Nomogram predicting leukopenia in osteosarcoma after high-dose methotrexate chemotherapy

Clinical pathological characteristics of patients

From January 2017 to December 2021, a total of 65 OS patients were recorded in Tianjin Medical University Cancer Institute and Hospital, Tianjin, China. Based on the inclusion and exclusion criteria, 35 OS patients were included in the construction cohort (19 males and 16 females with the median age of 16 years). A total of 128 cycles of MTX therapy were performed. Additionally, 21 OS patients from P.A. Hertsen Moscow Oncology Research Center between January 2019 to December 2021 were recorded, a total of 12 patients were included as the external validation cohort (7 males and 5 females with the median age of 16 years). A total of 37 cycles of MTX therapy were performed (Figure 1).

Among all OS lesions of the construction cohort, 19 were located in the distal femur, 12 were located in the proximal tibia, and a total of 4 were located in the proximal humerus, ilium, clavicle, and fibula. Regarding the pathological type of OS, 29 cases were osteogenic, 5 cases were chondrogenic, and 1 case was fibrogenic. Based on AJCC classification, 8 cases were grade IIA, 23 cases were grade IIB, and 4 cases were grade III. Three cases were accompanied by pathological fractures at the first diagnosis. The clinicopathological characteristics of the patients, who underwent a total of 128 chemotherapy cycles, are shown in Table 1.

Distribution of C_MTX

The total MTX doses in the LP and non-LP groups were 13.63±1.64 g and 14.48±1.44 g, respectively, with no significant difference between the 2 groups. Among the patients included in this study, the mean C_MTX levels immediately after 0 h and 6 h, 24 h, 48 h, and 72 h after MTX treatment were 980.05±373.53 μmol/L, 108.47±27.48 μmol/L, 1.02±0.84 μmol/L, 0.15±0.21 μmol/L, and 0.04±0.05 μmol/L, respectively. For patients in the post-chemotherapy LP group, the C_MTX levels at 0 h, 6 h, 24 h, 48 h and 72 h after MTX administration were 990.58±354.43 μmol/L, 121.99±24.86 μmol/L, 1.3±0.7 μmol/L, 0.18±0.18 μmol/L and 0.04±0.06 μmol/L, respectively. For patients in the post-chemotherapy non-LP group, the C_MTX levels at 0 h, 6 h, 24 h, 48 h and 72 h after MTX administration were 968.13±396.75 μmol/L, 93.15±21.76 μmol/L, 0.70±0.86 μmol/L, 0.12±0.23 μmol/L and 0.03±0.03 μmol/L, respectively. Six hours after MTX administration, the mean C_MTX value was 108.47±27.48 μmol/L. There was a significant difference in C_MTX at 6 h between the 2 groups (p<0.001). Twenty-four hours after MTX administration, the mean C_MTX was 1.02±0.84 μmol/L. There was a significant difference in C_MTX at 24 h between the 2 groups (p<0.001). The trend of the variation in C_MTX at different time points is shown in Figure 2A.

The relationship between C_MTX and postoperative WBC counts at different time points is shown in Figure 2B–2F. When the cut-off value for C_MTX was 100.0 μmol/L at 6 h, the sensitivity and specificity of predicting a WBC count below 3×10⁹/L after MTX administration were 83.8% and 58.3%, respectively. Among them, after 82 MTX treatments, C_MTX ranged from 100.26 to 211.49 μmol/L, and 57 cases of LP occurred; C_MTX was <100.0 μmol/L after the remaining 46 MTX treatments, and a total of 11 cases of LP occurred.

When the cut-off value for C_MTX was 1.0 μmol/L at 24 h, the sensitivity and specificity of predicting a WBC count below 3×10⁹/L were 72.1% and 83.3%, respectively. Among them, after 69 MTX treatments, C_MTX ranged from 0.05 to 0.94 μmol/L, and a total of 19 cases of LP occurred; C_MTX was >1.0 μmol/L after the remaining 59 MTX treatments, and a total of 49 cases of LP occurred. In this study, a total of 9 patients receiving MTX chemotherapy treatment had a C_MTX > 0.1 μmol/L after 72 h, suggesting delayed MTX excretion; among these patients, 8 cases of LP occurred.

By calculating the area under the receiver operating characteristic (ROC) curve (AUC), it was found that when the C_MTX cut-off value was 112 μmol/L at 6 h, the sensitivity and specificity of predicting a WBC count below 3×10⁹/L after MTX administration were 75.0% and 76.7%, respectively, with an AUC of 0.817. When the C_MTX cut-off was 1.01 μmol/L at 24h, the sensitivity and specificity were 72.1% and 85.0%, respectively, and the AUC was 0.797. There was no significant difference between 6h and 24h (p=0.721). The ROC curves are shown in Figure 3A.

Risk factors for LP after MTX administration

Based on the logistic multivariate analysis, females (OR=3.533, 95% CI 1.149–11.734, P=0.032), Ki-67 index > 20% (OR=4.797, 95% CI 1.457-18.146, P=0.014), average dose MTX >14 g (OR=0.494, 95% CI 0.176-1.324), C_MTX at 6h >112 μmol/L (OR=10.482, 95% CI 4.056–30.167, P<0.001), RBC (OR=0.828, 95% CI 0.285-2.324), PLT (OR=0.994, 95% CI 0.988–0.999, P=0.038), and AST concentration (OR=1.057, 95% CI 1.004-1.118, P=0.040) were risk factors for LP after chemotherapy. All the indicators were shown in Figure 3B–3D.

Establishing a nomogram to predict the risk of LP

Based on multivariate logistic regression model results, we selected the following variables to construct prediction spectra of the occurrence of LP in OS patients after MTX treatment: sex, adMTX, Ki-67, C_MTX at 6h, RBC, PLT and AST before neoadjuvant chemotherapy (Figure 4A). The nomogram showed good accuracy in predicting the risk of LP, with a C-index of 0.869 (95% CI: 0.810-0.929). The AUC of this prediction model was 86.9%, suggesting that the model has a relatively satisfactory prediction ability (Figure 4B). In addition, the calibration curves for the nomogram prediction model were all close to the 45-degree line, and the predicted values were in good agreement with the observed values (Figure 4C).

External validation of the prediction model

Detailed information on the clinicopathological characteristics of the external validation cohort was shown in Table 1. As shown in Figure 4D, the AUC of the predictive nomogram based on the external validation cohort were 0.8322 (95% CI: 0.695-0.970).

Inclusion and exclusion criteria

We retrospectively analysed 65 patients with OS who received comprehensive treatment between January 2017 and December 2020 at Tianjin Medical University Cancer Institute and Hospital, Tianjin, China. Among these patients, 35 patients were assigned to the construction cohort. Additionally, 21 patients with OS diagnosed between January 2019 to December 2021 in P.A. Hertsen Moscow Oncology Research Center, Moscow, Russia were recorded, and in total of 12 patients were included as the external validation cohort to validate the constructed model.

The inclusion criteria were as follows: patients with primary OS confirmed by pathological diagnosis and who received 2 complete preoperative rounds of MAP (MTX, cisplatin and doxorubicin) sequential neoadjuvant chemotherapy; routine blood test results before each round of chemotherapy suggesting no significant bone marrow function; and patients with detailed data.

The exclusion criteria were as follows: patients with no clear pathological diagnosis and/or multiple malignancies; OS patients who did not receive neoadjuvant chemotherapy; patients with parosteal OS or secondary OS; patients with bone marrow dysfunction suggested by routine blood results before chemotherapy; and patients with missing data.

Based on the clinical and pathological characteristics of the patients, the following characteristics were included in this study: age, sex, body surface area, lesion site, pathological subtype, presence of pathological fractures, American Joint Committee on Cancer (AJCC) clinical stage, average dose MTX, tumour necrosis rate, Ki-67 index, pre-chemotherapy erythrocyte count, haemoglobin count, white blood cell (WBC) count, platelet count (PLT), alanine aminotransferase (ALT) concentration, aspartate aminotransferase (AST) concentration, total bilirubin concentration, albumin concentration, serum creatinine concentration, alkaline phosphatase concentration, lactate dehydrogenase (LDH) concentration, C_MTX at various monitoring time points during chemotherapy, and WBC count within 1 week after chemotherapy.

All patients’ guardians in this study were informed, and a detailed data collection sheet was filled. All the information gathered from the patients or their caregivers.

High-dose MTX treatment regimen and C_MTX monitoring

Patients were hydrated and alkalized 12 h before high-dose MTX treatment: 500 mL of 0.9% NaCl + 500 mL of 0.9% NaCl + 250 mL of 5% NaHCO₃. Patients received continuous administration of the following during MTX administration: (1) 500 mL of 5% NaCl + 500 mL of 0.9% NaCl + 10 mL of 15% KCl; (2) 500 mL of 5% NaHCO₃; (3) 2 mg of vincristine + 50 mL of 0.9% NaCl; (4) continuous administration of 1000 mL of 0.9% NaCl + 8~12 g/m² MTX for 5 h; and (5) 100 mL of 0.9% NaCl + 5 mg of tropisetron, 250 mL of 0.9% NaCl + 200 mg of vitamin B6, 100 mL of 0.9% NaCl + 1 mg of dexamethasone, and 100 mL of 0.9% NaCl + furosemide; the total fluid volume ranged from 3200 to 4000 mL, depending on each patient’s body mass index. The urine volume within 24 h was recorded, and the urine pH was measured to maintain the urine pH at 7-9. After the completion of MTX administration, hydration and alkalization treatment was continued for 2 days. During the entire MTX chemotherapy period, the patients received oral administration of NaHCO₃ (1.0 g, tid) and allopurinol (200 mg, tid).

Two millilitres of blood were drawn from the peripheral venous at 0, 6, 12, 24, 48 and 72 h after MTX infusion. Blood samples were centrifuged at 3000g for 5 min at room temperature in tubes without anticoagulant. MTX concentration was measured using fluorescence polarization immunoassay (TDX, ABBOTT, USA) with a quantification limit of 0.01 μmol/L. Leucovorin calcium and diuretic detoxification therapy was started at 6 h. The leucovorin calcium detoxification regimen was as follows: intramuscular injection of 12-15 mg/m² (q6h, d1 to d3), and then, individualized leucovorin calcium detoxification therapy was performed based on C_MTX until peripheral blood concentration of MTX was below 0.05 μmol/L, after which detoxification treatment was stopped [18].

Toxicity analysis and statistical analysis

In this study, routine blood tests, liver and kidney function tests, and electrocardiograms were performed before MTX treatment, and routine blood tests and liver and kidney function were monitored within 1 week after sequential chemotherapy. Post-chemotherapy LP was defined as a WBC count<3.0×10⁹/L from day 1 to week 1 after chemotherapy [19–21].

Data are presented as frequencies (percentages) for categorical variables and medians (interquartile ranges [IQRs]) for continuous variables. Differences between groups were assessed by univariate analyses, specifically the chi-squared test for categorical variables and the Kruskal-Wallis test for continuous variables, respectively. The patients were divided into a normal group and an LP group. Cut-off values for C_MTX were determined by receiver operating characteristic (ROC) curve analysis. To further narrow the scope of the candidate explanatory variables, we adopted the least absolute shrinkage and selection operator (LASSO) logistic regression to select variables. We utilized ten-fold cross-validation and select the optimal penalty term, lambda.1se, which represents largest lambda that is still within one standard error of the minimum binomial deviance. Then, the multivariate logistic regression analysis was used to assess the selected covariates for the development of LP, and the odds ratio (OR) and 95% confidence interval (CI) of relevant indicators were calculated. A clinical prediction nomogram to assess the prognostic model of LP was constructed based on the results from the final multivariable logistic regression. In addition, the nomogram was subjected to bootstrapping validation (1000 bootstrap resamples) for internal validation to assess predictive accuracy. R 3.6.3 software was used for data processing and analysis. The validity and accuracy of the proposed models were further tested by the external validation cohort. P<0.05 was considered statistically significant.

Availability of data and material

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval

The present study complied with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards, and the Research Ethics Board of the Tianjin Medical University Cancer Institute and Hospital approved the study.