3.32	0.190
ALP (U/L)	109.54(91.88, 143.81)	100.46(83.96,131.51)	101.96(94.45,116.58)	146.20(122.64,218.19)	14.23	<0.001
Ca (mmol/L)	2.21(2.13, 2.28)	2.20(2.10,2.28)	2.22(2.14,2.26)	2.19(2.14,2.29)	1.29	0.525
PHOS (mmol/L)	1.14(1.01, 1.28)	1.21(1.07,1.32)	1.11(0.97,1.15)	1.08(0.99,1.25)	3.42	0.181
Mg (mmol/L)	0.85(0.81, 0.91)	0.84(0.80,0.93)	0.86(0.83,0.91)	0.84(0.81,0.86)	0.92	0.631

Note: Measurement data are expressed as median (interquartile range). The urinary level of NTx in BCE is expressed as a ratio to urine Cr excretion.

Abbreviations: ALP, alkaline phosphatase; BCE, bone collagen equivalents; Ca, calcium; CEA, carcinoembryonic antigen; Mg, magnesium. NTx/Cr, N-telopeptide of type I collagen corrected by creatinine; PHOS, phosphorus.

3.4. ROC curves of each indicator individually and combined for diagnosis of bone metastasis

Fig. 1 shows the diagnostic efficacy of each indicator for bone metastasis in lung cancer: The ROC curve for NTx/Cr alone indicated an AUC of 0.780 for prediction of bone metastasis, while ALP and Ca yielded AUCs of 0.737 and 0.710, respectively (Fig. 1A&B). The ROC analysis for combined NTx/Cr + ALP + Ca (Joint testing I) yielded the largest AUC of 0.882, suggesting the highest diagnostic efficacy (Fig. 1C). The AUC for NTx/Cr + Ca (Joint testing II) also showed strong prediction for bone metastasis (Fig. 1D). The results indicate that the combined detection of multiple indicators can significantly improve the diagnostic accuracy of bone metastasis in lung cancer, especially the combination of NTx/Cr with ALP and Ca.

Fig. 1.

ROC curves of each indicator individually and combined for diagnosis of bone metastasis. (A) NTx/Cr alone for the diagnosis of bone metastasis. (B) Individual ROC curves with NTx/Cr, ALP, and Ca. (C) Individual and combined NTx/Cr, ALP and Ca (Joint testing I) ROC curves. (D) Individual and combined NTx/Cr, and Ca (Joint testing II) ROC curves.

3.5. Impact of prior systemic therapy on biomarker levels

To address potential confounding effects, we performed subgroup analyses stratified by prior systemic anti-cancer therapy history. Among the 98 LC-nonBM patients, 30 (30.6%) were treatment-naïve, whereas 68 (69.4%) had received prior therapy. In the 68 patients among LC-BM group, 37 (54.4%) were untreated and 31 (45.6%) had prior treatment.

As shown in Table 4, the LC-nonBM patients with prior systemic therapy exhibited significantly higher Mg levels and lower CEA levels compared to treatment-naïve patients (P < 0.05). Crucially, the core markers—NTx/Cr, ALP, and Ca—showed no significant differences between treated and untreated subgroups (P > 0.05). Similarly, in the LC-BM cohort, no significant differences were observed for NTx/Cr, ALP, Ca, Mg, or CEA between the two groups (P > 0.05), except for PHOS (P = 0.034).

Table 4.

Subgroup analysis of biomarker levels stratified by prior systemic therapy history in lung cancer patients.

Variable	LC-nonBM (n = 98)			LC-BM (n = 68)
	Untreated (n = 30)	Treated (n = 68)	P	Untreated (n = 37)	Treated (n = 31)	P
NTx/Cr (nM BCE/nM Cr)	44.75 (37.78, 58.50)	48.03 (37.85, 73.90)	0.289	104.53 (83.09, 122.79)	78.81 (57.03, 144.50)	0.178
ALP (U/L)	85.48 (74.53, 106.55)	85.48 (68.50, 98.59)	0.412	103.87 (94.46, 139.87)	116.30 (90.62, 143.35)	0.937
Ca (mmol/L)	2.27 (2.24, 2.32)	2.28 (2.24, 2.42)	0.239	2.18 (2.13, 2.26)	2.22 (2.12, 2.28)	0.718
PHOS (mmol/L)	1.15 (1.05, 1.26)	1.22 (1.08, 1.32)	0.191	1.09 (1.00, 1.22)	1.20 (1.12, 1.34)	0.034
Mg (mmol/L)	0.86 (0.81, 0.92)	0.81 (0.77, 0.86)	0.001	0.86 (0.82, 0.89)	0.84 (0.79, 0.92)	0.543
CEA (ng/mL)	3.06 (1.89, 4.19)	3.59 (2.67, 6.71)	0.039	5.96 (3.85, 12.71)	8.94 (2.91, 97.70)	0.191

Note: Measurement data are expressed as median (interquartile range). The urinary level of NTx in BCE is expressed as a ratio to urine Cr excretion.

Abbreviations: ALP, alkaline phosphatase; BCE, bone collagen equivalents; Ca, calcium; CEA, carcinoembryonic antigen; LC-BM, lung cancer with bone metastases; LC-nonBM, lung cancer without bone metastases; Mg, magnesium. NTx/Cr, N-telopeptide of type I collagen corrected by creatinine; PHOS, phosphorus.

Importantly, the key finding indicates that urinary NTx/Cr levels were consistently elevated in bone metastasis patients who remained unaffected by prior systemic therapy status. These results demonstrate that urinary uNTx/Cr is a robust biomarker for bone metastasis, with minimal confounding from prior systemic therapy history.

4. Discussion

This study systematically compared multiple biochemical indicators among healthy controls, lung cancer patients without bone metastases, and those with lung cancer and bone metastases. It represents a relatively large-scale cohort study in China to confirm the core clinical value of urinary N-telopeptide of type I collagen corrected for creatinine (NTx/Cr) in diagnosing bone metastasis in lung cancer. The results clearly demonstrated that NTx/Cr, CEA, ALP, Ca, PHOS, and Mg all showed statistically significant differences between the three groups (P < 0.001). Specifically, NTx/Cr, CEA, and ALP were significantly elevated in the bone metastasis group, while Ca was notably decreased. Importantly, ROC curve analysis revealed that the combination of NTx/Cr, ALP, and Ca yielded the largest area under the curve (AUC), with diagnostic performance significantly superior to that of any single marker alone. This finding provides direct evidence to support the establishment of an efficient bone metastasis screening strategy for patients with lung cancer in clinical practice.

Bone metastases represent the most common form of distant metastasis in lung cancer. Its core pathological mechanism involves tumor cells disrupting the balance of the bone microenvironment, inducing excessive activation of osteoclasts and leading to abnormally increased bone resorption [23], [24]. NTx, a specific degradation product of type I collagen, is a sensitive marker reflecting bone resorption activity [4]. When tumor cells invade bone tissue, local osteoclastic activity increases significantly, resulting in extensive breakdown of type I collagen. The released NTx enters the bloodstream and is excreted via the kidneys, making urinary NTx concentration an indirect indicator of systemic bone resorption [20]. This study found that urinary NTx/Cr levels were significantly higher in the lung cancer with bone metastasis group compared to both the non-metastatic group and the healthy controls and were closely correlated with tumor stage and the number of metastatic lesions—consistent with previous findings [25]. Multiple studies have confirmed that elevated urinary NTx levels not only indicate the presence of bone metastasis but are also associated with disease progression, skeletal-related events (SREs), and poor prognosis in lung cancer [26], [27], [28]. Several recent clinical trials have evaluated the percentage change from baseline to week 13 in urinary NTx/Cr levels as the primary endpoint for assessing the therapeutic response to denosumab biosimilars relative to reference denosumab in patients with bone metastases from solid tumors [29], [30], [31]. Therefore, urinary NTx/Cr serves not only as a diagnostic marker but also as a liquid biopsy indicator that reflects bone metastatic burden and dynamic disease changes, holding potential value for management throughout the entire disease continuum in lung cancer patients [32], [33].

Early exploratory studies have preliminarily revealed the potential of urinary NTx in screening for bone metastasis. As early as the beginning of the 21st century, researchers proposed that urinary NTx could serve as a complementary tool to bone scintigraphy for assisting in the diagnosis of bone metastases [34]. However, these earlier studies were limited by small sample sizes and a lack of standardized detection protocols, leading to considerable heterogeneity in results [26], [27], [35], [36]. In the present study, ROC curve analysis confirmed that urinary NTx/Cr alone demonstrated considerable diagnostic value (with an AUC significantly superior to that of any single traditional marker), and its diagnostic performance was further enhanced when combined with indicators such as ALP and Ca (yielding the maximum AUC). This aligns with recent reports suggesting that combined biomarker panels outperform single markers [37].A clear research trajectory can be observed from molecular mechanism to clinical translation: early studies focused on the biological characteristics of urinary NTx [38]; mid-phase research explored its application across different tumor types,with lung cancer being one of the most extensively studied [39], [40], [41]; while recent studies have shifted toward establishing standardized detection protocols and conducting multi-center validation [25].It is noteworthy that although most studies support the diagnostic value of urinary NTx/Cr, some reports indicate that its sensitivity may be lower than that of bone scintigraphy, and results may overlap in patients with early-stage bone metastasis, leading to false negatives [42], [43]. The large-sample design of this study partially mitigates this issue, yet the limitations of urinary NTx/Cr as a screening tool must still be acknowledged in the context of lung cancer.

In the spectrum of diagnostic markers for bone metastasis, traditional biochemical indicators such as serum ALP, Ca, and PHOS are widely used but lack specificity. The data from this study show that although ALP is elevated in the lung cancer with bone metastasis group, its diagnostic performance is significantly lower than that of urinary NTx/Cr. Multiple meta-analyses have confirmed that, compared to markers such as ALP and type I collagen carboxy-terminal telopeptide (ICTP), NTx demonstrates superior diagnostic performance in terms of both sensitivity and specificity [42], [44]. For example, a systematic review involving over 2000 patients reported that NTx achieved an overall sensitivity of 77% and a specificity of 80% for diagnosing bone metastasis, with an AUC of 0.86, whereas ALP had an AUC of only approximately 0.70 [37]. This conclusion is further supported by ROC curve comparisons in the present study, which revealed that the AUC value for urinary NTx/Cr was significantly higher than that for ALP alone. However, the diagnostic value of NTx from different matrix sources remains debated. Some studies comparing serum NTx with urinary NTx/Cr found that although the two are highly correlated, urinary NTx/Cr may be more stable, as creatinine correction can reduce the impact of fluctuations in urine concentration [45], [46]. Additionally, bone formation markers such as serum osteocalcin have limited diagnostic value in early bone metastatic lesions [47], while newer bone resorption markers like serum C-telopeptide of type I collagen (CTx), although similar in mechanism to NTx, still lack sufficient clinical application data [30]. Therefore, urinary NTx/Cr remains a core biomarker among existing bone turnover markers, with advantages including non-invasive detection and strong reproducibility.

The most important practical implication of this study lies in validating the superiority of a multi-marker diagnostic strategy for lung cancer patients. ROC curve analysis showed that the combined detection of urinary NTx/Cr + ALP + Ca (Joint testing I) achieved the largest AUC and the best diagnostic performance, while urinary NTx/Cr + Ca (Joint testing II) also demonstrated favorable diagnostic value. This aligns closely with current trends in translational medicine research—namely, that a single biomarker is often insufficient to meet the demands of accurate diagnosis and treatment for complex diseases, and developing multi-dimensional diagnostic models is key to improving accuracy. The enhanced performance of combined diagnosis likely stems from the complementary roles of the markers: urinary NTx/Cr specifically reflects the extent of bone destruction, ALP indicates compensatory osteoblastic activity, and abnormal Ca metabolism captures the end effect of imbalanced bone turnover. Together, they provide a multi-faceted view of the pathophysiological changes in bone metastasis, thereby reducing the risk of missed diagnoses. It is worth noting that although authoritative guidelines such as those from the European Society for Medical Oncology (ESMO) and the National Comprehensive Cancer Network (NCCN) emphasize multidisciplinary management of bone metastasis, they have yet to offer clear recommendations on combined biomarker testing strategies [48], [49]. This study provides empirical evidence to support such an approach in lung cancer. Future work should validate the stability of this combined model in prospective cohorts of lung cancer patients and explore more advanced predictive models that incorporate tumor-specific markers (e.g., CEA, prostate-specific antigen [PSA]) or molecular imaging data.

We further performed subgroup analyses stratified by prior systemic therapy to address potential confounding. Notably, despite the higher prevalence of stage IV disease in the LC-BM group (82.4% vs. 45.9% in LC-nonBM), urinary NTx/Cr elevation remained consistent regardless of treatment history, reinforcing its diagnostic robustness. The observed intergroup differences in Mg and CEA among LC-nonBM patients are likely attributable to treatment-related effects (e.g., EFGR inhibitors altered magnesium absorption) [50] or greater baseline tumor burden in those requiring therapy, rather than reflecting bone resorption, as evidenced by the unaffected NTx/Cr levels.

Several limitations of this study should be acknowledged. First, this was a single-center study with a relatively small sample size, which may limit the generalizability of our findings. Second, the cross-sectional design identifies established bone metastases rather than predicting their future development. Serial measurements of urinary NTx/Cr would be needed to determine whether rising levels precede imaging-detected metastases. Third, due to limited subgroup sizes, we did not analyze small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) separately; the diagnostic performance of the panel may differ between histological types. Fourth, although bone scintigraphy or PET/CT was used as the reference standard, these imaging modalities have known limitations. We attempted to mitigate this by requiring confirmation with additional imaging when possible. Fifth, all patients were recruited from a single tertiary hospital in Xinjiang, which may introduce selection bias. Future prospective, multi-center studies with larger cohorts are warranted to validate this diagnostic panel and to evaluate its role in serial monitoring and early warning of bone metastases.

5. Conclusion

This study confirms that urinary NTx/Cr is an effective biomarker for diagnosing bone metastases in lung cancer, with its levels showing a strong correlation with tumor stage and metastatic burden. A multi-indicator diagnostic model combining NTx/Cr, ALP, and Ca significantly improved diagnostic accuracy for lung cancer patients. Although limitations such as sample composition bias due to the single-center design and a lack of external validation exist, this research provides important evidence for the clinical translation of NTx/Cr in the context of lung cancer. Future efforts should focus on standardizing detection methods, conducting multi-center validation, and performing prospective intervention studies to facilitate its integration into routine clinical pathways for lung cancer patients. This will ultimately enhance early diagnosis and precision management for patients with bone metastases.

CRediT authorship contribution statement

Lan Jiang: Writing – original draft, Visualization, Investigation, Formal analysis, Data curation. Puyue Zhang: Visualization, Investigation, Conceptualization. Ziwei Wang: Visualization, Methodology, Conceptualization. Xuejiao Chang: Visualization, Methodology, Investigation. Pengfei Lu: Supervision, Investigation, Data curation, Conceptualization. Yuxiang Zhang: Validation, Investigation, Conceptualization. Guoyao Sang: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.

Ethics approval

The research protocol was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Ethics Approval Number: K202404–26).

Ethics approval and consent to participate

This study was performed in accordance with Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Ethics number: K202404–26).

Funding

This research is funded by the Horizontal Project of the First Affiliated Hospital of Xinjiang Medical University (Grant No. HX2024–17).

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Guoyao Sang reports financial support was provided by The First Affiliated Hospital of Xinjiang Medical University. Guoyao Sang reports a relationship with The First Affiliated Hospital of Xinjiang Medical University that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.