Adenoid cystic carcinoma (ACC) is a rare malignancy that can occur in multiple organ sites and is primarily found in the salivary gland. While the identification of recurrent fusions of the MYB-NFIB genes have begun to shed light on the molecular underpinnings, little else is known about the molecular genetics of this frequently fatal cancer. We have undertaken exome sequencing in a series of 24 ACC to further delineate the genetics of the disease. We identified multiple mutated genes that, combined, implicate chromatin deregulation in half of cases. Further, mutations were identified in known cancer genes, including PIK3CA, ATM, CDKN2A, SF3B1, SUFU, TSC1, and CYLD. Mutations in NOTCH1/2 were identified in 3 cases, and we identify the negative NOTCH signaling regulator, SPEN, as a new cancer gene in ACC with mutations in 5 cases. Finally, the identification of 3 likely activating mutations in the tyrosine kinase receptor FGFR2, analogous to those reported in ovarian and endometrial carcinoma, point to potential therapeutic avenues for a subset of cases.

Purpose The objective of this study was to determine the incidence of the MYB-MFIB fusion in salivary adenoid cystic carcinoma (ACC), to establish the clinicopathological significance of the fusion and to analyze the expression of MYB in ACCs in the context of the MYB-NFIB fusion. Experimental Design We performed an extensive analysis involving 123 cancers of the salivary gland, including primary and metastatic ACCs, and non-ACC salivary carcinomas. MYB-NFIB fusions were identified by reverse transcription-PCR (RT-PCR) and sequencing of the RT-PCR products, and confirmed by fluorescence in situ hybridization. MYB RNA expression was determined by quantitative RT-PCR and protein expression was analyzed by immunohistochemistry. Results The MYB-NFIB fusion was detected in 28% primary and 35% metastatic ACCs, but not in any of the non-ACC salivary carcinomas analyzed. Different exons in both MYB and NFIB genes were involved in the fusions, resulting in expression of multiple chimeric variants. Notably, MYB was overexpressed in the vast majority of the ACCs, although MYB expression was significantly higher in tumors carrying the MYB-NFIB fusion. The presence of the MYB-NFIB fusion was significantly associated (p = 0.03) with patients older than 50 years of age. No correlation with other clinicopathological markers, factors and survival was found. Conclusions We conclude that the MYB-NFIB fusion characterizes a subset of ACCs and contributes to MYB overexpression. Additional mechanisms may be involved in MYB overexpression in ACCs lacking the MYB-NFIB fusion. These findings suggest that MYB may be a specific novel target for tumor intervention in patients with ACC.

Regenerating islet-derived family, member 4 (Reg IV) is a candidate marker for cancer and inflammatory bowel disease. In the present study, immunohistochemical analysis of Reg IV was performed in various human neoplastic (n = 289) and non-neoplastic tissues. In the stomach, foveolar epithelium was negative for Reg IV, whereas goblet cells of intestinal metaplasia and neuroendocrine cells at the base of intestinal metaplasia expressed Reg IV. Neuroendocrine cells of the small intestine and colon showed strong expression of Reg IV, whereas goblet cells of the small intestine and colon showed weak or no expression of Reg IV. Insulin-producing beta cells of the endocrine pancreas were positive for Reg IV. Among 143 gastric adenocarcinomas, Reg IV expression was detected in 42 (29.4%) and was associated with both the intestinal mucin phenotype and neuroendocrine differentiation. No association was found between Reg IV expression and clinical characteristics such as tumour stage and patient prognosis. Of 36 colorectal adenocarcinomas, 13 (36.1%) were positive for Reg IV, which was associated with tumour stage (p = 0.0379, Fisher's exact test). Expression of Reg IV was detected in 14 (93.3%) of 15 colorectal carcinoid tumours. Reg IV expression was also detected in 5 (21.7%) of 23 ductal adenocarcinomas of the pancreas. In contrast, lung cancers (n = 30) and breast cancers (n = 30) did not express Reg IV. This is the first immunohistochemical analysis of the expression and distribution of Reg IV protein in human tumours. These data suggest that Reg IV is expressed by gastrointestinal and pancreatic tumours, including adenocarcinomas and carcinoid tumours, and that Reg IV is associated with intestinal and neuroendocrine differentiation of the stomach and gastric carcinoma.

Purpose 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. Experimental Design We performed whole genome sequencing in 21 salivary ACCs and conducted targeted molecular analyses in a validation set (81 patients). Microarray gene expression data was also analyzed to explore the biological differences between fusion positive and negative tumors. Results 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. Additionally, 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 biological importance of the C-terminal part of these fusions. Conclusion Our study defines new molecular subclasses of ACC characterized by MYBL1 rearrangements and 5’-NFIB gene fusions.

Objective To investigate the molecular-genetic heterogeneity associated with the t(6:9) in adenoid cystic carcinoma (ACC) and correlate the findings with patient clinical outcome. Experimental Design Multi-molecular and genetic techniques complemented with massive pair-ended sequencing and SNP array analyses were used on tumor specimens from 30 new and 52 previously RT-PCR analyzed fusion transcript negative ACCs. MYB mRNA expression level was determined by quantitative RT-PCR. The results of 102 tumors (30 new and 72 previously reported cases) were correlated with the clinicopathologic factors and patients’ survival. Results The FISH analysis showed 34/82 (41.5%) fusion positive tumors and molecular techniques identified fusion transcripts in 21 of the 82 (25.6%) tumors. Detailed FISH analysis of 11 out the 15 tumors with gene fusion without transcript formation showed translocation of NFIB sequences to proximal or distal sites of the MYB gene. Massive pair-end sequencing of a subset of tumors confirmed the proximal translocation to an NFIB sequence and led to the identification of a new fusion gene (NFIB-AIG1) in one of the tumors. Overall, MYB-NFIB gene fusion rate by FISH was in 52.9% while fusion transcript forming incidence was 38.2%. Significant statistical association between the 5′ MYB transcript expression and patient survival was found. Conclusions We conclude that: 1) t(6;9) results in a complex genetic and molecular alterations in ACC, 2) MYB-NFIB gene fusion may not always be associated with chimeric transcript formation, 3) non-canonical MYB, NFIB gene fusions occur in a subset of tumors, 4) high MYB expression correlates with worse patient survival.

PurposeAdenoid cystic carcinomas (ACCs) represent a heterogeneous group of chemotherapy refractory tumors, with a subset demonstrating an aggressive phenotype. We investigated the molecular underpinnings of this phenotype and assessed the Notch1 pathway as a potential therapeutic target.MethodsWe genotyped 102 ACCs that had available pathologic and clinical data. Notch1 activation was assessed by immunohistochemistry for Notch1 intracellular domain. Luciferase reporter assays were used to confirm Notch1 target gene expression in vitro. The Notch1 inhibitor brontictuzumab was tested in patient-derived xenografts from patients with ACC and in a patient with ACC who was enrolled in a phase I study.ResultsNOTCH1 mutations occurred predominantly (14 of 15 patients) in the negative regulatory region and Pro-Glu-Ser-Thr–rich domains, the same two hotspots seen in T-cell acute lymphoblastic leukemias, and led to pathway activation in vitro. NOTCH1-mutant tumors demonstrated significantly higher levels of Notch1 pathway activation than wild-type tumors on the basis of Notch1 intracellular domain staining (P = .004). NOTCH1 mutations define a distinct aggressive ACC subgroup with a significantly higher likelihood of solid subtype (P < .001), advanced-stage disease at diagnosis (P = .02), higher rate of liver and bone metastasis (P ≤ .02), shorter relapse-free survival (median, 13 v 34 months; P = .01), and shorter overall survival (median 30 v 122 months; P = .001) when compared with NOTCH1 wild-type tumors. Significant tumor growth inhibition with brontictuzumab was observed exclusively in the ACC patient-derived xenograft model that harbored a NOTCH1 activating mutation. Furthermore, an index patient with NOTCH1-mutant ACC had a partial response to brontictuzumab.ConclusionNOTCH1 mutations define a distinct disease phenotype characterized by solid histology, liver and bone metastasis, poor prognosis, and potential responsiveness to Notch1 inhibitors. Clinical studies targeting Notch1 in a genotype-defined ACC subgroup are warranted.

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