Genetic tumour heterogeneity, driven by clonal evolution, contributes to therapy resistance in many cancers. Most salivary gland cancers, including adenoid cystic carcinoma (AdCC) and myoepithelial carcinoma (MECA), lack effective systemic treatments, highlighting the need to characterise their evolutionary profiles. In this autopsy study, we reconstructed genetic heterogeneity and clonal evolution in two patients with metastatic AdCC and one patient with metastatic MECA. Radiology-guided autopsy was performed between 12 and 56 h after out-of-hospital death. One hundred forty-nine tumour samples were snap-frozen, of which 17 (4-7 per patient) were selected for whole-genome sequencing. Phylogenetic reconstruction was performed using CONIPHER. Autopsy revealed multiple metastatic sites not visible on antemortem or postmortem imaging. MYB-NFIB gene fusions were present across all tumour sites from the two AdCC patients, while a LIFR-PLAG1 fusion was detected in all samples from the MECA patient. All three cases showed extensive genetic tumour heterogeneity and branched phylogenies, suggestive of parallel evolution. Histologic growth patterns were consistent across metastatic sites. Overall, this study demonstrated marked genetic heterogeneity and branched evolution in these AdCC and MECA patients.

Abstract Background Salivary duct carcinoma (SDC) is a rare and aggressive subtype of salivary gland cancer, frequently associated with incurable recurrences and distant metastases (R/M). Proliferation of SDC relies on androgen receptor (AR) signalling, prompting the use of combined androgen blockade (CAB, i.e., luteinizing hormone-releasing hormone agonist and/or AR antagonists) to R/M SDC patients. However, only a subset of patients benefits from such treatments. We have shown that response to CAB is associated with steroid 5α-reductase 1 (SRD5A1) mRNA expression. SRD5A1 catalyses the intracellular conversion of testosterone into the more potent AR-agonist dihydrotestosterone. This conversion can be inhibited by dutasteride, a potent SRD5A1-inhibitor, which is currently prescribed for benign prostatic hyperplasia. We hypothesize that repurposing dutasteride to target AR signalling in SDC could enhance therapeutic response and clinical outcome in SDC patients. Methods This prospective, open-label, randomized controlled phase II clinical trial, is designed to investigate whether dutasteride as an adjunct drug to CAB improves response rate and clinical outcome in patients with AR-positive R/M SDC. Patients are divided in two cohorts based on their prior systemic treatments. In cohort A, CAB-naïve patients (n = 74) will be randomly assigned to either a control arm (Arm 1) receiving CAB (goserelin 10.8 mg/3m and bicalutamide 50 mg/OD) or an experimental arm (Arm 2) where dutasteride (0.5 mg/OD) is added to the CAB regimen. In cohort B, patients with disease progression after adjuvant or first-line palliative CAB therapy (max. n = 24) will receive goserelin, bicalutamide, and dutasteride to assess whether the addition of dutasteride can overcome therapy resistance. The primary endpoints are the objective response rate and duration of response. Secondary endpoints are progression-free survival, overall survival, clinical benefit rate, quality of life, and safety. Translational research will be performed to explore molecular target expression differences and their correlation with clinical outcome. Discussion The DUCT study addresses an unmet medical need by investigating the repurposing of dutasteride to enhance treatment response and improve clinical outcome for patients with R/M SDC, especially those with limited alternative treatment options, such as HER2-negative cases. By repurposing a registered low-cost drug, this trial’s findings could be readily applied into clinical practice. Trial registration Clinicaltrials.gov Identifier: NCT05513365. Date of registration: August 24, 2022. Protocol version Current protocol version 4.0, February 21, 2024.

Tumors of the major and minor salivary glands histologically encompass a diverse and partly overlapping spectrum of frequent diagnostically challenging neoplasms. Despite recent advances in molecular testing and the identification of tumor-specific mutations or gene fusions, there is an unmet need to identify additional diagnostic biomarkers for entities lacking specific alterations. In this study, we collected a comprehensive cohort of 363 cases encompassing 20 different salivary gland tumor entities and explored the potential of DNA methylation to classify these tumors. We were able to show that most entities show specific epigenetic signatures and present a machine learning algorithm that achieved a mean balanced accuracy of 0.991. Of note, we showed that cribriform adenocarcinoma is epigenetically distinct from classical polymorphous adenocarcinoma, which could support risk stratification of these tumors. Myoepithelioma and pleomorphic adenoma form a uniform epigenetic class, supporting the theory of a single entity with a broad but continuous morphologic spectrum. Furthermore, we identified a histomorphologically heterogeneous but epigenetically distinct class that could represent a novel tumor entity. In conclusion, our study provides a comprehensive resource of the DNA methylation landscape of salivary gland tumors. Our data provide novel insight into disputed entities and show the potential of DNA methylation to identify new tumor classes. Furthermore, in future, our machine learning classifier could support the histopathologic diagnosis of salivary gland tumors.

Salivary duct carcinoma (SDC) is one of the most aggressive subtypes of salivary gland cancer. It is a very rare cancer (incidence 0.1–0.2 per 100 000 people/ year), characterised by androgen receptor (AR) expression in almost all patients and HER2 expression in approximately 30% of patients. A typical characteristic of SDC is the high propensity for lymph node metastases, with a median number of tumour positive lymph nodes of 4 (range: 0–97). There is a tendency towards worse survival when more lymph nodes are involved. Here, we present cutaneous lymphangitis carcinomatosa (CLC) as a relatively frequently occurring manifestation of advanced SDC. With this report, we aim to increase the knowledge on this disease manifestation, because it is sometimes not recognised, although it is typical for this subtype of salivary gland cancer. In our tertiary referral centre, 11 SDC patients developed skin lesions in the neck and chest region. All patients had extensive lymph node metastases at diagnosis (table 1). Skin biopsies showed CLC and cutaneous metastases (figures 1 and 2). Two representative cases will be described in detail. Furthermore, we report on the molecular analysis of these 11 patients (online supplemental table 1).

The use of anticancer drugs targeting specific molecular tumor characteristics is rapidly increasing in clinical practice, but selecting patients to benefit from these remains a challenge. It has been suggested that organoid cultures would be ideally suited to test drug responses in vitro. Here we describe and characterize in depth a case of ETV6-NTRK3 gene fusion-positive secretory carcinoma of the salivary glands and corresponding organoid cultures that responded and subsequently acquired resistance to TRK targeting therapy with larotrectinib. This case-culture-characterization illustrates the advances made in precision oncology, but also exposes important caveats in using organoids to predict treatment response.

Objective: Three-dimensional organoid cell cultures have been established for a variety of human cancers. For most rare cancers, including salivary gland cancer (SGC), these models are lacking, despite the great unmet need to study cancer biology in these diseases. Therefore, we aimed to develop patient-derived organoid (PDO) models for different subtypes of SGC.Methods: Tumor samples of SGC patients were processed and embedded in Matrigel. Successful PDOs (expand-able > 1*106 cells) were phenotypically characterized using immunohistochemistry (IHC) and genotypically by gene fusion analysis and by targeted and whole-exome sequencing. Successfully established PDOs were subjected to small-scale drug screening.Results: Out of 37 attempts, 7 viable short-term PDOs were established (19 % success rate; 3 salivary duct car-cinoma, 3 adenoid cystic carcinoma and 1 mucoepidermoid carcinoma). Each PDO showed close phenotypical mimicry to parental tissue. Genotypic characterization revealed that in each PDO > 97.6 % of all COSMIC an-notated variants and all MYB, MYBL1 and NFIB gene rearrangements were retained. Drug screening was proven feasible in all PDOs.Conclusion: We present the first comprehensively characterized short-term SGC PDO models for three subtypes of SGC with close phenotypic and genotypic resemblance to parental tissue, which can be used for drug screening applications.

Objective: Salivary duct carcinoma (SDC) overexpresses Human Epidermal growth factor Receptor 2 (HER2) in 29-46% of cases, favoring anti-HER2 therapy. Here, we present the results of patients with recurrent or metastatic HER2-positive SDC treated with docetaxel, trastuzumab, and pertuzumab (DTP) as first line anti-HER2 therapy and subsequently ado-trastuzumab emtansine (T-DM1) in second line. Furthermore, we searched for potential biomarkers. Methods: Retrospective case series from a tertiary hospital. First line anti-HER2 treatment consisted of DTP, after progression T-DM1 was considered for patients with an adequate performance status. Objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were assessed and related to mRNA-based PI3K and MAPK signaling pathway activity scores. Results: Thirteen SDC HER2 + patients received DTP. In twelve evaluable patients, one complete response (CR) and six partial responses (PR) were observed (ORR 58%), with a median PFS of 6.9 months (95%-CI 5.3-8.5). Seven patients received subsequent T-DM1 in second line, resulting in four PR (ORR 57%), with a median PFS of 4.4 months (95%-CI 0-18.8). Median OS after start of DTP was 42.0 months (95%-CI 13.8-70.1). Grade >= 3 toxicity on DTP was seen in 39% of patients, and 14% on T-DM1. Highest combined PI3K and MAPK signaling was seen in the patient with CR and lowest in the patient with progressive disease on DTP. Conclusion: In R/M HER2-positive SDC patients DTP followed by T-DM1 upon progression are promising treatments, leading to responses in the majority (58%) of the patients at an acceptable toxicity profile.

Introduction: Salivary gland cancer (SGC) is a rare cancer for which systemic treatment options are limited. Therefore, it is important to characterize its genetic landscape in search for actionable aberrations, such as NTRK gene fusions. This research aimed to identify these actionable aberrations by combining NGS-based analysis of RNA (gene fusions) and DNA (single and multiple nucleotide variants, copy number variants, microsatellite instability and tumor mutational burden) in a large cohort of SGC patients. Methods: RNA and DNA were extracted from archival tissue of 121 patients with various SGC subtypes. Gene fusion analysis was performed using a customized RNA-based targeted NGS panel. DNA was sequenced using a targeted NGS panel encompassing 523 cancer-related genes. Cross-validation of NGS-based NTRK fusion detection and pan-TRK immunohistochemistry (IHC) was performed. Results: Fusion transcripts were detected in 50% of the cases and included both known (MYB-NFIB, MYBL1-NFIB, CRTC1-MAML2) and previously unknown fusions (including transcripts involving RET, BRAF or RAD51B). Only one NTRK fusion transcript was detected, in a secretory carcinoma case. Pan-TRK IHC (clone EPR17341) was false positive in 74% of cases. The proportion of patients with targets for genetically matched therapies differed among subtypes (salivary duct carcinoma: 82%, adenoid cystic carcinoma 28%, mucoepidermoid carcinoma 50%, acinic cell carcinoma 33%). Actionable aberrations were most often located in PIK3CA (n = 18, 15%), ERBB2 (n = 15, 12%), HRAS and NOTCH1 (both n = 9, 7%). Conclusions: Actionable genetic aberrations were seen in 53.7% of all SGC cases on the RNA and DNA level, with varying percentages between subtypes.

SDC is an aggressive subtype of salivary gland cancer. In R/M disease, androgen deprivation therapy (ADT) resulted in response rates (RR) of 18-53% in androgen receptor-positive SDC. SDCs are HER2-positive in 29-46% of cases and have shown RR of 70% on docetaxel and trastuzumab. Here, we present the results of HER2-positive SDC patients treated with DTP and subsequent T-DM1. Retrospective case series from a tertiary hospital. HER2 status was assessed by immunohistochemistry and in situ hybridization. DTP consisted of docetaxel (IV 75 mg/m2), trastuzumab (IV 8 mg/kg, followed by 6 mg/kg or SC 600mg), and pertuzumab (IV 840 mg, followed by 420 mg) given 3-weekly. After 6 cycles, only trastuzumab and pertuzumab were continued until disease progression or intolerable toxicity. After disease progression on DTP, T-DM1 (IV 3.6 mg/kg, 3-weekly) was considered for patients with clinical benefit on the first-line of anti-HER-2 therapy and a WHO PS 0-1. The overall RR (ORR) was scored by RECIST v1.1. Progression-free survival (PFS), overall survival (OS) and toxicity are presented. From 2015-2020, 13 HER2-positive SDC patients received DTP. Median age was 61 years (range: 48-75), 77% were male. Prior systemic therapy included adjuvant ADT (23%), palliative ADT (46%) and chemotherapy (8%). Twelve patients were RECIST evaluable, ORR was 58%: 1 complete response (CR) and 6 partial responses (PR). Median PFS was 6.8 months (range: 1.8-26.8+). Seven patients received second-line T-DM1. T-DM1 resulted in ORR of 57% (4 PR) and median PFS was 4.4 months (range: 1.2-17.3). After median follow-up from start of DTP of 15.4 months (range: 5.5-55.0) median OS was not reached. 5/13 patients (39%) developed grade ≥3 toxicity on DTP, including infections (n=3), neutropenia (n=1) and heart failure (n=1). 1/7 patient (14%) developed grade ≥3 toxicity on T-DM1, hyponatremia (n=1). In R/M HER2-positive SDC patients DTP followed by T-DM1 upon progression are promising treatment strategies, leading to responses in the majority of the patients at an acceptable toxicity profile. The median OS was not reached after a median follow-up of 15.4 months.

Patients suffering from recurrent or metastatic (R/M) salivary duct carcinoma (SDC) are often treated with combined androgen blockade (CAB). However, CAB frequently fails, resulting in a worse prognosis. Therefore, biomarkers that can predict treatment failure are urgently needed. mRNA from 76 R/M androgen receptor (AR)-positive SDC patients treated with leuprorelin acetate combined with bicalutamide was extracted from pre-treatment tumor specimens. AR, Notch, MAPK, TGFβ, estrogen receptor (ER), Hedgehog (HH), and PI3K signaling pathway activity scores (PAS) were determined based on the expression levels of target genes. Additionally, 5-alpha reductase type 1 (SRD5A1) expression was determined. These markers were related to clinical benefit (complete/partial response or stable disease ≥6 months) and progression-free and overall survival (PFS/OS). SRD5A1 expression had the highest general predictive value for clinical benefit and positive predictive value (PPV: 85.7%). AR PAS had the highest negative predictive value (NPV: 93.3%). The fitting of a multivariable model led to the identification of SRD5A1, TGFβ, and Notch PAS as the most predictive combination. High AR, high Notch, high ER, low HH PAS, and high SRD5A1 expression were also of prognostic importance regarding PFS and SRD5A1 expression levels for OS. AR, Notch PAS, and SRD5A1 expression have the potential to predict the clinical benefit of CAB treatment in SDC patients. SRD5A1 expression can identify patients that will and AR PAS patients that will not experience clinical benefit (85.7% and 93.3% for PPV and NPV, respectively). The predictive potential of SRD5A1 expression forms a rational basis for including SRD5A1-inhibitors in SDC patients’ treatment.

Background: Salivary duct carcinoma (SDC) is an aggressive subtype of salivary gland cancer. Approximately half of SDC patients will develop recurrences or metastases. Therapeutic palliative therapy is therefore often needed. The majority of SDC tumors expresses the androgen receptor (AR) and one-third expresses human epidermal growth factor receptor 2 (HER2), both are potential therapeutic targets. The aim of this paper is to systematically review and summarize the evidence on systemic palliative therapy for SDC and to provide treatment recommendations. Materials and methods: Electronic libraries were systematically searched with a broad search strategy to identify studies where SDC patients received systemic therapy. Due to the rarity of SDC no restrictions were placed on study designs. Results: The search resulted in 2014 articles of which 153 were full-text analyzed. Forty-five studies were included in the analysis, which included in total 256 SDC patients receiving systemic therapy. Two phase 2 trials primarily including SDC patients were identified. The majority of the studies were case series or case reports, resulting in an overall low quality of available evidence. Based on studies including >= 5 SDC patients, objective responses to HER2 targeting agents were observed in 60-70%, to AR pathway agents in 18-53% and to chemotherapy in 10-50%. Conclusion: For AR or HER2 positive SDC, agents targeting these pathways are the cornerstone for palliative treatment. Regarding chemotherapy, the combination of carboplatin combined with a taxane is best studied. Regarding other targeted agents and immunotherapy evidence is anecdotal, limiting formulation of treatment recommendations for these antineoplastic agents.

Salivary gland cancer is a malignancy that arises in the head and neck area. It is not only rare but its clinical course is also very heterogeneous. A total of 22 different subtypes can be distinguished, the symptoms, treatment and prognosis of which may differ greatly. This means that both the diagnosis and treatment of the disease are prone to error. This is illustrated by two cases: a 62-year-old man with a salivary duct carcinoma, and a 56-year-old man with an adenoid cystic carcinoma. These cases are used to illustrate the advances that have been made in the treatment of salivary gland cancer.

BackgroundExtracorporeally induced whole-body hyperthermia (eWBH) might be a beneficial treatment in cancer patients. Objectives of this pig study were to assess thermal distribution, (patho-)physiological effects, and safety of eWBH with a new WBH device. MethodsFourteen healthy adult pigs were anesthetized, mechanically ventilated, and cannulated; 12 were included in the analysis. Blood was heated in 11 pigs (one pig served as control) using a WBH device (Vither Hyperthermia B.V.) containing two separate fluidic circuits and a heat exchanger. Temperature was monitored on nine different sites, including the brain. Core temperature (average of 4 deep probes) was elevated to 42 degrees C for 2 hr. ResultsElevation of core body temperature to 42 degrees C took on average ( standard deviation) 38 +/- 8 min. Initially observed temperature spikes diminished after lowering maximal blood temperature to 45 degrees C. Hereafter, brain temperature spikes never exceeded 42.5 degrees C, mean brain temperature was at highest 41.9 degrees C during maintenance. WBH resulted in increased heart rates and decreased mean arterial pressures. The vast amounts of fluids required to counter hypotension tended to be smaller after corticosteroid administration. Hemodialysis was started in three animals (potassium increase prevention in two and hyperkalemia treatment in one). Severe rhabdomyolysis was observed in all pigs (including the control). All animals survived the procedure until planned euthanasia 1, 6, or 24 hr post procedure. ConclusionFast induction of eWBH with homogenous thermal distribution is feasible in pigs using the Vither WBH device. Severe hemodynamic disturbances, rhabdomyolysis, and hyperkalemia were observed.

Salivary gland cancer (SGC) is a rare malignancy consisting of 22 subtypes with different genetic, histological and clinical characteristics. This rarity and heterogeneity makes systemic treatment of recurrent or metastatic (R/M) disease challenging. Use of chemotherapy is scarcely studied and chemotherapy at best has moderate effects. New therapeutic strategies are therefore warranted, but advances made in SGC are lagging behind on advances made in more common cancers. By unraveling tumor characteristics of SGC, such as genetic alterations and protein expression profiles, therapeutic strategies tailored to the patient's tumor can be rationalized. This genomic profiling and mapping of immunohistochemical expression profiles is essential in the search for a suitable treatment approach. Thereby, it alleviates the paucity in systemic treatment options and can significantly alter the prognosis of patients with R/M SGC. This review aims to give a comprehensive overview of known genetic alterations and expression profiles amenable for targeted therapy in every histological subtype of SGC. We discuss the remaining knowledge gaps and the implications of these targets for future studies and personalized treatments, thereby aiding clinicians faced with this rare and heterogeneous type of cancer.

Whole-body hyperthermia (WBH) might be beneficial for patients with metastasized solid malignancies when combined with systemic therapy. This review identified and summarized the phase I/II studies (n = 13/14) conducted using this combination of therapies. Most of the phase II studies used radiant heating methods in a thermal dose of 41.8 °C (1 h). All studies used classic chemotherapy. Great inter-study heterogeneity was observed regarding treatment regimes, included patients and reported response rates (12-89%). Ovarian cancer, colorectal adenocarcinoma, lung cancer and sarcoma have been studied most. Most reported toxicity (grade 3/4) was myelosuppression. Treatment related mortality was present (4 patients) in three out 14 phase II studies (350 evaluable patients, over 966 cycles of WBH with chemotherapy). Absence of phase III trials makes the additive value of WBH highly speculative. As modern oncology offers many less invasive treatments options, it is unlikely WBH will ever find its way in routine clinical care.

Background Recently 3D organoid cell cultures have been established for a variety of human cancers. Salivary gland cancer (SGC) is a rare cancer with 22 different subtypes and few treatment options. Our aim is to generate a large repertoire of patient-derived SGC organoids with different phenotypes, which will facilitate pre-clinical and pharmacological studies. Methods Fresh tissues of resected primary tumours and/or metastases of SGC patients were collected. Tissues were minced and digested with collagenase type 2 and TrypLE to generate single cells. After washing, the cells were seeded in growth factor reduced Matrigel. Organoid medium, containing Rho kinase inhibitor Y27632, was refreshed twice a week. Viable organoids were characterized by immunohistochemistry and by DNA/RNA sequencing. Moreover, the organoids were used to evaluate various treatments that are or may be relevant for the treatment of SGC. Results Between 2016 and 2018 we obtained tissue of 17 SGC patients, of which 16 contained sufficient material for processing: 10 salivary duct carcinoma (SDC), 3 adenoid cystic carcinoma (ACC), 2 muco-epidermoid carcinoma (MEC) and 1 parotid adenocarcinoma not otherwise specified (NOS). For 5 tumors (2 SDC, 2 ACC, 1 adenocarcinoma NOS) viable organoids were established, which could be passaged at least three times. Organoids cultures derived from the other tumours stopped growing after the first passage. Viable organoids showed resemblance with the original primary tumour, based on morphology, protein expression, and growth pattern. Various drugs were tested at a single dose, and showed reduced organoid cell growth compared to untreated controls. Moreover, a dose-response relationship was established for an ALK-positive MEC organoid that was treated with crizotinib. Finally, epithelial-mesenchymal transition was shown in SDC organoids of a primary tumour and a lymph node metastasis of the same patient. Conclusions We are the first that have successfully developed and characterized long-term organoid cultures for ACC and SDC. These organoid cell lines will facilitate pre-clinical and pharmacological studies. Other SGC organoid cultures do not grow infinitely, but do provide an in vitro model to study different treatments during initial growth. Legal entity responsible for the study Radboud University Medical Center. Funding Dutch Salivary Gland Cancer Patient Platform. Disclosure All authors have declared no conflicts of interest.