Drug holiday of high-dose denosumab and recovery from osteoclast inhibition using immunohistochemical investigation of 7 patients with medication-related osteonecrosis of the jaw undergoing segmental mandibulectomy.

Introduction

Antiresorptive agents, such as bisphosphonate (BP) and denosumab (DMB), are widely used to prevent fractures in patients with osteoporosis or to treat bone-related events associated with bone metastases of malignant tumors and multiple myelomas. 1 Although antiresorptive agents have excellent bone resorption inhibitory effects, the onset of medication-related osteonecrosis of the jaw (MRONJ) is a serious adverse event. 2

The American Association of Oral and Maxillofacial Surgeons (AAOMS) position paper, 2014 3 and the Japanese position paper, 2016 4 recommend having drug holidays before tooth extraction in patients using low-dose antiresorptive agents to prevent MRONJ and withdrawing antiresorptive agents during MRONJ treatment. However, there are no evidence-based reports on the significance and duration of drug holidays. The 2022 revised position paper 5 states that they were unable to reach an agreement on drug holiday recommendations. The opinion that drug holidays should be considered for each case following previous recommendations was presented, as well as the differing opinion that drug holidays should not be recommended since the risks of potential adverse effects may outweigh the benefits.

Kawakita et al. 6 and Hasegawa et al. 7 reported that a 3-month drug holiday before tooth extraction in patients receiving low-dose oral BP did not reduce the risk of developing MRONJ. In addition, Hasegawa et al. 8 , 9 reported that a 2-month drug holiday before tooth extraction in patients with cancer receiving high-dose antiresorptive agents and a 1-month washout before tooth extraction in patients with cancer receiving high-dose denosumab did not reduce the incidence of MRONJ. Furthermore, Hayashida et al. 10 reported that the presence or absence of antiresorptive agent withdrawal during surgery for MRONJ did not affect the treatment outcomes. Otsuru et al. 11 also stated that drug holidays of 60, 90, and 120 days were not significant factors for sequestrum separation and did not influence the surgical outcomes of patients with cancer receiving high-dose antiresorptive agent therapy. Thus, whether antiresorptive agent withdrawal is recommended for both the prevention and treatment of MRONJ remains controversial.

As BP is deposited in bone tissue for a long period, its effect on osteoclasts seems to be sustained even after short drug withdrawal. 12 Recently, Morishita et al. 13 reported that osteoclast suppression persisted even after oral BP withdrawal for approximately 6 months. However, considering that DMB is not deposited in bone tissue, its half-life in blood is approximately 1 month, and the lifespan of osteoclasts is approximately 2 weeks. The effects of DMB are expected to be shorter than those of BP, and recovery of osteoclast activity should occur after a short DMB drug holiday. 14 This study investigated the relationship between the withdrawal period of DMB and osteoclast morphology using immunohistochemical examination in patients with MRONJ.

Materials and methods

Patients

Twenty-six patients with cancer receiving high-dose antiresorptive agent therapy who underwent surgical treatment for MRONJ under general anesthesia between January 2019 and December 2021 at the Department of Dentistry and Oral Surgery of our university were assessed. Among them, 16 patients developed MRONJ in the lower jaw, and seven underwent segmental mandibulectomy. These seven patients with cancer who underwent segmental mandibulectomy due to MRONJ participated in the study.

Examination of the clinical factors and histologic features

The clinical factors, including age, sex, primary disease, diabetes, administration of corticosteroids, type of antiresorptive agent, administration period, duration of preoperative drug holiday, preoperative leukocyte count, albumin and creatinine levels, and treatment outcomes, were investigated using medical records.

For the histological examination, decalcified sections were prepared from the mesial and distal bone tissues of the excised specimens, and hematoxylin and eosin staining and cathepsin K immunostaining were performed. Osteoclast suppression was determined as follows, according to the method of Omori et al. 15 Osteoclasts were classified into the following three types: 1) osteoclast suppression (+), no cathepsin K-positive cells or only cathepsin K-positive mononuclear or small multinucleated cells observed on the bone surface of both mesial and distal specimens; 2) osteoclast suppression (−), cathepsin K-positive polynuclear giant cells similar to normal osteoclasts found on the bone surface; and 3) osteoclast suppression (±), osteoclast suppression observed, but a small number of normal osteoclasts were mixed. The evaluation was performed blindly by two oral surgeons with at least 15 years of clinical experience and who were familiar with bone histology.

Cathepsin K staining

Fixed specimens were decalcified in 0.5 M ethylenediaminetetraacetic acid solution at 4 °C with gentle agitation. The decalcified samples were fixed with a fixative, dehydrated, and embedded in paraffin. Five-micrometer-thick sections were prepared and deparaffinized. The sections were placed in an autoclave at 121 °C for 15 min in 10 mM citrate buffer (Dako, Carpinteria, CA, USA) and incubated with rabbit anti-cathepsin K primary antibody (1:100, #ab19027, Abcam, Cambridge, MA, USA) at 4 °C overnight. The EnVision + system (Dako) was used to perform an immunohistochemical reaction according to the manufacturer's instructions. The sections were counterstained with Meyer's hematoxylin (Dako).

Ethics

The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and Ethical Guidelines for Medical and Health Research involving Human Subjects by the Ministry of Health, Labor, and Welfare of the country of the authors' home institution. This study was approved by the Institutional Review Board of the authors' home institute. Owing to the retrospective nature of the study, the research plan was published on the homepage of the institution's website with a guaranteed opt-out opportunity.

Results

The participants consisted of two males and five females, with an average age of 66.6 ± 9.96 years. The primary diseases were breast cancer in four patients and lung cancer, multiple myeloma, and prostate cancer in 1 patient each. Three patients were taking steroids and had no history of diabetes. The period of DMB administration was 5–72 months, with an average of 35.1 months. In four patients, DMB was discontinued for 8–20 months, and three patients did not undergo a drug holiday ( Table 1 ). Table 1 Summary of the seven patients. Table 1 Case Sex Age Primary disease Diabetes Drug DMB dosage Dosing period (month) Drug holiday (month) Osteoclast suppression 1 Female 66 Breast cancer None BP→DMB 120 mg 62 9 + 2 Female 76 Multiple myeloma None DMB 120 mg 8 None + 3 Female 48 Breast cancer None DMB 120 mg 14 8 ± 4 Male 72 Prostate cancer None DMB 120 mg 48 None + 5 Female 71 Breast cancer None DMB 120 mg 72 13 – 6 Male 59 Lung cancer None DMB 120 mg 5 20 – 7 Female 74 Breast cancer None DMB 120 mg 37 None + Abbreviations: BP, bisphosphonate; DMB, denosumab.

Regarding the histological findings, multiple osteoclasts were present on the surface of bone remodeling in two patients in whom osteoclast suppression was not observed ( Fig. 1 A.) In Case 3, with an 8-month drug holiday, few normal osteoclasts were observed; however, most of them had a small number of nuclei and small osteoclasts ( Fig. 1 B). In the three patients with no drug holidays and one patient with a 9-month drug holiday, osteoclasts were nearly absent in the entire specimen ( Fig. 1 C) or cathepsin K-positive mononuclear or small multinucleated cells were observed ( Fig. 1 D). Figure 1 The histological features of HE and cathepsin K staining of human mandibular specimens from a patient using denosumab. (A) Large normal osteoclasts with polynuclear cells are observed in a patient on a 13-month drug holiday (bar = 100 μm). (B) HE staining of a patient on an 8-month drug holiday. Small abnormal osteoclasts with a small number of nuclei are observed (bar = 100 μm). (C) Cathepsin K staining of a patient without a drug holiday. No osteoclasts are observed (bar = 100 μm). (D) Cathepsin K staining of a patient without a drug holiday. Small mononuclear or multinucleated cells are observed on the bone surface (bar = 100 μm). Abbreviations: HE, hematoxylin and eosin. Fig. 1

The relationship between drug holiday duration and osteoclast suppression is shown in Table 1 . Osteoclast suppression was observed in all three patients who continued DMB therapy. In contrast, osteoclast suppression recovered in two patients who had drug holidays for more than 1 year. One of the two patients with a drug holiday from 6 months to 1 year had osteoclast suppression (+), and the other had (±). These findings suggest that patients receiving high-dose DMB therapy require a drug holiday for at least 1 year in order to recover from osteoclast suppression.

Discussion

This study suggests that osteoclast inhibition may recover when DMB is withdrawn for 12 months or longer; however, osteoclast suppression continues when the drug holiday is lesser than 12 months.

Since the first report by Marx et al., in 2003, 2 MRONJ has been considered a challenging disease to cure; therefore, complete cure has not necessarily been the treatment goal for MRONJ. According to the AAOMS Position Paper 2014, 3 the major goals for MRONJ treatment are prioritization and support for continued oncologic treatment, preservation of quality of life through pain control, secondary infection, and prevention of MRONJ lesion extensions. The AAOMS Position Paper was revised in 2022 5 ; however, the description of the treatment goals was similar to that of the previous version.

As for treatment strategies, in the past, conservative treatment was often used for MRONJ treatment. Most of the papers that reviewed MRONJ treatment based on more than 100 cases reported surgical treatment to be more effective in MRONJ stages 2–3. 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 Although no randomized controlled trials have been reported, five papers have been reported in observational studies examining effective treatments by multivariate or propensity score matching analysis methods, all of which stated that surgical therapy was more curative than conservative therapy. 18 , 19 , 20 , 21 , 22 The AAOMS Position Paper 2014 3 recommended conservative treatments for MRONJ stages 1–2, such as antibacterial mouth rinse, systematic administration of oral antibiotics, and debridement to relieve soft tissue irritation and control infection. For a stage 3 lesion, surgical debridement or resection is described as well as an antibacterial mouth rinse, antibiotic therapy, and pain control. However, the 2022 edition of the AAOMS position paper 5 changed the descriptions of the treatment strategies. The main change is that both conservative and surgical therapies are acceptable, regardless of the stage, although there is no mention of which treatment is better.

In the AAOMS Position Paper 2014, 3 evidence was not sufficient to determine whether antiresorptive agents should be withdrawn before performing invasive dental procedures such as tooth extraction; however, a 2-month withdrawal period was recommended in high-risk cases of osteoporosis. In the AAOMS Position Paper 2022, 5 the working group was unable to reach a consensus on recommendations for drug withdrawal, which should be addressed on a case-by-case basis. In contrast, a multicenter observational study by Hasegawa et al. 7 of 2458 extracted teeth in patients receiving oral BP, reported that a 3-month withdrawal did not reduce the incidence of MRONJ after tooth extraction. An observational study also reported that a 2-month withdrawal did not decrease the incidence of MRONJ after tooth extraction in patients with cancer receiving injectable bone resorption inhibitors. 9

Special concerns should be considered when suspending DMB and receptor activator of nuclear factor kappa-Β ligand (RANKL) inhibitors in patients with osteoporosis. Several studies have demonstrated a rebound increase in bone resorption following DMB discontinuation, resulting in an increased risk of multilevel vertebral fractures. Considering DMB suspension, the duration and stage of the holiday should be optimized to minimize the risk. Scheduled oral surgery can be completed up to 3 or 4 months after the last dose of DMB when the level of osteoclast inhibition is reduced. DMB usage can resume 6–8 weeks after surgery. This management minimizes the length of the drug holiday while maintaining an ideal environment for bone tissue healing. 5

The need for drug withdrawal during MRONJ treatment remains controversial. The Japanese Society of Oral and Maxillofacial Surgeons Position Paper 4 indicated that discontinuation of antiresorptive agents in patients with cancer with progressive bone metastases is not recommended; however, for patients with osteoporosis, excluding those with high fracture risk, discontinuation of antiresorptive therapy may be needed until completion of MRONJ treatment. Hayashida et al. 10 reported that drug holidays during MRONJ treatment did not affect the treatment outcomes in surgical cases; however, in conservative treatment cases, the cure rate after long-term follow-up was higher when antiresorptive agents were withdrawn. Otsuru et al. 11 recently reported that drug holidays of ≥2, 3, and 4 months did not influence the treatment outcomes of patients with MRONJ receiving high-dose antiresorptive agent therapy. Morishita et al. 13 performed an immunohistochemical study and reported that osteoclast suppression did not recover even if oral BP was withdrawn for ≥6 months.

BP and DMB have different mechanisms of action. BP is tightly bound to bone hydroxyapatite and elicits its effects when taken up from the bone matrix into the osteoclast cytoplasm during bone resorption. BP targets mature osteoclasts, inhibits farnesyl pyrophosphate synthase, and inhibits osteoclast resorptive function and survival by disrupting intercellular signaling pathways. However, dysfunctional osteoclasts can persist. The release of BP from the bone matrix depends on the bone turnover, and BP may remain in the bone for weeks or years. 34 , 35 In contrast, DMB is a monoclonal antibody that selectively binds RANKL. DMB is not taken up by the bone tissue but circulates in the blood and exists in the extracellular fluid; and the DMB target cells are osteoclast precursors and mature osteoclasts. DMB prevents the formation, function, and survival of osteoclasts and is cleared by the reticuloendothelial system with a half-life of 26 days or less. 35 , 36

Since the half-life of DMB is significantly shorter than that of BP, DMB withdrawal may result in more rapid healing than BP withdrawal in patients with MRONJ. 13 Tamaki et al. reported that in an MRONJ-like mouse model administered DMB and cyclophosphamide, DMB withdrawal for 2 weeks induced partial osseous wound healing and significantly improved soft tissue wound healing of the extraction sockets. 37 However, Hasegawa et al. 7 , 8 reported that in patients receiving low-dose and high-dose DMB therapy, a drug holiday of 1–2 months before tooth extraction did not reduce the risk of developing MRONJ. Ottesen also stated that a drug holiday from a high-dose of BP and DMB did not prevent MRONJ development following tooth extraction and the patient-reported health state decline during a drug holiday compared with drug continuation. 38 It is unclear how long DMB should be withdrawn to recover from osteoclast inhibition.

In the current study, the dosing period did not affect osteoclast suppression if the drug holiday lasted for >12 months. Cases 5 and 6 showed no evidence of osteoclast suppression. However, long-term drug holidays of DMB, such as over 12 months, are not allowed on usual occasions. Therefore, explaining the risk of MRONJ is crucial, even when the withdrawal duration is > 3 months. In Case 2, although the drug holiday was 8 months, osteoclast suppression was observed. In contrast, the 9-month drug holiday was sufficient to recover osteoclast suppression in Case 1. Therefore, approximately 6–12 months would be required for osteoclast suppression recovery in cases of high-dose DMB therapy. Similarly, the background of the patients could affect the drug holiday required for recovery of osteoclasts activity.

This study had several limitations. First, this was a retrospective observational study with a small number of patients; therefore, statistical analysis could not be conducted. It was difficult to generalize the results of the study. Second, the recovery of osteoclast inhibition was evaluated using only the morphological evaluation of osteoclasts. We are currently considering local osteoclast markers using molecular biology techniques. Third, the samples used in this study were specimens that had already undergone MRONJ. The section used for the evaluation was collected in the vicinity of the sump of the excised necrotic bone, including the healthy tissue not affected by the lesion as much as possible; however, the effect of osteonecrosis on the jaw is unknown. Nevertheless, to the best of our knowledge, this is the first study to histologically investigate DMB drug holidays and recovery from osteoclast inhibition. In the future, we wish to study a large number of cases.

To the best of our knowledge, this was the first report of a DMB drug holiday period and osteoclast recovery in human mandibular specimens. Based on our results, a 3-month drug holiday may not be sufficient to recover osteoclast activity. Further studies with a larger number of cases are required.

Declaration of competing interest

The authors declare no conflict of interest.

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Acknowledgements

The authors thank Manabu Deguchi and Applied the Medical Research Laboratory for their technical work and advice.