Tailoring treatment of salivary duct carcinoma (SDC) by liquid biopsy: ARv7 expression in circulating tumor cells

We read with interest the paper by Fushimi et al. [1.Fushimi C. Tada Y. Takahashi H. et al.A prospective phase II study of combined androgen blockade in patients with androgen receptor-positive metastatic or locally advanced unresectable salivary gland carcinoma.Ann Oncol. 2018; 29: 979-984Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar] prospectively reporting for the first time activity and acceptable safety profile by combined androgen blockade (CAB) in patients with metastatic, androgen receptor (AR) positive, salivary duct carcinoma (SDC). Fushimi et al. found no correlations between treatment outcomes and clinicobiologic factors/biomarkers. Here we report our preliminary findings demonstrating that circulating tumor cells (CTCs) isolated in patients with SDC can be used to guide treatment with androgen blockade, thus offering a way to improve treatment outcomes by exploiting blood borne biomarkers in a paradigm similar to prostate cancer (PC) [2.Antonarakis E.S. Lu C. Luber B. et al.Clinical significance of androgen receptor splice variant-7 mRNA detection in circulating tumor cells of men with metastatic castration-resistant prostate cancer treated with first- and second-line abiraterone and enzalutamide.J Clin Oncol. 2017; 35: 2149-2156Crossref PubMed Scopus (311) Google Scholar]. Androgen blockade is hindered by the onset of resistance mechanisms linked, among others, to expression of AR splice-variants lacking the hormone binding domain and acting as ligand-independent transcription factors [3.Antonarakis E.S. Armstrong A.J. Dehm S.M. Luo J. Androgen receptor variant-driven prostate cancer: clinical implications and therapeutic targeting.Prostate Cancer Prostatic Dis. 2016; 19: 231-241Crossref PubMed Scopus (118) Google Scholar, 4.Ciccarese C. Santoni M. Brunelli M. et al.AR-V7 and prostate cancer: the watershed for treatment selection?.Cancer Treat Rev. 2016; 43: 27-35Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. The most frequently expressed splice variant in SDC, the ARv7, is associated with resistance to enzalutamide/bicalutamide in PC [2.Antonarakis E.S. Lu C. Luber B. et al.Clinical significance of androgen receptor splice variant-7 mRNA detection in circulating tumor cells of men with metastatic castration-resistant prostate cancer treated with first- and second-line abiraterone and enzalutamide.J Clin Oncol. 2017; 35: 2149-2156Crossref PubMed Scopus (311) Google Scholar]. Since in the advanced settings tissue from the primary or metastatic disease is not always available for molecular analyses, liquid biopsy could be a potential tool to explore the molecular profile of SDC and investigate the androgen-resistance mechanisms. Liquid biopsy holds promise in the arena of precision medicine and CTCs offer the unique opportunity of obtaining samples that can be used for comprehensive molecular analyses. This concept was challenged in a single patient treated within the EORTC 1206 trial (NCT01969578) and receiving CAB from July 2016 to April 2017 with a stable disease as best response. At progression (new brain lesions) in May 2017 he was enrolled in the phase II trial with abiraterone (NCT02867852). After having carried out whole brain radiotherapy (total dose = 30 Gy/10 fx), the first and second radiologic evaluations at 2 and 4 months showed stable disease, whereas the last imaging carried out at 6 months showed bone and liver PD. Using a commercial kits for enrichment (ProstateCancerSelect, QIAGEN, Hilden, Germany), detection (BreastCancerDetect kit QIAGEN) and molecular characterization (AR-V7 assay RT PCR, Bird, Monteriggioni, Italy), we report for the first time the presence of CTCs and the expression of full-length AR and of the splicing variant ARv7 in the CTCs isolated in blood collected before treatment with abiraterone. Both the full length AR and the splicing variant ARv7 were highly expressed, 344 and 77 copies/ml of blood, respectively in the CTC isolated before treatment. Although no comparative data are available for SDC, the reported ARv7 levels were definitely higher than the median ARv7 levels (35.5 copies/ml blood) observed in castration resistant PC patients undergoing progression under enzalutamide or abiraterone in our institutional experience (unpublished data). In keeping with this, in our SDC patient the CTC ARv7 expression predicted the onset of resistance with an anticipation of six months. Using in parallel an unbiased CTC-enrichment method (Parsortix) coupled with single-CTC identification and recovery by the DEPArray, which allows also identification of non-conventional CTC (ncCTC) lacking epithelial markers [5.Reduzzi C. Motta R. Bertolini G. et al.Development of a protocol for single-cell analysis of circulating tumor cells in patients with solid tumors.Adv Exp Med Biol. 2017; 994: 83-103Crossref PubMed Scopus (8) Google Scholar], three ncCTC harboring numerous genetic aberrations were identified (Figure 1). CNA-profiles suggested genomic heterogeneity among CTCs and interestingly a gain could be observed in the chromosome X region harboring the AR gene in keeping with the over-expression of AR observed by immunohistochemistry in the primary tumor. These findings support exploitation of liquid biopsy-based approach in clinical practice to improve management of SDC patients, by the early detection of androgen-resistance molecular mechanisms. The authors would like to thank the patient who participated in the study and Drs. Marta Vismara and Marco Silvestri for assistance in preparation of libraries for sequencing and in their analysis. The skilful help in sequencing experiments by the Functional Genomics Service from the DRAST_INTM is greatly appreciated This study was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro to LL (AIRC IG 15930-2014) and by a grant from the Italian Ministry of Health to MGD (RF-2013-02359692).