Multicentre, retrospective study of the efficacy and safety of nivolumab for recurrent and metastatic salivary gland carcinoma

Although immune-checkpoint inhibitors (ICIs) are effective against various cancers, little is known regarding their role in salivary gland carcinoma (SGC) treatment. Therefore, we evaluated the efficacy and safety of nivolumab monotherapy in patients with recurrent and/or metastatic SGC. In this multicentre retrospective study, nivolumab (240 mg) was administered every 2 weeks. The overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and safety were examined; the correlation between treatment outcomes and clinicopathological factors was analysed. Twenty-four patients were enrolled; the most common histopathology was salivary duct carcinoma. Eleven tumours were PD-L1-positive; no tumour was microsatellite instability-high. The ORR was 4.2%, and the median PFS and OS were 1.6 and 10.7 months, respectively. One patient continued nivolumab for 28 months without disease progression. One patient showed grade 4 increase in creatine phosphokinase levels and grade 3 myositis. Biomarker analysis revealed significantly increased OS in patients with performance status of 0; modified Glasgow prognostic score of 0; low neutrophil-to-lymphocyte ratio, lactate dehydrogenase, and C-reactive protein; and high lymphocyte-to-monocyte ratio and in patients who received systemic therapy following nivolumab. Although nivolumab’s efficacy against SGC was limited, some patients achieved long-term disease control. Further studies are warranted on ICI use for SGC.


Results
Patient characteristics and treatment. Twenty-four patients, identified in the cancer registry of the participating institutions during the study period, were enrolled in this study (Table 1). Written informed consent was obtained from all participants. The median follow-up period for all patients was 6.5 (range 0.6-28.2) months. All patients had received systemic therapy before nivolumab. The most common histopathological type of cancer was SDC (n = 20, 83%). Eleven tumours (46%) presented PD-L1 expression at a rate of ≥ 1%. Among them, three (13%) presented ≥ 50% PD-L1 positivity. Among 23 evaluable patients, none was classified as MSI-H.

Discussion
In the present retrospective study of nivolumab monotherapy in 24 patients with R/M SGC, the ORR was 4.2%, with the median PFS and OS of 1.6 and 10.7 months, respectively. The ORR of the 20 patients with SDC was 5.0% and the median PFS and OS were 1.5 and 11.3 months, respectively. Nivolumab was well tolerated by patients with SGC, and AEs associated with nivolumab was comparable with those associated with pembrolizumab 24 . In this study, the therapeutic effects were limited; however, some patients achieved considerably long-term disease control.
Prospective studies on pembrolizumab 24 and pembrolizumab combined with vorinostat 25 reported that the ORR of patients with multiple histopathological types of SGC was 12% and 16%, respectively, with the median PFS and OS of 4-6.9 and 13-14 months, respectively. The ORR and the median OS of patients with SGC to nivolumab in this study were comparable with those observed in patients with advanced SGC treated with pembrolizumab 24,25 . However, the median PFS of this study was shorter than that reported by the previous study on pembrolizumab. This could be because only patients with obvious progression within 6 months were included in this study, whereas the previous studies with pembrolizumab did not adopt this criterion.
In the present study, the results of the biomarker analysis revealed that most factors related to PS and inflammatory biomarkers such as the NLR, LMR, LDH, and CRP levels were associated with the prognosis of tumour in

Efficacy All patients (n = 24) Salivary duct carcinoma (n = 20)
Complete response, n (%) www.nature.com/scientificreports/ patients treated with nivolumab. To the best of our knowledge, this is the first study to demonstrate an association between inflammatory biomarkers and prognosis in patients with SGC treated with nivolumab. In particular, NLR showed an apparent negative dose-response relationship with the OS; the 1-year OS of patients with lower NLR was 85.7%. The NLR, LMR, PLR, CRP level, mGPS, and LDH level have also been reported to correlate with the therapeutic effects of ICIs in various cancers [46][47][48][49][50][51] . As a result, NLR, LMR, PLR, LDH, CRP, and mGPS are suggested to reflect the general conditions (immunological competence) of the host, and they can be used as biomarkers of ICI treatment response.
In malignant melanoma, non-small cell lung cancer, and head and neck squamous cell carcinoma, PD-L1 immunohistochemistry 22,23,28,29 including the presence of PD-L1-expressing immune cells in the tumour microenvironment 52,53 , mismatch repair (MMR)/MSI 29,31 , and TMB 29  www.nature.com/scientificreports/ TMB in SDC than those in other tumours [32][33][34][35][36][37][38][39][40][41] , higher therapeutic effects of ICIs are being expected. However, the efficacy of ICI monotherapy for SDC was limited in our cohort. This might be since no tumour was MSI-H and the PD-L1-positivity rate in the tumour cells was low in our SDC cohort. While previous studies on anti-HER2 antibody including trastuzumab 5-8 and androgen deprivation therapy (e.g., bicalutamide and leuprorelin) 9-12 for HER2-or AR-positive patients with SDC showed the ORR was 20-89%, the response rate of patients to nivolumab in this study was unsatisfactory. Thus, nivolumab monotherapy is not recommended for patients with HER2-or AR-positive advanced SDCs before anti-HER2-or AR-targeted therapy. In contrast, ICIs might be tried in patients with SGC without targetable molecules instead of conventional cytotoxic anticancer agents. Recent studies reported that cytotoxic anticancer agents seemed to achieve a higher ORR with higher toxicity than the ICIs 3,4,24,25 . However, it is difficult to directly compare those therapies as patient background (e.g., histological type) might differ. As our data suggest low nivolumab ORR and shorter survival in patients with increased systemic inflammatory markers (e.g., NLR), the use of cytotoxic anticancer agents may be prioritised in symptomatic patients (e.g., patients with pain and/or aggressive tumour growth) and patients with increased systemic inflammatory markers. Currently, a clinical trial on the combination of pembrolizumab and docetaxel in patients with thyroid cancer or SGC without standardof-care treatment is under progress (ClinicalTrials.gov Identifier: NCT03360890). Other ongoing clinical trials targeting patients with SGC include the combination of pembrolizumab and lenvatinib (ClinicalTrials.gov Identifier: NCT04209660), two ICIs (nivolumab and ipilimumab; ClinicalTrials.gov Identifier: NCT02834013, NCT03146650 and NCT03172624), and ICIs and AR-targeted therapy (pembrolizumab and goserelin acetate; ClinicalTrials.gov Identifier: NCT03942653).
This study had some limitations. First, owing to the retrospective nature and small sample size of the study, the superiority of nivolumab over other drugs was not examined. Second, the biomarkers identified in this study including NLR might be merely prognostic factors, which are associated with survival and might not predict Table 3. Reported adverse events, n (%). ALP alkaline phosphatase, ALT alanine aminotransferase, APTT activated partial thromboplastin time, AST aspartate aminotransferase, CPK creatinine phosphatase, γ-GTP γ-glutamyl transpeptidase, LDH lactate dehydrogenase, INR international normalised ratio.  www.nature.com/scientificreports/ www.nature.com/scientificreports/ response to nivolumab. Moreover, the optimal cut-off value for NLR was unknown. Thus, future clinical trials with a larger sample size should be performed to address these issues.
In the present study, the efficacy of nivolumab monotherapy for SGC was limited. However, some patients achieved long-term disease control with nivolumab. Further studies are warranted to elucidate a predictive factor of ICI in patients with advanced SGC.  were extracted from the database of the nation-wide cancer registry of each participating institution. This study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient and/or their legal guardians. Additionally, we obtained informed consent of the patients for publication of identifying images and photographs. Patients with ≥ 20% tumour growth within 6 months prior to treatment detected by computed tomography (CT) scan, magnetic resonance imaging, and/or positron emission tomography-CT were treated with nivolumab (240 mg) every 2 weeks. The treatment dose and duration were determined in accordance with the Japanese guidelines for head and neck cancer, including salivary gland cancer. A pathological review of all patients was performed by a pathologist with expertise in SGCs (T.N.). Carcinoma ex pleomorphic adenomas were classified into different histopathological types according to each carcinoma component instead of a separate category. Imaging tests were performed every 6-8 weeks.
Immunohistochemical and gene alteration analyses. The expression status of PD-L1, MSI, HER2, and androgen receptor (AR) in patients administered nivolumab was also obtained from the database of the participating institutes. The expression level of PD-L1 in the resected or biopsy specimens of tumours was analysed using the rabbit antihuman PD-L1 clone 28-8 using the automated immunohistochemical assay (PD-L1 IHC 28-8 pharmDx; Dako-Agilent Technologies, Santa Clara, CA, USA). PD-L1 expression was defined as the percentage (instead of intensity) of tumour cells exhibiting plasma membrane staining 22 .
The MSI test kit (product code: 4987931010017; FALCO Biosystems, Kyoto, Japan) was used to evaluate MSI as described previously 54 . Briefly, a polymerase chain reaction (PCR) of microsatellite markers at five loci (BAT25, BAT26, NR21, NR24, and MONO27) was conducted using DNA extracted from tumour specimens. In normal patients, the PCR products were in the quasi-monomorphic variation range (QVR). Specimens with the PCR products outside the QVR were classified as MSI-positive. Specimens with more than one positive locus were classified as MSI-high (MSI-H).
HER2 and AR statuses were assessed as described previously 5,10,55,56 . Briefly, specimens with 3 + HER2 immunoreactivity or HER2 gene amplification were classified as HER2-positive according to the guidelines for breast cancer by the American Society of Clinical Oncology and the College of American Pathologists 56 . AR was classified as positive if ≥ 20% of nuclei in tumour cells were immunoreactive.
Analysis of biomarkers of ICI response. An exploratory analysis of potential biomarkers of ICI response was performed 43,46 . The associations between prognosis and age, sex, the Eastern Cooperative Oncology Group (ECOG) performance status (PS), prior systemic therapy (present or absent), immune-related adverse events (irAE; present or absent), systemic therapy after nivolumab (present or absent), histopathological type (SDC or non-SDC), PD-L1 status, HER2 status, AR status, MSI status, mGPS, NLR, PLR, LMR, serum CRP, LDH, and absolute eosinophil count were examined. Statistical analysis. The therapeutic effect of nivolumab was evaluated according to the overall response rate (ORR), which was defined as the percentage of patients who achieved complete response (CR) or partial response (PR), clinical benefit rate [CBR, defined as the percentage of patients who achieved CR, PR, or stable disease (SD) for at least 24 weeks], disease control rate (DCR, defined as the percentage of patients who achieved CR, PR, or SD regardless of duration), median progression-free survival (PFS), and median overall survival (OS) 5,10,43,46 . Treatment efficacy was evaluated according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) 57 . PFS was defined as the time from the start of nivolumab treatment to the diagnosis of progressive disease (PD). OS was defined as the time from the start of nivolumab treatment to death from any cause. Safety was evaluated according to Common Terminology Criteria for Adverse Events (CTCAE) ver. 5.0 58 . The Kaplan-Meier method was used to estimate PFS and OS. The Cox proportional hazards model was used to calculate the hazard ratio (HR) with 95% confidence interval (CI). The results with a P value of < 0.05 were considered statistically significance. All analyses were performed using STATA ver. 16  www.nature.com/scientificreports/