Epithelial-myoepithelial Carcinomas Research Articles

Adjuvant Radiation Therapy for Salivary Duct and Epithelial Myoepithelial Salivary Carcinoma

Salivary gland cancers are rare in general and salivary duct carcinoma and epithelial myoepithelial carcinomas are rare subtypes. This topic discussion will review the characteristics of these uncommon cancers. Additionally, it will briefly discuss available guidelines for salivary cancers and summarize author opinions on the role of adjuvant radiation therapy for these cases.

46. Third time’s the charm; the elusive nature of Epithelial-myoepthelial carcinomas

Epithelial-myoepithelial carcinomas (EMCs) are a rare type of malignant tumour that most commonly affects the major salivary glands, and comprises of approximately 1-2% of all salivary neoplasms. Generally it is regarded as a low-to-intermediate grade malignancy with a favourable long term prognosis.

Glial Fibrillary Acidic Protein Expression Helps Distinguish Pleomorphic Adenoma from Histologic Mimics.

Pleomorphic adenoma (PA) is the most common biphasic type of salivary gland tumour to arise in adults. It is a biphasic tumour composed of both luminal (ductal) cells and abluminal (basal and myoepithelial) cells. Other biphasic salivary gland type tumours, both benign and malignant, can mimic PA, especially on small biopsies. Previous studies have shown that glial fibrillary acidic protein (GFAP) is preferentially expressed in PA and can be useful in the distinction from other salivary gland tumours. However, most of these studies were performed on a small subset of tumour types at a time when the classification of salivary gland type tumours was less refined. The purpose of this study was to assess the expression of glial fibrillary acidic protein (GFAP) in a broad group of both benign and malignant salivary gland tumours. The expression of GFAP was assessed in 99 tumours including 54 PAs, 5 basal cell adenomas, 1 myoepitheliomas, 5 adenoid cystic carcinomas, 6 epithelial-myoepithelial carcinomas (EMCA), 6 mucoepidermoid carcinomas, 7 salivary duct carcinomas, 1 adenocarcinomas NOS, 2 myoepithelial carcinomas, 4 basal cell adenocarcinomas, 5 acinic cell carcinomas and 3 polymorphous adenocarcinomas. Of the malignant cases, 8 were classified as carcinomas ex PA. GFAP was also assessed in 19 concurrent biopsy specimens. GFAP was expressed in the resections of 51 PAs examined (94%). Expression was predominantly strong and diffusely seen in myoepithelial cells. Strong and diffuse GFAP expression was also seen in two EMCAs (33%) and one myoepithelial carcinoma (50%). On biopsy specimens, 100% of PAs and basal cell adenomas expressed GFAP. GFAP was also seen in 1 out of 3 carcinomas ex PAs on biopsies. Almost all PAs show strong and diffuse expression of GFAP. In contrast, most malignant neoplasms that can mimic PA on biopsies show only rare, focal expression. Other benign tumours composed of abluminal/myoepithelial cells also show focal expression of GFAP, highlighting the spectrum these tumours share with PA. Overall, the presence of strong and diffuse GFAP expression can favour a benign neoplasm, specifically a PA, on limited biopsy specimens.

A tree-based machine learning model to approach morphologic assessment of malignant salivary gland tumors

Malignant salivary gland tumors represent a challenge for pathologists due to their low frequency and morphologic overlap. In recent years machine learning techniques have been applied to the field of pathology to improve diagnostic performance. In the present work, we fitted a machine learning algorithm to approach the diagnosis of malignant salivary gland tumors. Twelve morphologic variables were scored across 115 samples representing the most commonly encountered malignant salivary gland tumors. The sample was randomly split into a discovery and validation set. A recursive partitioning algorithm was used to systematically screen and organize candidate variables into a classification tree using the discovery set. A cross-validation strategy was used to tune the algorithm hyperparameters. Inter-observer concordance was calculated by independent evaluation of 26 randomly selected cases. The five-tiered tree built, required the evaluation of 6 morphological variables. Basaloid appearance, presence of mucous cells, necrosis, cribriform pattern, clear cells and keratinization were selected by the algorithm to build the tree. This diagnostic tool correctly classified 89.9% and 84.6% of the samples in the discovery and validation sets respectively. Misclassification pattern was consistent between both sets. Misclassified tumors belonged to one of three histologic types: epithelial-myoepithelial, polymorphous and mucoepidermoid carcinomas. Other histotypes demonstrated perfect recall in both the discovery and validation sets. Overall inter-observer concordance was good, with median kappa scores between the expert evaluator and training pathologists being 0.81. Overall, our classification tool developed using a recursive partitioning algorithm can effectively guide the morphological approach to malignant salivary gland tumors.

SS18 Break-Apart Fluorescence In Situ Hybridization is a Practical and Effective Method for Diagnosing Microsecretory Adenocarcinoma of Salivary Glands.

Molecular analysis has allowed for refinement of salivary gland tumor classification and, in some cases, the recognition of entirely new tumor types. Microsecretory adenocarcinoma (MSA) is a salivary gland tumor described in 2019 characterized by microcystic growth, bland cytomorphology, luminal secretions, fibromyxoid stroma, and S100/p63 positivity with negative p40. Most important, MSA is defined by MEF2C-SS18 fusion. While this fusion has, to this point, been detected by next-generation sequencing, this is a technique that is currently inaccessible in most diagnostic laboratories. On the other hand, SS18 break-apart fluorescence in situ hybridization (FISH) is widely available and frequently used as an adjunct for diagnosing synovial sarcoma. It is not known if SS18 break-apart FISH is positive in tumors with MEF2C-SS18, or if it is entirely specific for MSA.Break apart FISH for SS18 was performed on 4 cases of MSA, as well as 8 tissue microarrays (TMAs) containing 423 various salivary gland carcinomas: 26 acinic cell carcinomas, 35 adenocarcinomas not otherwise specified, 96 adenoid cystic carcinomas, 3 basal cell adenocarcinomas, 20 epithelial-myoepithelial carcinomas, 15 hyalinizing clear cell carcinomas, 3 intraductal carcinomas, 12 myoepithelial carcinomas, 117 mucoepidermoid carcinomas, 30 polymorphous adenocarcinomas, 45 salivary duct carcinomas, 19 secretory carcinomas, and 2 undifferentiated carcinomas. SS18 break-apart FISH was also performed on whole slides of 2 tumors from the TMAs.All MSA cases demonstrated classic split patterns on SS18 break-apart FISH. On the TMAs, 374 cases were evaluable by FISH, and 372 cases were clearly negative for SS18 rearrangement. Two cases, both mucoepidermoid carcinomas, had rare split signals below the positivity threshold of 12% on their TMA cores, so FISH was performed on whole sections. On the whole sections both tumors were unequivocally negative for SS18 rearrangement. Taken together, SS18 break-apart FISH was 100% sensitive and 100% specific for a diagnosis of MSA.SS18 break-apart FISH, a diagnostic tool widely available in pathology laboratories, appears to be a highly accurate method for diagnosing MSA of salivary glands. Accordingly, this new tumor type may be molecularly confirmed without needing to resort to highly specialized techniques like next-generation sequencing.

Homeobox transcription factor engrailed homeobox 1 is a possible diagnostic marker for adenoid cystic carcinoma and polymorphous adenocarcinoma.

Diagnostic utility of a homeobox transcription factor, engrailed homeobox 1 (En1) in the histopathology of salivary gland neoplasms was studied. The expression of En1 was immunohistochemically examined in 51 cases of adenoid cystic carcinoma (AdCC) and 143 cases of other salivary gland neoplasms. In all 51 AdCCs, En1 was expressed in 30-100% of tumor cells. In eight of nine polymorphous adenocarcinomas (PACs), En1 was expressed in 40-100% of tumor cells. Less than 5% of tumor cells expressed En1 in three of 12 epithelial-myoepithelial carcinomas, one of 17 basal cell adenomas (BCAs), and one of 34 pleomorphic adenomas (PAs). Among 55 other carcinoma cases, 1-30% of tumor cells expressed En1 in three salivary duct carcinomas (SDCs) ex PA. None of the myoepitheliomas and Warthin tumors expressed En1. When the cut-off value of the percentage of En1-expressing cells was set to 25%, all 51 AdCCs, eight of nine PACs and one SDC ex PA were En1-positive and the others were En1-negative. En1 is expressed consistently in AdCCs, frequently in PACs, but rarely in other salivary gland neoplasms. En1 is a possible diagnostic marker for AdCC and PAC in the histopathology of salivary gland neoplasms.

The Decline of Salivary Adenocarcinoma Not Otherwise Specified as a Tumor Entity

The classification of salivary gland carcinomas has become increasingly specific over the last decade with the definition of new tumor types, documentation of novel molecular and immunohistochemical findings, and development of more refined diagnostic criteria. In this setting, it is unclear how many salivary tumors still cannot be easily categorized—and whether such tumors represent undifferentiated malignancies or include additional definable entities. Relying largely on current classification schemes and contemporary immunohistochemical panels, we reassessed salivary tumors previously diagnosed as adenocarcinoma, not otherwise specified (ACA NOS) from 2 large academic medical centers. Fifty-seven ACA NOS (72%) could be reclassified as more specific entities including 31 salivary duct carcinomas (39%), 7 polymorphous adenocarcinomas (9%), 5 epithelial-myoepithelial carcinomas (6%), 4 myoepithelial carcinomas (5%), 4 secretory carcinomas (5%), 1 acinic cell carcinoma (1%), 1 basal cell adenocarcinoma (1%), 1 intraductal carcinoma (1%), and 1 clear cell carcinoma (1%) as well as 2 metastatic squamous cell carcinomas (3%). Of reclassified cases, 21 (37%) represented variant histologies within these categories. ACA NOS comprised 11% of salivary malignancies before reclassification, but only 4% after reclassification. The remaining 22 ACA NOS demonstrated heterogeneous features, with an association between histologic grade and clinical outcome. In effect, ACA NOS is becoming a bygone entity as modern classification schemes and ancillary techniques now permit more specific typing of a majority of these tumors, potentially facilitating more specific prognostication and treatment. Additional distinctive entities such as mucinous adenocarcinoma may still be definable within the ACA NOS category.

Ex vivo assessment of targeted therapies in a rare metastatic epithelial–myoepithelial carcinoma

Epithelial–myoepithelial carcinoma (EMC) is a rare subtype of salivary gland neoplasms. Since the initial description of the cancer, just over 300 cases have been reported. EMCs occupy a biphasic cellular differentiation-state defined by the constitution of two cell types representing epithelial and myoepithelial lineages, yet the functional consequence of the differentiation-state heterogeneity with respect to therapy resistance of the tumors remains unclear. The reported local recurrence rate of the cases is approximately 30%, and while distant metastases are rare, a significant fraction of these cases are reported to receive no survival benefit from radio- or chemotherapy given in addition to surgery. Moreover, no targeted therapies have been reported for these neoplasms. We report here the first use and application of ex vivo drug screening together with next generation sequencing to assess targeted treatment strategies for a rare metastatic epithelial–myoepithelial carcinoma. Results of the ex vivo drug screen demonstrate significant differential therapeutic sensitivity between the epithelial and myoepithelial intra-tumor cell lineages suggesting that differentiation-state heterogeneity within epithelial–myoepithelial carcinomas may present an outlet to partial therapeutic responses to targeted therapies including MEK and mTOR inhibitors. These results suggest that the intra-tumor lineage composition of EMC could be an important factor to be assessed when novel treatments are being evaluated for management of metastatic EMC.

Prognostic Impact of PD-L1 Expression in Malignant Salivary Gland Tumors as Assessed by Established Scoring Criteria: Tumor Proportion Score (TPS), Combined Positivity Score (CPS), and Immune Cell (IC) Infiltrate.

Background: Malignant neoplasms of the salivary glands are rare, and therapeutic options are limited. Results from recently published studies indicate a possible use for checkpoint inhibition in a subset of patients, but there are no established criteria for programme cell death ligand 1 (PD-L1) scoring in salivary gland carcinomas (SGCs). Methods: In this retrospective study, we present a cohort of 94 SGC patients with full clinical follow-up. We included 41 adenoid cystic carcinomas (AdCC), 21 mucoepidermoid carcinomas (MEC), 16 acinic cell carcinomas (ACC), 12 adenocarcinomas, not otherwise specified (AC, NOS), 2 epithelial-myoepithelial carcinomas (EMC), one salivary duct carcinoma (SDC), and one carcinoma ex pleomorphic adenoma (CA ex PA). Subsequent histopathological analysis was performed with special emphasis on the composition of the immune cell infiltrate (B-/T-lymphocytes). We assessed PD-L1 (SP263) on full slides by established scoring criteria: tumor proportion score (TPS), combined positivity score (CPS) and immune cell (IC) score. Results: We identified significantly elevated CD3+, TP, CP, and IC scores in AC, NOS compared to AdCC, MEC, and ACC. CPS correlated with node-positive disease. Moreover, AC, NOS displayed IC scores of 2 or 3 in the majority (67%) of cases (p = 0.0031), and was associated with poor prognosis regarding progression-free (PFS) (p < 0.0001) and overall survival (OS) (p < 0.0001). CPS correlated with strong nuclear or null p53 staining in AC, NOS but not in other SGCs. Long-lasting partial remission could be achieved in one AC, NOS patient who received Pembrolizumab as third-line therapy. Conclusions: The current study is the first to investigate the use of established scoring criteria for PD-L1 expression in malignant salivary gland tumors. Our findings identify unique characteristics for AC, NOS among the family of SGCs, as it is associated with poor prognosis and might represent a valuable target for immune checkpoint inhibition.

Immunohistochemical features of carcinoma ex pleomorphic adenoma and pleomorphic adenoma in the lacrimal gland.

To investigate C-myc, Ki-67, pan-cytokeratin, and vimentin immunohistochemical features of carcinoma ex pleomorphic adenoma (Ca-ex-PA) and pleomorphic adenoma (PA) in the lacrimal gland in order to find some clues in the differential diagnosis between them. We reviewed microscopic slides and clinical records of 64 cases of PA and 15 cases of Ca-ex-PA in the lacrimal gland. Immunohistochemical antibodies for C-myc, Ki-67, pan-cytokeratin, and vimentin were employed. Median age of PA was 43.2y (from 21 to 75). The 35 patients (54.7%) were male and 29 patients (45.3%) were female. For the PAs, the average positivity of C-myc was 4.6%; the average proliferation index of Ki-67 was 3.2%; pan-cytokeratin was positive in ductal cells, and vimentin was positive in myoepithelial cells. Median age of Ca-ex-PA was 54.3y (from 26 to 76). There were 7 male patients (46.7%) and 8 female patients (53.3%). Among 15 Ca-ex-PAs, there were 6 myoepithelial carcinomas, 4 adenocarcinomas, 3 epithelial-myoepithelial carcinomas, and 2 squamous cell carcinomas. For the Ca-ex-PAs, the average positivity of C-myc was 36.4%; the average proliferation index of Ki-67 was 29.2%; pan-cytokeratin was positive in all cases, and vimentin was positive in myoepithelial carcinomas. PA has a lower positivity of C-myc and Ki-67, while Ca-ex-PA had a higher positivity of these two biomarkers. These four biomarkers as a set could provide valuable clues in the differential diagnosis between Ca-ex-PA and PA. Our results indicate that the activation of C-myc could play an important role in the pathogenesis of Ca-ex-PA and PA.

Diagnostic Significance of HRAS Mutations in Epithelial-Myoepithelial Carcinomas Exhibiting a Broad Histopathologic Spectrum.

Epithelial-myoepithelial carcinoma (EMC) is a rare salivary gland tumor that is histologically characterized by biphasic tubular structures composed of inner ductal and outer clear myoepithelial cells. Because of its histologic variety, it is sometimes challenging to make an accurate diagnosis, and useful ancillary tests are essential for this purpose. We investigated 87 cases of EMC arising in the major and minor salivary glands and seromucinous glands in the nasal cavity or bronchus to describe the histologic features and mutation status of selected key oncogenes. Classic EMC accounted for 40.2% of all cases. Other cases showed various growth patterns and cytologic features in addition to the typical histology; cribriform patterns, a basaloid appearance, and sebaceous differentiation were relatively common (17.2% to 18.4%), whereas oncocytic/apocrine, papillary-cystic, double-clear, squamous, psammomatous, Verocay-like, and high-grade transformation were rare. HRAS mutations were found in 82.7% of EMCs and were concentrated in codon 61. There was no significant correlation between the HRAS mutation status and the histology. No EMC ex pleomorphic adenoma cases had HRAS mutations. PIK3CA and/or AKT1 mutations were the second most frequent mutations (20.7%, 6.5%, respectively) and almost always cooccurred with HRAS mutations. It is noteworthy that the HRAS mutation was not identified in any salivary gland tumor entities manifesting EMC-like features, including adenoid cystic carcinoma, pleomorphic adenoma, basal cell adenoma/adenocarcinoma, and myoepithelial carcinoma. We conclude that HRAS mutations are a frequent tumorigenic gene alteration in EMC, despite its histologic diversity. This study provides further insight into strategies for diagnosing EMC and discriminating it from its mimics.

SOX11: a potentially useful marker in surgical pathology: a systematic analysis of SOX11 expression in epithelial and non‐epithelial tumours

SOX11 is known as an essential transcription factor for regulating neurogenesis. Recently, SOX11 has been suggested to be a diagnostic marker and oncogene because of its significant expression in mantle cell lymphoma (MCL). However, SOX11 expression in other tumour types has not yet been extensively studied. Herein, we examined SOX11 expression in 2026 cases of neuroectodermal, germ cell, mesenchymal and epithelial tumours by immunohistochemistry. SOX11 was consistently expressed in all neuroectodermal tumours with neural differentiation, as well as in immature teratomas revealing neurogenesis. Less frequently, SOX11 expression was observed in only 50% of astrocytomas and 24% of malignant peripheral nerve sheath tumours, and was mainly sporadic and weak/intermediate. In epithelial tumours, significant SOX11 expression was identified in 97% of salivary ductal carcinomas (SDCs) and a high percentage of high-grade neuroendocrine carcinomas (NECs), especially the small-cell lung carcinomas (68%), and was absent in most other carcinomas, except for less and/or focal and weak expression in adenocarcinomas from the lung, genital tract and breast, and salivary adenoid cystic carcinomas and epithelial-myoepithelial carcinomas. In mesenchymal tumours, in addition to MCLs, prominent SOX11 expression was observed in 90% of rhabdomyosarcomas and all myxoid/round cell liposarcomas (MRCLs). Less frequent and/or focal and weak expression was observed in lymphoblastic, Burkitt and follicular lymphomas, synovial sarcoma and angiosarcoma. SOX11 showed prominent expression in neuroectodermal tumours with neural differentiation, high grade-NEC, SDC, rhabdomyosarcoma and MRCL. The high sensitivity and specificity of SOX11 in SDC and MRCL make it a useful diagnostic marker.

Epithelial-Myoepithelial Carcinoma: Frequent Morphologic and Molecular Evidence of Preexisting Pleomorphic Adenoma, Common HRAS Mutations in PLAG1-intact and HMGA2-intact Cases, and Occasional TP53, FBXW7, and SMARCB1 Alterations in High-grade Cases.

We hypothesized that there is a relationship between the preexisting pleomorphic adenoma [PA]), histologic grade of epithelial-myoepithelial carcinomas (EMCAs), and genetic alterations. EMCAs (n=39) were analyzed for morphologic and molecular evidence of preexisting PA (PLAG1, HMGA2 status by fluorescence in situ hybridization, FISH, and FGFR1-PLAG1 fusion by next-generation sequencing, NGS). Twenty-three EMCAs were further analyzed by NGS for mutations and copy number variation in 50 cancer-related genes. On the basis of combined morphologic and molecular evidence of PA, the following subsets of EMCA emerged: (a) EMCAs with morphologic evidence of preexisting PA, but intact PLAG1 and HMGA2 (12/39, 31%), (b) Carcinomas with PLAG1 alterations (9/39, 23%), or (c) HMGA2 alterations (10/39, 26%), and (d) de novo carcinomas, without morphologic or molecular evidence of PA (8/39, 21%). Twelve high-grade EMCAs (12/39, 31%) occurred across all subsets. The median disease-free survival was 80 months (95% confidence interval, 77-84 mo). Disease-free survival and other clinicopathologic parameters did not differ by the above defined subsets. HRAS mutations were more common in EMCAs with intact PLAG1 and HMGA2 (7/9 vs. 1/14, P<0.001). Other genetic abnormalities (TP53 [n=2], FBXW7 [n=1], SMARCB1 deletion [n=1]) were seen only in high-grade EMCAs with intact PLAG1 and HMGA2. We conclude that most EMCAs arose ex PA (31/39, 80%) and the genetic profile of EMCA varies with the absence or presence of preexisting PA and its cytogenetic signature. Progression to higher grade EMCA with intact PLAG1 and HMGA2 correlates with the presence of TP53, FBXW7 mutations, or SMARCB1 deletion.

Comprehensive genomic profiles of metastatic and relapsed salivary gland carcinomas are associated with tumor type and reveal new routes to targeted therapies

BackgroundRelapsed/metastatic salivary gland carcinomas (SGCs) have a wide diversity of histologic subtypes associated with variable clinical aggressiveness and response to local and systemic therapies. We queried whether comprehensive genomic profiling could define the tumor subtypes and uncover clinically relevant genomic alterations, revealing new routes to targeted therapies for patients with relapsed and metastatic disease.Patients and methodsFrom a series of 85 686 clinical cases, DNA was extracted from 40 µm of formalin-fixed paraffin embedded (FFPE) sections for 623 consecutive SGC. CGP was carried out on hybridization-captured, adaptor ligation-based libraries (mean coverage depth, >500×) for up to 315 cancer-related genes. Tumor mutational burden was determined on 1.1 Mb of sequenced DNA. All classes of alterations, base substitutions, short insertions/deletions, copy number changes, and rearrangements/fusions were determined simultaneously.ResultsThe clinically more indolent SGC including adenoid cystic carcinoma, acinic cell carcinoma, polymorphous low-grade adenocarcinoma, mammary analog secretory carcinoma, and epithelial–myoepithelial carcinomas have significantly fewer genomic alterations, TP53 mutations, and lower tumor mutational burden than the typically more aggressive SGCs including mucoepidermoid carcinoma, salivary duct carcinoma, adenocarcinoma, not otherwise specified, carcinoma NOS, and carcinoma ex pleomorphic adenoma. The more aggressive SGCs are commonly driven by ERBB2 PI3K pathway genomic alterations. Additional targetable GAs are frequently seen.ConclusionsGenomic profiling of SGCs demonstrates important differences between traditionally indolent and aggressive cancers. These differences may provide therapeutic options in the future.

HMGA2 is a specific immunohistochemical marker for pleomorphic adenoma and carcinoma ex-pleomorphic adenoma.

Accurate classification of salivary gland neoplasms may be challenging, owing to morphological overlap, particularly in small biopsies. Recurrent translocations involving the high-mobility group AT-hook 2 (HMGA2) gene are present in a subset of pleomorphic adenomas (PAs) and carcinoma ex-pleomorphic adenomas (CA ex-PAs). The aim of this study was to evaluate immunohistochemical HMGA2 expression in 225 salivary gland tumours, including 56 PAs, 37 CA ex-PAs, and 132 potential histological mimics, to determine its diagnostic utility. HMGA2 expression was identified in 19 PAs (33.9%) and nine CA ex-PAs (24.3%). Expression was strong and diffuse throughout all PAs, and in four of nine positive CA ex-PAs. In five CA ex-PAs, HMGA2 showed weak-to-strong multifocal staining within the carcinomatous component, and strong diffuse HMGA2 expression in the residual PA. Among histological mimics, six de-novo salivary duct carcinomas (28.5%), three epithelial-myoepithelial carcinomas (33.3%) and one case each of myoepithelioma and basal cell adenoma expressed HMGA2. Fluorescence in-situ hybridization for HMGA2 rearrangement performed on a subset of tumours that showed diffuse HMGA2 expression in PAs and CA ex-PAs was frequently associated with rearrangement of the HMGA2 locus, whereas cases of de-novo salivary duct carcinoma, or CA ex-PA with limited or no HMGA2 expression, had an intact HMGA2 locus. HMGA2 expression is a highly specific (96.2%), but low-sensitivity (29.8%), marker for PA and CA ex-PA when compared with histological mimics, and is frequently associated with rearrangement of the HMGA2 locus.

Abstract 3379: Massively parallel sequencing analysis of breast adenomyoepitheliomas reveals the heterogeneity of the disease and identifies a subset driven by HRAS hotspot mutations

Abstract Adenomyoepithelioma (AME) is a rare biphasic proliferative breast lesion, which may resemble salivary gland epithelial-myoepithelial carcinomas (EMCs). Most AMEs have an indolent clinical course, but malignant transformation and local and distant recurrences have been reported. We sought to define the mutational landscape of AMEs and investigate the functional impact of recurrent likely pathogenic mutations identified in these tumors. Nineteen AMEs were subjected to whole-exome massively parallel sequencing (MPS, n=7) or targeted capture MPS using MSK-IMPACT assay (n=12). Somatic genetic alterations and the cancer cell fraction of mutations were defined using state-of-the-art bioinformatics algorithms. Selected genes (i.e. HRAS and PIK3CA) were subjected to Sanger sequencing in a series of 17 additional AMEs (total n=36). Non-tumorigenic mammary epithelial cells (i.e. MCF10A, MCF10A with the PIK3CAH1047R mutation and MCF12A), which are estrogen receptor (ER)-negative, were utilized for 2D and 3D functional studies. Of 36 cases, 22 were ER-positive and 14 were ER-negative. MPS analysis revealed a low mutation burden and HRASQ61 and PIK3CA hotspot mutations in 6/19 (32%) and 11/19 (58%) AMEs, respectively. All HRASQ61 and all but one PIK3CA mutations were clonal. ER-positive and ER-negative AMEs were fundamentally histologically and genetically distinct. Whilst ER-positive AMEs displayed recurrent PIK3CA mutations (50%, 11/22) but lacked HRAS mutations, ER-negative AMEs displayed, in addition to PIK3CA mutations (57%, 8/14), recurrent HRASQ61 mutations (57%, 8/14). HRASQ61 mutations co-occurred with PIK3CA mutations (50%, 4/8), PIK3R1 deletions (12.5%, 1/8) and/or CDKN2A homozygous deletions (25%, 2/8). HRASQ61 mutations, but not PIK3CA mutations, were significantly associated with ER-negativity (100% vs 21%), concurrent carcinoma (50% vs 7%), axillary metastases (38% vs 0%), high proliferation (63% vs 4%), necrosis (63% vs 11%) and nuclear pleomorphism (75% vs 29%). In vitro forced HRASQ61R expression in MCF10A and MCF12A cells resulted in increased proliferation and transformation. In 3D organotypic cell cultures, forced HRASQ61R resulted in a highly disorganized growth pattern, a partial loss of epithelial phenotype and acquisition of aberrant myoepithelial differentiation, which was more overt in PIK3CA-mutant MCF10A cells. In conclusion, AMEs are phenotypically and genetically heterogeneous. Whilst PIK3CA hotspot mutations occur across the spectrum of lesions, HRASQ61 hotspot mutations are restricted to ER-negative AMEs, which should arguably be classified as breast EMCs. Our genomic and functional analyses are consistent with the notion that HRASQ61 mutations are driver events in the pathogenesis of ER-negative AMEs and may be sufficient for the acquisition of myoepithelial differentiation in breast cells. Citation Format: Felipe C. Geyer, Kathleen A. Burke, Anqi Li, Anastasios D. Papanastatiou, Fresia Pareja, Anne S. Schulteis, Charlotte K. Ng, Salvatore Piscuoglio, Marcia Edelweiss, Luciano G. Martelotto, Pier Selenica, Maria R. Filippo, Gabriel S. Macedo, Achim Jungbluth, Hannah Y. Wen, Juan Palazzo, Zsuzsanna Varga, Emad Rakha, Ian O. Ellis, Brian Rubin, Britta Weigelt, Jorge S. Reis-Filho. Massively parallel sequencing analysis of breast adenomyoepitheliomas reveals the heterogeneity of the disease and identifies a subset driven by HRAS hotspot mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3379. doi:10.1158/1538-7445.AM2017-3379

Distinctive Patterns of CTNNB1 (β-Catenin) Alterations in Salivary Gland Basal Cell Adenoma and Basal Cell Adenocarcinoma.

Salivary gland basaloid neoplasms are diagnostically challenging. Limited publications report that some basal cell adenomas harbor CTNNB1 mutations, and nuclear β-catenin expression is prevalent. We evaluated β-catenin expression in basal cell adenomas and adenocarcinomas in comparison with salivary tumors in the differential diagnosis and performed targeted genetic analysis on a subset of cases. β-catenin immunohistochemistry was performed on formalin-fixed, paraffin-embedded whole sections from 73 tumors. Nuclear staining was scored semiquantitatively by extent and intensity. DNA was extracted from 6 formalin-fixed, paraffin-embedded samples (5 basal cell adenomas, 1 basal cell adenocarcinoma) for next-generation sequencing. Nuclear β-catenin staining was present in 18/22 (82%) basal cell adenomas; most were diffuse and strong and predominant in the basal component. Two of 3 basal cell adenocarcinomas were positive (1 moderate focal; 1 moderate multifocal). All adenoid cystic carcinomas (0/20) and pleomorphic adenomas (0/20) were negative; 2/8 epithelial-myoepithelial carcinomas showed focal nuclear staining. Most β-catenin-negative tumors showed diffuse membranous staining in the absence of nuclear staining. Four of 5 basal cell adenomas had exon 3 CTNNB1 mutations, all c.104T>C (p.I35T). Basal cell adenocarcinoma showed a more complex genomic profile, with activating mutations in PIK3CA, biallelic inactivation of NFKBIA, focal CYLD deletion, and without CTNNB1 mutation despite focal β-catenin expression. Nuclear β-catenin expression has moderate sensitivity (82%) for basal cell adenoma but high specificity (96%) in comparison with its morphologic mimics. CTNNB1 mutation was confirmed in most basal cell adenomas tested, and findings in basal cell adenocarcinoma suggest possible tumorigenic mechanisms, including alterations in PI3K and NF-κB pathways and transcriptional regulation.

Targeted next generation sequencing of parotid gland cancer uncovers genetic heterogeneity.

Salivary gland cancer represents a heterogeneous group of malignant tumors. Due to their low incidence and the existence of multiple morphologically defined subtypes, these tumors are still poorly understood with regard to their molecular pathogenesis and therapeutically relevant genetic alterations.Performing a systematic and comprehensive study covering 13 subtypes of salivary gland cancer, next generation sequencing was done on 84 tissue samples of parotid gland cancer using multiplex PCR for enrichment of cancer related gene loci covering hotspots of 46 cancer genes.Mutations were identified in 22 different genes. The most frequent alterations affected TP53, followed by RAS genes, PIK3CA, SMAD4 and members of the ERB family. HRAS mutations accounted for more than 90% of RAS mutations, occurring especially in epithelial-myoepithelial carcinomas and salivary duct carcinomas. Additional mutations in PIK3CA also affected particularly epithelial-myoepithelial carcinomas and salivary duct carcinomas, occurring simultaneously with HRAS mutations in almost all cases, pointing to an unknown and therapeutically relevant molecular constellation. Interestingly, 14% of tumors revealed mutations in surface growth factor receptor genes including ALK, HER2, ERBB4, FGFR, cMET and RET, which might prove to be targetable by new therapeutic agents. 6% of tumors revealed mutations in SMAD4.In summary, our data provide novel insight into the fundamental molecular heterogeneity of salivary gland cancer, relevant in terms of tumor classification and the establishment of targeted therapeutic concepts.

Primary epithelial-myoepithelial carcinoma of the lung: A case report demonstrating high-grade transformation-like changes.

Primary salivary gland-type tumors of the lung are rare; among them, epithelial-myoepithelial carcinomas (EMC) represent a minor histological subtype. The present case documents an EMC that occluded the B8 segment of the left lung in a 72-year-old woman. Macroscopically, the tumor was well-demarcated; however, microscopic examination demonstrated that it had infiltrated the lung parenchyma. The majority of the tumor mass was composed of a myoepithelial overgrowth in conjunction with conventional bilayered ductal structures comprising epithelial and myoepithelial cells. At the advancing edge of the tumor, the myoepithelial overgrowth was observed to be gradually transitioning to a higher-grade component, which demonstrated venous invasion. The Ki-67 labeling index was reduced compared with high-grade transformation (HGT) of salivary gland EMC; p53 was sparsely observed on immunostaining. However, cyclin D1, which is reported to be overexpressed in certain subtypes of salivary gland carcinomas with HGT, was overexpressed in the higher-grade component of the tumor, indicating a potential HGT initiation. The surgical margin was tumor free, and no recurrence has been observed for 4 months. A thorough follow-up is required considering the HGT-like changes and venous invasion of the tumor. Additional studies are required to elucidate the characteristics of pulmonary EMC, with an emphasis on detecting HGT or HGT-like changes.

HRAS Mutations in Epithelial–Myoepithelial Carcinoma

The molecular profile of epithelial-myoepithelial carcinomas (EMCa) has not been well studied, though a recent association with Harvey rat sarcoma viral oncogene homolog (HRAS) mutations has been noted. To confirm and validate this, we surveyed fifteen EMCa for HRAS codon 61 mutations and correlated HRAS status with clinicopathologic parameters. There were 11 females and 4 males and mean patient age was 64 (range 49-90). Parotid gland was most commonly involved (n=10) and the most common histologic appearance was that of a 'classic' EMCa (7/15). Four of fifteen (26.7%) cases demonstrated local recurrence, while 2/15 (13.3%) demonstrated distant metastases. Other variant morphologies included EMCa arising from pleomorphic adenoma (3/15), and high grade EMCa (2/15). HRAS exon 3, codon 61 mutations, p.Q61R (n=3) and p.Q61K (n=1) were identified in 4 of 15 successfully tested EMCAs (14 patients). Two cases were classic type, while the other cases consisted of one oncocytic variant, and one tumor with myoepithelial overgrowth, the latter of which showed the same mutation in both the primary and recurrence. Of note, the high grade EMCa and EMCa ex pleomorphic adenoma were negative for mutations. Given the small number of cases, there were no significant differences between mutation positive and mutation negative cases in terms of age, gender and outcome.