Patients’ characteristics

A total of 27 adult patients with XLH were enrolled (Table 1). Patients had a mean age of 42 years (range 18–62), and 48% were female. In 23 patients, there was a confirmed PHEX gene mutation, while genetic analysis was not available for 4 patients, who instead had a positive family history. The clinical manifestations associated with XLH in patients are also reported in Table 1. Serum phosphate levels were significantly below the lower limit of normal (median 1.5 mg/dL, IQR 1.3–1.8), as was TmP/GFR (median 1.3 mg/dL, IQR 0.9–1.5). Serum and urinary calcium, serum creatinine, and eGFR were within the normal range in all patients. All patients were receiving phosphate and/or calcitriol therapy prior to initiating burosumab treatment. Common side effects associated with conventional therapy include secondary hyperparathyroidism, nephrolithiasis, and nephrocalcinosis. In our cohort, no patient had a diagnosis of nephrocalcinosis, while nephrolithiasis was present in 33% (9/27) of patients (Table 1). After withdrawal from any phosphate or active vitamin D analogue therapies, all patients began treatment with burosumab at a median dose of 60 (IQR 53–78) mg, corresponding to 1.00 (IQR 0.96–1.03) mg per kg, every 4 weeks. No dose adjustments occurred during the observation period, and no significant adverse events were reported (e.g., hypersensitivity reactions, hyperphosphatemia, kidney stones).

Table 1 Patients’ baseline characteristicsWhole group analysisTrend of serum phosphate over 24 weeks of burosumab treatment

After the first administration of burosumab, serum phosphate levels increased from a baseline of 1.5 mg/dL to a peak of 2.7 mg/dL (IQR 2.4–3.3) at week 2, and then decreased to 2.4 mg/dL (IQR 2.2–2.8) by week 4. By the end of the observation period, serum phosphate at week 24 was 2.0 mg/dL (IQR 1.7–2.4). Serum phosphate levels at all considered timepoints during burosumab treatment were significantly higher than baseline (Fig. 1, p < 0.05 for all comparisons).

Fig. 1

Trends in serum phosphate up to week 24, comparing each timepoint with baseline. * represents a p-value of < 0.05, ** = p < 0.01 and *** = p < 0.001

As in the pivotal trial by Insogna et al. [17], we decided to calculate, for each patient, the mean of serum phosphate levels at midpoints over the 24 weeks of treatment as a laboratory efficacy outcome. The median value of this variable for the entire cohort was 2.7 mg/dL (IQR 2.3–3.3), with a median percentage increase of 86% (IQR 85–132%) compared to baseline. We also observed that 17 patients (63%) achieved a mean serum phosphate at midpoints of 2.5 mg/dL or higher. Consequently, we divided the patients into two groups based on their mean serum phosphate at midpoints: lower (< 2.5 mg/dL) and higher (≥ 2.5 mg/dL). Supplementary Fig. 1 shows the different trends of serum phosphate in the two groups. We observed that serum phosphate levels were consistently more elevated in the latter group at all considered timepoints (p < 0.05 for all comparisons). Compared to serum baseline phosphate, in the former group we observed a median percentage increase of 51% (IQR 42–75), while in the latter group, the increase was 95% (IQR 73–135).

Factors associated with serum phosphate levels at midpoints during 24 weeks of burosumab treatment

We analyzed baseline factors associated with mean serum phosphate levels at midpoints using a linear regression model. As shown in Table 2, baseline serum phosphate levels were positively associated with mean serum phosphate at midpoints, while TmP/GFR exhibited a similar but non-significant trend. As described in the Methods section, PTH and FGF23 values were ZLOG-transformed using the respective laboratory reference ranges. Both baseline PTH and FGF23 levels were negatively associated with mean serum phosphate at midpoints. A multivariable regression analysis was subsequently performed to further explore the factors associated with mean serum phosphate at midpoints. Due to the sample size and the established influence of serum PTH and FGF23 on serum phosphate, only these two variables were included in the model. ZLOG PTH was not significantly associated with serum phosphate (b = − 0.138, p = 0.298), whereas ZLOG FGF23 showed a significant negative association (b = − 0.165, p = 0.020). The adjusted R-squared value for the model was 0.528, indicating that approximately 52.8% of the variance in mean serum phosphate levels at midpoints was explained by the included predictors.

Table 2 Univariable linear regression analysis with mean serum phosphate levels at midpoints as the dependent variableSubgroup analysis

We then conducted a subgroup analysis using data only from the 11 patients evaluated at the University Hospital of Padova, who were followed for 48 weeks. No significant differences were observed between these patients and patients enrolled in the other Centers in demographic, anthropometric, clinical and laboratory parameters, except for a slight difference in baseline serum phosphate and TmP/GFR values was observed (Table 3).

Table 3 Baseline characteristics of patients by cohortLaboratory test trends over 48 weeks of burosumab treatment

Even in this analysis, after the first administration of burosumab, serum phosphate increased from a baseline of 1.3 mg/dL (IQR 1.0–1.5) to a maximum value of 2.6 mg/dL (IQR 2.4–2.9) at week 2 and decreased to 2.1 mg/dL (IQR 1.9–2.4) at week 4, remaining significantly higher than baseline throughout the dosing interval. Although showing a slight decreasing trend, median serum phosphate levels remained higher than baseline throughout the observation period, both at midpoints and endpoints of dosing intervals, with the observed value at week 48 being 2.0 mg/dL (IQR 1.7–2.7) (Fig. 2A).

Fig. 2

Effect of burosumab on serum phosphate (A), TmP/GFR (B), serum calcium (C) and bALP (D) up to week 48. * represents a p-value of < 0.05, ** = p < 0.01 and *** = p < 0.001. bALP = bone alkaline phosphatase, TmP/GFR = tubular maximum phosphate reabsorption per glomerular filtration rate

Similarly, TmP/GFR increased from a baseline of 0.9 mg/dL (IQR 0.8–1.1) and reached higher values at each endpoint of the dosing interval, with the observed value at week 48 being 1.8 mg/dL (IQR 1.2–2.5) (Fig. 2B). No significant difference in the trend of serum calcium levels was observed between the endpoints of dosing intervals (Fig. 2C). Conversely, for bALP (Fig. 2D), there seemed to be an initial upward trend (median 45.7 µg/L [IQR 29.5–53.6 µg/L]) followed by a decline (median 31.8 µg/L IQR [20.9–38.7 µg/L]), although this was not statistically significant (p = 0.199). No difference was also observed between baseline PTH levels (median 29.5 ng/L [IQR 27.2–53.8 ng/L]) versus 48 weeks (median 37.3 ng/L [IQR 36.4–44.6 ng/L]), (p = 0.377).

Factors associated with serum phosphate at midpoints in subgroup analysis

Consistent with the analysis conducted in the entire cohort, the mean serum phosphate at midpoints during the first 24 weeks was also calculated for patients from the University Hospital of Padova. Four patients had mean serum phosphate values below 2.5 mg/dL, and seven had values equal to or greater than 2.5 mg/dL. In particular, there was a significant (p < 0.01) difference between the two groups with respective median values of 2.1 mg/dL (IQR 1.9–2.3) and 2.8 mg/dL (IQR 2.7–3.4). Different trends in serum phosphate in the two groups at the midpoints and endpoints of the dosing interval up to week 48 are shown in Supplementary Fig. 2.

The two groups—lower (< 2.5 mg/dL) and higher (≥ 2.5 mg/dL) mean serum phosphate at 24-week midpoints—did not significantly differ in terms of age, sex, anthropometric parameters, serum phosphate, TmP/GFR, serum and urinary calcium, creatinine and 25-OH-vitamin D (p > 0.05 for all comparisons). However, both PTH (63 ng/L, IQR 34–85 vs. 29 ng/L, IQR 24–38, p = 0.04) and iFGF23 (240 pg/mL, IQR 144–399 vs. 103 pg/mL, IQR 67–161, p = 0.04) were higher in subjects with low mean serum phosphate at midpoints. A univariable regression analysis was performed using mean serum phosphate at 24-week midpoints as the dependent variable. These results revealed significant associations for both PTH (b = − 0.014, p = 0.043) and iFGF23 (b = − 0.0026, p = 0.035), indicating that higher levels of these variables are predictive of lower mean serum phosphate at midpoints. The small sample size precluded the possibility to perform multivariable regression analysis.

We next performed ROC curve analysis to assess the discriminative power of PTH and iFGF23 in identifying patients with lower (< 2.5 mg/dL) versus higher (≥ 2.5 mg/dL) mean serum phosphate at 24-week midpoint. The AUC for PTH is 0.79, while for iFGF23 it is 0.88. The optimal cutoffs were determined to be 43.1 ng/L (reference range: 6.5–36.8 ng/L) for PTH and 209 pg/mL (reference range: 23.2–95.4 pg/mL) for iFGF23.

Finally, in this subgroup we also calculated the mean serum phosphate levels at midpoints over 48 weeks. Only one patient, who was categorized in the higher (≥ 2.5 mg/dL) group at 24 weeks, transitioned to the lower (< 2.5 mg/dL) group at 48 weeks, while all other patients remained in their original categories.

Patient-reported outcome trends during treatment with burosumab in subgroup analysis

Treatment with burosumab significantly improved patient-reported outcomes at week 48 compared to baseline. These outcomes were collected from 8 patients out of 11 from Padova. Regarding the WOMAC scale, there was an improvement in Pain, Stiffness, Physical Function, and Overall scores (Fig. 3A–D). Specifically, the Overall score improved from a median of 45.0 (IQR 31.1–72.2) at week 0 to a median of 25.5 (IQR 21.4–51.0) at week 48, with a median percentage improvement of 20.0% (IQR 10.5–45.4, p = 0.03). For the WOMAC Pain score, the median improved from 9.0 (IQR 6.4–13.6) at week 0 to 4.5 (IQR 3.0–9.1) at week 48, with a median percentage improvement of 39.3% (IQR 16.7–61.5, p = 0.02).

Fig. 3

Patient-reported outcome scores after burosumab treatment are shown at baseline and at week 48 for the following measures: WOMAC Pain (A), WOMAC Stiffness (B), WOMAC Physical Function (C), WOMAC Overall (D), BPI Worst Pain (E), BPI Average Pain (F), BPI Pain Interference (G), HAQ (H), RAPID3 Function (I), RAPID3 Pain (J), RAPID3 Patient Global Estimate (K), and RAPID3 Overall (L). BPI = The Brief Pain Inventory, HAQ = Health Assessment Questionnaire, RAPID3 = Routine Assessment of Patient Index Data 3, WOMAC = Western Ontario and McMaster Universities Osteoarthritis Index. * represents a p-value of < 0.05, ** = p < 0.01 and *** = p < 0.001

Similarly, the WOMAC Stiffness and Physical Function scores showed significant improvements with median percentage changes of 29.1% (IQR 20.2–59.7, p < 0.01) and 17.3% (IQR 7.1–37.7, p = 0.04), respectively. Additionally, significant reductions were observed in BPI Worst Pain, BPI Average Pain, and BPI Pain Interference scores (Fig. 3E–G). For BPI Worst Pain, the median score improved from 7.5 (IQR 5.8–8.6) at week 0 to 5.5 (IQR 4.4–6.0) at week 48, with a median percentage improvement of 29.1% (IQR 18.8–47.0, p < 0.01).

BPI Average Pain and BPI Pain Interference scores also showed notable improvements with median percentage changes of 26.8% (IQR 20.9–50.0, p < 0.01) and 11.8% (IQR 6.3–41.0, p = 0.04), respectively. HAQ and RAPID3 scores (Fig. 3H–L) also showed significant improvements. The HAQ score improved from a median of 1.0 (IQR 0.4–1.4) at week 0 to 0.5 (IQR 0.2–1.2) at week 48, with a median percentage improvement of 37.0% (IQR 13.3–52.5, p < 0.01). RAPID3 Function, Pain, Patient Global Estimate, and Overall scores demonstrated similar trends, with significant reductions and median percentage improvements of 19.5% (IQR 8.7–36.8, p < 0.01), 16.7% (IQR 11.7–37.5, p < 0.001), 16.7% (IQR 13.2–36.1, p < 0.01), and 16.0% (IQR 11.6–38.1, p < 0.001), respectively. Age was not correlated with any of the baseline scores nor with the extent of variation at 48 weeks (p > 0.05 for all comparisons). Similarly, no significant differences between the two sexes were found in baseline scores and the extent of variation at 48 weeks (p > 0.05 for all comparisons).

Regarding baseline laboratory tests, neither serum phosphate, TmP/GFR, PTH, nor FGF23 levels were correlated with the baseline score values; similarly, serum phosphate at week 48 was not correlated with the extent of score variation (p > 0.05 for all comparisons).

On the contrary, the extent of the change in scores for some measures was positively correlated with the increase in serum phosphate at week 48 compared to baseline, specifically WOMAC Pain (r = 0.94, p = 0.02), WOMAC Physical Function (r = 0.89, p = 0.03), WOMAC Overall (r = 0.94, p = 0.02), BPI Worst Pain (r = 0.98, p < 0.001), and BPI Average Pain (r = 0.88, p = 0.02).