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PURPOSE The androgen receptor (AR) is expressed (+) in a subset of salivary gland cancers (SGCs). This phase II trial evaluated the efficacy of the antiandrogen enzalutamide in AR+ SGC. METHODS Patients with locally advanced/unresectable or metastatic AR+ SGCs were enrolled. Enzalutamide (160 mg) was given orally once daily. The primary end point was the best overall response rate per RECIST v1.1 within eight cycles. Confirmed responses in ≥ 5 of 41 patients would be considered promising. Secondary end points were progression-free survival, overall survival, and safety. RESULTS Forty-six patients were enrolled; 30 (65.2%) received prior systemic therapy, including 13 (28.3%) with AR-targeted drugs. Of seven (15.2%) partial responses (PRs), only two (4.3%) were confirmed per protocol and counted toward the primary end point. Twenty-four patients (52.2%) had stable disease; 15 (32.6%) had progression of disease as best response. Twenty-six patients (56.5%) experienced tumor regression in target lesions; 18 (39.1%) had partial response/stable disease ≥ 6 months. Tumor regressions were observed in female patients (5 of 6 [83.3%]) and those who received prior AR– (6 of 13 [46.2%]) or human epidermal growth factor receptor 2–targeted therapies (5 of 8 [62.5%]). Three patients remained on treatment at data cutoff (duration, 32.2-49.8 months). The median progression-free survival was 5.6 months (95% CI, 3.7 to 7.5); the median overall survival was 17.0 months (95% CI, 11.8 to 30.0). The most common adverse events were fatigue, hypertension, hot flashes, and weight loss. Total and free testosterone levels increased by a mean of 61.2% and 48.8%, respectively, after enzalutamide. CONCLUSION Enzalutamide demonstrated limited activity in AR+ SGC, failing to meet protocol-defined success in part because of a lack of response durability. Strategies to enhance the efficacy of antiandrogen therapy are needed.

Targeted inhibition of BRAF V600E achieves tumor control in a subset of advanced thyroid tumors. Nearly all tumors develop resistance, and some have been observed to subsequently undergo dedifferentiation. The molecular alterations associated with thyroid cancer dedifferentiation in the setting of BRAF inhibition are unknown. We analyzed targeted next-generation sequencing data from 639 advanced, recurrent and/or metastatic thyroid carcinomas, including 15 tumors that were treated with BRAF inhibitor drugs and had tissue sampled during or posttreatment, 8 of which had matched pretherapy samples. Pre- and posttherapy tissues from one additional patient were profiled with whole-exome sequencing and RNA expression profiling. Mutations in genes comprising the SWI/SNF chromatin remodeling complex and the PI3K–AKT–mTOR, MAPK, and JAK–STAT pathways all increased in prevalence across more dedifferentiated thyroid cancer histologies. Of 7 thyroid cancers that dedifferentiated after BRAF inhibition, 6 had mutations in these pathways. These mutations were mostly absent from matched pretreatment samples and were rarely detected in tumors that did not dedifferentiate. Additional analyses in one of the vemurafenib-treated tumors before and after anaplastic transformation revealed the emergence of an oncogenic PIK3CA mutation, activation of ERK signaling, dedifferentiation, and development of an immunosuppressive tumor microenvironment. These findings validate earlier preclinical data implicating these genetic pathways in resistance to BRAF inhibitors, and suggest that genetic alterations mediating acquired drug resistance may also promote thyroid tumor dedifferentiation.Implications:The possibility that thyroid cancer dedifferentiation may be attributed to selective pressure applied by BRAF inhibitor–targeted therapy should be investigated further.

Supplemental Figure 1. Increased clonal heterogeneity in anaplastic metastases (ATC Met 1, 5 clones Supplemental Figure 2. Copy-number alterations for the pre-vemurafenib papillary thyroid carcinoma sample Supplemental Figure 3. Differentially expressed gene analyses comparing pre-treatment PTC sample and ATC metastases in MSK-THY1. Supplemental Figure 4. Prevalence of genetic alterations linked to BRAF inhibitor resistance and/or ATC pathogenesis in primary TCGA PTCs and metastatic/recurrent PTCs, PDTCs, and ATCs sequenced by MSK-IMPACT. Supplemental Figure 5. Histology and immunohistochemical studies for MSK-THY6. Supplemental Figure 6. Mutational landscape of tumors that underwent dedifferentiation subsequent to BRAF inhibitor targeted therapy with vemurafenib or dabrafenib.

Supplemental Table 1. Genomic alterations observed in MSK-THY1 Supplemental Table 2. Cellular prevalence of single nucleotide variants in MSK-THY Supplemental Table 3. Normalized gene expression data from patient MSK-THY1 Supplemental Table 4. Mutational data for 639 thyroid tumors sequenced by MSK-IMPACT Supplemental Table 5. Mutational data for BRAFi treated thyroid cancers with post-treatment sequencing

Targeted inhibition of BRAF V600E achieves tumor control in a subset of advanced thyroid tumors. Nearly all tumors develop resistance, and some have been observed to subsequently undergo dedifferentiation. The molecular alterations associated with thyroid cancer dedifferentiation in the setting of BRAF inhibition are unknown. We analyzed targeted next-generation sequencing data from 639 advanced, recurrent and/or metastatic thyroid carcinomas, including 15 tumors that were treated with BRAF inhibitor drugs and had tissue sampled during or posttreatment, 8 of which had matched pretherapy samples. Pre- and posttherapy tissues from one additional patient were profiled with whole-exome sequencing and RNA expression profiling. Mutations in genes comprising the SWI/SNF chromatin remodeling complex and the PI3K–AKT–mTOR, MAPK, and JAK–STAT pathways all increased in prevalence across more dedifferentiated thyroid cancer histologies. Of 7 thyroid cancers that dedifferentiated after BRAF inhibition, 6 had mutations in these pathways. These mutations were mostly absent from matched pretreatment samples and were rarely detected in tumors that did not dedifferentiate. Additional analyses in one of the vemurafenib-treated tumors before and after anaplastic transformation revealed the emergence of an oncogenic PIK3CA mutation, activation of ERK signaling, dedifferentiation, and development of an immunosuppressive tumor microenvironment. These findings validate earlier preclinical data implicating these genetic pathways in resistance to BRAF inhibitors, and suggest that genetic alterations mediating acquired drug resistance may also promote thyroid tumor dedifferentiation. Implications: The possibility that thyroid cancer dedifferentiation may be attributed to selective pressure applied by BRAF inhibitor–targeted therapy should be investigated further.

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Purpose:Salivary gland carcinomas (SGC) are rare, aggressive cancers with high rates of recurrence and distant metastasis. These factors, and a lack of active systemic therapies, contribute to poor clinical outcome. Response rates with immune checkpoint blockade have been low, although clinical data remain sparse. To improve the efficacy of therapies, a more comprehensive understanding of relevant molecular alterations and immunologic processes is needed.Experimental Design:To characterize the immune microenvironment and neoantigen landscape of SGCs, we performed RNA sequencing (RNA-seq) in 76 tumors representing the three most lethal histologies: adenoid cystic carcinoma (ACC), myoepithelial carcinoma (MECA), and salivary duct carcinoma (SDC). We analyzed transcriptomic profiles, tumor-infiltrating immune cell populations, and measures of T-cell activation/dysfunction. In 37 cases also undergoing exome sequencing, we analyzed somatic mutations and neoantigens.Results:SDCs exhibited high levels of immune infiltration, with corresponding higher levels of T-cell dysfunction, and higher mutational load. In contrast, ACCs were characterized by an immune-excluded microenvironment, the presence of M2-polarized macrophages and myeloid-derived suppressor cells, and very low mutational load. MECAs were more heterogeneous, with both immune-low and immune-high phenotypes represented. Across all SGCs, levels of immune infiltration were associated with mutation- and fusion-derived neoantigens, and with aggressive clinical behavior.Conclusions:These findings provide new insights into the immune microenvironment and neoantigen landscape of SGCs, showing that mechanisms of immune escape appear to differ by histology. These data nominate potential immunologic vulnerabilities and may help guide the next steps of investigation in precision immunotherapy for these difficult-to-treat cancers.

Anti-tumor immunity is driven by self versus non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole-genome sequencing and RNA sequencing, we identified a novel gene fusion and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint-inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration. Fusion proteins in cancers with low mutational burden represent functional neoantigens that elicit T cell activation and mediate responses to immunotherapy.

Purpose: Salivary gland carcinomas (SGC) are rare, aggressive cancers with high rates of recurrence and distant metastasis. These factors, and a lack of active systemic therapies, contribute to poor clinical outcome. Response rates with immune checkpoint blockade have been low, although clinical data remain sparse. To improve the efficacy of therapies, a more comprehensive understanding of relevant molecular alterations and immunologic processes is needed. Experimental Design: To characterize the immune microenvironment and neoantigen landscape of SGCs, we performed RNA sequencing (RNA-seq) in 76 tumors representing the three most lethal histologies: adenoid cystic carcinoma (ACC), myoepithelial carcinoma (MECA), and salivary duct carcinoma (SDC). We analyzed transcriptomic profiles, tumor-infiltrating immune cell populations, and measures of T-cell activation/dysfunction. In 37 cases also undergoing exome sequencing, we analyzed somatic mutations and neoantigens. Results: SDCs exhibited high levels of immune infiltration, with corresponding higher levels of T-cell dysfunction, and higher mutational load. In contrast, ACCs were characterized by an immune-excluded microenvironment, the presence of M2-polarized macrophages and myeloid-derived suppressor cells, and very low mutational load. MECAs were more heterogeneous, with both immune-low and immune-high phenotypes represented. Across all SGCs, levels of immune infiltration were associated with mutation- and fusion-derived neoantigens, and with aggressive clinical behavior. Conclusions: These findings provide new insights into the immune microenvironment and neoantigen landscape of SGCs, showing that mechanisms of immune escape appear to differ by histology. These data nominate potential immunologic vulnerabilities and may help guide the next steps of investigation in precision immunotherapy for these difficult-to-treat cancers.

Nucleolin (NCL) is an abundant stress-responsive, RNA-binding phosphoprotein that controls gene expression by regulating either mRNA stability and/or translation. NCL binds to the AU-rich element (ARE) in the 3'UTR of target mRNAs, mediates miRNA functions in the nearby target sequences, and regulates mRNA deadenylation. However, the mechanism by which NCL phosphorylation affects these functions and the identity of the deadenylase involved, remain largely unexplored. Earlier we demonstrated that NCL phosphorylation is vital for cell cycle progression and proliferation, whereas phosphorylation-deficient NCL at six consensus CK2 sites confers dominant-negative effect on proliferation by increasing p53 expression, possibly mimicking cellular DNA damage conditions. In this study, we show that NCL phosphorylation at those CK2 consensus sites in the N-terminus is necessary to induce deadenylation upon oncogenic stimuli and UV stress. NCL-WT, but not hypophosphorylated NCL-6/S*A, activates poly (A)-specific ribonuclease (PARN) deadenylase activity. We further demonstrate that NCL interacts directly with PARN, and under non-stress conditions also forms (a) complex (es) with factors that regulate deadenylation, such as p53 and the ARE-binding protein HuR. Upon UV stress, the interaction of hypophosphorylated NCL-6/S*A with these proteins is favored. As an RNA-binding protein, NCL interacts with PARN deadenylase substrates such as TP53 and BCL2 mRNAs, playing a role in their downregulation under non-stress conditions. For the first time, we show that NCL phosphorylation offers specificity to its protein-protein, protein-RNA interactions, resulting in the PARN deadenylase regulation, and hence gene expression, during cellular stress responses.

Myoepithelial carcinoma (MECA) is an aggressive salivary gland cancer with largely unknown genetic features. Here we comprehensively analyze molecular alterations in 40 MECAs using integrated genomic analyses. We identify a low mutational load, and high prevalence (70%) of oncogenic gene fusions. Most fusions involve the PLAG1 oncogene, which is associated with PLAG1 overexpression. We find FGFR1-PLAG1 in seven (18%) cases, and the novel TGFBR3-PLAG1 fusion in six (15%) cases. TGFBR3-PLAG1 promotes a tumorigenic phenotype in vitro, and is absent in 723 other salivary gland tumors. Other novel PLAG1 fusions include ND4-PLAG1; a fusion between mitochondrial and nuclear DNA. We also identify higher number of copy number alterations as a risk factor for recurrence, independent of tumor stage at diagnosis. Our findings indicate that MECA is a fusion-driven disease, nominate TGFBR3-PLAG1 as a hallmark of MECA, and provide a framework for future diagnostic and therapeutic research in this lethal cancer., Myoepithelial carcinoma (MECA) is a rare aggressive salivary gland cancer. Here, the authors analyze the genomic landscape of MECA and identify a high prevalence of oncogenic gene fusions, primarily PLAG1 fusions, highlighting TGFBR3-PLAG1 as a potential hallmark of MECA.

Myoepithelial carcinoma (MECA) is an aggressive type of salivary gland cancer with largely unknown molecular features. MECA may arise de novo or result from oncogenic transformation of a pre-existing pleomorphic adenoma (MECA ex-PA). We comprehensively analyzed the molecular alterations in MECA with integrated genomic analyses. We identified a low mutational load (0.5/MB), but a high prevalence of fusion oncogenes (28/40 tumors; 70%). We foundFGFR1-PLAG1in 7 (18%) cases, and the novelTGFBR3-PLAG1fusion in 6 (15%) cases.TGFBR3-PLAG1was specific for MECA de novo tumors or the malignant component of MECA ex-PA, was absent in 723 other salivary gland tumors, and promoted a tumorigenic phenotype in vitro. We discovered other novelPLAG1fusions, includingND4-PLAG1,which is an oncogenic fusion between mitochondrial and nuclear DNA. One tumor harbored anMSN-ALKfusion, which was tumorigenic in vitro, and targetable with ALK inhibitors. Certain gene fusions were predicted to result in neoantigens with high MHC binding affinity. A high number of copy number alterations was associated with poorer prognosis. Our findings indicate that MECA is a fusion-driven disease, nominateTGFBR3-PLAG1as a hallmark of MECA, and provide a framework for future steps of diagnostic and therapeutic research in this lethal cancer.