Molecular profile of driver genes in lung adenocarcinomas of Brazilian patients who have never smoked: implications for targeted therapies.

INTRODUCTION: Lung cancer in never-smoker (LCINS) patients accounts for 20% of lung cancer cases, and its biology remains poorly understood, particularly in genetically admixed populations. We elucidated the molecular profile of driver genes in Brazilian LCINS. METHODS: The mutational and gene fusion status of 119 lung adenocarcinomas from self-reported never-smoker patients, was assessed using targeted sequencing (NGS), nCounter, and immunohistochemistry. A panel of 46 ancestry-informative markers determined patients' genetic ancestry. RESULTS: The most frequently mutated gene was EGFR (49.6%), followed by TP53 (39.5%), ALK (12.6%), ERBB2 (7.6%), KRAS (5.9%), PIK3CA (1.7%), and less than 1% alterations in RET, NTRK1, MET∆ex14, PDGFRA, and BRAF. Except for TP53 and PIK3CA, all other alterations were mutually exclusive. Genetic ancestry analysis revealed a predominance of European (71.1%), and a higher African ancestry was associated with TP53 mutations. CONCLUSION: Brazilian LCINS exhibited a similar molecular profile to other populations, except the increased ALK and TP53 alterations. Importantly, 73% of these patients have actionable alterations that are suitable for targeted treatments.

Comprehensive microRNA expression analysis of pediatric gonadal germ cell tumors: unveiling novel biomarkers and signatures.

microRNAs (miRNAs) are small endogenous noncoding RNAs, and alterations in their expression may contribute to oncogenesis. Discovering a unique miRNA pattern holds the potential for early detection and novel treatment possibilities in cancer. This study aimed to evaluate miRNA expression in pediatric patients with gonadal germ cell tumors (GCTs), focusing on characterizing the miRNA profiles of each histological subtype and identifying a distinct histological miRNA signature for a total of 42 samples of pediatric gonadal GCTs. The analysis revealed distinct miRNA expression profiles for all histological types, regardless of the primary site. We identified specific miRNA expression signatures for each histological type, including 34 miRNAs for dysgerminomas, 13 for embryonal carcinomas, 25 for yolk sac tumors, and one for immature teratoma, compared to healthy controls. Furthermore, we identified 26 miRNAs that were commonly expressed in malignant tumors, with six miRNAs (miR-302a-3p, miR-302b-3p, miR-371a-5p, miR-372-3p, miR-373-3p, and miR-367-3p) showing significant overexpression. Notably, miR-302b-3p exhibited a significant association with all the evaluated clinical features. Our findings suggest that miRNAs have the potential to aid in the diagnosis, prognosis, and management of patients with malignant GCTs.

Identifying malignant mesothelioma by a pathological survey using the São Paulo state hospital cancer registry, Brazil.

OBJECTIVE: To review the pathological diagnosis of possible cases and/or hidden cases of malignant mesothelioma (MM) between 2000 and 2012 using the Hospital-Based Cancer Registry database in the state of São Paulo, Brazil. METHODS: Possible cases were retrieved by assessing the database. Inclusion criteria were being older than 30 years of age and having ICD-O-3 topography and morphology codes related to MM. A board of expert pathologists reviewed the pathology reports and requested paraffin blocks in cases that demanded revision. After staining with calretinin, D2-40, WT-1 (as positive MM markers) and Ber-EP4 and MOC31 (as negative MM markers), cases were divided and studied independently by a pair of pathologists to confirm or discard the diagnosis of MM. RESULTS: Our sample comprised 482 cases from 25 hospitals, and 130 needed further histological revision. We received 73 paraffin blocks with adequate material. After board analysis, there were 9 cases with a definitive diagnosis of MM, improving the diagnostic rate in 12%. Two cases of previously diagnosed MM were discarded by review. CONCLUSIONS: Our results confirm that part of MM underdiagnosis and underreporting in Brazil is due to incomplete or mistaken pathological diagnosis.

Identifying malignant mesothelioma by a pathological survey using the São Paulo state hospital cancer registry, Brazil

ABSTRACT Objective: To review the pathological diagnosis of possible cases and/or hidden cases of malignant mesothelioma (MM) between 2000 and 2012 using the Hospital-Based Cancer Registry database in the state of São Paulo, Brazil. Methods: Possible cases were retrieved by assessing the database. Inclusion criteria were being older than 30 years of age and having ICD-O-3 topography and morphology codes related to MM. A board of expert pathologists reviewed the pathology reports and requested paraffin blocks in cases that demanded revision. After staining with calretinin, D2-40, WT-1 (as positive MM markers) and Ber-EP4 and MOC31 (as negative MM markers), cases were divided and studied independently by a pair of pathologists to confirm or discard the diagnosis of MM. Results: Our sample comprised 482 cases from 25 hospitals, and 130 needed further histological revision. We received 73 paraffin blocks with adequate material. After board analysis, there were 9 cases with a definitive diagnosis of MM, improving the diagnostic rate in 12%. Two cases of previously diagnosed MM were discarded by review. Conclusions: Our results confirm that part of MM underdiagnosis and underreporting in Brazil is due to incomplete or mistaken pathological diagnosis.

Disruptive and Truncating TP53 Mutations Are Associated with African-Ancestry and Worse Prognosis in Brazilian Patients with Lung Adenocarcinoma.

INTRODUCTION: TP53 is the most frequently mutated gene in lung tumors, but its prognostic role in admixed populations, such as Brazilians, remains unclear. In this study, we aimed to evaluate the frequency and clinicopathological impact of TP53 mutations in non-small cell lung cancer (NSCLC) patients in Brazil. METHODS: We analyzed 446 NSCLC patients from Barretos Cancer Hospital. TP53 mutational status was evaluated through targeted next-generation sequencing (NGS) and the variants were biologically classified as disruptive/nondisruptive and as truncating/nontruncating. We also assessed genetic ancestry using 46 ancestry-informative markers. Analysis of lung adenocarcinomas from the cBioportal dataset was performed. We further examined associations of TP53 mutations with patients' clinicopathological features. RESULTS: TP53 mutations were detected in 64.3% (n = 287/446) of NSCLC cases, with a prevalence of 60.4% (n = 221/366) in lung adenocarcinomas. TP53 mutations were associated with brain metastasis at diagnosis, tobacco consumption, and higher African ancestry. Disruptive and truncating mutations were associated with a younger age at diagnosis. Additionally, cBioportal dataset revealed that TP53 mutations were associated with younger age and Black skin color. Patients harboring disruptive/truncating TP53 mutations had worse overall survival than nondisruptive/nontruncating and wild-type patients. CONCLUSION: TP53 mutations are common in Brazilian lung adenocarcinomas, and their biological characterization as disruptive and truncating mutations is associated with African ancestry and shorter overall survival.

Quality Assessment of Cryopreserved Human Biological Samples from the Biobank of Barretos Cancer Hospital.

Background: Biobanks process, store, and supply biological materials for research. Preanalytical factors, especially storage time and temperature, must be controlled and standardized at all stages when handling biospecimen samples, especially because the literature reports highly contradictory optimal parameters. As large-sample studies are required to better understand the influence of time and temperature on cryopreserved samples' quality for genomic research, this study evaluated the integrity and quality of cryopreserved samples stored for up to 9 years at the biobank of Barretos Cancer Hospital, one of the largest biobanks in Latin America. Methods: We randomly selected 447 samples with tumor tissue paired with buffy coat or peripheral blood mononuclear cells (PBMCs) that were stored from 2008 to 2016. The genetic material quality was evaluated based on RNA integrity (RIN) and DNA integrity (DIN) ≥7, which indicated undegraded samples, and compared with storage time, which means that for DNA storage time, samples <8.1 and ≥8.1 years and for RNA <4.5 and ≥4.5 were used. Results: A total of 190 tumor tissues were eligible for DNA and RNA extraction. Those stored for 8 years had lower DIN (68%) than those stored for a shorter period (92%). A similar pattern, based on storage time (<8.1 and ≥8.1 years), was observed in the buffy coat (74% and 95%, respectively) and PBMCs (54% and 96%, respectively). For RNA extracted from tumor tissues, we observed lower RIN in samples stored for 4.5 years (17%) than in samples stored for a shorter period (45%). Buffy coat and PBMC samples stored at -30°C exhibited greater degradation (26%) than those stored at -80°C (1%). The DIN (p = 0.15) and RNA (p = 0.18) were unrelated to topography type. Conclusion: The temperature, particularly cryopreservation methodology, and storage time were the main factors that affected nucleic acid integrity, especially RNA, during cryopreservation of biospecimens.

Proposing Specific Neuronal Epithelial-to-Mesenchymal Transition Genes as an Ancillary Tool for Differential Diagnosis among Pulmonary Neuroendocrine Neoplasms.

Pulmonary neuroendocrine neoplasms (PNENs) are currently classified into four major histotypes, including typical carcinoid (TC), atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC), and small cell lung carcinoma (SCLC). This classification was designed to be applied to surgical specimens mostly anchored in morphological parameters, resulting in considerable overlapping among PNENs, which may result in important challenges for clinicians' decisions in the case of small biopsies. Since PNENs originate from the neuroectodermic cells, epithelial-to-mesenchymal transition (EMT) gene expression shows promise as biomarkers involved in the genotypic transformation of neuroectodermic cells, including mutation burden with the involvement of chromatin remodeling genes, apoptosis, and mitosis rate, leading to modification in final cellular phenotype. In this situation, additional markers also applicable to biopsy specimens, which correlate PNENs subtypes with systemic treatment response, are much needed, and current potential candidates are neurogenic EMT genes. This study investigated EMT genes expression and its association with PNENs histotypes in tumor tissues from 24 patients with PNENs. PCR Array System for 84 EMT-related genes selected 15 differentially expressed genes among the PNENs, allowing to discriminate TC from AC, LCNEC from AC, and SCLC from AC. Functional enrichment analysis of the EMT genes differentially expressed among PNENs subtypes showed that they are involved in cellular proliferation, extracellular matrix degradation, regulation of cell apoptosis, oncogenesis, and tumor cell invasion. Interestingly, four EMT genes (MAP1B, SNAI2, MMP2, WNT5A) are also involved in neurological diseases, in brain metastasis, and interact with platinum-based chemotherapy and tyrosine-kinase inhibitors. Collectively, these findings emerge as an important ancillary tool to improve the strategies of histologic diagnosis in PNENs and unveil the four EMT genes that can play an important role in driving chemical response in PNENs.

ERBB2 exon 20 insertions are rare in Brazilian non-small cell lung cancer.

ERBB2 exon 20 insertions may impact the clinical management of lung cancer patients. However, the frequency of ERBB2 exon 20 insertions in lung cancer patients in Brazil is scarce. Here, we analyzed 722 Brazilian non-small cell lung cancer (NSCLC) patients from Barretos Cancer Hospital that were indicated to require routine lung cancer molecular testing. ERBB2 exon 20 insertions were evaluated by a targeted panel using next-generation sequencing (NGS). Clinicopathological and molecular data were collected from patient medical records. Among the 722 NSCLC patients, 85.2% had lung adenocarcinomas, 53.9% were male, 66.8% were quitter or current smokers, and 63.2% were diagnosed at an advanced stage of the disease. We identified 0.8% (6/722) of patients who harbored the insertion p.(Tyr772_Ala775dup) at exon 20 of the ERBB2 gene. All ERBB2 mutated patients were diagnosed with lung adenocarcinoma, were never smokers, and wild-type for EGFR, KRAS, and ALK hotspot alterations. Less than 1% of Brazilian NSCLC patients harbor ERBB2 exon 20 insertions, yet they could benefit in future from the new drugs in development.

EGFR Mutations and PD-L1 Expression in Early-Stage Non-Small Cell Lung Cancer: A Real-World Data From a Single Center in Brazil.

BACKGROUND: Targeted and immunotherapies are currently moving toward early-stage settings for patients with non-small cell lung cancer (NSCLC). Predictive biomarkers data are scarce in this scenario. We aimed to describe the frequency of EGFR mutations and PD-L1 expression levels in early-stage non-squamous patients with NSCLC from a large, single Brazilian oncology center. METHODS: We retrospectively evaluated patients with NSCLC diagnosed at an early-stage (IB to IIIA-AJCC seventh edition) at Barretos Cancer Hospital (n = 302). EGFR mutational status was assessed in FFPE tumor tissues using distinct methodologies (NGS, Cobas, or Sanger sequencing). PD-L1 expression was evaluated by immunohistochemistry (clone 22C3) and reported as Tumor Proportion Score (TPS), categorized as <1%, 1-49%, and ≥50%. We evaluated the association between EGFR mutational status and PD-L1 expression with sociodemographic and clinicopathological parameters by Fisher's test, qui-square test, and logistic regression. Survival analysis was assessed by the Kaplan-Meier method and Cox regression model. RESULTS: EGFR mutations were detected in 17.3% (n = 48) of cases and were associated with female sex, never smokers, and longer overall and event-free survival. PD-L1 positivity was observed in 36.7% (n = 69) of cases [TPS 1-49% n = 44(23.4%); TPS ≥50% n = 25(13.3%)]. PD-L1 positivity was associated with smoking, weight loss, and higher disease stages (IIB/IIIA). CONCLUSION: The frequencies of EGFR mutations and PD-L1 positivity were described for early-stage non-squamous patients with NSCLC. These results will be essential for guiding treatment strategies with the recent approvals of osimertinib and immunotherapy in the adjuvant setting.

Microsatellite Instability Is Rare in the Admixed Brazilian Population of Non-Small Cell Lung Cancer: A Cohort of 526 Cases.

BACKGROUND: Microsatellite instability (MSI) in non-small cell lung cancer (NSCLC) is uncommon; however, most studies refer to European and Asian populations. There are currently no data on MSI frequency in highly admixed populations, such as the one represented by Brazilian NSCLC patients. AIM: This study aimed to evaluate the frequency of MSI in Brazilian NSCLC patients. METHODS: We evaluated 526 patients diagnosed with NSCLC at the Barretos Cancer Hospital (Brazil). The molecular MSI evaluation was performed using a hexa-plex marker panel by polymerase chain reaction followed by fragment analysis. The mutation profile of MSI-positive cases was performed using next-generation sequencing. RESULTS: Only 1 patient was MSI positive (0.19%). This patient was a female, white, and active smoker, and she was diagnosed with clinical stage IV lung adenocarcinoma at 75 years old. The molecular profile exhibited 4 Tumor Protein p53 (TP53) mutations and the absence of actionable mutations in the Epidermal Growth Factor Receptor (EGFR), Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS), or V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) genes. CONCLUSIONS: The frequency of MSI in Brazilian NSCLC patients is equally rare, a finding that is consistent with the current literature based on other populations such as Europeans, North Americans, and Asians.

Pulmonary Neuroendocrine Neoplasms Overexpressing Epithelial-Mesenchymal Transition Mechanical Barriers Genes Lack Immune-Suppressive Response and Present an Increased Risk of Metastasis.

Typical carcinoids (TC), atypical carcinoids (AC), large cell neuroendocrine carcinomas (LCNEC), and small cell lung carcinomas (SCLC) encompass a bimodal spectrum of metastatic tumors with morphological, histological and histogenesis differences, The hierarchical structure reveals high cohesiveness between neoplastic cells by mechanical desmosomes barrier assembly in carcinoid tumors and LCNEC, while SCLC does not present an organoid arrangement in morphology, the neoplastic cells are less cohesive. However, the molecular mechanisms that lead to PNENs metastasis remain largely unknown and require further study. In this work, epithelial to mesenchymal transition (EMT) transcription factors were evaluated using a set of twenty-four patients with surgically resected PNENs, including carcinomas. Twelve EMT transcription factors (BMP1, BMP7, CALD1, CDH1, COL3A1, COL5A2, EGFR, ERBB3, PLEK2, SNAI2, STEAP1, and TCF4) proved to be highly expressed among carcinomas and downregulated in carcinoid tumors, whereas upregulation of BMP1, CDH2, KRT14 and downregulation of CAV2, DSC2, IL1RN occurred in both histological subtypes. These EMT transcription factors identified were involved in proliferative signals, epithelium desmosomes assembly, and cell motility sequential steps that support PNENs invasion and metastasis in localized surgically resected primary tumor. We used a two-stage design where we first examined the candidate EMT transcription factors using a whole-genome screen, and subsequently, confirmed EMT-like changes by transmission electron microscopy and then, the EMT-related genes that were differentially expressed among PNENs subtypes were predicted through a Metascape analysis by in silico approach. A high expression of these EMT transcription factors was significantly associated with lymph node and distant metastasis. The sequential steps for invasion and metastasis were completed by an inverse association between functional barrier created by PD-L1 immunosuppressive molecule and EMT transcriptional factors. Our study implicates upregulation of EMT transcription factors to high proliferation rates, mechanical molecular barriers disassembly and increased cancer cell motility, as a critical molecular event leading to metastasis risk in PNENs thus emerging as a promising tool to select and customize therapy.

Expression of Glycolysis-Related Proteins in Cancer of Unknown Primary Origin.

INTRODUCTION: Cancer of unknown primary origin (CUP) is defined as metastatic cancer without identification of the primary site. Considering that only 15-20% of patients with CUP show a favorable outcome, identifying biomarkers may help improve the clinical management of patients who do not respond well to conventional therapies. In this context, the study of the metabolic profile of CUP may pave the way to establish new biomarkers and/or therapeutic targets; therefore, this study aimed to characterize the expression of metabolism-related proteins in CUP. MATERIALS AND METHODS: The expression of monocarboxylate transporters MCT1, MCT2 and MCT4, their chaperone CD147, the glucose transporter GLUT1 and the pH regulator CAIX was evaluated by immunohistochemistry in a series of 118 CUP patients, and the results were associated with the available clinicopathological information. RESULTS: The metabolism-related proteins MCT1, MCT4, CD147, GLUT1 and CAIX were expressed in a critical portion of the CUP (approximately 20 to 70%). MCT1 and CD147 were both more frequently expressed in cases with lymph nodes as metastasis dominant sites (p = 0.001) as well as in samples from lymph nodes (p <0.001 and p = 0.002, respectively), while MCT1 expression was more frequently expressed in squamous cell carcinomas (p = 0.045). A higher overall survival was observed in patients with tumors positive for GLUT1 and CAIX expression (p = 0.011 and p = 0.041, respectively), but none of the proteins was an independent prognostic factor for overall survival in multivariable analysis. CONCLUSION: The results suggest that a portion of CUPs present a hyperglycolytic phenotype, which is associated with higher overall survival.

Simultaneous analysis of ALK, RET, and ROS1 gene fusions by NanoString in Brazilian lung adenocarcinoma patients.

BACKGROUND: Gene fusions have been successfully employed as therapeutic targets for lung adenocarcinoma. However, tissue availability for molecular testing of multiples alterations is frequently unfeasible. We aimed to detect the presence of ALK, RET, and ROS1 rearrangements by a RNA-based single assay in Brazilian lung adenocarcinomas and to associate with clinicopathological features and genetic ancestry. METHODS: From a FFPE series of 444 molecularly characterized lung adenocarcinomas, 253 EGFR/KRAS wild-type cases were eligible for gene rearrangement analysis. Following RNA isolation, ALK, RET, and ROS1 rearrangements were simultaneously analyzed employing the ElementsXT Custom panel (NanoString Technologies). Rearrangements were further associated with clinicopathological features and genetic ancestry of the patients. RESULTS: The NanoString platform was performed in subset of 142 cases. Gene fusion results were conclusive for 94.4% (n=134) cases (failure rate =5.6%). ALK rearrangements were observed in 21 out of 134 cases, and associated with younger, never smokers, metastatic disease, and metastases in the central nervous system. RET and ROS1 fusions were detected in two and one out of 134 cases, respectively. Genetic ancestry was not associated with gene fusions. Overall, considering all cases for which a molecular analysis was conclusive (EGFR/KRAS/ALK/RET/ROS1), ALK fusions frequency was observed in 6.5% (21/325), RET in 0.6% (2/325), and ROS1 in 0.3% (1/325). CONCLUSIONS: This study successfully used a RNA-based single assay for the simultaneous analysis of ALK, RET, and ROS1 fusions employing routine biopsies from Brazilian patients lung adenocarcinoma allowing an extensive molecular testing for actionable rearrangements contributing to guide clinical strategies.

PD-L1 expression by Tumor Proportion Score (TPS) and Combined Positive Score (CPS) are similar in non-small cell lung cancer (NSCLC).

BACKGROUND: For non-small cell lung cancer (NSCLC) the most used method for analysing programmed cell death ligand 1 (PD-L1) expression is the Tumor Proportion Score (TPS). Nevertheless, for other tumour types, the Combined Positive Score (CPS) has been the method of choice. AIM: Evaluate and compare the predictive value of both CPS and TPS as predictors of immunotherapy response in NSCLC, and to evaluate the agreement intra-observer between both methods and inter-observer between two expert lung pathologists. METHODS: 56 NSCLC patients who were treated with anti-programmed cell death 1 (PD-1)/PD-L1 therapy were included. Two pathologists evaluated all cases independently, considering the sample's adequacy for analysis, and the PD-L1 expression by TPS and CPS. RESULTS: The Kappa coefficient for adequacy was 0.82 (95% CI 0.67 to 0.97). There was a high agreement between TPS and CPS and a high agreement between pathologists concerning the two methods. The Kappa coefficient between TPS and CPS was 0.85 for both pathologists, and between pathologists was 0.94 and 0.93 for TPS and CPS, respectively. CONCLUSIONS: Both methods proved to be equally predictive of response to anti-PD-1/PD-L1 therapy. There was both a high intra-observer agreement between the two methods and a high inter-observer agreement between pathologists. This study suggests that CPS could also be used in a routine setting for immunotherapy decision in NSCLC.

Mutational Profiling of Driver Tumor Suppressor and Oncogenic Genes in Brazilian Malignant Pleural Mesotheliomas.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly lethal disease comprising a heterogeneous group of tumors with challenging to predict biological behavior. The diagnosis is complex, and the histologic classification includes 2 major subtypes of MPM: epithelioid (∼60% of cases) and sarcomatous (∼20%). Its identification depends upon pathological investigation supported by clinical and radiological evidence and more recently ancillary molecular testing. Treatment options are currently limited, with no known targeted therapies available. OBJECTIVES: To elucidate the mutation profile of driver tumor suppressor and oncogenic genes in a cohort of Brazilian patients. METHODS: We sequenced 16 driver genes in a series of 43 Brazilian malignant mesothelioma (MM) patients from 3 distinct Brazilian centers. Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor tissue blocks, and the TERT promoter region was amplified by PCR followed by direct capillary sequencing. The Illumina TruSight Tumor 15 was used to evaluate 250 amplicons from 15 genes associated with solid tumors (AKT1, GNA11, NRAS, BRAF, GNAQ, PDGFRA, EGFR, KIT, PIK3CA, ERBB2, KRAS, RET, FOXL2, MET,and TP53). Library preparation with the TruSight Tumor 15 was performed before sequencing at the MiSeq platform. Data analysis was performed using Sophia DDM software. RESULTS: Out of 43 MPM patients, 38 (88.4%) were epithelioid subtype and 5 (11.6%) were sarcomatoid histotype. Asbestos exposure was present in 15 (39.5%) patients with epithelioid MPM and 3 (60%) patients with sarcomatoid MPM. We found a TERT promoter mutation in 11.6% of MM, and the c.-146C>T mutation was the most common event. The next-generation sequencing was successful in 33 cases. A total of 18 samples showed at least 1 pathogenic, with a median of 1.8 variants, ranging from 1 to 6. The most mutated genes were TP53 and ERBB2 with 7 variants each, followed by NRAS BRAF, PI3KCA, EGFR and PDGFRA with 2 variants each. KIT, AKT1, and FOXL2 genes exhibited 1 variant each. Interestingly, 2 variants observed in the PDGFRA gene are classic imatinib-sensitive therapy. CONCLUSIONS: We concluded that Brazilian MPM harbor mutation in classic tumor suppressor and oncogenic genes, which might help in the guidance of personalized treatment of MPM.

Odontogenic Carcinoma With Dentinoid in Long-Term Follow-up With 2 Recurrences.

Dentinoid has been mentioned as a frequent component in several types of benign odontogenic tumors; however, there are some other very rare dentinoid-producing odontogenic tumors that have been described, which are not recognized in the current World Health Organization Histological Classification of Odontogenic Tumours. In this context, we report an unusual malignant odontogenic tumor containing dentinoid located in the left maxilla of a 41-year-old man. The lesion was initially diagnosed and treated as a cemento-ossifying fibroma. After 7 years, a tumor was noted at the same location and was diagnosed as pleomorphic adenoma. The patient developed a new lesion 2 years later. Histological features included an epithelial proliferation of basaloid and clear cells, some with peripheral palisading, which were scattered both in a fibrous stroma and within an amorphous eosinophilic dentinoid product. Because of doubts about the first 2 diagnoses and the current situation, all histopathological slides were reviewed in our service as a consultation case, and the findings were consistent with the diagnosis of an odontogenic carcinoma with dentinoid. Immunohistochemical analysis was performed and an ultrastructural study by scanning electronic microscopy and energy-dispersive X-ray microanalysis was made to characterize dentinoid material. After 1 year of follow-up, the patient is alive and free of the disease. This case highlights the wide variability regarding cytological evidence of malignancy, and adds a new case of odontogenic carcinoma with dentinoid, which represents a distinct entity with locally aggressive behavior and should be considered be included in a future World Health Organization Histological Classification of Tumours.

Mutational profile of Brazilian lung adenocarcinoma unveils association of EGFR mutations with high Asian ancestry and independent prognostic role of KRAS mutations.

Lung cancer is the deadliest cancer worldwide. The mutational frequency of EGFR and KRAS genes in lung adenocarcinoma varies worldwide per ethnicity and smoking. The impact of EGFR and KRAS mutations in Brazilian lung cancer remains poorly explored. Thus, we investigated the frequency of EGFR and KRAS mutations in a large Brazilian series of lung adenocarcinoma together with patients' genetic ancestry, clinicopathological and sociodemographic characteristics. The mutational frequency of EGFR was 22.7% and KRAS was 20.4%. The average ancestry proportions were 73.1% for EUR, 13.1% for AFR, 6.5% for AME and 7.3% for ASN. EGFR mutations were independently associated with never-smokers, high-Asian ancestry, and better performance status. KRAS mutations were independently associated with tobacco exposure and non-Asian ancestry. EGFR-exon 20 mutations were associated with worse outcome. The Cox regression model indicated a worse outcome for patients whose were older at diagnosis (>61 y), solid histological subtype, loss of weight (>10%), worse performance status (≥2), and presence of KRAS mutations and EGFR mutational status in TKi non-treated patients. In conclusion, we assessed the clinicopathological and ethnic impact of EGFR and KRAS mutations in the largest series reported of Brazilian lung adenocarcinomas. These findings can support future clinical strategies for Brazilian lung cancer patients.

Molecular Classification of Thyroid Nodules with Indeterminate Cytology: Development and Validation of a Highly Sensitive and Specific New miRNA-Based Classifier Test Using Fine-Needle Aspiration Smear Slides.

Background: Thyroid nodules can be identified in up to 68% of the population. Fine-needle aspiration (FNA) cytopathology classifies 20%-30% of nodules as indeterminate, and these are often referred for surgery due to the risk of malignancy. However, histological postsurgical reports indicate that up to 84% of cases are benign, highlighting a high rate of unnecessary surgeries. We sought to develop and validate a microRNA (miRNA)-based thyroid molecular classifier for precision endocrinology (mir-THYpe) with both high sensitivity and high specificity, to be performed on the FNA cytology smear slide with no additional FNA. Methods: The expression of 96 miRNA candidates from 39 benign/39 malignant thyroid samples, (indeterminate on FNA) was analyzed to develop and train the mir-THYpe algorithm. For validation, an independent set of 58 benign/37 malignant FNA smear slides (also classified as indeterminate) was used. Results: In the training set, with a 10-fold cross-validation using only 11 miRNAs, the mir-THYpe test reached 89.7% sensitivity, 92.3% specificity, 90.0% negative predictive value and 92.1% positive predictive value. In the FNA smear slide validation set, the mir-THYpe test reached 94.6% sensitivity, 81.0% specificity, 95.9% negative predictive value, and 76.1% positive predictive value. Bayes' theorem shows that the mir-THYpe test performs satisfactorily in a wide range of cancer prevalences. Conclusions: The presented data and comparison with other commercially available tests suggest that the mir-THYpe test can be considered for use in clinical practice to support a more informed clinical decision for patients with indeterminate thyroid nodules and potentially reduce the rates of unnecessary thyroid surgeries.

MGMT and CALCA promoter methylation are associated with poor prognosis in testicular germ cell tumor patients.

Testicular germ cell tumors (TGCT) represent the second main cause of cancer-related death in young men. Despite high cure rates, refractory disease results in poor prognosis. Epigenetic reprogramming occurs during the development of seminomas and non-seminomas. Understanding the molecular and genetic basis of these tumors would represent an important advance in the search for new TGCT molecular markers. Hence the frequency of methylation of a gene panel (VGF, MGMT, ADAMTS1, CALCA, HOXA9, CDKN2B, CDO1 and NANOG) was evaluated in 72 primary TGCT by quantitative methylation specific PCR. A high frequency of MGMT (90.9%, 20/22; p=0.019) and CALCA (90.5%, 19/21; p<0.026) methylation was associated with non-seminomatous tumors while CALCA methylation was also associated with refractory disease (47.4%, 09/19; p=0.005). Moreover, promoter methylation of both genes predicts poor clinical outcome for TGCT patients (5-year EFS: 50.5% vs 77.1%; p=0.032 for MGMT and 51.3% vs 77.0%; p=0.029 for CALCA). The findings of this study indicate that methylation of MGMT and CALCA are frequent and could be used as new molecular markers of prognosis in TGCT.

Hotspot TERT promoter mutations are rare events in testicular germ cell tumors.

The abnormal activation of telomerase, codified by the telomerase reverse transcriptase (TERT) gene, is related to one of cancer hallmarks. Hotspot somatic mutations in the promoter region of TERT, specifically the c.-124:C>T and c.-146:C>T, were recently identified in a range of human cancers and have been associated with a more aggressive behavior. Testicular germ cell tumors frequently exhibit a good prognosis; however, the development of refractory disease is still a clinical challenge. In this study, we aim to evaluate for the first time the presence of the hotspot telomerase reverse transcriptase gene promoter mutations in testicular germ cell tumors. A series of 150 testicular germ cell tumor cases and four germ cell tumor cell lines were evaluated by PCR followed by direct Sanger sequencing and correlated with patient's clinical pathological features. Additionally, we genotyped the telomerase reverse transcriptase gene promoter single nucleotide polymorphism rs2853669 (T>C) located at -245 position. We observed the presence of the TERT promoter mutation in four patients, one exhibited the c.-124:C>T and three the c.-146:C>T. No association between TERT mutation status and clinicopathological features could be identified. The analysis of the rs2853669 showed that variant C was present in 22.8 % of the cases. In conclusion, we showed for the first time that TERT promoter mutations occur in a small subset (~3 %) of testicular germ cell tumors.