A phase 2 study of MK-2206 in patients with incurable adenoid cystic carcinoma (Alliance A091104).

BACKGROUND: Recurrent/metastatic adenoid cystic carcinoma (ACC) is a rare, incurable disease. MYB is a putative oncogenic driver in ACC that is often overexpressed through an MYB-NFIB rearrangement. The authors hypothesized that AKT inhibition with the allosteric inhibitor MK-2206 could decrease MYB expression and induce tumor regression in patients with incurable ACC (ClinicalTrials.gov identifier NCT01604772). METHODS: Patients with progressive, incurable ACC were enrolled and received MK-2206 150 mg weekly; escalation to 200 mg was allowed. The primary end point was confirmed response. Secondary end points were progression-free survival, overall survival, and safety. An exploratory analysis evaluating the effect of MK-2206 on MYB expression was conducted in a subset of patients. RESULTS: Sixteen patients were enrolled, and 14 were evaluable for efficacy. No confirmed responses were observed. Thirteen patients had stable disease, and one had disease progression as their best response. The median progression-free survival was 9.7 months (95% CI, 3.8-11.8 months), and the median overall survival was 18.0 months (95% CI, 11.8-29.9 months). Nine of 16 patients (56%) had at least one grade 3 treatment-related adverse event, and the most common were rash (38%), fatigue (19%), decreased lymphocyte count (13%), and hyperglycemia (13%). Twelve of 14 tumors (86%) had detectable MYB expression by immunohistochemistry, and seven of 14 tumors (50%) had an MYB-NFIB gene rearrangement. Serial biopsies revealed decreased MYB levels with MK-2206 in four of five patients. CONCLUSIONS: MK-2206 failed to induce clinical responses in patients with incurable ACC. AKT inhibition may diminish MYB protein levels, although the effect was highly variable among patients. Novel approaches to target MYB in ACC are needed.

Adenoid cystic carcinoma of the Bartholin's gland is underpinned by MYB- and MYBL1- rearrangements.

OBJECTIVE: Adenoid cystic carcinoma (AdCC) of the Bartholin's gland (AdCC-BG) is a very rare gynecologic vulvar malignancy. AdCC-BGs are slow-growing but locally aggressive and are associated with high recurrence rates. Here we sought to characterize the molecular underpinning of AdCC-BGs. METHODS: AdCC-BGs (n = 6) were subjected to a combination of RNA-sequencing, targeted DNA-sequencing, reverse-transcription PCR, fluorescence in situ hybridization (FISH) and MYB immunohistochemistry (IHC). Clinicopathologic variables, somatic mutations, copy number alterations and chimeric transcripts were assessed. RESULTS: All six AdCC-BGs were biphasic, composed of ductal and myoepithelial cells. Akin to salivary gland and breast AdCCs, three AdCC-BGs had the MYB::NFIB fusion gene with varying breakpoints, all of which were associated with MYB overexpression by IHC. Two AdCC-BGs were underpinned by MYBL1 fusion genes with different gene partners, including MYBL1::RAD51B and MYBL1::EWSR1 gene fusions, and showed MYB protein expression. Although the final AdCC-BG studied had MYB protein overexpression, no gene fusion was identified. AdCC-BGs harbored few additional somatic genetic alterations, and only few mutations in cancer-related genes were identified, including GNAQ, GNAS, KDM6A, AKT1 and BCL2, none of which were recurrent. Two AdCC-BGs, both with a MYB::NFIB fusion gene, developed metastatic disease. CONCLUSIONS: AdCC-BGs constitute a convergent phenotype, whereby activation of MYB or MYBL1 can be driven by the MYB::NFIB fusion gene or MYBL1 rearrangements. Our observations further support the notion that AdCCs, irrespective of organ site, constitute a genotypic-phenotypic correlation. Assessment of MYB or MYBL1 rearrangements may be used as an ancillary marker for the diagnosis of AdCC-BGs.

The Landscape of MYB/MYBL1- and Peri-MYB/MYBL1-Associated Rearrangements in Adenoid Cystic Carcinoma.

Approximately 60% of adenoid cystic carcinoma (AdCC) cases are positive for MYB::NFIB or MYBL1::NFIB, whereas MYB/MYBL1 oncoprotein, a key driver of AdCC, is overexpressed in most cases. Juxtaposition of superenhancer regions in NFIB and other genes into the MYB/MYBL1 locus is an attractive oncogenic hypothesis for AdCC cases, either negative or positive for MYB/MYBL1::NFIB. However, evidence supporting this hypothesis is insufficient. We examined 160 salivary AdCC cases for rearrangements in MYB/MYBL1 loci and peri-MYB/MYBL1 areas (centromeric and telomeric areas of 10 Mb each) using formalin-fixed, paraffin-embedded tumor sections. For the detection of the rearrangements, we employed conventional fluorescence in situ hybridization split and fusion assays and a 5 Mb fluorescence in situ hybridization split assay. The latter is a novel assay that enabled us to detect any possible splits within a 5 Mb distance of a chromosome. We found MYB/MYBL1- and peri-MYB/MYBL1-associated rearrangements in 149/160 patients (93%). AdCC cases positive for rearrangements in MYB, MYBL1, the peri-MYB area, and the peri-MYBL1 area numbered 105 (66%), 20 (13%), 19 (12%), and 5 (3%), respectively. In 24 peri-MYB/MYBL1 rearrangement-positive cases, 14 (58%) were found to have a juxtaposition of the NFIB or RAD51B locus into the MYB/MYBL1 loci. On comparing with a tumor group positive for MYB::NFIB, a hallmark of AdCC, other genetically classified tumor groups had similar features of overexpression of the MYB transcript and MYB oncoprotein as detected by semiquantitative RT-qPCR and immunohistochemistry, respectively. In addition, clinicopathological and prognostic features were similar among these groups. Our study suggests that peri-MYB/MYBL1 rearrangements may be a frequent event in AdCC and may result in biological and clinicopathological consequences comparable to MYB/MYBL1 rearrangements. The landscape of MYB/MYBL1 and peri-MYB/MYBL1 rearrangements shown here strongly suggests that juxtaposition of superenhancers into MYB/MYBL1 or peri-MYB/MYBL1 loci is an alteration that acts as a key driver for AdCC oncogenesis and may unify MYB/MYBL1 rearrangement-positive and negative cases.

A Review of the Molecular Landscape of Adenoid Cystic Carcinoma of the Lacrimal Gland.

Adenoid cystic carcinoma (ACC) has a worldwide incidence of three to four cases per million population. Although more cases occur in the minor and major salivary glands, it is the most common lacrimal gland malignancy. ACC has a low-grade, indolent histological appearance, but is relentlessly progressive over time and has a strong proclivity to recur and/or metastasise. Current treatment options are limited to complete surgical excision and adjuvant radiotherapy. Intra-arterial systemic therapy is a recent innovation. Recurrent/metastatic disease is common due to perineural invasion, and it is largely untreatable as it is refractory to conventional chemotherapeutic agents. Given the rarity of this tumour, the molecular mechanisms that govern disease pathogenesis are poorly understood. There is an unmet, critical need to develop effective, personalised targeted therapies for the treatment of ACC in order to reduce morbidity and mortality associated with the disease. This review details the evidence relating to the molecular underpinnings of ACC of the lacrimal gland, including the MYB-NFIB chromosomal translocations, Notch-signalling pathway aberrations, DNA damage repair gene mutations and epigenetic modifications.

A clinicopathological and molecular analysis of cervical carcinomas with basaloid features.

AIMS: To investigate the relationship between adenoid basal carcinoma (ABC), adenoid cystic carcinoma (ACC) and squamous cell carcinoma (SCC) in the uterine cervix. METHODS AND RESULTS: We analysed the clinicopathological and molecular features in two pure ABCs, 15 SCCs with ABC-/ACC-like features and seven basaloid SCCs (BSCCs) by chart review, immunohistochemistry, human papillomavirus (HPV) RNA in-situ hybridisation and fluorescence in-situ hybridisation. All patients were alive with no evidence of disease, except for one patient with ACC-like features who died of disease at 18 months post diagnosis. The mixed carcinomas comprised variable SCCs and ABC-/ACC-like components displaying vague transitional zones. All components consistently showed diffuse p16, p63 and SOX2, variable cytokeratin (CK)7 and CK17 and rare Ber-EP4 and MYB expression; there was a substantially lower Ki67 index in pure ABCs and the ABC-like components. The ACC-like components showed no myoepithelial differentiation (SMA, calponin and S100) and MYB gene fusions. CK7, CK17 and Ber-EP4 were characteristically stronger in BSCCs than in the mixed carcinomas (P < 0.01). High-risk HPV (HR-HPV) E6/E7 mRNA was detected in 12 mixed carcinomas and seven BSCCs, but not in pure ABCs. The HR-HPV mRNA expression was higher in the SCC components and BSCCs than in the ABC-like components of mixed carcinomas (P < 0.05). CONCLUSIONS: The ACC-like components in mixed carcinomas probably represent the morphological mimics of salivary ACCs. ABC-like components may be the potential precursor of the ACC-like and SCC components. HR-HPV oncogenes may play a role in the pathogenesis of SCCs with ABC-/ACC-like features.

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.

Study of MYB-NFIB chimeric gene expression, tumor angiogenesis, and proliferation in adenoid cystic carcinoma of salivary gland.

Adenoid cystic carcinoma (ACC) is one of the common malignant tumors in salivary glands, and the clinical prognosis is poor with frequent distant metastasis which may lead to death. Expression of the MYB-NFIB chimeric gene in ACC has been reported recently. MYB is an oncogene with transcription regulating functions, and NFIB encodes nuclear transcription factor although detailed functions are unknown. This study investigated whether MYB-NFIB chimeric gene expression affects tumor angiogenesis and proliferation in salivary gland ACC. In 26 salivary gland ACC cases, MYB-NFIB chimeric gene expression was analyzed by RT-PCR and direct sequencing. Immunohistochemical studies for CD31, vascular endothelial growth factor (VEGF) and Ki-67 were performed. Tumor angiogenesis was evaluated by blood vessel (CD31-positive) density and tumor proliferation by Ki-67 labeling index, and the relationship with MYB-NFIB chimeric gene expression was analyzed. MYB-NFIB chimeric gene expression was detected in nine of 26 ACC cases. Blood vessel density was significantly higher in chimeric gene-expressing cases compared to non-expressing cases. VEGF score tended to be higher in chimeric gene-expressing cases than in non-expressing cases, while Ki-67 labeling index was not significantly different. The number of chimeric gene-expressing cases increased with age, peaking in the sixties age group and declining thereafter, while the number of non-expressing cases increased with age continuously. In ACC, blood vessel density was significantly higher in MYB-NFIB chimeric gene-expressing cases compared to non-expressing cases, which may be due to higher VEGF production capability. MYB-NFIB chimeric gene expression may also be related to the onset age of ACC.

Overexpression of MYB drives proliferation of CYLD-defective cylindroma cells.

Cutaneous cylindroma is an adnexal tumour with apocrine differentiation. A predisposition to multiple cylindromas is seen in patients with Brooke-Spiegler syndrome, who carry germline mutations in the tumour suppressor gene CYLD. Previous studies of inherited cylindromas have highlighted the frequent presence of bi-allelic truncating CYLD mutations as a recurrent driver mutation. We have previously shown that sporadic cylindromas express either MYB-NFIB fusion transcripts or show evidence of MYB activation in the absence of such fusions. Here, we investigated inherited cylindromas from several families with germline CYLD mutations for the presence of MYB activation. Strikingly, none of the inherited CYLD-defective (n = 23) tumours expressed MYB-NFIB fusion transcripts. However, MYB expression was increased in the majority of tumours (69%) and global gene expression analysis revealed that well-established MYB target genes were up-regulated in CYLD-defective tumours. Moreover, knock-down of MYB expression caused a significant reduction in cylindroma cell proliferation, suggesting that MYB is also a key player and oncogenic driver in inherited cylindromas. Taken together, our findings suggest molecular heterogeneity in the pathogenesis of sporadic and inherited cutaneous cylindromas, with convergence on MYB activation. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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.

Molecular signature of salivary gland tumors: potential use as diagnostic and prognostic marker.

Salivary gland tumors are a highly heterogeneous group of lesions with diverse microscopic appearances and variable clinical behavior. The use of clinical and histological parameters to predict patient prognosis and survival rates has been of limited utility, and the search for new biomarkers that could not only aid in a better understanding of their pathogenesis but also be reliable auxiliaries for prognostic determination and useful diagnostic tools has been performed in the last decades with very exciting results. Hence, gene rearrangements such as CRTC1-MAML2 in mucoepidermoid carcinomas have shown excellent specificity, and more than that, it has been strongly correlated with low-grade tumors and consequently with an increased survival rate and better prognosis of patients affected by neoplasms carrying this translocation. Moreover, MYB-NFIB and EWSR1-ATF1 gene fusions were shown to be specifically found in cases of adenoid cystic carcinomas and hyalinizing clear cell carcinomas, respectively, in the context of salivary gland tumors, becoming reliable diagnostic tools for these entities and potential therapeutic targets for future therapeutic protocols. Finally, the identification of ETV6-NTRK3 in cases previously diagnosed as uncommon acinic cell carcinomas, cystadenocarcinomas, and adenocarcinomas not otherwise specified led to the characterization of a completely new and now widely accepted entity, including, therefore, mammary analogue secretory carcinoma in the list of well-recognized salivary gland carcinomas. Thus, further molecular investigations of salivary gland tumors are warranted, and the recognition of other genetic abnormalities can lead to the acknowledgment of new entities and the acquirement of reliable biomarkers.

[New developments in molecular diagnostics of carcinomas of the salivary glands: "translocation carcinomas"]. / Novinky v molekulární diagnostice karcinomu slinných zláz: "translokacní karcinomy".

In recent years the discovery of translocations and the fusion oncogenes that they result in has changed the way diagnoses are made in salivary gland pathology. These genetic aberrations are recurrent; and at the very least serve as powerful diagnostic tools in salivary gland tumors diagnosis and classification. They also show promise as prognostic markers and hopefully as targets of therapy. In this review the 4 carcinomas currently known to harbor translocations will be discussed, namely mucoepidermoid carcinoma, adenoid cystic carcinoma, mammary analogue secretory carcinoma, and hyalinizing clear cell carcinoma. The discovery and implications of each fusion will be highlighted and how they have helped to reshape the current classification of salivary gland tumors.

Development and Characterization of MYB-NFIB Fusion Expression in Adenoid Cystic Carcinoma.

Adenoid cystic carcinoma (ACC) is the second most common cancer type arising from the salivary gland. The frequent occurrence of chromosome t(6;9) translocation leading to the fusion of MYB and NFIB transcription factor genes is considered a genetic hallmark of ACC. This inter-chromosomal rearrangement may encode multiple variants of functional MYB-NFIB fusion in ACC. However, the lack of an ACC model that harbors the t(6;9) translocation has limited studies on defining the potential function and implication of chimeric MYB-NFIB protein in ACC. This report aims to establish a MYB-NFIB fusion protein expressing system in ACC cells for in vitro and in vivo studies. RNA-seq data from MYB-NFIB translocation positive ACC patients' tumors and MYB-NFIB fusion transcript in ACC patient-derived xenografts (ACCX) was analyzed to identify MYB breakpoints and their frequency of occurrence. Based on the MYB breakpoint identified, variants of MYB-NFIB fusion expression system were developed in a MYB-NFIB deficient ACC cell lines. Analysis confirmed MYB-NFIB fusion protein expression in ACC cells and ACCXs. Furthermore, recombinant MYB-NFIB fusion displayed sustained protein stability and impacted transcriptional activities of interferon-associated genes set as compared to a wild type MYB. In vivo tumor formation analysis indicated the capacity of MYB-NFIB fusion cells to grow as implanted tumors, although there were no fusion-mediated growth advantages. This expression system may be useful not only in studies to determine the functional aspects of MYB-NFIB fusion but also in evaluating effective drug response in vitro and in vivo settings.

MYB-NFIB Translocation by FISH in Adenoid Cystic Carcinoma of the Head and Neck in Nigerian Patients: A Preliminary Report.

Adenoid cystic carcinoma (AdCC) is a relatively rare malignancy of head and neck sites such as the salivary glands, lacrimal gland, sinonasal region, and pharynx and may arise in other exocrine glands. The oncologic event in AdCC is the translocation between MYB proto-oncogene transcription factor (MYB) and nuclear factor I/B (NFIB) resulting in t(6;9)(q22-23;p23-24). We carried out a preliminary evaluation of MYB-NFIB translocation by fluorescence in-situ hybridization on seven archived formalin-fixed paraffin-embedded tissues of AdCC of Nigerian patients and its clinicopathologic features. Only 3 of the 7 cases were successfully hybridized, all featuring MYB-NFIB translocations with a range of 14.7-83.3% of translocated cells in 60 cells examined. The 3 translocation positive cases were located in the maxillary sinus, buccal mucosa and parotid. Their morphologic appearances were cribriform-solid (1) & cribriform (2) and classified as grades III (1) & I (2), respectively. These patients may potentially benefit from MYB-targeted anti-neoplastic therapy.

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

BACKGROUND: Adenoid 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. METHODS: A 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. RESULTS: Of 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. CONCLUSION: We 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.

Prognostic significance of 1p36 locus deletion in adenoid cystic carcinoma of the salivary glands.

Adenoid cystic carcinoma (AdCC) of the salivary glands is characterized by MYB-NFIB or MYBL1-NFIB fusion, prolonged but relentlessly progressive clinical course with frequent recurrences, and development of distant metastasis resulting in high long-term mortality. Currently, no effective therapy is available for patients with advanced non-resectable and/or metastatic disease. Complicating the clinical management of this patient group is the lack of prognostic markers. The purpose of this study is to investigate the prognostic value of 1p36 loss in patients with AdCC. The presence of 1p36 deletion and gene fusions involving the MYB, NFIB, and MYBL1 genes in a cohort of 93 salivary gland AdCCs was studied using fluorescence in situ hybridization. These results were statistically correlated with clinical data and outcome. Deletion of 1p36 in AdCC was identified in 13 of 85 analyzable cases (15.29%). MYB-NFIB fusion was detected in 57/85 (67.1%), MYBL1-NFIB fusion in 12/85 (14.1%), MYB-X fusion in 4/85 (4.7%), MYBL1-X in 4/85 (4.7%), and NFIB-X in 2/85 (2.4%) of AdCC cases. None of the 1p36-deleted samples showed MYBL1 rearrangement. Statistical analysis demonstrated a significant correlation between 1p36 deletion and advanced tumor stage and solid histology (p = 0.0061 and 0.0007, respectively). Kaplan-Meier survival curves showed statistically significant correlations between 1p36 deletion and decreased overall survival, disease-specific survival, recurrence-free interval, and recurrence-free survival, all of which were maintained in multivariate analysis. We demonstrate that 1p36 deletion can serve as an indicator of unfavorable outcome of patients with salivary gland AdCC.

UM-HACC-2A: MYB-NFIB fusion-positive human adenoid cystic carcinoma cell line.

OBJECTIVES: Limited availability of validated human adenoid cystic carcinoma (ACC) cell lines has hindered the mechanistic understanding of the pathobiology of this malignancy and the development of effective therapies. The purpose of this work was to generate and characterize a human ACC cell line. MATERIAL AND METHODS: Immediately after surgery, a tumor fragment from a minor salivary gland from the tongue of a female Caucasian was minced, dissociated, and a single cell suspension was plated in fibronectin-coated flasks. A culture medium containing bovine brain extract and rhEGF was optimized for these cells. Whole exome sequencing was used to evaluate the presence of MYB-NFIB translocation. RESULTS: The University of Michigan-Human Adenoid Cystic Carcinoma (UM-HACC)-2A cells showed continuous growth in monolayers for at least 180 in vitro passages while maintaining epithelial morphology. Short-tandem repeat (STR) profiling confirmed a 100% match to patient DNA. Whole exome sequencing revealed the presence of the MYB-NFIB fusion in UM-HACC-2A cells, which was confirmed by PCR analysis. Western blots revealed high expression of epithelial markers (e.g. E-cadherin, EGFR, pan-cytokeratin) and proteins associated with ACC (e.g. c-Myb, p63). Developmental therapeutic studies showed that UM-HACC-2A cells were resistant to cisplatin (IC50 = 44.7 µM) while more responsive to paclitaxel (IC50 = 0.0006 µM). In a pilot study, we observed that UM-HACC-2A cells survived orthotopic transplantation into the submandibular gland. Notably, one of the mice injected with UM-HACC-2A cells exhibited lung metastasis after 6 months. CONCLUSION: UM-HACC-2A is a MYB-NFIB fusion-positive ACC cell line that is suitable for mechanistic and developmental therapeutics studies.

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.

Unusual pleomorphic adenoma of the lacrimal Gland: Immunohistochemical demonstration of PLAG1 and HMGA2 oncoproteins.

Painless low-grade right proptosis with 20/25 visual acuity developed slowly in a 49-year-old woman with a past history of breast cancer. Imaging studies disclosed an oval-to-round superotemporal mass in the right lacrimal fossa without bone erosion. Excisional biopsy revealed a pseudoencapsulated, bosselated tumor with a spindled, hypocellular, and heavily periodic acid Schiff-positive stroma constituted of abundant basement membrane material and collagen. Scattered lumens and focal cribriform cellular clusters were present in the peripheries of several of the lobules. Immunohistochemistry showed epithelial membrane antigen+ and cytokeratin (CK) 7+ in many small luminal structures. The spindled cells were calponin+, CK5/6+, CK14+, and p63+, confirming their myoepithelial nature. The Ki67 proliferation index was 2-3%, and upregulation of nuclear p53, a tumor suppressor gene product which may be aberrantly overexpressed in malignancy, was observed in rare cells. Immunohistochemical probes for HMGA2 and PLAG1 oncoproteins, characteristic of pleomorphic adenoma, were stained intensely and less intensely, respectively. MYB and c-KIT (CD117) were negative, thereby strongly arguing against the diagnosis of adenoid cystic carcinoma. In atypical epithelial tumors of the lacrimal gland, genetic probes identifying distinctive gene translocations or their oncoprotein products complement traditional immunohistochemical biomarkers such as cytokeratins and other structural or secretory molecules. Characteristic genetic abnormalities demonstrated by immunohistochemistry for their upregulated protein products, or by in situ hybridization for translocations, are increasingly being relied on for diagnostic precision.

The impact of the MYB-NFIB fusion proto-oncogene in vivo.

Recurrent fusion of the v-myb avian myelobastosis viral oncogene homolog (MYB) and nuclear factor I/B (NFIB) generates the MYB-NFIB transcription factor, which has been detected in a high percentage of individuals with adenoid cystic carcinoma (ACC). To understand the functional role of this fusion protein in carcinogenesis, we generated a conditional mutant transgenic mouse that expresses MYB-NFIB along with p53 mutation in tissues that give rise to ACC: mammary tissue, salivary glands, or systemically in the whole body. Expression of the oncogene in mammary tissue resulted in hyperplastic glands that developed into adenocarcinoma in 27.3% of animals. Systemic expression of the MYB-NFIB fusion caused more rapid development of this breast phenotype, but mice died due to abnormal proliferation in the glomerular compartment of the kidney, which led to development of glomerulonephritis. These findings suggest the MYB-NFIB fusion is oncogenic and treatments targeting this transcription factor may lead to therapeutic responses in ACC patients.

A subset of prostatic basal cell carcinomas harbor the MYB rearrangement of adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a basaloid tumor consisting of myoepithelial and ductal cells typically arranged in a cribriform pattern. Adenoid cystic carcinoma is generally regarded as a form of salivary gland carcinoma, but it can arise from sites unassociated with salivary tissue. A rare form of prostate carcinoma exhibits ACC-like features; it is no longer regarded as a true ACC but rather as prostatic basal cell carcinoma (PBCC) and within the spectrum of basaloid prostatic proliferations. True ACCs often harbor MYB translocations resulting in the MYB-NFIB fusion protein. MYB analysis could clarify the true nature of prostatic carcinomas that exhibit ACC features and thus help refine the classification of prostatic basaloid proliferations. Twelve PBCCs were identified from the pathology consultation files of Johns Hopkins Hospital. The histopathologic features were reviewed, and break-apart fluorescence in situ hybridization for MYB was performed. All 12 cases exhibited prominent basaloid histology. Four were purely solid, 7 exhibited a cribriform pattern reminiscent of salivary ACC, and 1 had a mixed pattern. The MYB rearrangement was detected in 2 (29%) of 7 ACC-like carcinomas but in none (0%) of the 5 PBCCs with a prominent solid pattern. True ACCs can arise in the prostate as is evidenced by the presence of the characteristic MYB rearrangement. When dealing with malignant basaloid proliferations in the prostate, recommendations to consolidate ACCs with other tumor types may need to be reassessed, particularly in light of the rapidly advancing field of biologic therapy where the identification of tumor-specific genetic alterations presents novel therapeutic targets.