Contributed equally
Immune checkpoint inhibitors (ICIs) have been an encouraging treatment method in non-small cell lung cancer (NSCLC). However, bone and liver metastases are considered to restrain immunotherapy efficacy. Since serum alkaline phosphatase (ALP) is associated with bone and liver metastases, it was investigated whether serum ALP could be a novel biomarker to predict the efficacy of ICIs treatment. In the present study, 143 patients with NSCLC receiving ICIs treatment were retrospectively analyzed. The objective response rate (ORR) was compared between the ALP high and low groups, bone metastasis and non-bone metastasis groups, and liver metastasis or non-liver metastasis groups. The associations between clinical characteristics, including ALP level, bone or liver metastasis and median progression-free survival (mPFS) time were analyzed by univariate and multivariate Cox regression analysis. It was found that bone metastasis was associated with a lower ORR (24 vs. 43%; P<0.05) and shorter mPFS (10.2 vs. 17.3 months; P=0.010) in patients with NSCLC receiving ICIs. Liver metastasis was associated with lower ORR (22 vs. 38%; P<0.05), but not with mPFS (P=0.119). The ALP level was higher in patients with bone or liver metastasis than in those without (119.6 or 103.6 vs. 83.3 U/l, respectively; P<0.05). Higher ALP levels were also associated with bone or liver metastasis, lower ORR (20 vs. 39%; P<0.05) and shorter mPFS (8.5 vs. 15.4 months; P=0.009). Cox regression analysis demonstrated that ALP was an independent prognostic indicator of mPFS (hazard ratio, 1.856; 95% confidence interval, 1.030-3.343; P=0.040). In conclusion, pretreatment levels of serum ALP might be a predictive indicator of clinical outcome in patients with NSCLC after ICIs treatment.
Lung cancer is the leading cause of cancer-related mortality worldwide (
At present, PD-L1 expression, tumor mutational burden (TMB) and microsatellite instability (MSI) status have been recognized as reliable indicators to predict the response to ICIs in NSCLC, hepatocellular carcinoma, urothelial carcinoma and other tumors (
ALP is a glycoprotein that catalyzes hydrolytic and phospho-transfer reactions. Elevated serum ALP has been reported in bone and liver-related diseases (
In the present study it was found that bone or liver metastasis was associated with poor prognosis in patients with NSCLC receiving ICIs. Furthermore, elevated serum ALP levels were associated with bone and liver metastases, indicating that ALP levels might be an independent prognostic indicator of ICIs treatment efficacy in patients with NSCLC.
Between January 2018 and December 2020, patients with NSCLC receiving ICIs treatment were investigated at the Institute of Cancer, Xinqiao Hospital, Third Military Medical University (Chongqing, China). Patients who met the following criteria were included in the present study: i) Pathologically diagnosed lung squamous cancer or adenocarcinoma without receiving an operation; ii) not harboring driver gene mutations, including EGFR, ALK, ROS1 and MET; iii) receiving ICIs treatment (including PD-1, PD-L1 or CTLA-4 antibody) for at least 2–3 cycles (6 weeks); iv) complete image examination of evaluable foci pretreatment and post-treatment; and v) complete record of pretreatment ALP levels and complete record of follow-up. Patients with mutations of driver genes, or without records of pretreatment ALP levels, or receiving ICIs treatment <6 weeks were excluded. Overall, 143 patients were included and data regarding their clinical characteristics [age, sex, histology type, TNM stage (according to the 8th AJCC staging system) (
ALP was detected using the endpoint spectrophotometric method in the peripheral blood of patients <1 week before treatment with ICIs, and the ALP level data were collected from medical records. The normal level of ALP is between 0 and 110 U/l (
Patients were evaluated during follow-up by CT of the lungs and abdomen or MRI of the brain. The response to ICIs treatment was classified according to iRECIST criteria (
The ORR was defined as the proportion of patients with response of iPR and iCR. PFS was defined as the time from the beginning of immunotherapy until recurrence of disease or death due to any cause. Telephone follow-up was performed every 6 months, and the last follow-up date was August 31, 2021.
The ALP level data are presented as the median and interquartile range. The mPFS data are presented as Kaplan-Meier curves. The composition of patients or response rate data are presented as n (%), n or the proportion. The results of univariate and multivariate Cox regression analyses are presented as the hazard ratio and 95% CI. The difference in ALP levels was analyzed using an unpaired Student's t-test or one-way ANOVA with a Bonferroni post hoc test. The associations between ALP levels and clinical characteristics were analyzed using a χ2 test and Fisher's exact test if the expected count was <5. The percentages of clinical response of patients to ICIs treatment in two groups were compared using a χ2 test. Kaplan-Meier survival curves and the log-rank test were used to estimate mPFS, and univariate and multivariate Cox regression analyses were used to determine the associations between clinicopathological factors and survival. A forest plot was used to show the results of multivariate Cox regression analysis using Prism GraphPad 9.0 software (GraphPad Software, Inc.). All statistical analyses were conducted using SPSS 20.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.
A total of 143 patients with NSCLC receiving ICIs were included, and their baseline characteristics are presented in
Previous preclinical and clinical evidence has demonstrated that bone or liver metastases can restrain immunotherapy efficacy (
ALP has been recognized as a valuable marker for skeletal and hepatobiliary disorders, including bone and liver metastases in patients with cancer (
To determine the predictive value of ALP, the ALP levels in responders and non-responders to ICIs treatment were first compared. It was found that the levels of ALP were higher in non-responders than in responders (110.1 vs. 88.0 U/l; P<0.01;
Immunotherapy, especially ICIs treatment, has been one of the most promising treatment approaches in NSCLC (
Numerous studies have demonstrated the mechanisms of primary resistance in ICIs treatment (
Primary resistance cannot be completely explained by currently recognized biomarkers, such as PD-L1 expression, MSI status or TMB level. Thus, studies are increasingly focusing on other biomarkers to predict the response to ICIs treatment (
As aforementioned, bone or liver metastasis, and high ALP levels were associated with poor prognosis in patients with NSCLC receiving ICIs treatment in the present study. However, in the present study, it was found that only high ALP levels, rather than bone or liver metastasis, were an independent prognostic factor using multivariate analysis, rather than bone or liver metastasis. This difference indicated that other factors affecting ALP levels, such as benign liver and bone diseases, might also restrain the efficacy of ICIs treatment. Furthermore, it was found that higher ALP levels were associated with a shorter PFS time in patients without bone metastasis (data not shown). However, this hypothesis should be verified in further studies.
There were a number of limitations in the present study. Firstly, this was a retrospective study in a single center with a small number of cases. Secondly, the lack of overall survival data might affect the reliability of the conclusions made. Thirdly, the PD-L1 expression, TMB and MSI status in patients might also affect the outcomes of ICIs treatment (
In conclusion, serum ALP levels might be an independent predictor of the response to ICIs treatment in patients with NSCLC. Due to a number of limitations of the present study, prospective studies are required to determine the actual prognostic significance of ALP levels in patients with NSCLC.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
FL and JY conceived and designed the study. KY and TS developed the methodology. TY, JC and SF acquired the data. TY and JC confirmed the authenticity of all the raw data. TY and JC analyzed and interpreted the data. TY and JC wrote the original draft. JY and FL reviewed and revised the manuscript. FL and JY supervised the study. TY, JC and TS were involved in funding acquisition. All authors read and approved the final manuscript.
The study was approved by the Ethics Committee of Xinqiao Hospital, Third Military Medical University (Chongqing, China). All patients or their legal representatives gave written informed consent for the treatment and inquiries related to this study.
The patients provided written informed consent for publication of any associated data.
The authors declare that they have no competing interests.
alkaline phosphatase
cytotoxic T lymphocyte-associated antigen-4
immune checkpoint inhibitors
microsatellite instability
non-small cell lung cancer
objective response rate
programmed cell death-1
programmed cell death-ligand 1
progression-free survival
tumor mutational burden
Bone and liver metastasis restrain efficacy of ICIs treatment. (A) Response to ICIs treatment of patients with NSCLC with or without bone metastasis. (B) mPFS of patients with NSCLC with or without bone metastasis after ICIs treatment. (C) Response to ICIs treatment of patients with NSCLC with or without liver metastasis. (D) mPFS of patients with NSCLC with or without liver metastasis after ICIs treatment. (E) ALP levels in patients with NSCLC with or without bone metastasis and liver metastasis. *P<0.05, **P<0.01. ALP, alkaline phosphatase; ICIs, immune checkpoint inhibitors; iCPD, progressive disease; iPR, partial response; iSD, stable disease; NSCLC, non-small cell lung cancer; mPFS, median progression-free survival.
ALP levels predict efficacy of ICIs treatment in patients with NSCLC. (A) ALP levels in responders and non-responders to ICIs treatment. (B) Response to ICIs treatment of patients with NSCLC with or without high ALP levels. (C) Progression-free survival of patients with NSCLC with or without high ALP levels after ICIs treatment. *P<0.05, **P<0.01. ALP, alkaline phosphatase; ICIs, immune checkpoint inhibitors; iCPD, progressive disease; iSD, stable disease; NSCLC, non-small cell lung cancer; iPR, partial response.
Forest plot of univariate analyses of different parameters for progression-free survival. AD, adenocarcinoma; ALP, alkaline phosphatase; HR, hazard ratio; SC, squamous cancer.
Baseline characteristics of 143 patients with lung cancer.
Characteristics | No. (%) |
---|---|
Age, years | |
≥60 | 61 (42.7) |
<60 | 82 (57.3) |
Sex | |
Male | 113 (79.0) |
Female | 30 (21.0) |
Histology type | |
SC | 74 (51.7) |
AD | 69 (48.3) |
TNM stage | |
III | 18 (12.6) |
IV | 125 (87.4) |
Metastatic sites | |
Bone only | 45 (31.4) |
Liver only | 10 (7.0) |
Both bone and liver | 17 (11.9) |
Neither bone or liver | 71 (49.7) |
Treatment lines | |
1st | 85 (59.4) |
≥2nd | 58 (40.6) |
Combined therapy | |
Yes | 65 (45.5) |
No | 78 (54.5) |
AD, adenocarcinoma; SC, squamous cancer.
Association between clinical variables and serum ALP levels.
Pretreatment ALP levels | |||
---|---|---|---|
Characteristics | ≥110 U/l, n | <110 U/l, n | P-value |
Age, years | 0.809 | ||
≥60 | 16 | 45 | |
<60 | 23 | 59 | |
Sex | 0.402 | ||
Male | 29 | 84 | |
Female | 10 | 20 | |
Histology type | 0.931 | ||
SC | 21 | 53 | |
AD | 18 | 51 | |
Stage | 0.100 | ||
III | 2 | 16 | |
IV | 37 | 88 | |
Bone metastasis | <0.001 | ||
Yes | 34 | 28 | |
No | 5 | 76 | |
Liver metastasis | 0.036 | ||
Yes | 10 | 17 | |
No | 29 | 87 | |
Bone and liver metastasis | <0.001 | ||
Both | 10 | 7 | |
Neither | 4 | 67 | |
Treatment lines | 0.945 | ||
1st | 23 | 62 | |
≥2nd | 16 | 42 | |
Combined therapy | 0.304 | ||
Yes | 15 | 50 | |
No | 24 | 54 |
AD, adenocarcinoma; ALP, alkaline phosphatase; SC, squamous cancer.
Univariate and multivariate analyses of different parameters for progression-free survival.
Univariate | Multivariate | |||||
---|---|---|---|---|---|---|
Variables | HR | 95% CI | P-value | HR | 95% CI | P-value |
Sex (male vs. female) | 1.274 | 0.731-2.219 | 0.417 | |||
Age (≥60 vs. <60 years) | 1.028 | 0.653-1.617 | 0.903 | |||
Histology (SC vs. AD) | 1.402 | 0.889-2.209 | 0.134 | 0.511 | 0.302-0.863 | 0.012 |
Stage (III vs. IV) | 0.343 | 0.195-0.603 | 0.006 | 3.104 | 1.213-7.944 | 0.018 |
Bone metastasis (no vs. yes) | 0.563 | 0.349-0.907 | 0.010 | 1.174 | 0.856-2.641 | 0.103 |
Liver metastasis (no vs. yes) | 0.658 | 0.356-1.217 | 0.119 | |||
Treatment lines (1st vs. ≥2nd) | 0.898 | 0.548-1.472 | 0.662 | |||
Combined therapy (yes vs. no) | 1.453 | 0.804-2.262 | 0.199 | |||
ALP (<110 vs. ≥110 U/l) | 1.838 | 1.609-3.191 | 0.009 | 1.856 | 1.030-3.343 | 0.040 |
AD, adenocarcinoma; ALP, alkaline phosphatase; HR, hazard ratio; SC, squamous cancer.