Analysis of B7-H4 Expression Across Salivary Gland Carcinomas Reveals Adenoid Cystic Carcinoma-Specific Prognostic Relevance.

B7-H4 (VTCN1), a member of the B7 family, is overexpressed in several types of cancer. Here we investigated the pattern of expression of B7-H4 in salivary gland carcinomas (SGC) and assessed its potential as a prognostic marker and therapeutic target. Immunohistochemistry (IHC) analyses were performed in a cohort of 340 patient tumors, composed of 124 adenoid cystic carcinomas (ACC), 107 salivary duct carcinomas (SDC), 64 acinic cell carcinomas, 36 mucoepidermoid carcinomas (MEC), 9 secretory carcinomas (SC), as well as 20 normal salivary glands (controls). B7-H4 expression was scored and categorized into negative (<5% expression of any intensity), low (5%-70% expression of any intensity or >70% with weak intensity), or high (>70% moderate or strong diffuse intensity). The associations between B7-H4 expression and clinicopathologic characteristics, as well as overall survival, were assessed. Among all tumors, B7-H4 expression was more prevalent in ACC (94%) compared with those of SC (67%), MEC (44%), SDC (32%), and acinic cell carcinomas (0%). Normal salivary gland tissue did not express B7-H4. High expression of B7-H4 was found exclusively in ACC (27%), SDC (11%), and MEC (8%). In SDC, B7-H4 expression was associated with female gender (P = .002) and lack of androgen receptor expression (P = .012). In ACC, B7-H4 expression was significantly associated with solid histology (P < .0001) and minor salivary gland primary (P = .02). High B7-H4 expression was associated with a poorer prognosis in ACC, regardless of clinical stage and histologic subtype. B7-H4 expression was not prognostic in the non-ACC SGC evaluated. Our comparative study revealed distinct patterns of B7-H4 expression according to SGC histology, which has potential therapeutic implications. B7-H4 expression was particularly high in solid ACC and was an independent prognostic marker in this disease but not in the other SGC assessed.

Comprehensive molecular characterization of adenoid cystic carcinoma reveals tumor suppressors as novel drivers and prognostic biomarkers.

Adenoid cystic carcinoma (ACC) is a MYB-driven head and neck malignancy with high rates of local recurrence and distant metastasis and poor long-term survival. New effective targeted therapies and clinically useful biomarkers for patient stratification are needed to improve ACC patient survival. Here, we present an integrated copy number and transcriptomic analysis of ACC to identify novel driver genes and prognostic biomarkers. A total of 598 ACCs were studied. Clinical follow-up was available from 366 patients, the largest cohort analyzed to date. Copy number losses of 1p36 (70/492; 14%) and of the tumor suppressor gene PARK2 (6q26) (85/343; 25%) were prognostic biomarkers; patients with concurrent losses (n = 20) had significantly shorter overall survival (OS) than those with one or no deletions (p < 0.0001). Deletion of 1p36 independently predicted short OS in multivariate analysis (p = 0.02). Two pro-apoptotic genes, TP73 and KIF1B, were identified as putative 1p36 tumor suppressor genes whose reduced expression was associated with poor survival and increased resistance to apoptosis. PARK2 expression was markedly reduced in tumors with 6q deletions, and PARK2 knockdown increased spherogenesis and decreased apoptosis, indicating that PARK2 is a tumor suppressor in ACC. Moreover, analysis of the global gene expression pattern in 30 ACCs revealed a transcriptomic signature associated with short OS, multiple copy number alterations including 1p36 deletions, and reduced expression of TP73. Taken together, the results indicate that TP73 and PARK2 are novel putative tumor suppressor genes and potential prognostic biomarkers in ACC. Our studies provide new important insights into the pathogenesis of ACC. The results have important implications for biomarker-driven stratification of patients in clinical trials. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Frequent PTEN loss and differential HER2/PI3K signaling pathway alterations in salivary duct carcinoma: Implications for targeted therapy.

BACKGROUND: Patients with advanced primary and recurrent salivary duct carcinoma (SDC), a rare and lethal malignancy, have limited therapeutic options. Novel small-molecule agents aimed at targeting critical signaling associated with SDC tumorigenesis may lead to new therapeutic options for patients with these tumors. The human epidermal growth factor receptor 2 (HER2)/phosphoinositide 3-kinase (PI3K) axis, an important oncogenic pathway, has been targeted for therapy in several solid tumors. Currently, little is known about the role and clinical implications of alterations of the HER2/PI3K pathway in patients with SDC. METHODS: The authors investigated the clinicopathologic features, genetic alterations, and expression of key members of the HER2/PI3K pathway in 43 primary tumors and conducted in vitro functional and targeted drug-response analyses on cell lines derived from salivary epithelial carcinomas. RESULTS: In primary tumors, loss of phosphatase and tensin homolog (PTEN) expression was identified in 22 of 43 tumors (51%), overexpression of HER2 was observed in 12 of 43 tumors (28%), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations were identified in 12 of 43 tumors (28%). Phosphorylated protein kinase B (p-AKT) was highly expressed in most tumors. Most tumors (70%) displayed mutually exclusive alterations of PI3K members, whereas 8 tumors (19%) had 2 or more concurrent abnormalities. In vitro studies demonstrated a direct association between PTEN loss and PI3K pathway activation and evidence of response to combined PI3Kα and PI3Kß and/or pan-PI3K inhibitors. CONCLUSIONS: The current analyses reveal frequent PTEN loss and mutually exclusive alterations of key PI3K pathway members in SDC and demonstrate in vitro evidence of a response to pan-PI3K inhibitors. These results provide a framework for a biomarker-based substratification of patients with SDC in future targeted therapy. Cancer 2018;124:3523-32. © 2018 American Cancer Society.

Frequent and differential mutations of the CYLD gene in basal cell salivary neoplasms: linkage to tumor development and progression.

Basal cell salivary neoplasms display similar cyto-morphologic features and are classified into adenoma and adenocarcinoma based on the presence or absence of tumor invasion at diagnosis. These neoplasms also share considerable phenotypic resemblance and co-exist with certain dermal adnexal tumors harboring the CYLD gene mutations inferring common genetic association. We sequenced the CYLD gene in both basal cell adenomas and adenocarcinomas and correlated the findings with CYLD, NF-κB, and ß-catenin expression levels and clinicopathologic factors. Twenty mutations were identified and comprised of 3 synonymous and 17 non-synonymous (missense) types involving the coding exons of the CYLD gene. Mutations in exons 9-11 were identified in both adenomas and adenocarcinomas, while mutations in exons 12-20, encoding the USP domain, were exclusively found in carcinomas. Although no significant correlation between CYLD mutations and expression levels of CYLD, NF-κB, and ß-catenin or clinicopathologic parameters was found, basal cell adenocarcinomas with multiple mutations showed reduction in CYLD protein expression and pursued aggressive clinical behavior. Our study revealed high incidence and sequential CYLD mutations in both basal cell adenoma and adenocarcinoma supporting a single neoplastic continuum for their evolution and provides evidence for potential diagnostic and therapeutic utility.

Detailed genome-wide SNP analysis of major salivary carcinomas localizes subtype-specific chromosome sites and oncogenes of potential clinical significance.

The molecular genetic alterations underlying the development and diversity of salivary gland carcinomas are largely unknown. To characterize these events, comparative genomic hybridization analysis was performed, using a single-nucleotide polymorphism microarray platform, of 60 fresh-frozen specimens that represent the main salivary carcinoma types: mucoepidermoid carcinoma (MEC), adenoid cystic carcinoma (ACC), and salivary duct carcinoma (SDC). The results were correlated with the clinicopathologic features and translocation statuses to characterize the genetic alterations. The most commonly shared copy number abnormalities (CNAs) in all types were losses at chromosomes 6q23-26 and the 9p21 region. Subtype-specific CNAs included a loss at 12q11-12 in ACC and a gain at 17q11-12 in SDC. Focal copy number losses included 1p36.33-p36-22 in ACC, 9p13.2 in MEC, and 3p12.3-q11-2, 6q21-22.1, 12q14.1, and 12q15 in SDC. Tumor-specific amplicons were identified at 11q23.3 (PVRL1) in ACC, 11q13.3 (NUMA1) in MEC, and 6p21.1 (CCND3), 9p13.2 (PAX5), 12q15 (CNOT2/RAB3IP), 12q21.1 (GLIPR1L1), and 17q12 (ERBB2/CCL4) in SDC. A comparative CNA analysis of fusion-positive and fusion-negative ACCs and MECs revealed relatively lower CNAs in fusion-positive tumors than in fusion-negative tumors in both tumor types. An association between CNAs and high grade and advanced stage was observed in MECs only. These findings support the pathogenetic segregation of these entities and define novel chromosomal sites for future identification of biomarkers and therapeutic targets.

Intratumoral microbiome of adenoid cystic carcinomas and comparison with other head and neck cancers.

Adenoid cystic carcinoma (ACC) is a rare, usually slow-growing yet aggressive head and neck malignancy. Despite its clinical significance, our understanding of the cellular evolution and microenvironment in ACC remains limited. We investigated the intratumoral microbiomes of 50 ACC tumor tissues and 33 adjacent normal tissues using 16S rRNA gene sequencing. This allowed us to characterize the bacterial communities within the ACC and explore potential associations between the bacterial community structure, patient clinical characteristics, and tumor molecular features obtained through RNA sequencing. The bacterial composition in the ACC was significantly different from that in adjacent normal salivary tissue, and the ACC exhibited diverse levels of species richness. We identified two main microbial subtypes within the ACC: oral-like and gut-like. Oral-like microbiomes, characterized by increased diversity and abundance of Neisseria, Leptotrichia, Actinomyces, Streptococcus, Rothia, and Veillonella (commonly found in healthy oral cavities), were associated with a less aggressive ACC-II molecular subtype and improved patient outcomes. Notably, we identified the same oral genera in oral cancer and head and neck squamous cell carcinomas. In both cancers, they were part of shared oral communities associated with a more diverse microbiome, less aggressive tumor phenotype, and better survival that reveal the genera as potential pancancer biomarkers for favorable microbiomes in ACC and other head and neck cancers. Conversely, gut-like intratumoral microbiomes, which feature low diversity and colonization by gut mucus layer-degrading species, such as Bacteroides, Akkermansia, Blautia, Bifidobacterium, and Enterococcus, were associated with poorer outcomes. Elevated levels of Bacteroides thetaiotaomicron were independently associated with significantly worse survival and positively correlated with tumor cell biosynthesis of glycan-based cell membrane components.

Spatial Immunoprofiling of Adenoid Cystic Carcinoma Reveals B7-H4 Is a Therapeutic Target for Aggressive Tumors.

PURPOSE: Adenoid cystic carcinoma (ACC) is a heterogeneous malignancy, and no effective systemic therapy exists for metastatic disease. We previously described two prognostic ACC molecular subtypes with distinct therapeutic vulnerabilities, ACC-I and ACC-II. In this study, we explored the ACC tumor microenvironment (TME) using RNA-sequencing and spatial biology to identify potential therapeutic targets. EXPERIMENTAL DESIGN: Tumor samples from 62 ACC patients with available RNA-sequencing data that had been collected as part of previous studies were stained with a panel of 28 validated metal-tagged antibodies. Imaging mass cytometry (IMC) was performed using the Fluidigm Helios CyTOF instrument and analyzed with Visiopharm software. The B7-H4 antibody-drug conjugate AZD8205 was tested in ACC patient-derived xenografts (PDX). RESULTS: RNA deconvolution revealed that most ACCs are immunologically "cold," with approximately 30% being "hot." ACC-I tumors with a poor prognosis harbored a higher density of immune cells; however, spatial analysis by IMC revealed that ACC-I immune cells were significantly restricted to the stroma, characterizing an immune-excluded TME. ACC-I tumors overexpressed the immune checkpoint B7-H4, and the degree of immune exclusion was directly correlated with B7-H4 expression levels, an independent predictor of poor survival. Two ACC-I/B7-H4-high PDXs obtained 90% complete responses to a single dose of AZD8205, but none were observed with isotype-conjugated payload or in an ACC-II/B7-H4 low PDX. CONCLUSIONS: Spatial analysis revealed that ACC subtypes have distinct TMEs, with enrichment of ACC-I immune cells that are restricted to the stroma. B7-H4 is highly expressed in poor-prognosis ACC-I subtype and is a potential therapeutic target.

Phase II Clinical Trial of Axitinib and Avelumab in Patients With Recurrent/Metastatic Adenoid Cystic Carcinoma.

PURPOSE: We conducted a phase II trial evaluating the efficacy of VEGFR inhibitor axitinib and PD-L1 inhibitor avelumab in patients with recurrent/metastatic adenoid cystic carcinoma (R/M ACC). PATIENTS AND METHODS: Eligible patients had R/M ACC with progression within 6 months before enrollment. Treatment consisted of axitinib and avelumab. The primary end point was objective response rate (ORR) per RECIST 1.1; secondary end points included progression-free survival (PFS), overall survival (OS), and toxicity. Simon's optimal two-stage design tested the null hypothesis of ORR ≤5% versus ORR ≥20% at 6 months; ≥4 responses in 29 patients would reject the null hypothesis. RESULTS: Forty patients enrolled from July 2019 to June 2021; 28 were evaluable for efficacy (six screen failures; six evaluable for safety only). The confirmed ORR was 18% (95% CI, 6.1 to 36.9); there was one unconfirmed partial response (PR). Two patients achieved PR after 6 months; thus, the ORR at 6 months was 14%. The median follow-up time for surviving patients was 22 months (95% CI, 16.6 to 39.1 months). The median PFS was 7.3 months (95% CI, 3.7 to 11.2 months), 6-month PFS rate was 57% (95% CI, 41 to 78), and median OS was 16.6 months (95% CI, 12.4 to not reached months). Most common treatment-related adverse events (TRAEs) included fatigue (62%), hypertension (32%), and diarrhea (32%). Ten (29%) patients had serious TRAEs, all grade 3; four patients (12%) discontinued avelumab, and nine patients (26%) underwent axitinib dose reduction. CONCLUSION: The study reached its primary end point with ≥4 PRs in 28 evaluable patients (confirmed ORR of 18%). The potential added benefit of avelumab to axitinib in ACC requires further investigation.

Expression and regulation of the ΔN and TAp63 isoforms in salivary gland tumorigenesis clinical and experimental findings.

The TP63 gene, a TP53 homologue, encodes for two main isoforms by different promoters: one retains (TA) and the other lacks (ΔN) the transactivation domain. p63 plays a critical role in the maintenance of basal and myoepithelial cells in ectodermally derived tissues and is implicated in tumorigenesis of several neoplastic entities. However, the biological and regulatory roles of these isoforms in salivary gland tumorigenesis remain unknown. Our results show a reciprocal expression between TA and ΔN isoforms in both benign and malignant salivary tumors. The most dominantly expressed were the ΔN isoforms, whereas the TA isoforms showed generally low levels of expression, except in a few tumors. High ΔNp63 expression characterized tumors with aggressive behavior, whereas tumors with high TAp63 expression were significantly smaller and less aggressive. In salivary gland cells, high expression of ΔNp63 led to enhanced cell migration and invasion and suppression of cell senescence independent of TAp63 and/or TP53 gene status. We conclude the following: i) overexpression of ΔNp63 contributes to salivary tumorigenesis, ii) ΔNp63 plays a dominant negative effect on the TA isoform in the modulation of cell migration and invasion, and iii) the ΔN isoform plays an oncogenic role and may represent an attractive target for therapeutic intervention in patients with salivary carcinomas.

Rearrangements, Expression, and Clinical Significance of MYB and MYBL1 in Adenoid Cystic Carcinoma: A Multi-Institutional Study.

Adenoid cystic carcinoma (ACC) is an aggressive head and neck malignancy characterized by a t (6;9) translocation resulting in an MYB-NFIB gene fusion or, more rarely, an MYBL1 fusion. The true frequency and clinical significance of these alterations are still unclear. Here, we have used tissue microarrays and analyzed 391 ACCs and 647 non-ACC salivary neoplasms to study the prevalence, expression, and clinical significance of MYB/MYBL1 alterations by FISH and immunohistochemistry. Alterations of MYB or MYBL1 were found in 78% of the cases, of which 62% had MYB alterations and 16% had MYBL1 rearrangements. Overexpression of MYB/MYBL1 oncoproteins was detected in 93% of the cases. MYB split signal, seen in 39% of the cases, was specific for ACC and not encountered in non-ACC salivary tumors. Loss of the 3'-part of MYB was enriched in grade 3 tumors and was a significant independent prognostic biomarker for overall survival in multivariate analyses. We hypothesize that loss of the 3'-part of MYB results from an unbalanced t(6;9) leading to an MYB-NFIB fusion with concomitant loss of the segment distal to the MYB breakpoint in 6q23.3. Our study provides new knowledge about the prevalence and clinical significance of MYB/MYBL1 alterations and indicates the presence of genes with tumor suppressive functions in 6q23.3-qter that contribute to poor prognosis and short overall survival in ACC.

Whole-Genome Sequencing of Common Salivary Gland Carcinomas: Subtype-Restricted and Shared Genetic Alterations.

PURPOSE: Salivary gland carcinomas (SGCs) are pathologically classified into several widely diverse subtypes, of which adenoid cystic carcinoma (ACC), mucoepidermoid carcinoma (MEC), and salivary duct carcinoma (SDC) are the most commonly encountered. A comparative genetic analysis of these subtypes provides detailed information on the genetic alterations that are associated with their tumorigenesis and may lead to the identification of biomarkers to guide tumor-specific clinical trials. EXPERIMENTAL DESIGN: Whole-genome sequencing of 58 common SGCs (20 ACCs, 20 SDCs, and 18 MECs) was performed to catalog structural variations, copy number, rearrangements, and driver mutations. Data were bioinformatically analyzed and correlated with clinicopathologic parameters, and selected targets were validated. RESULTS: Novel and recurrent type-specific and shared genetic alterations were identified within and among 3 subtypes. Mutually exclusive canonical fusion and nonfusion genomic alterations were identified in both ACC and MEC. In ACCs, loss of chromosome 12q was dominant in MYB or MYBL1 fusion-positive tumors and mutations of NOTCH pathway were more common in these fusion negatives. In MECs, CRTC1-MAML2 fusion-positive tumors showed frequent BAP1 mutation, and tumors lacking this fusion were enriched with LRFN1 mutation. SDCs displayed considerable genetic instability, lacked recurrent chromosomal rearrangements, and demonstrated nonoverlapping TP53 mutation and ERBB2 amplification in a subset of tumors. Limited genetic alterations, including focal amplifications of 8q21-q23, were shared by all subtypes and were associated with poor survival. CONCLUSIONS: This study delineates type-specific and shared genetic alterations that are associated with early phenotypic commitment and the biologic progression of common SGCs. These alterations, upon validation, could serve as biomarkers in tumor-specific clinical trials.

LMW cyclin E and its novel catalytic partner CDK5 are therapeutic targets and prognostic biomarkers in salivary gland cancers.

Salivary gland cancers (SGCs) are rare yet aggressive malignancies with significant histological heterogeneity, which has made prediction of prognosis and development of targeted therapies challenging. In majority of patients, local recurrence and/or distant metastasis are common and systemic treatments have minimal impact on survival. Therefore, identification of novel targets for treatment that can also be used as predictors of recurrence for multiple histological subtypes of SGCs is an area of unmet need. In this study, we developed a novel transgenic mouse model of SGC, efficiently recapitulating the major histological subtype (adenocarcinomas of the parotid gland) of human SGC. CDK2 knock out (KO) mice crossed with MMTV-low molecular weight forms of cyclin E (LMW-E) mice generated the transgenic mouse models of SGC, which arise in the parotid region of the salivary gland, similar to the common site of origin seen in human SGCs. To identify the CDK2 independent catalytic partner(s) of LMW-E, we used LMW-E expressing cell lines in mass spectrometric analysis and subsequent biochemical validation in pull down assays. These studies revealed that in the absence of CDK2, LMW-E preferentially binds to CDK5. Molecular targeting of CDK5, using siRNA, resulted in inhibition of cell proliferation of human SGCs overexpressing LMW-E. We also provide clinical evidence of significant association of LMW-E/CDK5 co-expression and decreased recurrence free survival in human SGC. Immunohistochemical analysis of LMW-E and CDK5 in 424 patients representing each of the four major histological subtypes of human salivary cancers (Aci, AdCC, MEC, and SDC) revealed that LMW-E and CDK5 are concordantly (positive/positive or negative/negative) expressed in 70% of these patients. The co-expression of LMW-E/CDK5 (both positive) robustly predicts the likelihood of recurrence, regardless of the histological classification of these tumors. Collectively, our results suggest that CDK5 is a novel and targetable biomarker for the treatment of patients with SGC presenting with LMW-E overexpressing tumors.

Proteogenomic Analysis of Salivary Adenoid Cystic Carcinomas Defines Molecular Subtypes and Identifies Therapeutic Targets.

PURPOSE: Salivary gland adenoid cystic carcinoma (ACC) has heterogeneous clinical behavior. Currently, all patients are treated uniformly, and no standard-of-care systemic therapy exists for metastatic ACC. We conducted an integrated proteogenomic analyses of ACC tumors to identify dysregulated pathways and propose a classification with therapeutic implications. EXPERIMENTAL DESIGN: RNA/DNA sequencing of 54 flash-frozen salivary ACCs and reverse phase protein array (RPPA) in 38 specimens were performed, with validation by Western blotting and/or IHC. Three independent ACC cohorts were used for validation. RESULTS: Both unbiased RNA sequencing (RNA-seq) and RPPA analysis revealed two molecular subtypes: ACC-I (37%) and ACC-II (63%). ACC-I had strong upregulation of MYC, MYC target genes, and mRNA splicing, enrichment of NOTCH-activating mutations, and dramatically worse prognosis. ACC-II exhibited upregulation of TP63 and receptor tyrosine kinases (AXL, MET, and EGFR) and less aggressive clinical course. TP63 and MYC were sufficient to assign tumors to ACC subtypes, which was validated in one independent cohort by IHC and two additional independent cohorts by RNA-seq. Furthermore, IHC staining for MYC and P63 protein levels can be used to identify ACC subtypes, enabling rapid clinical deployment to guide therapeutic decisions. Our data suggest a model in which ACC-I is driven by MYC signaling through either NOTCH mutations or direct amplification, which in turn suppress P63 signaling observed in ACC-II, producing unique therapeutic vulnerabilities for each subtype. CONCLUSIONS: Cooccurrence of multiple actionable protein/pathways alterations in each subtype indicates unique therapeutic vulnerabilities and opportunities for optimal combination therapy for this understudied and heterogeneous disease.

Reciprocal and Autonomous Glucocorticoid and Androgen Receptor Activation in Salivary Duct Carcinoma.

PURPOSE: To determine the expression of glucocorticoid receptor (GR) and androgen receptor (AR) in salivary duct carcinoma (SDC) and to analyze the role of these proteins in the development and management of this disease entity. EXPERIMENTAL DESIGN: We performed a phenotypic assessment of GR and AR localization and expression, and determined their association with clinicopathologic factors in 67 primary SDCs. In vitro functional and response analysis of SDC cell lines was also performed. RESULTS: Of the 67 primary tumors, 12 (18%) overexpressed GR protein, 30 (45%) had constitutive expression, and 25 (37%) had complete loss of expression. Reciprocal GR and AR expression was found in 32 (48%) tumors, concurrent constitutive GR and AR expression in 23 (34%), and simultaneous loss of both receptors and high GR with AR expressions were found in 12 (18%). GR overexpression was significantly associated with worse clinical outcomes. In vitro ligand-independent AR activation was observed in both male- and female-derived cell lines. GR antagonist treatment resulted in decreased cell proliferation and survival in GR-overexpressing cells, irrespective of AR status. Reciprocal GR- and AR-knockdown experiments revealed an independent interaction. CONCLUSIONS: Our study, for the first time, demonstrates differential GR and AR expressions, autonomous GR and AR activation, and ligand-independent AR expression and activation in SDC cells. The findings provide critical information on the roles of GR and AR steroid receptors in SDC tumorigenesis and development of biomarkers to guide targeted steroid receptor therapy trials in patients with these tumors.

Oncogenic Orphan Nuclear Receptor NR4A3 Interacts and Cooperates with MYB in Acinic Cell Carcinoma.

Acinic cell carcinoma (AcCC) is a morphologically distinctive salivary gland malignancy often associated with chromosome rearrangements leading to overexpression of the NR4A3 transcription factor. However, little is known about how NR4A3 contributes to AcCC biology. Detailed RNA-sequencing of 21 archived AcCC samples revealed fusion reads arising from recurrent t(4;9), t(9;12), t(8;9) or t(2;4) chromosomal translocations, which positioned highly active enhancers adjacent to the promoter of the NR4A3 gene or the closely related NR4A2 gene, resulting in their aberrant overexpression. Transcriptome analyses revealed several distinct subgroups of AcCC tumors, including a subgroup that overexpressed both NR4A3 and MSANTD3. A poor survival subset of the tumors with high-grade transformation expressed NR4A3 and POMC as well as MYB, an oncogene that is the major driver in a different type of salivary gland tumor, adenoid cystic carcinoma. The combination of NR4A3 and MYB showed cooperativity in regulating a distinct set of genes. In addition, the ligand binding domain of NR4A3 directly bound the Myb DNA binding domain. Transformation assays indicated that, while overexpressed NR4A3 was sufficient to generate transformed colonies, the combination of NR4A3 plus Myb was more potent, leading to anchorage-independent growth and increased cellular invasiveness. The results confirm that NR4A3 and NR4A2 are the main driver genes of AcCC and suggest that concurrent overexpression of NR4A3 and MYB defines a subset of AcCC patients with high-grade transformation that display exceptionally poor outcome.

DeltaNp63alpha-dependent expression of Id-3 distinctively suppresses the invasiveness of human squamous cell carcinoma.

p63 is a member of the p53 family and DeltaNp63alpha is the dominant-expressing isoform of p63 in basal layer of normal stratified epithelium and human squamous cell carcinoma (SCC) cells. We have previously reported that down-regulation of p63 was accompanied with epithelial-to-mesenchymal transition (EMT) by Snail-expressing SCC cells, in which re-expression of DeltaNp63alpha diminished their invasiveness (Higashikawa K, Yoneda S, Tobiume K, Taki M, Shigeishi H, Kamata N. Snail-induced down-regulation of DeltaNp63alpha acquires invasive phenotype of human squamous cell carcinoma. Cancer Res 2007;67:9207-13). In this study, we found that DeltaNp63alpha positively regulated inhibitor of differentiation-3 (Id-3) expression. Id is a dominant negative regulator of E2A which is a transcriptional repressor of E-cadherin. Enforced expression of Id-3 was incapable of invoking E-cadherin expression in the SCC cells with EMT phenotype, whereas it significantly impaired their invasiveness with down-regulation of matrix-metalloproteinase-2 (MMP-2) expression. Reporter gene assay revealed that the Ets-1-induced MMP-2 promoter activity was suppressed by the Id-3, while the Id-3-dependent E-cadherin promoter activity was remarkably reduced in the presence of Snail. Furthermore, knockdown of p63 in SCC cells significantly decreased Id-3 expression, in which up-regulation of MMP-2 expression was concomitant with the acquired invasiveness. These findings propose a particular role of the off-signaling of the DeltaNp63alpha-Id-3 axis incident to Snail-mediated EMT for the MMP-2-dependent invasiveness in SCC cells.

N-Terminal Truncated Myb with New Transcriptional Activity Produced Through Use of an Alternative MYB Promoter in Salivary Gland Adenoid Cystic Carcinoma.

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland tumor that frequently displays perineural invasion and is often associated with translocations or overexpression of the MYB oncogene. Detailed analyses of MYB transcripts from ACC patient samples revealed that ACC tumors utilize an alternative MYB promoter, which is rarely used in normal cells or other tumor types. The alternative promoter transcripts produce N-terminally truncated Myb proteins lacking a highly conserved and phosphorylated domain, which includes the pS11 epitope that is frequently used to detect Myb proteins. In RNA-seq assays, Myb isoforms lacking the N-terminal domain displayed unique transcriptional activities, regulating many genes differently than full-length Myb. Thus, a regulatory pathway unique to ACC activates the alternative MYB promoter, leading to the production of a truncated Myb protein with altered transcriptional activities. This could provide new therapeutic opportunities for ACC patients.

Expression of epiregulin, a novel epidermal growth factor ligand associated with prognosis in human oral squamous cell carcinomas.

We examined the expression of epiregulin and amphiregulin mRNA in 39 oral SCCs, 2 epithelial dysplasias and 7 normal gingivae by real-time RT-PCR. The mean expression level of epiregulin mRNA was higher in oral SCCs (0.29+/-0.50) than normal gingivae (0.01+/-0.007) and epithelial dysplasias (0.01+/-0.001). The expression level of epiregulin mRNA was significantly higher in oral SCCs than normal gingivae (Mann-Whitney U test, P=0.023). Epiregulin mRNA was higher in stage III/IV than in stage I/II oral SCCs. However, a significant association was not found. The mean expression level of amphiregulin mRNA was higher in oral SCCs (0.18+/-0.24) than normal gingivae (0.002+/-0.003) and epithelial dysplasias (0.01+/-0.001). Amphiregulin mRNA was significantly higher in oral SCCs than normal gingivae (Mann-Whitney U test, P=0.001). We then examined the expression of four EGF receptor mRNA in oral SCCs. The expression levels of HER1, HER2, HER3 and HER4 mRNA in oral oral SCCs were increased compared to those in normal gingivae. A significant correlation was found between the mRNA expression levels of epiregulin and HER2, HER3 and HER4 (Spearman's correlation coefficient by rank test, P=0.031, P=0.004 and P=0.027, respectively). Patients with oral SCC that have a high expression of epiregulin had a significantly shorter survival than those with low a expression (log-rank test, P<0.05). These results indicate that human epiregulin is closely linked to the increased or abnormal cell proliferation in human oral SCC.

Transcriptomes define distinct subgroups of salivary gland adenoid cystic carcinoma with different driver mutations and outcomes.

The relative rarity of salivary gland adenoid cystic carcinoma (ACC) and its slow growing yet aggressive nature has complicated the development of molecular markers for patient stratification. To analyze molecular differences linked to the protracted disease course of ACC and metastases that form 5 or more years after diagnosis, detailed RNA-sequencing (RNA-seq) analysis was performed on 68 ACC tumor samples, starting with archived, formalin-fixed paraffin-embedded (FFPE) samples up to 25 years old, so that clinical outcomes were available. A statistical peak-finding approach was used to classify the tumors that expressed MYB or MYBL1, which had overlapping gene expression signatures, from a group that expressed neither oncogene and displayed a unique phenotype. Expression of MYB or MYBL1 was closely correlated to the expression of the SOX4 and EN1 genes, suggesting that they are direct targets of Myb proteins in ACC tumors. Unsupervised hierarchical clustering identified a subgroup of approximately 20% of patients with exceptionally poor overall survival (median less than 30 months) and a unique gene expression signature resembling embryonic stem cells. The results provide a strategy for stratifying ACC patients and identifying the high-risk, poor-outcome group that are candidates for personalized therapies.