A next-generation sequencing approach for the simultaneous study of Wilms tumors and parathyroid tumours

Wilms Tumor (WT), also known as nephroblastoma, is a rare kidney condition which affects 1 in ~10.000 infants and children. This tumor is of complex etiology with underlying causes still incompletely understood. The majority of known mutations in WT are of somatic origin, with approximately one-third of patients displaying mutations in WT1, CTNNB1, AMER1 (WTX) and/or TP53 genes. In contrast, familial predisposition mutations are very rare. Recently, we used Next-Generation Sequencing (NGS) technology and a customized amplicon panel (TruSeq Custom Amplicon, Illumina) to detect somatic mutations of WT1, AMER1, TP53 and CTNNB1 (exon 4) genes, and identified mutations in 11 of 36 patients (30.5%). These results prompted us to design a new sequencing panel which incorporates a larger set of genes and addresses the problems encountered with low or inexistent coverage for some amplicons. In addition to those 4 genes, the new panel also includes genes involved in the SIX1/SIX2 pathway, which are frequently mutated in WT patients with blastemal-type histology(1), microRNA-processing genes (DROSHA, DICER1, DGCR8, XPO5 and TARBP2), which were found to be mutated in over 10% of WT patients(2), as well as other genes (e.g., MYCN) previously known to be involved in Wilms tumorigenesis. Moreover, we included 6 other genes (CDC73, CTR9, PAF1, LEO1, RTF1 and WDR61), which encode subunits of the PAF1 complex. Among these genes, CTR9 is already known to be a WT-predisposition gene(3), and CDC73 is associated with the pathogenesis of the hereditary hyperparathyroidism-jaw tumour syndrome, and is also frequently mutated in sporadic parathyroid carcinomas. Using this new sequencing approach it will be possible to sequence genes involved in different types of familial cancer syndromes and sporadic tumours, simultaneously. Moreover, this strategy may also lead to identification of novel mutations of PAF1 complex genes, not yet known to be implicated in WT and parathyroid tumour development.N/

Chronic hypocalcemia due to anti-calcium sensing receptor antibodies

Introduction: Hypoparathyroidism is an entity associated with hypocalcemia, more frequently a consequence of neck surgery. An autoimmune etiology is rare and its diagnosis difficult to establish. Clinical report: 52 year-old woman, with irrelevant past medical history and no significant familial conditions, referred because of hypocalcemia and basal ganglia calcifications, detected in the course of investigation of myalgias. Besides hypocalcemia (4.6 mg/ dL), hyperphosphatemia (8.7 mg/dL), undetectable parathyroid hormone and low urinary calcium, phosphorus and magnesium were present. Molecular analysis of CaSR gene excluded germinal mutations. Anti-calcium sensing receptor antibodies (anti-CaSR) were present. The patient is asymptomatic and normocalcemic under treatment with calcium and vitamin D. Discussion: Although rare, hypocalcemia due to anti-CaSR hypoparathyroidism must be considered in the absence of previous neck surgery, hypocalcemic drugs, familial history or phenotype suggesting a genetic disorder. Low or undetectable parathyroid hormone excludes pseudohypoparathyroidism and anti-CaSR positivity establishes the diagnosis.publishersversionpublishe

a case report

personBACKGROUND: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive solid tumors. ATC is frequently diagnosed at advanced stages with unresectable disease and palliative care is often indicated. Recently, several patient-tailored therapies for ATC are emerging due to advances in molecular profiling of these tumors. Entrectinib is a potent oral selective inhibitor of neutrotrophic tropomyosin receptor kinase (NTRK), ROS1, and anaplastic lymphoma kinase fusions. The experience regarding ATC and other thyroid carcinomas, particularly in the neoadjuvant setting, is minimal. CASE REPORT: We present a case of a 51-year-old female patient presenting with a bulky mass of the left thyroid lobe measuring 100 × 108 × 80 mm that was considered surgically unresectable. While waiting for next-generation sequence (NGS) profiling, lenvatinib was initiated. There was an initial clinical and imagiologic response; however, progression occurred after 12 weeks, and at this time NGS identified an ETV6-NTRK3 fusion and entrectinib was started. After 12 weeks, tumor diameters reduced to a minimum of 68×60×49 mm, and the patient underwent total thyroidectomy plus central lymphadenectomy. Histological diagnosis confirmed an ATC (pT4a R2 N1a). Adjuvant radiotherapy (RT) (60 Grays) with weekly paclitaxel (45 mg/m2) was then administered followed by maintenance entrectinib 600 mg daily. Fluorodeoxyglucose positron emission tomography performed 3 months after completion of RT showed only non-specific uptake in the posterior wall of the hypopharynx and larynx, suggestive of inflammation. CONCLUSION: We report the first case of an ATC with a dramatic response to neoadjuvant therapy with entrectinib, which enabled surgical resection of an ab initio unresectable tumor.publishersversionpublishe

Coincidence or correlation?

Rationale:Multiple Endocrine Neoplasia type 1 (MEN1) is a familial syndrome that results from the disruption of a tumor suppressor protein called MENIN. Its management is challenging, as MEN1 affects different endocrine tissues and predisposes to both benign and malignant tumors. MENIN-deficient cells have recently been recognized to play a role in triggering autoimmunity. Herein, we present a case of MEN1 with multiple endocrine and autoimmune disorders.Patient concerns:A 50 years old female with a 25 years history of complicated nephrolithiasis presented with primary hyperparathyroidism.Diagnoses:Over several decades, she was diagnosed with recurrent primary hyperparathyroidism, autoimmune thyroiditis, multinodular goiter, pernicious anemia, metastatic gastric type 1 neuroendocrine tumor, macroprolactinemia, gonadotropin deficiency, mucosa-associated lymphoid tissue lymphoma of the thyroid gland, positive anti-calcium sensor receptor antibodies, and BRCA 1/2-negative invasive breast cancer. The autoimmune regulator gene was sequenced, but no pathogenic variants were found. Next-generation sequencing revealed both a pathogenic MEN1 mutation and a benign CDC73 gene variant. Familial genetic screening revealed a large kindred with multiple carriers of one or both genetic variants (MEN1 = 19; CDC73 = 7).Interventions:The patient underwent surgical excision of three parathyroid glands, total thyroidectomy and breast tumorectomy plus tamoxifen, and monthly injections of octreotide. The patient and family members with the MEN1 mutation are under a life-long surveillance program for MEN1 prototypic tumors.Outcomes:The patient was stable and alive during a 24-years follow-up period.Lessons:With the present case, the authors highlight a new interplay between MENIN and the immune system, which may have implications for future targeted life-long surveillance and treatment of MEN1 patients.publishersversionpublishe

a clinical and molecular study

Funding This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associated Laboratory LS4FUTURE (LA/P/0087/ 2020), by Associação de Endocrinologia Oncológica (AEO), and by Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG). Carolina Pires was granted with a PhD scholarship by FCT - 2020.07120.BD. Ricardo Rodrigues was granted with a PhD scholarship by iNOVA4Health Research Unit - UIDP/04462/2020; UI/BD/ 154256/2022.OBJECTIVES: Anaplastic thyroid carcinoma (ATC) has a poor survival. The combination of Dabrafenib plus Trametinib (DT) had a significant impact in survival of BRAF p.V600E patients. However, durable responses may be compromised by resistance. We aim to present our experience with DT in BRAF positive ATC patients and compare the outcomes with usual therapy, and to study tumor molecular alterations in the DT group. METHODS: Patients treated between May 2018 and April 2022 in a tertiary referral center, assessed for BRAF status were included. Patients were divided in three groups: BRAF p.V600E treated with DT, BRAF wild type (WT) under multimodal therapy (MT), and BRAF WT under compassionate care (CC). Response was assessed monthly in the first 6 months and every 3 months afterwards, by RECIST 1.1. Overall survival (OS) and progression-free survival (PFS) were estimated with the Kaplan-Meier method and compared with the log-rank test. RESULTS: Twenty-seven ATC patients were included (DT = 9, MT = 8, and CC = 10). Median OS was 475 days for DT, 156 days for MT, and 39 days for CC (P < .001). At 12 months, only patients in the DT group were alive (71%). Median PFS was 270 days, in the DT group, compared with less than 32 days in BRAF WT (P < .001). No severe adverse events were reported. Molecular profiling showed that in one of the four clinical progressions, a pathogenic NRAS mutation was found. CONCLUSIONS: Our results show a significant real-world efficacy of Dabrafenib plus Trametinib in both survival and recurrence compared with standard treatment, with a good safety profile.publishersversionpublishe

Clinical and molecular characterization of parathyroid carcinoma in multiple endocrine neoplasia type 1

Nineteen cases of parathyroid carcinoma in patients with multiple endocrine neoplasia type 1 have been reported in the literature, of which 11 carry an inactivating germline mutation in the MEN1 gene. Somatic genetic abnormalities in these parathyroid carcinomas have never been detected. In this paper, we aimed to describe the clinical and molecular characterization of a parathyroid carcinoma ident ified in a patient with MEN1. A 60-year-old man was diagnosed with primary hyperparathyroidism during the postoperative period of lung carcinoid surgery. Serum calcium and parathyroid hormone levels were 15.0 mg/dL (8.4–10.2) and 472 pg/mL (12–65), respectively. The patient underwent parathyroid surgery, and histological findings were consistent with parathyroid carcinoma. Analysis of the MEN1 gene by next-generation sequencing (NGS) identified a novel germline heterozygous nonsense pathogen ic variant (c.978C>A; p.(Tyr326*)), predicted to encode a truncated protein. Genetic analysis of the parathyroid carcinoma revealed a c.307del, p.(Leu103Cysfs*16) frameshift truncating somatic MEN1 variant in the MEN1 gene, which is consistent with MEN1 tumor-suppressor role, confirming its involvement in parathyroid carcinoma etiology. Genetic analysis of CDC73, GCM2, TP53, RB1, AKT1, MTOR, PIK3CA and CCND1 genes in the parathyroid carcinoma DNA did not detect any somatic mutations. To our knowledge, this is the firs t report of a PC case presenting both germline (first-hit) and somatic (second-hit) in activation of the MEN1 gene

SPRY4 as a Potential Mediator of the Anti-Tumoral Role of Macrophages in Anaplastic Thyroid Cancer Cells

Funding Information: This work was funded by MERCK in collaboration with Grupo de Estudos da Tiroide (GET) from Sociedade Portuguesa de Endocrinologia Diabetes e Metabolismo (SPEDM) (MERCK/GET/SPEDM/2017), by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associated Laboratory LS4FUTURE (LA/P/0087/2020), by Associação de Endocrinologia Oncológica (AEO/2017), and by Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG/2017). Marta Pojo was granted by Liga Portuguesa Contra o Cancro—Núcleo Regional do Sul (LPCC-NRS/2017). Carolina Pires was granted with a Ph.D. scholarship by FCT—2020.07120.BD. Ricardo Rodrigues was granted with a Ph.D. scholarship by iNOVA4Health Research Unit—UIDP/04462/2020; UI/BD/154256/2022. Publisher Copyright: © 2023 by the authors.Anaplastic thyroid carcinoma (ATC) is the most lethal subtype of thyroid cancer, with high invasive and metastatic potential, not responding to conventional treatments. Its aggressiveness may be influenced by macrophages, which are abundant cells in the tumor microenvironment. To investigate the role of macrophages in ATC aggressiveness, indirect co-cultures were established between ATC cell lines and THP-1-derived macrophages. Macrophages significantly increased both the migration and invasion of T235 cells (p < 0.01; p < 0.01), contrasting with a decrease in C3948 (p < 0.001; p < 0.05), with mild effects in T238 migration (p < 0.01) and C643 invasion (p < 0.05). Flow cytometry showed upregulation of CD80 (pro-inflammatory, anti-tumoral) and downregulation of CD163 (anti-inflammatory, pro-tumoral) in macrophages from co-culture with T235 (p < 0.05) and C3948 (p < 0.05), respectively. Accordingly, we found an upregulation of secreted pro-inflammatory mediators (e.g., GM-CSF, IL-1α; p < 0.05) in C3948–macrophage co-cultures. Proteomic analysis showed the upregulation of SPRY4, an inhibitor of the MAPK pathway, in C3948 cells from co-culture. SPRY4 silencing promoted cancer cell invasion, reverting the reduced invasion of C3948 caused by macrophages. Our findings support that macrophages play a role in ATC cell aggressiveness. SPRY4 is a possible modulator of macrophage–ATC cell communication, with a tumor suppressor role relevant for therapeutic purposes.publishersversionpublishe

TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas

Context: Telomerase promoter mutations (TERT) were recently described in follicular cell-derived thyroid carcinomas (FCDTC) and seem to be more prevalent in aggressive cancers. Objectives: We aimed to evaluate the frequency of TERT promoter mutations in thyroid lesions and to investigate the prognostic significance of such mutations in a large cohort of patients with differentiated thyroid carcinomas (DTCs). Design: This was a retrospective observational study. Setting and Patients: We studied 647 tumors and tumor-like lesions. A total of 469 patients with FCDTC treated and followed in five university hospitals were included. Mean follow-up (±SD) was 7.8 ± 5.8 years. Main Outcome Measures: Predictive value of TERT promoter mutations for distant metastasization, disease persistence at the end of follow-up, and disease-specific mortality. Results: TERT promoter mutations were found in 7.5% of papillary carcinomas (PTCs), 17.1% of follicular carcinomas, 29.0% of poorly differentiated carcinomas, and 33.3% of anaplastic thyroid carcinomas. Patients with TERT-mutated tumors were older (P < .001) and had larger tumors (P = .002). In DTCs, TERT promoter mutations were significantly associated with distant metastases (P < .001) and higher stage (P < .001). Patients with DTC harboring TERT promoter mutations were submitted to more radioiodine treatments (P = .009) with higher cumulative dose (P = .004) and to more treatment modalities (P = .001). At the end of follow-up, patients with TERT-mutated DTCs were more prone to have persistent disease (P = .001). TERT promoter mutations were significantly associated with disease-specific mortality [in the whole FCDTC (P < .001)] in DTCs (P < .001), PTCs (P = .001), and follicular carcinomas (P < .001). After adjusting for age at diagnosis and gender, the hazard ratio was 10.35 (95% confidence interval 2.01–53.24; P = .005) in DTC and 23.81 (95% confidence interval 1.36–415.76; P = .03) in PTCs. Conclusions: TERT promoter mutations are an indicator of clinically aggressive tumors, being correlated with worse outcome and disease-specific mortality in DTC. TERT promoter mutations have an independent prognostic value in DTC and, notably, in PTC.We acknowledge GENZYME for funding our work through a research project. This study was supported by the Portuguese Foundation for Science and Technology through PhD Grant SFRH/BD/81940/2011 (to J.V.); PhD Grant SFRH/BD/87887/2012 (to C.T.); PhD Grant SFRH/BD/79135/2011 (to A.A.); and the Scientific Investigation Project PIC/IC/83037/2007. Further funding was obtained from the project “Microenvironment, Metabolism and Cancer,” partially supported by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referência Estratégico Nacional, and through the European Regional Development Fund. The work of J.M.C.-T. was supported by Grant PI12/00749-FEDER from the Instituto de Salud Carlos III, the Ministry of Economy and Competitiveness (Madrid, Spain). The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) is an associate laboratory of the Portuguese Ministry of Science, Technology, and Higher Education, which is partially supported by the Foundation for Science and Technology

TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas

Funding: This study was supported by the Portuguese Foundation for Science and Technology through PhD Grant SFRH/BD/81940/ 2011 (to J.V.); PhD Grant SFRH/BD/87887/2012 (to C.T.); PhD Grant SFRH/BD/79135/2011 (to A.A.); and the Scientific Investigation Project PIC/IC/83037/2007. Further funding was obtained from the project “Microenvironment, Metabolism and Cancer,” partially supported by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referência Estratégico Nacional, and through the European Regional Development Fund. The work of J.M.C.-T. was supported by Grant PI12/00749-FEDER from the Instituto de Salud Carlos III, the Ministry of Economy and Competitiveness (Madrid, Spain). The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) is an associate laboratory of the Portuguese Ministry of Science, Technology, and Higher Education, which is partially supported by the Foundation for Science and Technology.Context: Telomerase promoter mutations (TERT) were recently described in follicular cell-derived thyroid carcinomas (FCDTC) and seem to be more prevalent in aggressive cancers. Objectives:Weaimed to evaluate the frequency of TERT promoter mutations in thyroid lesions and to investigate the prognostic significance of such mutations in a large cohort of patients with differentiated thyroid carcinomas (DTCs). Design: This was a retrospective observational study. Setting and Patients: We studied 647 tumors and tumor-like lesions. A total of 469 patients with FCDTC treated and followed in five university hospitals were included. Mean follow-up (±SD) was 7.8 ± 5.8 years. Main Outcome Measures: Predictive value of TERT promoter mutations for distant metastasization, disease persistence at the end of follow-up, and disease-specific mortality. Results: TERT promoter mutations were found in 7.5% of papillary carcinomas (PTCs), 17.1% of follicular carcinomas, 29.0% of poorly differentiated carcinomas, and 33.3% of anaplastic thyroid carcinomas. Patients with TERT-mutated tumors were older (P < .001) and had larger tumors (P = .002). In DTCs, TERT promoter mutations were significantly associated with distant metastases (P< .001) and higher stage (P < .001). Patients with DTC harboring TERT promoter mutations were submitted to more radioiodine treatments (P = .009) with higher cumulative dose (P = .004) and to more treatment modalities (P=.001). At the end of follow-up, patients with TERT-mutated DTCs were more prone to have persistent disease (P=.001). TERT promoter mutations were significantly associated with disease-specific mortality [in the whole FCDTC (P < .001)] in DTCs (P < .001), PTCs (P = .001), and follicular carcinomas (P < .001). After adjusting for age at diagnosis and gender, the hazard ratio was 10.35 (95% confidence interval 2.01-53.24; P = .005) in DTC and 23.81 (95% confidence interval 1.36-415.76; P = .03) in PTCs. Conclusions: TERT promoter mutations are an indicator of clinically aggressive tumors, being correlated with worse outcome and disease-specific mortality in DTC. TERT promoter mutations have an independent prognostic value in DTC and, notably, in PTC.publishersversionpublishe

FLOCK-REPROD non hormonal insemination protocols for goats

Projekt je razvio inovativna rješenja za proizvodnju hormone free kozjeg mlijeka tijekom cijele godine. FLOCK-REPROD (Hormone free non-seasonal or seasonal goat reproduction for a sustainable European goat-milk market), uz potporu 7. okvirnog programa, iznosi nehormonska rješenja koja omogućuju kontrolu sezonosti uz primjenu umjetnog osjemenjivanja (UO). Na taj način FLOCK-REPROD pomaže uzgajivačima proizvesti više mlijeka i osigurava alternativni put koji je u skladu sa zakonskom regulativom EU koja ograničava uporabu hormona. Razvijeni su novi progestagen free UO protokoli (PG1, PG1, HF) koji uključuju postupke temeljene na učinku mužjaka i svjetlosnim režimima u svrhu indukcije i sinkronizacije ovulacije tijekom cijele godine. PG1 i PG2 temelje se na jednoj ili dvije injekcije prostaglandina (nisu podvrgnute rezidualnim ograničenjima). HF protokol je hormone free te može biti primijenjen i na organskim uzgojima. Novi protokoli UO testirani su u terenskim uvjetima. Najbolji su rezultati dobiveni s HF (58 % gravidnosti, slično klasičnom hormonskom protokolu HT), a zatim s PG2 (54 %) te PG1 (45 %). Osnovni problem za implementaciju protokola PG1 i HF jest visoka varijabilnost plodnosti među farmama. Novi protokoli UO manje su učinkoviti glede utrošenih radnih sati i ulaznih troškova u usporedbi s HT. PG1 je protokol koji zahtijeva najveći utrošak vremena, a nakon njega to su HF i PG2. HF se pokazao najskupljim protokolom, dok je PG2 jeftiniji od PG1. Veći radni angažman i viši troškovi koje stvaraju novi UO protokoli nastaju najviše zbog potrebe za dodatnim brojem jarčeva nužnih za provođenje utjecaja mužjaka (veći troškovi hranidbe, utrošak vremena za baratanje mužjacima).The project has developed innovative solutions for the production of hormone-free goat milk throughout the year. FLOCK-REPROD (“Hormone-free non-seasonal or seasonal goat reproduction for a sustainable European goat-milk market”), supported by the 7th Framework Programme, created non-hormonal solutions that enable seasonal control of reproduction, which include the use of artificial insemination (AI). In this way, FLOCK-REPROD helps farmers to produce more milk and provides an alternative in line with the EU legislation which restricts the use of hormones. New “progestagen free” AI protocols (PG1, PG2, HF) have been developed, which include protocols based on the male effect and light treatment in order to provide induction and synchronization of blokiovulation throughout the year. PG1 and PG2 are based on one or two injections of prostaglandins (not subject to residual restrictions so far). The HF protocol is hormone-free and can be applied even in organic farming systems. New AI protocols have been tested in field conditions. The best results were obtained with HF (58% pregnancy, similar to classical hormonal protocol HT results), and then with PG2 (54%) and PG1 (45%). The main problem for the implementation of protocols PG1 and HF is the high variability of fertility between goat farms. New AI protocols are less effective with regard to working hours and input costs compared with HT protocols. PG1 is a protocol that requires the greatest working hour input, followed by the HF and PG2 protocols. HF has proven to be the most expensive protocol, while the PG 2 is cheaper than the PG1 protocol. The greater work engagement and higher input costs created by new AI protocols arise mainly due to the need for additional bucks to perform the male effect (higher feeding costs, more time spent in handling males)