MYB-NFIB Fusion Gene Research Articles

The Role of Novel Immunohistochemical Markers for Special Types of Breast Carcinoma.

Some histologic special types of breast carcinoma harbor specific recurrent genetic alterations that are not seen in other types of breast carcinoma (no special type), namely adenoid cystic carcinoma, secretory carcinoma, and tall cell carcinoma with reversed polarity. These tumors have unique morphologic features, are triple-negative, that is, do not express hormone receptors or HER2, and are generally associated with a favorable prognosis. Adenoid cystic carcinoma, like its counterpart in other organs, shows a MYB-NFIB fusion gene that is the result of a recurrent t(6;9)(q22-23;p23-24) translocation. Other MYB alterations have been described that result in overexpression of MYB . Secretory carcinoma is characterized by an ETV6-NTRK3 gene fusion that is the result of recurrent (12;15);(p13;q25) translocation, which is also seen in mammary analog secretory carcinoma of the salivary gland. Tall cell carcinoma with reversed polarity shows IDH2 p.Arg172 hotspot mutations. Immunohistochemical antibodies have emerged that identify the underlying genetic alterations in these tumors and serve as useful diagnostic tools. This review will provide an update on the molecular features and diagnostic immunohistochemical markers that have become increasingly popular to aid in diagnosing these uncommon triple-negative breast tumors.

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

Simple SummaryAdenoid cystic carcinoma (ACC) is an aggressive glandular cancer with poor prognosis that preferentially occurs in the head and neck. The MYB and MYBL1 oncogenes are main oncogenic drivers, but the true frequency and clinical significance of these alterations are unclear. Here, we have used tissue microarrays to study these genes in a multi-institutional study of close to 400 ACCs, the largest study to date. We found alterations of MYB/MYBL1 in 78% of the cases and overexpression of the MYB/MYBL1 proteins in 93% of the cases. Importantly, we show that patients with loss of one part of the MYB gene and its neighboring sequences on chromosome 6 have a significantly shorter overall survival compared to those without loss. Our study provides new knowledge about the frequency and clinical significance of MYB/MYBL1 alterations and identifies genes with tumor suppressive functions on chromosome 6 that contribute to poor prognosis in ACC.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.

Heterogeneity of Genetic Landscapes in Salivary Gland Tumors and Their Critical Roles in Current Management.

Salivary gland neoplasms (SGNs) are rare and heterogeneous tumors in the head and neck region. Although progress has been recently made in revealing the molecular landscape of salivary glands tumors, it is limited and appears to be the tip of the iceberg. Some genetic aberrations include chromosomal translocations, such as CRTC1/3-MAML2 in mucoepidermoid carcinoma, g MYB-NFIB gene fusions in adenoid cystic carcinoma, and PLAG1-HMGA2 gene changes in pleomorphic adenoma and carcinoma ex pleomorphic adenoma. These chromosomal translocations provide fresh insights into the molecular etiology of diverse SGNs and aid in their classification and in approaching treatment. In future, these genetic variations may serve as critical tools for diagnosing salivary gland tumors and optimizing the management as well as prognosis of patients. This review presents the most recent advances in the molecular pathology of salivary gland cancers, with an emphasis on distinguishing molecular features that can be used for optimizing current patient management.

Abstract 1611: MYB and the MYB-NFIB fusion induce adenoid cystic carcinoma

Abstract Adenoid cystic carcinoma (ACC) is the second most common malignancy of the salivary glands, also found at lower frequency rates in other secretory glands such as the mammary and lacrimal glands. Salivary ACCs are slow-growing but aggressive neoplasms, with propensity to perineural invasion and high recurrence rates. MYB is the most frequently altered gene in ACCs, as approximately 40% of the ACCs contain MYB-NFIB gene fusions, and the full-length MYB is overexpressed in over 50% of the fusion-negative ACCs. However, it is still unclear whether MYB activities promote ACC development. To assess the oncogenic potential of MYB in vivo, we generated transgenic mice that overexpress MYB using the TetO system. In these mice, focal MYB overexpression was directed to salivary and mammary glands using the activator line MMTV-tTA. We found that 25% of these mice developed tumors with an average latency of 23 months. Remarkably, all the tumors that developed in these mice were ACCs, and the three main phenotypic variants of human ACCs (cribriform, tubular and solid) were represented in this cohort. Using the same transgenic system, we found that the MYB-NFIB fusion also induced ACCs, with similar incidence and lower latency. Immunohistochemical analysis of these mouse ACCs for several biomarkers of human ACC, including p63 and c-kit, provided additional evidence of the similarity of the mouse ACCs that developed in these models, and human ACCs. RNAseq global expression profiling uncovered the activation of several cancer-associated pathways in MYB-induced mouse ACCs. Most notably, activation NOTCH signaling was highly enriched in these tumors. Additional analysis for the presence of the NOTCH intracellular domain demonstrated that all of the MYB- and MYB-NFIB-induced ACCs contain NOTCH activation. These findings, and the recent identification of activating mutations in NOTCH1 and NOTCH2 in human ACCs, suggest that NOTCH activities may contribute to ACC development. To test this hypothesis, ongoing studies in the lab are directed to assess the role of NOTCH signaling in ACC development, by generating mouse models in which constitutive activation of NOTCH is induced in mice alone or in combination with MYB alterations. Overall, these studies demonstrate that MYB activities are the main oncogenic driver of ACC. Genomic rearrangements that result in MYB-NFIB fusions may enhance the oncogenic potential of MYB. Citation Format: Yuan Hu, Xiaoyuan Zhu, Han Luo, Adel El-Naggar, Carlos Caulin. MYB and the MYB-NFIB fusion induce adenoid cystic carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1611.

MYB-NFIB gene fusion in prostatic basal cell carcinoma: clinicopathologic correlates and comparison with basal cell adenoma and florid basal cell hyperplasia

Prostatic basal cell carcinoma is a malignant neoplasm composed of basaloid cells forming infiltrative nests and tubules, which may potentially be misdiagnosed as benign basal cell proliferations (i.e., florid basal cell hyperplasia or basal cell adenoma) and also closely resembles adenoid cystic carcinoma of the salivary gland. MYB-NFIB gene rearrangement occurs in 30–86% of salivary gland adenoid cystic carcinomas. We sought to further characterize MYB gene rearrangement in prostatic basal cell carcinoma and correlate MYB-NFIB fusion status with other clinicopathologic characteristics. To this end, FISH analysis for MYB-NFIB gene fusion using fusion probes was performed on formalin-fixed, paraffin-embedded tissue sections from prostatic basal cell carcinoma (n = 30), florid basal cell hyperplasia (n = 18), and basal cell adenoma (n = 4). Fourteen of 30 (47%) cases of basal cell carcinoma were positive for MYB-NFIB gene fusion FISH, and no cases of benign basal cell proliferations were positive (p < 0.05). FISH-positive patients (mean age = 63 years, range: 35–81) tended to be younger than FISH-negative patients (mean age = 70 years, range: 55–93). Most FISH-positive cases demonstrated adenoid cystic carcinoma-like morphology (57%), and most FISH-negative cases demonstrated nonadenoid cystic carcinoma-like morphology (93%); one case (FISH-positive) demonstrated areas with both adenoid cystic carcinoma-like and nonadenoid cystic carcinoma-like morphology. FISH-positive cases more frequently demonstrated perineural invasion (50% vs. 14%, p < 0.05) compared to FISH-negative cases. Conversely, tall basal cells (i.e., neoplastic cells at least two times taller than wide) were more frequent in FISH-negative cases than FISH-positive cases (93% vs. 36%, p < 0.05). Approximately, 50% of prostatic basal cell carcinoma harbor MYB-NFIB gene fusion. The majority of these cases were characterized by adenoid cystic carcinoma-like morphology, perineural invasion, and lack tall basal cells. Florid basal cell hyperplasia and basal cell adenoma are negative for MYB-NFIB gene fusion.

MYB-NFIB gene fusions identified in archival adenoid cystic carcinoma tissue employing NanoString analysis: an exploratory study

BackgroundAdenoid cystic carcinoma (ACC) is a slow growing salivary gland malignancy that is molecularly characterized by t(6:9)(q22–23;p23–24) translocations which predominantly result in MYB-NFIB gene fusions in nearly half of tumours. Detection of MYB-NFIB transcripts is typically performed with fresh ACC tissue using conventional RT-PCR fragment analysis or FISH techniques, which are prone to failure when only archival formalin fixed paraffin embedded (FFPE) tissue is available. The purpose of this pilot study was to evaluate the utility of NanoString probe technology for the detection of MYB-NFIB transcripts in archival ACC tissue.MethodsA NanoString probeset panel was designed targeting the junctions of three currently annotated MYB-NFIB fusion genes as well as 5′/3′ MYB probesets designed to detect MYB gene expression imbalance. RNA isolated from twenty-five archival ACC specimens was profiled and analyzed. RT-qPCR and sequencing were performed to confirm NanoString results. MYB protein expression was analyzed by immunohistochemistry.ResultsOf the 25 samples analyzed, 11/25 (44%) expressed a high degree of MYB 5′/3′ imbalance and five of these samples were positive for at least one specific MYB-NFIB variant in our panel. MYB-NFIB variant detection on NanoString analysis was confirmed by direct cDNA sequencing. No clinical correlations were found to be associated with MYB fusion status.ConclusionWe conclude that the application of NanoString digital probe counting technology is well suited for the detection and quantification of MYB-NFIB fusion transcripts in archival ACC specimens.

Genomic Alterations Landscape in Adenoid Cystic Carcinoma of Head and Neck

Introduction: Adenoid cystic carcinoma (ACC) accounts for 1% of all head and neck cancers and 10% to 22% of all malignant tumors of the salivary glands. ACC is associated with MYB- nuclear factor I/B-type (NFIB) gene fusion in about 50% of the cases. The genetic alteration (GA) landscape of ACC of head and neck region in this aspect has not been much studied, mainly due to small cohorts. We aim to describe the GA landscape of MYB-NFIB gene fusions, copy number alterations, and mutations of the related gene MYBL1 in human ACC by studying its association with regard to demographics and histopathological parameters. Materials and Methods: Using the GA data of ACC from a peer-reviewed web portal, we studied the presence of MYB-NFIB gene fusion with respect to age and gender distribution, site involved (major/minor salivary gland, lacrimal gland, cancer metastasis, others), histopathological diagnosis, perineural invasion (PNI), and the overall number of mutations. Descriptive statistics and Chi square test were performed. Data were entered and analyzed using SPSS version 23. The mutation counts between the type of gene fusions involved and the gender of patients was studied using analysis of variance tests. Results: Of the 214 reported ACC cases, 65 (30%) cases had MYB-NFIB fusion alone, 15 (7%) other had rare fusions, and 12 (6%) other had GAs. Perineural invasion was found to be associated with MYB gene fusion cases with or without copy number alterations (P = 0.022). The number of mutations were significantly associated with the gender of patient (P = 0.003) and NFIB gene fusions (P = 0.001). The other factors had no statistical significance. Conclusion: MYB-NFIB gene fusion and associated GA correlates with perineural involvement in ACC.

Abstract P2-05-03: Novel driver genetic alterations in MYB-NFIB-negative breast adenoid cystic carcinomas

Abstract Introduction: Breast adenoid cystic carcinoma (AdCC) is a rare type of triple-negative breast cancer associated with an indolent clinical behavior. AdCCs provide a clear example of genotypic-phenotypic correlation with the majority harboring the MYB-NFIB fusion gene. In this study, we sought to identify alternative driver genetic alterations in breast AdCCs lacking the MYB-NFIB fusion gene. Methods: Nucleic acids obtained from four breast AdCCs lacking the MYB-NFIB fusion gene as defined by reverse transcription (RT)-PCR and/or fluorescence in situ hybridization (FISH) were subjected to RNA-sequencing (n=3), whole-genome (n=2) and/or targeted (n=1) massively parallel sequencing. Sequencing data were analyzed using state-of-the-art bioinformatics algorithms, and potential alternative driver genetic alterations were validated using orthogonal sequencing and molecular pathology methods. Results: RNA-sequencing revealed the presence of MYBL1-ACTN1 or MYBL1-NFIB fusion genes in two breast AdCCs, which were validated by whole-genome sequencing and/or MYBL1 FISH analysis. Both MYBL1 fusion gene-positive cases were found to overexpress MYBL1 as defined by quantitative RT-PCR analysis. In the third MYB-NFIB-negative breast AdCC studied, a high-level MYB gene amplification coupled with overexpression of MYB at the mRNA and protein levels was identified. In the fourth breast AdCC, which expressed high levels of MYB, whole-genome and RNA-sequencing revealed no definite alternative driver alteration, however, a MYBL2 intronic mutation was found in this case, which was associated with high levels of MYBL2 mRNA expression. In this case, single sample gene set enrichment analysis revealed activation of pathways similar to those activated in AdCCs harboring the MYB-NFIB or MYBL1 fusions genes. Conclusion: We demonstrate that in breast AdCCs lacking the MYB-NFIB fusion gene MYBL1 rearrangements and MYB amplification are likely alternative driver genetic events. Given that activation of MYB/MYBL1 and their downstream targets can be driven by the MYB-NFIB fusion gene, MYBL1 rearrangements, MYB amplification or other yet to be validated mechanisms (e.g. MYBL2 non-coding mutations), our findings further suggest that breast AdCCs constitute a convergent phenotype. Citation Format: Kim J, Geyer FC, Martelotto LG, Ng CKY, Lim RS, Selenica P, Li A, Pareja F, Fusco N, Edelweiss M, Mariani O, Badve S, Vincent-Salomon A, Norton L, Reis-Filho JS, Weigelt B. Novel driver genetic alterations in MYB-NFIB-negative breast adenoid cystic carcinomas [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-03.

The Role of Molecular Testing in the Differential Diagnosis of Salivary Gland Carcinomas.

Salivary gland neoplasms are a morphologically heterogenous group of lesions that are often diagnostically challenging. In recent years, considerable progress in salivary gland taxonomy has been reached by the discovery of tumor type-specific fusion oncogenes generated by chromosome translocations. This review describes the clinicopathologic features of a selected group of salivary gland carcinomas with a focus on their distinctive genomic characteristics. Mammary analog secretory carcinoma is a recently described entity characterized by a t(12;15)(p13;q25) translocation resulting in an ETV6-NTRK3 fusion. Hyalinizing clear cell carcinoma is a low-grade tumor with infrequent nodal and distant metastasis, recently shown to harbor an EWSR1-ATF1 gene fusion. The CRTC1-MAML2 fusion gene resulting from a t(11;19)(q21;p13) translocation, is now known to be a feature of both low-grade and high-grade mucoepidermoid carcinomas associated with improved survival. A t(6;9)(q22-23;p23-34) translocation resulting in a MYB-NFIB gene fusion has been identified in the majority of adenoid cystic carcinomas. Polymorphous (low-grade) adenocarcinoma and cribriform adenocarcinoma of (minor) salivary gland origin are related entities with partly differing clinicopathologic and genomic profiles; they are the subject of an ongoing taxonomic debate. Polymorphous (low-grade) adenocarcinomas are characterized by hot spot point E710D mutations in the PRKD1 gene, whereas cribriform adenocarcinoma of (minor) salivary glands origin are characterized by translocations involving the PRKD1-3 genes. Salivary duct carcinoma (SDC) is a high-grade adenocarcinoma with morphologic and molecular features akin to invasive ductal carcinoma of the breast, including HER2 gene amplification, mutations of TP53, PIK3CA, and HRAS and loss or mutation of PTEN. Notably, a recurrent NCOA4-RET fusion has also been found in SDC. A subset of SDC with apocrine morphology is associated with overexpression of androgen receptors. As these genetic aberrations are recurrent they serve as powerful diagnostic tools in salivary gland tumor diagnosis, and therefore also in refinement of salivary gland cancer classification. Moreover, they are promising as prognostic biomarkers and targets of therapy.

MYBL1 rearrangements and MYB amplification in breast adenoid cystic carcinomas lacking the MYB-NFIB fusion gene.

Breast adenoid cystic carcinoma (AdCC), a rare type of triple-negative breast cancer, has been shown to be driven by MYB pathway activation, most often underpinned by the MYB-NFIB fusion gene. Alternative genetic mechanisms, such as MYBL1 rearrangements, have been reported in MYB-NFIB-negative salivary gland AdCCs. Here we report on the molecular characterization by massively parallel sequencing of four breast AdCCs lacking the MYB-NFIB fusion gene. In two cases, we identified MYBL1 rearrangements (MYBL1-ACTN1 and MYBL1-NFIB), which were associated with MYBL1 overexpression. A third AdCC harboured a high-level MYB amplification, which resulted in MYB overexpression at the mRNA and protein levels. RNA-sequencing and whole-genome sequencing revealed no definite alternative driver in the fourth AdCC studied, despite high levels of MYB expression and the activation of pathways similar to those activated in MYB-NFIB-positive AdCCs. In this case, a deletion encompassing the last intron and part of exon 15 of MYB, including the binding site of ERG-1, a transcription factor that may downregulate MYB, and the exon 15 splice site, was detected. In conclusion, we demonstrate that MYBL1 rearrangements and MYB amplification probably constitute alternative genetic drivers of breast AdCCs, functioning through MYBL1 or MYB overexpression. These observations emphasize that breast AdCCs probably constitute a convergent phenotype, whereby activation of MYB and MYBL1 and their downstream targets can be driven by the MYB-NFIB fusion gene, MYBL1 rearrangements, MYB amplification, or other yet to be identified mechanisms. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genomic imbalances and MYB fusion in synchronous bilateral adenoid cystic carcinoma and invasive lobular carcinoma of the breast.

The incidence of synchronous bilateral breast carcinomas (BBCs) has increased with a more frequent use of magnetic resonance imaging screening of the contralateral breast in women with newly diagnosed breast cancer. A total of 30% of all BBCs occur synchronously. In the present study, we describe a unique case of synchronous BBC in a 59-year-old previously healthy woman with no known family history of breast or ovarian cancer. At the time of diagnosis the patient had an invasive lobular carcinoma (ILC) in the right breast and an adenoid cystic carcinoma (ACC) in the left breast. To the best of our knowledge, this is the first published case of bilateral, simultaneously occurring ACC and ILC of the breast. Genome-wide genomic profiling of the tumors revealed that they had distinctly different genomic imbalances. The ACC had a 5.7 Mb interstitial 6q deletion with a breakpoint located in the 3′-part of MYB, resulting in loss of the last coding exon of MYB and its 3′-UTR. RT-PCR analysis confirmed that the tumor expressed an ACC-specific MYB-NFIB fusion transcript. In contrast, the ILC had no rearrangements of 6q or MYB-NFIB gene fusion but showed instead gain of 1q21.1-qter, loss of 16q11.2-qter, and 22q12.2-q12.3 as the sole genomic imbalances. Notably, concurrent gains of 1q and losses of 16q are characteristic features of ILC. Collectively, our findings indicate that the ACC and ILC had originated independently of each other and that the MYB-NFIB fusion is a specific biomarker for breast ACC.

Targeting the Oncogenic Transcriptional Regulator MYB in Adenoid Cystic Carcinoma by Inhibition of IGF1R/AKT Signaling.

Adenoid cystic carcinoma (ACC) is an aggressive cancer with no curative treatment for patients with recurrent/metastatic disease. The MYB-NFIB gene fusion is the main genomic hallmark and a potential therapeutic target. Oncogenic signaling pathways were studied in cultured cells and/or tumors from 15 ACC patients. Phospho-receptor tyrosine kinase (RTK) arrays were used to study the activity of RTKs. Effects of RTK inhibition on cell proliferation were analyzed with AlamarBlue, sphere assays, and two ACC xenograft models (n = 4-9 mice per group). The molecular effects of MYB-NFIB knockdown and IGF1R inhibition were studied with quantitative polymerase chain reaction, immunoblot, and gene expression microarrays. All statistical tests were two-sided. The MYB-NFIB fusion drives proliferation of ACC cells and is crucial for spherogenesis. Intriguingly, the fusion is regulated through AKT-dependent signaling induced by IGF1R overexpression and is downregulated upon IGF1R-inhibition (% expression of control ± SD = 27.2 ± 1.3, P < .001). MYB-NFIB regulates genes involved in cell cycle control, DNA replication/repair, and RNA processing. The transcriptional program induced by MYB-NFIB affects critical oncogenic mediators normally controlled by MYC and is reversed by pharmacological inhibition of IGF1R. Co-activation of epidermal growth factor receptor (EGFR) and MET promoted proliferation of ACC cells, and combined targeting of IGFR1/EGFR/MET induced differentiation and synergistically inhibited the growth of patient-derived xenografted ACCs (ACCX5M1, % growth of control ± SD = 34.9 ± 20.3, P = .006; ACCX6, % growth of control ± SD = 24.1 ± 17.5, P = .04). MYB-NFIB is an oncogenic driver and a key therapeutic target in ACC that is regulated by AKT-dependent IGF1R signaling. Our studies uncover a new strategy to target an oncogenic transcriptional master regulator and provide new important insights into the biology and treatment of ACC.

Genetic Characterization of Adenoid Cystic Carcinoma of the Minor Salivary Glands: A Potential Familial Occurrence in First-Degree Relatives

Adenoid cystic carcinoma (AdCC) is a malignant salivary gland tumor. To date, no cases of AdCC in first-degree relatives have been reported in the literature. We present a 50-year-old female (Case 1) and this patients' father (Case 2), both of whom were diagnosed with AdCC of the minor salivary glands. Histology of Case 1 demonstrated a tubulocribriform AdCC whereas Case 2 primarily was an AdCC of solid type. Both cases harbored the MYB-NFIB gene fusion as demonstrated by FISH and RNA-sequencing. After filtering and selection of putative deleterious variants, whole exome sequencing identified 18 germline variants in common between Case 1 and Case 2. However, none of the variants were associated with AdCC or other head and neck cancers. To our knowledge, we present the first potential case of familial AdCC. The presented genetic data may contribute to further investigations of the underlying genetic mechanisms for AdCC susceptibility.

Genetic events in the progression of adenoid cystic carcinoma of the breast to high-grade triple-negative breast cancer

AbstractAdenoid cystic carcinoma of the breast is a rare histological type of triple-negative breast cancer with an indolent clinical behavior, often driven by the MYB-NFIB fusion gene. Here we sought to define the repertoire of somatic genetic alterations in two adenoid cystic carcinomas associated with high-grade triple-negative breast cancer. The different components of each case were subjected to copy number profiling and massively parallel sequencing targeting all exons and selected regulatory and intronic regions of 488 genes. Reverse transcription PCR and fluorescence in situ hybridization were employed to investigate the presence of the MYB-NFIB translocation. The MYB-NFIB fusion gene was detected in both adenoid cystic carcinomas and their associated high-grade triple-negative breast cancer components. Although the distinct components of both cases displayed similar patterns of gene copy number alterations, massively parallel sequencing analysis revealed intratumor genetic heterogeneity. In case 1, progression from the trabecular adenoid cystic carcinoma to the high-grade triple-negative breast cancer was found to involve clonal shifts with enrichment of mutations affecting EP300, NOTCH1, ERBB2 and FGFR1 in the high-grade triple-negative breast cancer. In case 2, a clonal KMT2C mutation was present in the cribriform adenoid cystic carcinoma, solid adenoid cystic carcinoma and high-grade triple-negative breast cancer components, whereas a mutation affecting MYB was present only in the solid and high-grade triple-negative breast cancer areas and additional three mutations targeting STAG2, KDM6A and CDK12 were restricted to the high-grade triple-negative breast cancer. In conclusion, adenoid cystic carcinomas of the breast with high-grade transformation are underpinned by the MYB-NFIB fusion gene and, akin to other forms of cancer, may be constituted by a mosaic of cancer cell clones at diagnosis. The progression from adenoid cystic carcinoma to high-grade triple-negative breast cancer of no special type may involve the selection of neoplastic clones and/or the acquisition of additional genetic alterations.

Abstract 3983: Highly sensitive detection of MYB-NFIB fusion transcripts in adenoid cystic carcinoma

Abstract In recent years, the understanding of genomic events underlying head and neck cancer has significantly advanced. Several of the newly discovered genomic aberrations observed in salivary gland neoplasms were found to be recurrent and uniquely associated with specific disease entities. Adenoid cystic carcinoma (ACC), one of the most common salivary gland malignancies, demonstrates a particular challenge in head and neck oncology due its relentless growth, high local recurrence rate and poor long-term prognosis. Recent identification of the MYB-NFIB gene fusion as a recurrent and pathognomonic feature of ACC created new opportunities for the molecular diagnostics of this malignancy. Liquid biopsy techniques coupled with droplet digital PCR (ddPCR) platform may be feasible aiming for non-invasive detection of disease-specific genetic markers such as gene fusions. Given the prevalence of MYB-NFIB transcript in ACC, detection of the fusion event in patients’ saliva may present a unique opportunity for an accurate diagnostics as well as for long-term patient monitoring. Our goal is to develop a highly-sensitive ddPCR-based assay that would precisely detect and identify MYB-NFIB transcripts in tumor tissue and patients’ bodily fluids. RNA sequencing (RNAseq) performed in fresh frozen ACC samples identified MYB-NFIB fusion in 56% (10/18) of the cases and revealed large variability in MYB gene break sites and alternative splicing of NFIB component. 84% of ACC cases demonstrated high expression of 5’ MYB fragment by ddPCR based RT-PCR, 62.5% of which were MYB-NFIB fusion positive in RNAseq. We developed a panel of RT-PCR assays to detect a range of different MYB-NFIB transcripts observed by our RNAseq data and/or previously reported. We identified all ACC cases harboring the most commonly reported MYB-NFIB fusions (9/9) and validated the fusion products with conventional Sanger sequencing. Additionally, our preliminary results indicate that the assays are feasible for PCR multiplexing retaining the high sensitivity and specificity observed in singleplex experiments. Evaluation of the assay performance in ddPCR and assessing the detection limit is currently in progress. Once the methodology is validated, we will be able to evaluate a cohort of salivary gland neoplasms and matched RNA samples isolated from saliva. The detection of MYB-NFIB fusion in bodily fluids using a highly sensitive ddPCR platform will open a new avenue for the diagnosis and the clinical management of patients with ACC. Furthermore, our work creates the possibility of a liquid biopsy-based detection of other disease-specific gene fusions increasingly identified in different solid tumors. Citation Format: Piotr T. Wysocki, Shizhang Ling, Chunbo Shao, Marietta Tan, David Sidransky, Patrick Ha, Mariana Brait. Highly sensitive detection of MYB-NFIB fusion transcripts in adenoid cystic carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3983.

Novel MYBL1 Gene Rearrangements with Recurrent MYBL1-NFIB Fusions in Salivary Adenoid Cystic Carcinomas Lacking t(6;9) Translocations.

Adenoid cystic carcinoma (ACC) is an indolent salivary gland malignancy, characterized by t(6;9) translocations and MYB-NFIB gene fusions in approximately 50% of the tumors. The genetic alterations underlying t(6;9)-negative and t(6;9)-positive/MYB-NFIB fusion-negative ACC remain unknown. To uncover the genetic alterations in ACC lacking the canonical translocation and fusion transcript and identify new abnormalities in translocation positive tumors. We performed whole-genome sequencing in 21 salivary ACCs and conducted targeted molecular analyses in a validation set (81 patients). Microarray gene-expression data were also analyzed to explore the biologic differences between fusion positive and negative tumors. We identified a novel MYBL1-NFIB gene fusion as a result of t(8;9) translocation and multiple rearrangements in the MYBL1 gene in 35% of the t(6;9)-negative ACCs. All MYBL1 alterations involved deletion of the C-terminal negative regulatory domain and were associated with high MYBL1 expression. Reciprocal MYB and MYBL1 expression was consistently found in ACCs. In addition, 5'-NFIB fusions that did not involve MYB/MYBL1 genes were identified in a subset of t(6;9)-positive/fusion-negative tumors. We also delineated distinct gene-expression profiles in ACCs associated with the length of the MYB or MYBL1 fusions, suggesting a biologic importance of the C-terminal part of these fusions. Our study defines new molecular subclasses of ACC characterized by MYBL1 rearrangements and 5'-NFIB gene fusions.

Adenoid cystic carcinoma: current therapy and potential therapeutic advances based on genomic profiling.

Adenoid cystic carcinoma (ACC) is a rare cancer with high potential for recurrence and metastasis. Efficacy of current treatment options, particularly for advanced disease, is very limited. Recent whole genome and exome sequencing has dramatically improved our understanding of ACC pathogenesis. A balanced translocation resulting in the MYB-NFIB fusion gene appears to be a fundamental signature of ACC. In addition, sequencing has identified a number of other driver genes mutated in downstream pathways common to other well-studied cancers. Overexpression of oncogenic proteins involved in cell growth, adhesion, cell cycle regulation, and angiogenesis are also present in ACC. Collectively, studies have identified genes and proteins for targeted, mechanism-based, therapies based on tumor phenotypes, as opposed to nonspecific cytotoxic agents. In addition, although few studies in ACC currently exist, immunotherapy may also hold promise. Better genetic understanding will enable treatment with novel targeted agents and initial exploration of immune-based therapies with the goal of improving outcomes for patients with ACC.

Genomic landscape of adenoid cystic carcinoma of the breast.

Adenoid cystic carcinoma (AdCC) is a rare type of triple-negative breast cancer (TNBC) characterized by the presence of the MYB-NFIB fusion gene. The molecular underpinning of breast AdCCs other than the MYB-NFIB fusion gene remains largely unexplored. Here we sought to define the repertoire of somatic genetic alterations of breast AdCCs. We performed whole-exome sequencing, followed by orthogonal validation, of 12 breast AdCCs to determine the landscape of somatic mutations and gene copy number alterations. Fluorescence in situ hybridization and reverse-transcription PCR were used to define the presence of MYB gene rearrangements and MYB-NFIB chimeric transcripts. Unlike common forms of TNBC, we found that AdCCs have a low mutation rate (0.27 non-silent mutations/Mb), lack mutations in TP53 and PIK3CA and display a heterogeneous constellation of known cancer genes affected by somatic mutations, including MYB, BRAF, FBXW7, SMARCA5, SF3B1 and FGFR2. MYB and TLN2 were affected by somatic mutations in two cases each. Akin to salivary gland AdCCs, breast AdCCs were found to harbour mutations targeting chromatin remodelling, cell adhesion, RNA biology, ubiquitination and canonical signalling pathway genes. We observed that, although breast AdCCs had rather simple genomes, they likely display intra-tumour genetic heterogeneity at diagnosis. Taken together, these findings demonstrate that the mutational burden and mutational repertoire of breast AdCCs are more similar to those of salivary gland AdCCs than to those of other types of TNBCs, emphasizing the importance of histological subtyping of TNBCs. Furthermore, our data provide direct evidence that AdCCs harbour a distinctive mutational landscape and genomic structure, irrespective of the disease site of origin.

MYB rearrangement and clinicopathologic characteristics in head and neck adenoid cystic carcinoma.

Salivary gland adenoid cystic carcinoma (ACC) is rare, aggressive, and challenging to treat. Many ACCs have a t(6;9) chromosomal translocation resulting in a MYB-NFIB fusion gene, but the clinical significance is unclear. The purposes of this study were to describe the clinicopathologic factors impacting survival and to determine the prevalence and clinical significance of MYB-NFIB fusion. Case series. Medical records of patients treated for ACC of the head and neck from 1974 to 2011 were reviewed and clinicopathologic data recorded. Fluorescence in situ hybridization (FISH) was used to detect MYB rearrangement in archival tumor tissue as a marker of MYB-NFIB fusion. One hundred fifty-eight patients were included, with median follow-up 75.1 months. Median overall survival was 171.5 months (95% confidence interval [CI] = 131.9-191.6), and median disease-free survival was 112.0 months (95% CI = 88.7-180.4). Advanced stage was associated with decreased overall survival (adjusted ptrend < 0.001), and positive margins were associated with decreased disease-free survival (adjusted hazard ratio [aHR] = 8.80, 95% CI = 1.25-62.12, P = 0.029). Ninety-one tumors were evaluable using FISH, and 59 (65%) had evidence of a MYB-NFIB fusion. MYB-NFIB positive tumors were more likely than MYB-NFIB negative tumors to originate in minor salivary glands (adjusted prevalence ratios = 1.51, 95% CI = 1.07-2.12, P = 0.019). MYB-NFIB tumor status was not significantly associated with disease-free or overall survival (hazard ratio [HR] = 1.53, 95% CI = 0.77-3.02, P = 0.22 and HR = 0.91, 95% CI = 0.46-1.83, P = 0.80, respectively, for MYB-NFIB positive compared with MYB-NFIB negative tumors). Stage and margin status were important prognostic factors for ACC. Tumors with evidence of MYB-NFIB fusion were more likely to originate in minor salivary glands, but MYB-NFIB tumor status was not significantly associated with prognosis. 4.

Pathogenic Biomarkers for Perineural Invasion in Adenoid Cystic Carcinoma

Objectives: Perineural invasion (PNI) has emerged as an adverse pathological feature of adenoid cystic carcinoma (ACC). Despite their importance, the local control factors involved in PNI remain unknown. Recent research has suggested that nerve growth factor (NGF) may be one of the important factors for PNI in other malignancies. Furthermore, in ACC, Myb overexpression related to the MYB–NFIB fusion gene has been correlated with PNI; however, this concept has been highly controversial. In this study, we examined the overexpression of NGF, its receptors (TrkA and p75NRT), and Myb in ACC cancer cells and investigated the relationship with PNI and local control. Methods: We retrospectively analyzed 47 patients with ACC who were surgically treated from 1991 to 2011. We reviewed NGF, TrkA, p75NRT, and Myb overexpression in the surgical specimens and examined the correlation with PNI, local control, and disease-specific survival. Results: The overexpression rates of NGF, TrkA, p75NRT, and Myb were 66%, 66%, 30%, and 57%, respectively. NGF and TrkA were significantly overexpressed in the PNI-positive group (NGF overexpression rate: PNI-positive vs PNI-negative, 88% vs 20%, P &lt; .001; TrkA overexpression rate: PNI-positive vs PNI-negative, 81% vs 33%, P &lt; .01). Moreover, patients with NGF overexpression had a significantly poorer 10-year local control rate (75% vs 13%, P &lt; .05); however, p75NRT and Myb overexpression were not related to PNI. Conclusions: NGF and TrkA overexpression may contribute to PNI and cause local recurrence in ACC. However, Myb overexpression was not related to PNI in our analysis.