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Alpha-particle emitting radionuclides continue to be the subject of medical research because of their high energy and short range of action that facilitate effective cancer therapies. Radium-224 (224Ra) is one such candidate that has been considered for use in combating micrometastatic disease. In our prior studies, a suspension of 224Ra-labeled calcium carbonate (CaCO3) microparticles was designed as a local therapy for disseminated cancers in the peritoneal cavity. The progenies of 224Ra, of which radon-220 (220Rn) is the first, together contribute three of the four alpha particles in the decay chain. The proximity of the progenies to the delivery site at the time of decay of the 224Ra-CaCO3 microparticles can impact its therapeutic efficacy. In this study, we show that the diffusion of 220Rn was reduced in labeled CaCO3 suspensions as compared with cationic 224Ra solutions, both in air and liquid volumes. Furthermore, free-floating lead-212 (212Pb), which is generated from released 220Rn, had the potential to be re-adsorbed onto CaCO3 microparticles. Under conditions mimicking an in vivo environment, more than 70% of the 212Pb was adsorbed onto the CaCO3 at microparticle concentrations above 1 mg/mL. Further, the diffusion of 220Rn seemed to occur whether the microparticles were labeled by the surface adsorption of 224Ra or if the 224Ra was incorporated into the bulk of the microparticles. The therapeutic benefit of differently labeled 224Ra-CaCO3 microparticles after intraperitoneal administration was similar when examined in mice bearing intraperitoneal ovarian cancer xenografts. In conclusion, both the release of 220Rn and re-adsorption of 212Pb are features that have implications for the radiotherapeutic use of 224Ra-labeled CaCO3 microparticles. The release of 220Rn through diffusion may extend the effective range of alpha-particle dose deposition, and the re-adsorption of the longer lived 212Pb onto the CaCO3 microparticles may enhance the retention of this nuclide in the peritoneal cavity.

Pheochromocytoma and paragangliomas (PPGLs) are rare neuroendocrine tumors carrying 25–40% pathogenic germline gene variants (PGVs). We evaluated clinical, laboratory, and germline molecular profile of 115 patients with pathologic (14 patients were relatives from 8 different families recruited for genetic survey) confirmed PPGL followed in our institution. Patients with classic MEN2A/MEN2B phenotypes and at-risk relatives underwent direct analysis of RET proto-oncogene, and the remaining had samples submitted to complete next-generation sequencing aiming 23 PPGL-related genes: ATM, ATR, CDKN2A, EGLN1, FH, HRAS, KIF1B, KMT2D, MAX, MDH2, MERTK, MET, NF1, PIK3CA, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TMEM127, TP53, and VHL. We also developed a clinical judgment score (CJS) to determine the probability of patients having a potentially hereditary disease. The resulting genetic landscape showed that 67 patients (58.3%) had variants in at least one gene: 34 (50.7%) had exclu sively pathogenic or likely pathogenic variants, 13 (19.4%) had pathogenic or likely pathogenic variants and variant of undetermined significance (VUS), and 20 (29.8%) carried only VUS. PGVs were found in RET (n = 18; 38.3%), VHL (n = 10; 21.3%), SDHB and NF1 (n = 8; 17% each), and MAX, SDHD, TMEM127, and TP53 (n = 1; 2.1% each). Direct genetic testing disclosed 91.3% sensitivity, 81.2% specificity, and 76.4% and 93.3% positive predictive value (PPV) and negative predictive values (NPV), respectively. The CJS to identify patients who would not benefit from genetic testing had 75% sensitivity, 96.4% specificity, and 60% and 98.2% PPV and NPV, respectively. In summary, the landscape of PPGL germline gene variants from 115 Brazilian patients resulted in slightly higher prevalent pathogenic and likely pathogenic variants, especially in the RET gene. We suggest a CJS to identify PPGL patients who would not require initial genetic evaluation, improving test specificity and reducing costs.

Hypertension (HT) during pregnancy is not an infrequent obstetric problem, reaching a prevalence of 5-10%. This condition is highly associated with both maternal and fetal complications if not precisely diagnosed and managed. Even though primary HT, obesity, and preeclampsia are the main causes of HT in this period, other less familiar conditions must be considered during the investigation. Pheochromocytoma and paraganglioma (PPGL) are chromaffin cell tumors that produce, store, and secrete catecholamines, leading to HT and other adrenergic manifestations. Recognition of PPGL is crucial since misdiagnosis and improper management can lead to high morbidity and mortality, particularly during pregnancy. We report on two cases of PPGL diagnosed during pregnancy with different managements. Case 1 is a 25-year-old female at 31 weeks of first pregnancy, whose severe HT and life-threatening symptoms prompted an emergency delivery without previous confirmation or medical treatment of a suspected PPGL. After confirmation, a right adrenal PPGL was surgically resected 4 months later, following 15 days of medical therapy. Case 2 is a 22-year-old female at 18 weeks of pregnancy whose symptomatic PPGL was resected in the second trimester. A next-generation sequencing panel, including 23 PPGL-related genes, found no germline pathogenic variants (GPVs) in case 1 and an exon 1-4 germinative heterozygous deletion of the MAX gene in case 2. Despite the different medical approaches, both cases had satisfactory outcomes. Although uncommon, PPGL should be considered in the differential diagnosis of HT in pregnancy since missing the diagnosis and failing to introduce appropriate and timely treatment may lead to dramatic consequences for the mother and fetus. PPGL diagnosed during reproductive age is likely to result from GPV, prompting genetic investigation and counseling.

ABSTRACT Purpose: To determine molecular events involved in the tumorigenesis of phyllodes tumors (PT) and the role of each stromal (SC) and epithelial (EC) cell. Methods: Frozen breast samples enriched with epithelial and stromal cells from three fibroadenomas and 14 PT were retrieved and laser microdissected. Sanger and polymerase chain reaction-based sequencing of exon 2 MED12 and TERT promoter hotspot mutations were performed; 44K microarray platform was used to analyze gene expression. Results: All three fibroadenomas (FAs) presented mutations in MED12, but not in TERT, whose mutation was observed in five of the 14 PTs. EC and SC of each affected tumor displayed identical alterations. Of the total differentially expressed genes (DEG) (EC = 1,543 and SC = 850), 984 were EC-eDEGs and 291 were SC-eDEGs. We found a high similarity of diseases and functions enriched by both cell types, but dissimilarity in the number of enriched canonical pathways. Three signaling canonical pathways overlapping with EC and SC were predicted to be activated in one cell type and inactivated in the other, while no overlap in eDEGs was assigned to them. We also identified 13 EC-eDEGs and five SC-eDEGs enriched networks, in which the SC-eDEGs were able to segregate FA from PT samples. Conclusions: Identical TERT mutations from both SC and ES origins might affect the PTs tumorigenesis. Gene expression differences suggest coordinated molecular processes between these components with determinant differences acquired by SC, able to fully distinguish PTs from FAs lesions.

Molecular profile of breast cancer in Latin-American women was studied in five countries: Argentina, Brazil, Chile, Mexico, and Uruguay. Data about socioeconomic characteristics, risk factors, prognostic factors, and molecular subtypes were described, and the 60-month overall cumulative survival probabilities (OS) were estimated. From 2011 to 2013, 1,300 eligible Latin-American women 18 years or older, with a diagnosis of breast cancer in clinical stage II or III, and performance status ≦̸1 were invited to participate in a prospective cohort study. Face-to-face interviews were conducted, and clinical and outcome data, including death, were extracted from medical records. Unadjusted associations were evaluated by Chi-squared and Fisher’s exact tests and the OS by Kaplan–Meier method. Log-rank test was used to determine differences between cumulative probability curves. Multivariable adjustment was carried out by entering potential confounders in the Cox regression model. The OS at 60 months was 83.9%. Multivariable-adjusted death hazard differences were found for women living in Argentina (2.27), Chile (1.95), and Uruguay (2.42) compared with Mexican women, for older (≥60 years) (1.84) compared with younger (≤40 years) women, for basal-like subtype (5.8), luminal B (2.43), and HER2-enriched (2.52) compared with luminal A subtype, and for tumor clinical stages IIB (1.91), IIIA (3.54), and IIIB (3.94) compared with stage IIA women. OS was associated with country of residence, PAM50 intrinsic subtype, age, and tumor stage at diagnosis. While the latter is known to be influenced by access to care, including cancer screening, timely diagnosis and treatment, including access to more effective treatment protocols, it may also influence epigenetic changes that, potentially, impact molecular subtypes. Data derived from heretofore understudied populations with unique geographic ancestry and sociocultural experiences are critical to furthering our understanding of this complexity.

PurposesMost molecular-based published studies on breast cancer do not adequately represent the unique and diverse genetic admixture of the Latin American population. Searching for similarities and differences in molecular pathways associated with these tumors and evaluating its impact on prognosis may help to select better therapeutic approaches.Patients and MethodsWe collected clinical, pathological, and transcriptomic data of a multi-country Latin American cohort of 1,071 stage II-III breast cancer patients of the Molecular Profile of Breast Cancer Study (MPBCS) cohort. The 5-year prognostic ability of intrinsic (transcriptomic-based) PAM50 and immunohistochemical classifications, both at the cancer-specific (OSC) and disease-free survival (DFS) stages, was compared. Pathway analyses (GSEA, GSVA and MetaCore) were performed to explore differences among intrinsic subtypes.ResultsPAM50 classification of the MPBCS cohort defined 42·6% of tumors as LumA, 21·3% as LumB, 13·3% as HER2E and 16·6% as Basal. Both OSC and DFS for LumA tumors were significantly better than for other subtypes, while Basal tumors had the worst prognosis. While the prognostic power of traditional subtypes calculated with hormone receptors (HR), HER2 and Ki67 determinations showed an acceptable performance, PAM50-derived risk of recurrence best discriminated low, intermediate and high-risk groups. Transcriptomic pathway analysis showed high proliferation (i.e. cell cycle control and DNA damage repair) associated with LumB, HER2E and Basal tumors, and a strong dependency on the estrogen pathway for LumA. Terms related to both innate and adaptive immune responses were seen predominantly upregulated in Basal tumors, and, to a lesser extent, in HER2E, with respect to LumA and B tumors.ConclusionsThis is the first study that assesses molecular features at the transcriptomic level in a multicountry Latin American breast cancer patient cohort. Hormone-related and proliferation pathways that predominate in PAM50 and other breast cancer molecular classifications are also the main tumor-driving mechanisms in this cohort and have prognostic power. The immune-related features seen in the most aggressive subtypes may pave the way for therapeutic approaches not yet disseminated in Latin America.Clinical Trial RegistrationClinicalTrials.gov (Identifier: NCT02326857).

Abstract Next-generation sequencing (NGS) has altered clinical genetic testing by widening the access to molecular diagnosis of genetically determined rare diseases. However, physicians may face difficulties selecting the best diagnostic approach. Our goal is to estimate the rate of possible molecular diagnoses missed by different targeted gene panels using data from a cohort of patients with rare genetic diseases diagnosed with exome sequencing (ES). For this purpose, we simulated a comparison between different targeted gene panels and ES: the list of genes harboring clinically relevant variants from 158 patients was used to estimate the theoretical rate of diagnoses missed by NGS panels from 53 different NGS panels from eight different laboratories. Panels presented a mean rate of missed diagnoses of 64% (range 14%-100%) compared to ES, representing an average predicted sensitivity of 36%. Metabolic abnormalities represented the group with highest mean of missed diagnoses (86%), while seizure represented the group with lowest mean (46%). Focused gene panels are restricted in covering select sets of genes implicated in specific diseases and they may miss molecular diagnoses of rare diseases compared to ES. However, their role in genetic diagnosis remains important especially for well-known genetic diseases with established genetic locus heterogeneity.

The majority of the hereditary triple-negative breast cancers (TNBCs) are associated with BRCA1 germline mutations. Nevertheless, the understanding of the role of BRCA1 deficiency in the TNBC tumorigenesis is poor. In this sense, we performed whole-exome sequencing of triplet samples (leucocyte, tumor, and normal-adjacent breast tissue) for 10 cases of early-onset TNBC, including 5 hereditary (with BRCA1 germline pathogenic mutation) and 5 sporadic (with no BRCA1 or BRCA2 germline pathogenic mutations), for assessing the somatic mutation repertoire. Protein-affecting somatic mutations were identified for both mammary tissues, and Ingenuity Pathway Analysis was used to investigate gene interactions. BRCA1 and RAD51C somatic promoter methylation in tumor samples was also investigated by bisulfite sequencing. Sporadic tumors had higher proportion of driver mutations (≥25% allele frequency) than BRCA1 hereditary tumors, whereas no difference was detected in the normal breast samples. Distinct gene networks were obtained from the driver genes in each group. The Cancer Genome Atlas data analysis of TNBC classified as hereditary and sporadic reinforced our findings. The data presented here indicate that in the absence of BRCA1 germline mutations, a higher number of driver mutations are required for tumor development and that different defective processes are operating in the tumorigenesis of hereditary and sporadic TNBC in young women.

Objectives: Approximately 60% of lung adenocarcinomas (LAs) carry mutations that can guide treatment with tyrosine-kinase inhibitors (TKI) and other targeted therapies. Data on activating mutations in EGFR and other tyrosine-kinase receptor (TKR) genes in highly admixed populations, such as that of Brazil, are scarce. In this study, we comprehensively analyzed the actionable alteration profile of LA in Brazilian patients.Materials and Methods:EGFR driver mutation data were collected from a large Brazilian LA cohort covering an 8-year period of molecular testing in a single institution. Tests were performed using three distinct methods, and demographic and histopathological data were analyzed. For a subset of patients, driver mutations in KRAS, NRAS, and BRAF and gene fusions involving TKR genes (before TKI treatment) and EGFR T790M (after TKI treatment) were assessed.Results:EGFR mutations were detected in 25% of 1,316 LAs evaluated, with exon 19 deletions and exon 21 L858R TKI sensitizing mutations representing 72.5% of all mutations. Mutation rates were higher in women and non-smokers (p < 0.001). Next-generation sequencing was very sensitive, with a lower rate of inconclusive results compared with Sanger sequencing and pyrosequencing. EGFR/RAS/BRAF hotspot gene panels were applied in 495 LA cases and detected oncogenic mutations in 51.3% of samples, most frequently in EGFR (22.4%) and KRAS (26.9%). In subgroups of 36 and 35 patients, gene fusions were detected in 11.1% of tumors and EGFR T790M resistance mutations were detected in 59% of plasma samples from patients previously treated with TKI, respectively.Conclusion: This report provides the first comprehensive actionable alteration portrait of LA in Brazil. The high rate of actionable alterations in EGFR and other driver genes in LA reinforces the need to incorporate TKI guided by molecular diagnostics into clinical routines for patients in both public and private healthcare systems.

Abstract Next-generation sequencing (NGS) platforms allow the analysis of hundreds of millions of molecules in a single sequencing run, revolutionizing many research areas. NGS-based microRNA studies enable expression quantification in unprecedented scale without the limitations of closed-platforms. Yet, whereas a massive amount of data produced by these platforms is available, comparisons of quantification/discovery capabilities between platforms are still lacking. Here we compare two NGS-platforms: SOLiD and PGM, by evaluating their microRNA identification/quantification capabilities using two breast-derived cell-lines. A high expression correlation (R2 > 0.9) was achieved, encompassing 97% of the miRNAs, and the few discrepancies in miRNA counts were attributable to molecules that have very low expression. Quantification divergences indicative of artefactual representation were seen for 14 miRNAs (higher in SOLiD-reads) and another 10 miRNAs more abundant in PGM-data. An inspection of these revealed an increased and statistically significant count of uracyls and uracyl-stretches for PGM-enriched miRNAs, compared to SOLiD and to the miRBase. In parallel, adenines and adenine-stretches were enriched for SOLiDderived miRNA reads. We conclude that, whereas both platforms are overall consistent and can be used interchangeably for microRNA expression studies, particular sequence features appear to be indicative of specific platform bias, and their presence in microRNAs should be considered for database-analyses.

Chondrosarcoma originates in the bones of the head and neck. It is an unusual neoplasm that is slow-growing and represents only 1–3% of all cases of chondrosarcoma. Here, we report a case of a 45-year-old male Caucasian patient treated at Hospital Amaral Carvalho with a history of swelling of the face and a tumoral mass in the right maxilla with infiltration into the skin, which had been present for 4 months. A computerized tomography (CT) of the face and sinuses demonstrated a lesion in the right maxilla. A maxilectomia without orbital exenteration was performed. It was diagnosed as a grade III chondrosarcoma, with infiltration into the subjacent bone, anterior wall of the maxillary sinus and floor of the orbit. The patient presented with recurrence of the tumor after adjuvant therapies. A molecular study on the present case showed an unusually large number of abnormalities. This finding demonstrated extreme chromosomal instability, which was likely due to the undifferentiation of the tumor. Although there are no cases in the literature with which to compare, these findings may elucidate potential therapeutic targets for advanced tumors without other therapeutic options. Keywords: Chondrosarcoma, Head and neck, Maxilectomia, Molecular study, Chromosomal instability, Tumor undifferentiation, Pathology, Cytology

We report the first quantitative and qualitative analysis of the poly (A)⁺ transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.