Molecular B-cell clonality assay in minor salivary glands as a useful tool for the lymphoma risk assessment in Sjögren's syndrome.

OBJECTIVES: Non-Hodgkin's lymphoma (NHL) risk assessment is crucial in Sjögren's syndrome (SS). We studied the prevalence of clonal immunoglobulin gene rearrangements in minor salivary glands (MSG) and their correlations with lymphoma occurrence and with previously established NHL predictors. METHODS: Molecular B-cell expansion was studied in fresh-frozen MSG of 207 patients with either suspected SS or with suspected lymphoma during SS, using a standardised multiplex PCR assay combined with heteroduplex analysis by microcapillary electrophoresis. The assignation of clonal cases was based on EuroClonality consortium guidelines. RESULTS: Among 207 studied patients, 31 (15%) had MSG monoclonal B-cell infiltration. Monoclonality was significantly more frequent in patients with SS (28/123, 22.8%) compared with patients without SS (3/84, 3.6%, P<0.001). Monoclonal B-cell infiltration in MSG of SS patients correlated significantly with ongoing salivary gland NHL, salivary gland swelling, CD4+ T-cell lymphopenia, rheumatoid factor (RF) activity, low complement levels and type 2 mixed cryoglobulinemia. The accumulation of biological risk factors was associated with a higher rate of MSG B-cell monoclonality given that patients with only positive RF had no probability of MSG B-cell monoclonality, RF-positive patients with 1 or 2 other risk factors had a 25.0% and 85.7% probability of MSG B-cell monoclonality, respectively. CONCLUSION: The detection of MSG monoclonal B-cell expansion by this easy-to-perform molecular assay is useful, both at the time of diagnosis and during the course of SS. Monoclonal B-cell expansion is associated with a subset of SS patients presenting either ongoing lymphoma or other established lymphoma predictive factors.

A minority of cases of acinic cell carcinoma of the salivary glands are driven by an NR4A2 rearrangement: the diagnostic utility of the assessment of NR4A2 and NR4A3 alterations in salivary gland tumors.

Acinic cell carcinoma (AciCC) is a common salivary gland malignancy, typically composed of neoplastic acinic cells with zymogen granules. The vast majority of cases are driven by a t(4;9)(q13;q31) leading to enhancer hijacking and upregulation of the NR4A3 gene. However, a minority of cases do not display NR4A3 overexpression on immunohistochemical examination and are negative for the rearrangement involving the NR4A3 gene when tested by FISH. Such cases overexpress NR4A2, and the protein product is detectable by immunohistochemistry. In this study, we aimed to assess the utility of NR4A2 and NR4A3 immunohistochemistry in the differential diagnosis of salivary gland tumors. Eighty-five cases of classic low-grade ACiCC, as well as 36 cases with high-grade transformation (HGT) and 7 high-grade AciCC cases were included in the analysis. NR4A3 was at least focally positive in 105/128 (82%) cases. Out of the 23 cases that were immunohistochemically negative for NR4A3, 6 displayed nuclear immunopositivity with the NR4A2 antibody. The NR4A3 rearrangement was confirmed by FISH in 38/52 (73%) cases. In addition, this is the first report of an NR4A2 rearrangement being detected by FISH in 2 AciCC cases that were negative for the NR4A3 rearrangement. Our analysis confirms that the majority of AciCC, including high-grade cases and cases with HGT, are immunopositive for NR4A3, and suggests that NR4A3 immunohistochemistry is a powerful tool in the differential diagnosis of salivary gland tumors. However, its utility is limited in sub-optimally fixed samples which often display weaker and focal positivity. Our study also indicates that in a minority of cases, AciCC might be negative for NR4A3 immunostaining, because the pathogenic genetic event in these cases is instead a rearrangement involving the NR4A2 gene.

Potential role of hybrid positron emission tomography in pre-operative assessment of primary salivary gland carcinomas.

OBJECTIVE: The added value of hybrid positron emission tomography is increasingly recognised in head and neck cancer. However, its potential role in salivary gland carcinomas has been scarcely investigated. METHODS: A consecutive cohort of 45 salivary gland carcinoma patients who underwent pre-therapeutic hybrid positron emission tomography and surgical resection was reviewed. This study investigated whether maximum standardised uptake value correlated with tumour phenotype. RESULTS: Tumours of high-grade disease on histology (salivary duct carcinoma, carcinoma ex pleomorphic adenoma) had higher maximum standardised uptake value (Kruskal-Wallis test, p = 0.011) than low-grade tumours (adenoid cystic carcinoma and acinic cell carcinoma). Patients with pathologically confirmed node-positive disease had significantly higher maximum standardised uptake value of the primary tumour than patients with pathologically confirmed node-negative disease (Kruskal-Wallis test, p = 0.012). CONCLUSION: Maximum standardised uptake value of the primary tumour may guide clinical decision-making in patients with salivary gland carcinomas, as a high maximum standardised uptake value is associated with high-grade tumour histology and the presence of lymph node metastases. Clinicians may consider more aggressive surgery for these patients.

Assessment of angiogenesis using endoglin in salivary gland tumors - An immunohistochemical study.

Background: Endoglin, a co-receptor of transforming growth factor (TGF)-ß1 and TGF-ß2, is indispensable for endothelial cell proliferation and modulation of tumor promotion activities of TGF-ß1. The assessment of neovascularization using endoglin expression has been considered a potential predictor of prognosis in various solid malignancies. Aims and Objectives: To analyze the expression of endoglin by immunohistochemistry in both benign and malignant salivary gland tumors. Materials and Methods: Fifteen cases of benign salivary gland tumors and seventeen cases of malignant salivary gland tumors were included in the study, and immunohistochemistry was performed using anti-CD105 antibody using standard protocol. Results and Conclusion: The study demonstrated that there is increased endoglin expression in malignant tumors as compared to their benign counterparts which is suggestive of increased angiogenic activity in tumor areas and could be responsible for the aggressive behavior of the malignancies. The highest density of endoglin-positive blood vessels was observed in the inflammatory tumor stromal areas. Furthermore, a significant increase in endoglin expression was evident as the grade of malignant salivary gland tumor increased. The results of the study indicate that the increased expression of endoglin in high-grade malignancies contributes to their aggressive nature.

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.

Assessment of Risk of Malignancy of Fine-needle Aspiration Cytology in Salivary Gland Lesions Using the Milan System for Reporting Salivary Gland Cytopathology Categorization: A Systematic Review and Meta-analysis.

BACKGROUND: Fine-needle aspiration cytology (FNAC) of the salivary gland is crucial in the identification of salivary gland lesions, but the variation in morphological pattern and the overlap of morphological traits can result in erroneous interpretation and affect treatment, making FNAC of the salivary gland problematic. The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) was created to address these problems. OBJECTIVES: To ascertain whether the FNAC method using MSRSGC was reliable in predicting the risk of malignancy (ROM) in each category of salivary gland lesions. MATERIALS AND METHODS: The databases PubMed-MEDLINE, Web of Science, Cochrane, Scopus, and Google Scholar were all searched using pertinent keywords, reference searches, and citation searches. A fixed effect model was used to determine the pooled proportion with a 95% confidence interval (CI). All statistical analyses were performed using Meta Disc and R version 4.0.2 (R Foundation for Statistical Computing). RESULTS: After reviewing the submissions' abstracts and titles, 58 documents that satisfied the necessary inclusion and exclusion criteria were ultimately selected. A total of 19,652 samples from 19,408 individuals was analyzed, out of which 9,958 samples were available for histopathological follow-up. The pooled ROM for category I was 10%, category II was 5%, category III was 28%, category IV A was 2%, Category IV B was 34%, category V was 91%, and category VI was 99%. CONCLUSION: Milan System for Reporting Salivary Gland Cytopathology is useful for risk stratification and quality control, confirming its validity and diagnostic utility. Widespread use of MSRSGC would improve the accuracy of salivary gland cytology and lead to better patient care and improved treatment strategies. The results of this study are in consonance with reported values as per MSRSGC except for category V. CLINICAL SIGNIFICANCE: The MSRSGC which was first reported in 2018 is a very useful tool for proper stratification of ROM in salivary gland FNAC. This study allowed us to validate the ROM values in different categories as reported in MSRSGC.

The Milan System for Reporting Salivary Gland Cytopathology: Assessment of Cytohistological Concordance and Risk of Malignancy.

INTRODUCTION: The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) was proposed by the American Society of Cytopathology and the International Academy of Cytology to bring uniformity in the reporting system and the treatment protocol. A wide range of risk of malignancy for each category has been reported by various authors by applying the system. AIM: We intend to study the cytohistological concordance and the ROM for each of the diagnostic categories of the Milan system. MATERIALS AND METHODS: The study included 292 cases of fine-needle aspiration cytology (FNAC) of salivary gland lesions over a period of 3 years. The diagnosis of these cases was reclassified into the 6 categories of the Milan system. The cytohistological concordance and ROM for each category of the Milan system were calculated based on the clinical and histopathological follow-up. RESULTS: The patients' age ranged from 3 to 81 years with the mean of 42.65 ± 16.3 years. The cases included 189 (64.7%) parotid, 82 (28.1%) submandibular, and 21 (7.2%) cases of minor salivary gland swellings. Follow-up histopathological diagnosis for 102 cases was available. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were calculated to be 64.28, 97.01, 90, 86.67, and 87.37%, respectively. After reclassification, the number of cases in each category was as follows: category I: 31 (10.62%), category II: 80 (27.4%), category III: 2 (0.68%), category IVA: 143 (48.97%), category IVB: 1 (0.34%), category V: 13 (4.45%), and category VI: 22 (7.53%). The calculated ROM was as follows: category I: 42.86%, category II: 26.67%, category III: 100% category IVA: 10.17%, category IVB: 0%, category V: 71.42%, category VI: 100%. CONCLUSION: FNAC is an excellent procedure to differentiate benign from malignant tumors, and MSRSGC is a useful system for risk assessment and deciding the further treatment protocol. Our findings also suggest that in addition to the surgical follow-up, inclusion of the clinical and radiological follow-up may be a better strategy for calculation of ROM, especially for categories I and II.

Impact of the Milan System for Reporting Salivary Gland Cytology on risk assessment when used in routine practice in a real-time setting.

INTRODUCTION: Several retrospective studies across the world have validated the role of the Milan System for Reporting Salivary Gland Cytology (MSRSGC) in improving communication between pathologists and clinicians. In this study, we evaluated the applications of MSRSGC in a real-time setting for 2 years. MATERIALS AND METHODS: All salivary gland lesions that underwent fine-needle aspiration (FNA) from January 2018 to December 2020 were categorized according to MSRSGC guidelines. The risk of malignancy (ROM) was calculated for each category and compared with the ROM proposed by MSRSGC and recent retrospective studies. RESULTS: A total of 160 FNA of salivary gland lesions were categorized as: nondiagnostic (ND) 30 (18%), non-neoplastic (NN) 7 (10.6%), atypia of undetermined significance (AUS) 5 (3.1%), benign neoplasm (BN) 59 (36.8%), salivary gland of uncertain malignant potential (SUMP) 21 (13%), suspicious for malignancy (SM) 3 (1.84%), and malignant (M) 25 (15.6%). Histopathologic follow-up was available for 94 (57.5%) cases. The ROM for each category was ND 54%, NN 0%, AUS 66%, BN 0%, SUMP 37.56%, SM 100%, and M 100%. CONCLUSION: With strict adherence to the diagnostic criteria and MSRSGC guidelines, a ROM of 100% in SM and M categories and a ROM of 0% in NN can be achieved in a real-time setting. The high ROM in the ND category in our study highlights the value of repeat FNA/biopsy for this category. High ROM for AUS indicates the tendency to classify high-grade tumors as AUS, calling for refinement in its criteria.

Primary De novo ductal adenocarcinoma of the lacrimal gland.

BACKGROUND: Primary ductal adenocarcinoma of the lacrimal gland is a rare and aggressive malignant epithelial lacrimal gland neoplasm, morphologically and phenotypically resembles salivary duct carcinoma, and both strongly resemble infiltrating ductal carcinoma of breast. METHOD: Retrospective Chart review of cases of malignant lacrimal gland tumors from 2013 July to 2020 July. Authors describe the clinico radiological, morphological and immunohistochemical features of primary ductal adenocarcinoma (PDA) of lacrimal gland. Extensive review of literature of PDA of lacrimal gland and salivary gland ductal carcinoma has been performed. RESULTS: Retrospective chart review of the last 7 years yielded 22 malignant lacrimal gland neoplasms of which 4 cases demonstrated features of primary ductal adenocarcinoma of lacrimal gland, 2/4 cases showed an evidence of a pre existing pleomorphic adenoma and 2 were found to be de novo ductal adenocarcinomas. PDA of lacrimal gland showed expression of CK7, CK19, AR, HER2, cyclin D1 and were negative for CK5/14, CK 20, ER, PR, PSA, TTF-1, S-100 and SMA. Expression of GCDFP-15 was noted in one case. The presence of multiple events of loco-regional recurrences and/or distant metastasis necessitated a multidisciplinary approach. CONCLUSIONS: Authors have expressed the need of clinical correlation; thorough tissue sampling and extensive immunohistochemical work up in identification of de novo PDA's and their molecular subtypes. A multi-institutional study might help in formulating the diagnostic criteria, identification of actionable targets, and thus study the role of targeted therapy in this rare and aggressive tumor which may result in better patient outcomes.

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.

Distant metastasis of salivary gland cancer: Incidence, management, and outcomes.

BACKGROUND: Distant metastases (DMs) are the primary cause of treatment failure in patients with salivary gland carcinoma. There is no consensus on the standard treatment. METHODS: Patients with DMs were identified from an institutional database of 884 patients with salivary gland cancer who underwent resection of the primary tumor between 1985 and 2015. Survival outcomes for patients with DMs were determined with the Kaplan-Meier method. Univariate and multivariate analyses were performed to identify factors associated with DM. RESULTS: Of the 884 patients identified, 137 (15%) developed DMs during follow-up. Most of the primary tumors (n = 77 [56%]) were located in a major salivary gland. At clinical presentation, 53% of the tumors were classified as T3 or T4, and 32% had clinical node metastases. The median time to DM was 20.3 months. The factors associated with shorter distant recurrence-free survival were male sex, high-risk tumor histology, and advanced pathological T and N classifications. Patients with bone metastases had a lower survival rate than patients with lung metastases. The total number of DMs in a patient was inversely associated with survival. Patients who underwent surgical resection of DMs had a significantly higher 5-year rate of metastatic disease-specific survival than patients who underwent observation or nonsurgical treatment (44%, 29%, and 19%, respectively; P = .003). CONCLUSIONS: In patients with DMs of salivary gland carcinoma, survival is negatively associated with high-grade histology, bone DMs, and the total number of DMs. Metastasectomy can help to lengthen disease-free survival.

Risk Assessment of Salivary Gland Cytological Categories of the Milan System: A Retrospective Cytomorphological and Immunocytochemical Institutional Study.

OBJECTIVE: The Milan System for Reporting Salivary Gland Cytology (MSRSGC) has been recently published to help communication between cytopathologists and clinicians. The aim was to assess our institutional experience with salivary gland fine needle aspiration cytology (FNAC) and the potential applicability of the MSRSGC for the estimation of the risk of neoplasm (RON) and risk of malignancy (ROM) for each category. MATERIAL AND METHOD: Salivary gland FNAC procedures performed at NCI, Cairo University in a three-year period from 2016 to 2018 and had a corresponding histopathological diagnosis were included in the current study. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were estimated. Histopathological final diagnosis was the gold standard. Cytological diagnoses were re-stratified according to MSRSGC with estimation of RON and ROM for each category. RESULTS: A total of 118 cases were included in the current work. Sensitivity, specificity, PPV, NPV and accuracy were 84.6%, 88.2%, 78.6%, 91.8% and 87%, respectively. Cytological diagnoses were re-classified as non-diagnostic (2.5%), non-neoplastic (14.4%), atypia of undetermined significance (AUS) (6.8%), benign neoplasm (40.7%), salivary gland neoplasm of uncertain malignant potential (SUMP) (7.6%), suspicious for malignancy (8.5%), and malignancy (19.5%). The RON and ROM for each category were as follows: non-diagnostic (100%, 33.3%), non-neoplastic (17.6%, 11.8%), AUS (50%, 37.5%), benign neoplasm (97.9%, 2.1%), SUMP (88.9%, 44.4%), suspicious (90%, 60%), and malignancy (100% for each). CONCLUSION: The Milan System for Reporting Salivary Gland Cytology is a helpful classification system. The calculated ROM for each category of the studied cases was slightly above the published MSRSGC rates but still supported the recommended management for the patient.

Retrospective assessment of the effectiveness of the Milan system for reporting salivary gland cytology: A systematic review and meta-analysis of published literature.

INTRODUCTION: Fine needle aspiration (FNA) has been widely utilized in establishing the nature of salivary gland lesions and guiding the clinical management. This study aimed to determine the accuracy of FNA in detecting salivary gland neoplasms and malignancies, employing the "Milan System for Reporting Salivary Gland Cytopathology" (MSRSGC). METHOD: A systematic search was conducted. The data on FNA and histologic diagnosis were extracted and categorized based on the MSRSGC and risk of malignancy (ROM) was calculated. The risk of publication bias and level of heterogeneity were evaluated. A mixed-effects model was used to estimate FNA accuracy. Meta-regression was conducted to assess the potential effect of different variables on FNA accuracy. RESULTS: Ninety-two studies with a total of 16 456 FNA with surgical follow-up were included. ROM was estimated as 17%, 8%, 34%, 4%, 42%, 58%, and 91%, in nondiagnostic, nonneoplastic, atypia of undetermined significance, benign neoplasm, salivary gland neoplasm of uncertain malignant potential, suspicious for malignancy, and malignant groups, respectively. High level of heterogeneity was detected (P-value <.001). Including cases with definite FNA diagnosis of neoplasm or malignancy, summary estimates of FNA sensitivity, specificity, diagnostic odds ratio, and positive and negative likelihood ratio in detecting neoplasms and malignancies were 96.9%, 95.3%, 636.8, 20.5, and 0.03, and 80.5%, 97.9%, 189.5, 37.8, and 0.2, respectively. Meta-regression showed several variables significantly impacting FNA accuracy; however, subgroup analysis did not reduce the level of heterogeneity. CONCLUSION: FNA can be used as a reliable diagnostic tool in the preoperative evaluation and management of salivary glands lesions. Concise of abstract is using Milan system for reporting salivary gland FNA could increase FNA reliability, facilitate communication, and improve patient care.

Assessment of CTNNB1 gene mutations and ß-catenin immunoexpression in salivary gland pleomorphic adenomas and adenoid cystic carcinomas.

ß-Catenin exerts multiple functions in several neoplasms, playing a major role in cell signaling and tumor progression. This study analyzed possible CTNNB1 mutations in salivary gland pleomorphic adenomas (PAs) and adenoid cystic carcinomas (ACCs), and determined possible differences in ß-catenin immunoexpression in relation to these mutations, as well as histopathological aspects of these tumors. Twenty-four PAs (15 cell-rich and 9 cell-poor tumors) and 24 ACCs (10 tubular, 8 cribriform, and 6 solid tumors) were selected for the analysis of ß-catenin distribution and cellular localization. Furthermore, ß-catenin expression was evaluated using the H-score scoring system. Mutations in CTNNB1 exon 3 were investigated by the single-strand conformational polymorphism test. Diffuse ß-catenin expression was more frequently observed in ACCs compared to PAs (P = 0.008). No significant difference in ß-catenin cellular localization was observed between these tumors (P = 0.098). Comparisons between PA and ACC cases revealed a higher median H-score in the latter (P = 0.036). Cell-rich PAs exhibited a trend for higher H-score than cell-poor tumors (P = 0.060), whereas lower H-scores were observed in cribriform ACCs when compared to tubular and solid ACCs (P = 0.042). Mutations in CTNNB1 were observed in 6 PAs and 7 ACCs, with no significant difference in H-scores for ß-catenin according to mutation status (P = 0.135). ß-Catenin is important in the pathogenesis of salivary gland PAs and ACCs. In addition, CTNNB1 exon 3 mutations do not seem to significantly influence ß-catenin cytoplasmic/membranous expression or nuclear translocation in these tumors.

Assessment of BRAF V600E (VE1) protein expression and BRAF gene mutation status in codon 600 in benign and malignant salivary gland neoplasms.

BACKGROUND: The role of BRAF mutations in cancerogenesis has been demonstrated in several solid tumor types. However, in salivary gland tumors, this genetic alteration is very uncommon, and its role still remains unclear. Thus, the aim of this study was to analyze BRAF V600E (VE1) protein expression with BRAF mutation status in codon 600, in malignant and benign salivary gland tumors. METHODS: Studies were performed on archived formalin-fixed paraffin-embedded tissue sections derived from 95 patients who underwent surgery for tumors of the salivary gland. Immunohistochemical staining (IHC) on tissue microarray slides was performed for evaluation of BRAF V600E (VE1) protein expression, and the automatic molecular diagnostics platform was used for the evaluation of mutations in codon 600 of BRAF gene. RESULTS: IHC cytoplasmic expression of BRAF V600E (VE1) protein was found in two of 95 cases: one case of adenocarcinoma NOS (one of three; 33%) and one case of carcinoma ex pleomorphic adenoma (one of five; 20%). Although, in IHC studies, nuclear BRAF V600E (VE1) protein expression was found in 14 (15%) of the analyzed cases: nine of 28 (32%) cases of pleomorphic adenoma, three of five (60%) cases of ductal carcinoma, one of nine (11%) case of mucoepidermoid carcinoma, and in one of five (20%) case of carcinoma ex pleomorphic adenoma. All cases were negative for polymerase chain reaction PCR-based analyses of BRAF mutations in codon 600. CONCLUSIONS: In studied salivary gland cancers, no PCR-based prove mutations of BRAF V600 were detected. Further molecular analyses are necessary to rapid molecular arrays for the identification of specific mutations, optimal for individualized targeted therapies.

Pathological aspects of the assessment of head and neck cancers: United Kingdom National Multidisciplinary Guidelines.

This is the official guideline endorsed by the specialty associations involved in the care of head and neck cancer patients in the UK. It introduces the current best practice in histopathology and cytopathology as it pertains to head and neck and thyroid cancers. Recommendations • Accurate diagnosis of the type of malignancy is a key component of effective management. (R) • Surgeons and oncologists should understand the scope and limitations of cellular pathology in order to inform multidisciplinary discussions. (R) • A clinically suspected diagnosis of malignancy should be confirmed by biopsy or cytology before operation. (R) • Cytopathological diagnoses should be discussed with surgeons and radiologists to maximise the information gained from each modality of investigation. (R) • Pathological investigations are the basis for accurate cancer staging and stratification of clinical outcomes. (R).

Avaliação da via do c-kit no adenoma pleomórfico e carcinoma adenoide cístico das glândulas salivares: estudo da expressão das proteínas da via, das mutações e da regulação pós-transcricional do gene kit / c-Kit signaling cascade in pleomorphic adenoma and adenoid cystic carcinoma of salivary glands: evaluation of cascade proteins, mutations and post-transcriptional regulation of gene Kit

Introdução: As neoplasias das glândulas salivares têm amplo espectro histológico resultante da múltipla diferenciação celular tumoral. O adenoma pleomórfico (AP) e o carcinoma adenoide cístico (CAC) são as mais comuns neoplasias benignas e malignas provenientes do ducto intercalado, respectivamente, além de serem compostas por estruturas luminais e células mioepiteliais. Em estudo realizado previamente pelo nosso grupo, detectamos que a proteína c-kit está envolvida nos processos da morfogênese das glândulas salivares e no adenoma pleomórfico. A proteína c-Kit tem papel importante no desenvolvimento de muitos processos embrionários, incluindo a gametogênese, melanogênese e hematopoiese, e também na biologia de tumores. Sua ativação induz diversas respostas intracelulares através de cascatas de sinalização de vias como PI3K/AKT e MAPK. Em tumores da glândula salivar ainda há poucos estudos sobre as alterações do gene KIT e das proteínas relacionadas a sua via de sinalização, assim como sua regulação pós-transcricional, realizada principalmente por meio dos microRNAs. O presente estudo avaliou, em APs e CACs (a) a localização das proteínas das vias PI3K/AKT/mTOR e MAPK por meio da técnica de imunoistoquímica; (b) a expressão dos microRNAs 221 e 222, relacionados ao gene KIT (c) a associação dos achados laboratoriais com variáveis clínicas, patológicas e sobrevida. Resultados: Nos casos de AP a proteína c-Kit foi identificada em formações luminais e em raras células isoladas no parênquima tumoral. Já nos CAC, observou-se positividade na membrana das células ductais. Para a via de PI3K/AKT/mTOR, no AP, a proteína PI3K beta mostrou-se parcialmente positiva no citoplasma das células próximas à capsula tumoral, e as proteínas AKT e mTOR fosforiladas, foram expressas especialmente nas células epiteliais e em poucas células mioepiteliais. Já no CAC, a proteína PI3K beta e AKT fosforilada mostraram-se negativas na maioria dos casos, e a proteína mTOR fosforilada foi expressa no citoplasma das células epiteliais e em algumas células mioepiteliais. Para a via MAPK, as proteínas RAS, MEK-1 fosforilada e ERK 1/2 foram negativas na maioria dos AP e CAC; B-Raf e MEK-2 fosforilada foram observadas nas células luminais dos AP. Nos CAC, estruturas luminais neoplásicas foram positivas para a proteína MEK-2 fosforilada; B-Raf foi positivo nas células luminais e mioepiteliais. Além disso, os pacientes que expressaram as proteínas mTOR e MEK-2 fosforilada apresentaram sobrevida câncer-específica significativamente aumentada (p=0,040 e p=0,005, respectivamente). Na análise do microRNAs, a expressão do miR-221 foi variável nas 13 amostras analisadas, tendo baixa expressão em 30,77% dos casos, expressão normal em 38,46 e expressão aumentada em 30,77% dos casos. Já nos APs o miR-221 foi detectado em 19 amostras, sendo 36,84% com baixa expressão, 52,63% com expressão normal e expressão aumentada foi vista em 10,53% dos casos. A expressão do miR-222 foi detectada em 14 CACs, sendo que a maioria dos casos (8 casos ­ 57,1%) a expressão do miR-222 foi semelhante ao observado nas amostras não neoplásicas. Nos APs, o miR-222 foi detectado em 22 amostras, sendo 31,8% com baixa expressão, 31,8% com expressão normal e 36,4% com expressão aumentada. Conclusão: Apesar de a proteína c-Kit ser expressa em ambas as neoplasias ­ AP e CAC, sua influência sobre as vias de sinalização MAPK e PI3K/AKT/mTOR ainda permanece por ser estabelecida. Ainda, os microRNAs 221 e 222 não mostram correlação consistente com a expressão de c-Kit nos tipos tumorais estudados.

Introduction: Salivary gland tumors present broad histological spectrum resulting from multiple tumor cell differentiation. Pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) are the commonest benign and malignant salivary gland neoplasms originated from the intercalated duct region, respectively, and are composed by luminal structures and myoepithelial cells. In a previous study we detected that protein c-kit is involved in the process of salivary gland morphogenesis and PA. c-Kit protein is important during embryogenesis, including gametogenesis, melanogeneis and hematopoiesis as well as in tumorigenesis. Its activation induces various intracellular responses through pathways such as MAPK and PI3K/AKT/mTOR signaling cascades. In salivary gland neoplasms, only a few reports have shown that alterations in KIT gene are present and proteins related to its signaling pathway as well as its post-transcriptional regulation. This study has aimed at evaluating in PA and ACC: (a) the proteins location of PI3K/AKT/mTOR and MAPK pathways using immunohistochemistry (IHC); (b) expression of miR-221 and miR-222, related to KIT gene; and (c) the association of these findings with clinical, pathological and survival data of patients. Results: In PA c-kit was positive in isolated luminal cells; in ACC, neoplastic luminal structures were positive for c-Kit. In PA, PI3K beta protein was shown to be partially positive in the cytoplasm of cells near the tumor capsule and phosphor AKT and phospho mTOR, are specifically expressed in epithelial cells and in a few myoepithelial. In ACC, PI3K and phosphor AKT protein showed to be negative in most of cases. Phospho mTOR protein was expressed in the cytoplasm of epithelial cells and some myoepithelial cells. In MAPK pathway, Ras, ERK1/2 and phosphor MEK-1 proteins were negative in most PAs and CACs; B-Raf and phospho MEK-2 were detected in luminal cells of PA. In ACC neoplastic luminal structures were positive for phospho MEK-2; B-Raf was also positive in myoepithelial and epithelial cells. In addition, cases with expressed phospho-mTOR and phosphor MEK-2 proteins were significantly associated with higher cancer-specific survival (p = 0.040 and p = 0.005, respectively). Moreover, expression of miR-221 was detected in 13 CAC samples and 19 PA samples. In CAC, expression of miR-221 was downregulated in 30,77% of the samples, upregulated in 30,77% samples, and normal in 38,46% samples. In PA, miR-221 expression was downregulated in 36,84% samples, upregulated in 10,53% samples, and normal in 52,63% samples. Expression of miR-222 was detected in 14 CAC samples and 22 PA samples. In the majority of CAC samples, the expression of miR-222 was similar to that observed in non-neoplastic samples. In PA samples, expression of miR-222 was downregulated in 31,8% samples, upregulated in 36,4% samples, and normal in 31,8% samples. Conclusion: Although c-Kit expression is detected in PA and ACC, its influence on the MAPK e PI3K/AKT/mTOR signaling cascades remains to be established. miR-221 e -222 did not show a robust correlation with c-Kit expression in the tumors studied.

Ki-67 and p53 immunostaining assessment of proliferative activity in salivary tumors.

Salivary gland tumors are rare neoplasias with approximately 34 different histological types. Because they have a considerably histological and biological behavior variability, salivary gland tumors represent a challenge both for the pathologist and the surgeon regarding their diagnosis, prognosis and treatment. Evaluation of mitotic index in case of Ki-67 and p53 expression has proved to be useful in predicting the biological aggressiveness in many tumors. In this study, we have analyzed the p53 and Ki-67 immunohistochemical expressions in 40 cases of salivary gland tumors, their correlations with clinicopathological factors and the prognostic relevance and diagnostic value of the results obtained. We analyzed eight pleomorphic adenomas (PA), seven Warthin tumors (WT), five basal cell adenomas (BA), four carcinomas ex pleomorphic adenoma (CEPA), four mucoepidermoid carcinomas (MEC), four acinic cell carcinomas (AC), four adenoid cystic carcinomas (ACC) and four adenocarcinomas not otherwise specified (ADK NOS). p53 positive staining was detected in 18 of the 40 cases studied, with higher expression in the malignant salivary tumors investigated. Ki-67 was expressed in 29 cases. High p53 and Ki-67 expression was noted in 3/4 CEPA, 3/4 ADK NOS and 2/4 MEC. Also, 2/8 PA, 3/7 WT and 2/5 BA were p53 positive and 2/7 WT and 2/5 BA had high Ki-67 mitotic index. The investigation of p53 and Ki-67 expression is useful in identifying highly proliferative forms of salivary tumors, with aggressive potential of evolution. The evaluation of these proliferative markers seems to have a prognostic value for CEPA, ADK NOS and MEC types of salivary tumors.

Diagnostic yield of ThinPrep preparation in the assessment of fine-needle aspiration biopsy of salivary gland neoplasms.

BACKGROUND: Fine-needle aspiration (FNA) has been widely recognized as an important modality in assessment of salivary gland neoplasms, and specimens are often processed as conventional smears. We conducted the current study to evaluate the diagnostic utility of ThinPrep preparation as an alternative method for assessment of salivary gland neoplasms. METHODS: A computer SNOMED search from the pathology database at our institution between July 1999 and June 2012 was conducted to identify FNA cytology specimens of salivary gland lesions for which follow-up surgical specimens revealed neoplasms. The FNA specimens were divided into two cohorts: one cohort consisted solely of the specimens in which all needle passes were collected into CytoLyt solution and only ThinPrep slides were prepared; and the other cohort included the specimens prepared with conventional smears. Diagnostic performance of the two cohorts was compared. RESULTS: Nondiagnostic rate of ThinPrep preparation was significantly higher than that of conventional smears (40% vs.18%; P <0.001). Among the diagnostic specimens, although more indeterminate diagnoses were generated in ThinPrep preparation compared to conventional smears (40% vs. 26%; P = 0.024), absolute cytohistologic concordant rate for the positive cases (type of neoplasms specified) is similar between the two preparations (80% vs. 86%; P = 0.354). Furthermore, there is no significant difference in rate of accurate diagnosis (correct typing of benign versus malignant neoplasm) between the two preparations (70% vs. 81%; P = 0.057). CONCLUSIONS: ThinPrep may be considered as another practical method of specimen preparation in the assessment of salivary gland neoplasms, particularly when FNA is performed without immediate assistance from cytology.