Anti-inflammatory role of APRIL by modulating regulatory B cells in antigen-induced arthritis.

APRIL (A Proliferation-Inducing Ligand), a member of the TNF superfamily, was initially described for its ability to promote proliferation of tumor cells in vitro. Moreover, this cytokine has been related to the pathogenesis of different chronic inflammatory diseases, such as rheumatoid arthritis. This study aimed to evaluate the ability of APRIL in regulating B cell-mediated immune response in the antigen-induced arthritis (AIA) model in mice. AIA was induced in previously immunized APRIL-transgenic (Tg) mice and their littermates by administration of antigen (mBSA) into the knee joints. Different inflammatory cell populations in spleen and draining lymph nodes were analyzed using flow cytometry and the assay was performed in the acute and chronic phases of the disease, while cytokine levels were assessed by ELISA. In the acute AIA, APRIL-Tg mice developed a less severe condition and a smaller inflammatory infiltrate in articular tissues when compared with their littermates. We also observed that the total cellularity of draining lymph nodes was decreased in APRIL-Tg mice. Flow cytometry analysis revealed an increase of CD19+IgM+CD5+ cell population in draining lymph nodes and an increase of CD19+CD21hiCD23hi (B regulatory) cells in APRIL-Tg mice with arthritis as well as an increase of IL-10 and CXCL13 production in vitro.

Tracheal compression by brachiocephalic arterial trunk: a rare cause of failed endotracheal intubation.

Encountering and managing an unanticipated difficult airway are among the many challenges faced by anaesthesiologists. Due to the intimate anatomical relationship between the thoracic vasculature and the trachea, an anatomical variation could potentially lead to airway compression. This clinical case report documents a failed intubation in an adult patient caused by undiagnosed extrinsic tracheal compression from the brachiocephalic arterial trunk, a rare condition. After a thorough investigation and diagnostic clarification, a safe anaesthetic plan following the predictable difficult airway guidelines was established to enable surgery. Anaesthesiologists should consider rare vascular causes as potential contributors to difficult airway scenarios, thereby enhancing their expertise.

Multiple TP53 p.R337H haplotypes and implications for tumor susceptibility.

The germline TP53 p.R337H mutation is reported as the most common germline TP53 variant. It exists at a remarkably high frequency in the population of southeast Brazil as founder mutation in two distinct haplotypes with the most frequent co-segregating with the p.E134∗ variant of the XAF1 tumor suppressor and an increased cancer risk. Founder mutations demonstrate linkage disequilibrium with neighboring genetic polymorphic markers that can be used to identify the founder variant in different geographic regions and diverse populations. We report here a shared haplotype among Brazilian, Portuguese, and Spanish families and the existence of three additional distinct TP53 p.R337H alleles. Mitochondrial DNA sequencing and Y-STR profiling of Brazilian carriers of the founder TP53 p.R337H allele reveal an excess of Native American haplogroups in maternal lineages and exclusively European haplogroups in paternal lineages, consistent with communities established through male European settlers with extensive intermarriage with Indigenous women. The identification of founder and independent TP53 p.R337H alleles underlines the importance for considering the haplotype as a functional unit and the additive effects of constitutive polymorphisms and associated variants in modifier genes that can influence the cancer phenotype.

Frequency of CDH1, CTNNA1 and CTNND1 Germline Variants in Families with Diffuse and Mixed Gastric Cancer.

The most well-characterized hereditary form of gastric cancer is hereditary diffuse gastric cancer (HDGC), an autosomal dominant syndrome characterized by an increased risk of diffuse gastric and lobular breast cancer. HDGC is predominantly caused by germline pathogenic variants in the CDH1 gene, and more rarely in the CTNNA1 gene. Furthermore, the International Gastric Cancer Linkage Consortium (IGCLC) guidelines do not clarify whether or not mixed gastric cancer (with a diffuse component) should be considered in the HDGC genetic testing criteria. We aimed to evaluate the contribution of CTNNA1 and CTNND1 germline variants to HDGC. Additionally, we also intended to compare the frequencies of CDH1 and CTNNA1 (and eventually CTNND1) germline variants between patients with diffuse and mixed gastric carcinomas to evaluate if genetic testing for these genes should or should not be considered in patients with the latter. We analyzed the CDH1 gene in 67 cases affected with early-onset/familial mixed gastric carcinomas and the CTNNA1 and CTNND1 genes in 208 cases with diffuse or mixed gastric cancer who had tested negative for CDH1 pathogenic germline variants. A deleterious CTNNA1 germline variant was found in 0.7% (1/141) of diffuse gastric cancer patients meeting the 2020 IGCLC criteria, as compared to the rate of 2.8% of CDH1 deleterious variants found by us in this setting. No deleterious variants were found in CTNND1, but six variants of uncertain significance were identified in this gene. We did not find any pathogenic CDH1, CTNNA1 or CTNND1 variant in index patients with early-onset/familial mixed gastric cancer, so there is no evidence that supports including this tumor type in the testing criteria for germline variants in these genes. The role of the CTNND1 gene in inherited gastric cancer predisposition is still unclear.

Combined germline and tumor mutation signature testing identifies new families with NTHL1 tumor syndrome.

NTHL1 tumor syndrome is an autosomal recessive rare disease caused by biallelic inactivating variants in the NTHL1 gene and which presents a broad tumor spectrum. To contribute to the characterization of the phenotype of this syndrome, we studied 467 index patients by KASP assay or next-generation sequencing, including 228 patients with colorectal polyposis and 239 patients with familial/personal history of multiple tumors (excluding multiple breast/ovarian/polyposis). Three NTHL1 tumor syndrome families were identified in the group of patients with polyposis and none in patients with familial/personal history of multiple tumors. Altogether, we identified nine affected patients with polyposis (two of them diagnosed after initiating colorectal cancer surveillance) with biallelic pathogenic or likely pathogenic NTHL1 variants, as well as two index patients with one pathogenic or likely pathogenic NTHL1 variant in concomitance with a missense variant of uncertain significance. Here we identified a novel inframe deletion classified as likely pathogenic using the ACMG criteria, supported also by tumor mutational signature analysis. Our findings indicate that the NTHL1 tumor syndrome is a multi-tumor syndrome strongly associated with polyposis and not with multiple tumors without polyposis.

Implementation of upfront DPYD genotyping with a low-cost and high-throughput assay to guide fluoropyrimidine treatment in cancer patients.

OBJECTIVES: Genetic variants in the dihydropyrimidine dehydrogenase (DPYD ) gene are associated with reduced dihydropyrimidine dehydrogenase enzyme activity and can cause severe fluoropyrimidine-related toxicity. We assessed the frequency of the four most common and well-established DPYD variants associated with fluoropyrimidine toxicity and implemented a relatively low-cost and high-throughput genotyping assay for their detection. METHODS: This study includes 457 patients that were genotyped for the DPYD c.1129-5923C>G, c.1679T>G, c.1905 + 1G>A and c.2846A>T variants, either by Sanger sequencing or kompetitive allele specific PCR (KASP) technology. Of these, 172 patients presented toxicity during treatment with fluoropyrimidines (post-treatment group), and 285 were tested before treatment (pretreatment group). RESULTS: Heterozygous DPYD variants were identified in 7.4% of the entire series of 457 patients, being the c.2846A>T the most frequent variant. In the post-treatment group, 15.7% of the patients presented DPYD variants, whereas only 2.5% of the patients in the pretreatment group presented a variant. The KASP assays designed in this study presented 100% genotype concordance with the results obtained by Sanger sequencing. CONCLUSIONS: The combined assessment of the four DPYD variants in our population increases the identification of patients at high risk for developing fluoropyrimidine toxicity, supporting the upfront routine implementation of DPYD variant genotyping. Furthermore, the KASP genotyping assay described in this study presents a rapid turnaround time and relatively low cost, making upfront DPYD screening feasible in clinical practice.

Genetic landscape of homologous recombination repair genes in early-onset/familial prostate cancer patients.

Prostate cancer (PrCa) is one of the three most frequent and deadliest cancers worldwide. The discovery of PARP inhibitors for the treatment of tumors with deleterious variants in homologous recombination repair (HRR) genes has placed PrCa on the roadmap of precision medicine. However, the overall contribution of HRR genes to the 10%-20% of carcinomas arising in men with early-onset/familial PrCa has not been fully clarified. We used targeted next-generation sequencing (T-NGS) covering eight HRR genes (ATM, BRCA1, BRCA2, BRIP1, CHEK2, NBN, PALB2, and RAD51C) and an analysis pipeline querying both small and large genomic variations to clarify their global and relative contribution to hereditary PrCa predisposition in a series of 462 early-onset/familial PrCa cases. Deleterious variants were found in 3.9% of the patients, with CHEK2 and ATM being the most frequently mutated genes (38.9% and 22.2% of the carriers, respectively), followed by PALB2 and NBN (11.1% of the carriers, each), and finally by BRCA2, RAD51C, and BRIP1 (5.6% of the carriers, each). Using the same NGS data, exonic rearrangements were found in two patients, one pathogenic in BRCA2 and one of unknown significance in BRCA1. These results contribute to clarify the genetic heterogeneity that underlies PrCa predisposition in the early-onset and familial disease, respectively.

Practical lessons learned from real-world implementation of the molecular classification for endometrial carcinoma.

OBJECTIVES: This study aimed to explore the practical organisational aspects and difficulties in the implementation of the molecular classification of endometrial carcinoma (EC), and to demonstrate its potential impact in prognostic risk group classification. METHODS: We conducted a multicentre, retrospective cohort study of 230 patients with EC diagnosed between 2019 and 2022. Sample processing, clinicopathological, treatment and follow-up data were collected. Molecular classification was obtained by p53 and mismatch repair proteins immunohistochemistry, and POLE next-generation sequencing. RESULTS: Implementation was achieved through centralization of molecular analysis. In practice, it was possible to optimise turnaround times of complete integrative reports for hysterectomy specimens to a median time of 18 workdays. If genetic study was started in endometrial biopsies before surgery, 82.0% were available at the time of multidisciplinary tumour board, compared to 8.4% if performed in hysterectomy. ECs were classified as follows: 37.8% no specific molecular profile, 31.7% p53 abnormal, 24.3% mismatch repair deficient, and 6.1% POLE mutant. Integration of these results with traditional clinicopathologic factors led to a change in prognostic risk group in 15 (6.5%) patients, most being initially allocated to high-intermediate (n = 8) and low (n = 5) risk groups. Eight patients changed to a higher risk, and 7 to a lower risk group, whereas 2 remained in the same group. CONCLUSIONS: Centralization of EC molecular classification is a feasible option for countries with limited resources. Optimization of workflows may be achieved by earlier analysis in biopsies and prioritisation of patients whose results imply changes in risk group classification.

Genotype-first approach to identify associations between CDH1 germline variants and cancer phenotypes: a multicentre study by the European Reference Network on Genetic Tumour Risk Syndromes.

BACKGROUND: Truncating pathogenic or likely pathogenic variants of CDH1 cause hereditary diffuse gastric cancer (HDGC), a tumour risk syndrome that predisposes carrier individuals to diffuse gastric and lobular breast cancer. Rare CDH1 missense variants are often classified as variants of unknown significance. We conducted a genotype-phenotype analysis in families carrying rare CDH1 variants, comparing cancer spectrum in carriers of pathogenic or likely pathogenic variants (PV/LPV; analysed jointly) or missense variants of unknown significance, assessing the frequency of families with lobular breast cancer among PV/LPV carrier families, and testing the performance of lobular breast cancer-expanded criteria for CDH1 testing. METHODS: This genotype-first study used retrospective diagnostic and clinical data from 854 carriers of 398 rare CDH1 variants and 1021 relatives, irrespective of HDGC clinical criteria, from 29 institutions in ten member-countries of the European Reference Network on Tumour Risk Syndromes (ERN GENTURIS). Data were collected from Oct 1, 2018, to Sept 20, 2022. Variants were classified by molecular type and clinical actionability with the American College of Medical Genetics and Association for Molecular Pathology CDH1 guidelines (version 2). Families were categorised by whether they fulfilled the 2015 and 2020 HDGC clinical criteria. Genotype-phenotype associations were analysed by Student's t test, Kruskal-Wallis, χ2, and multivariable logistic regression models. Performance of HDGC clinical criteria sets were assessed with an equivalence test and Youden index, and the areas under the receiver operating characteristic curves were compared by Z test. FINDINGS: From 1971 phenotypes (contributed by 854 probands and 1021 relatives aged 1-93 years), 460 had gastric and breast cancer histology available. CDH1 truncating PV/LPVs occurred in 176 (21%) of 854 families and missense variants of unknown significance in 169 (20%) families. Multivariable logistic regression comparing phenotypes occurring in families carrying PV/LPVs or missense variants of unknown significance showed that lobular breast cancer had the greatest positive association with the presence of PV/LPVs (odds ratio 12·39 [95% CI 2·66-57·74], p=0·0014), followed by diffuse gastric cancer (8·00 [2·18-29·39], p=0·0017) and gastric cancer (7·81 [2·03-29·96], p=0·0027). 136 (77%) of 176 families carrying PV/LPVs fulfilled the 2015 HDGC criteria. Of the remaining 40 (23%) families, who did not fulfil the 2015 criteria, 11 fulfilled the 2020 HDGC criteria, and 18 had lobular breast cancer only or lobular breast cancer and gastric cancer, but did not meet the 2020 criteria. No specific CDH1 variant was found to predispose individuals specifically to lobular breast cancer, although 12 (7%) of 176 PV/LPV carrier families had lobular breast cancer only. Addition of three new lobular breast cancer-centred criteria improved testing sensitivity while retaining high specificity. The probability of finding CDH1 PV/LPVs in patients fulfilling the lobular breast cancer-expanded criteria, compared with the 2020 criteria, increased significantly (AUC 0·92 vs 0·88; Z score 3·54; p=0·0004). INTERPRETATION: CDH1 PV/LPVs were positively associated with HDGC-related phenotypes (lobular breast cancer, diffuse gastric cancer, and gastric cancer), and no evidence for a positive association with these phenotypes was found for CDH1 missense variants of unknown significance. CDH1 PV/LPVs occurred often in families with lobular breast cancer who did not fulfil the 2020 HDGC criteria, supporting the expansion of lobular breast cancer-centred criteria. FUNDING: European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme.

Immunoparalysis in critically ill children.

Immunoparalysis is associated with poorer outcomes in the paediatric intensive care unit (PICU) setting. We aimed to determine the group of patients with higher chances of immunoparalysis and correlate this status with increased risks of nosocomial infection and adverse clinical parameters. We conducted an exploratory study with prospective data collection in a university-affiliated tertiary medical, surgical, and cardiac PICU. Fifteen patients with multiple organ dysfunction syndrome were included over a period of 6 months. Monocyte's human leucocyte antigen (HLA)-DR expression and tumour necrosis factor (TNF)-α and interleukin (IL)-6 production were measured by flow-cytometry at three time points (T1 = 1-2 days; T2 = 3-5 days; T3 = 6-8 days). Using the paediatric logistic organ dysfunction-2 score to assess initial disease severity, we established the optimal cut-off values of the evaluated parameters to identify the subset of patients with a higher probability of immunoparalysis. A comparative analysis was performed between them. Sixty per cent were males; the median age was 4.1 years. Considering the presence of two criteria in T1 (classical monocytes mean fluorescence intensity [MFI] for HLA-DR ≤ 1758.5, area under the curve (AUC) = 0.775; and frequency of monocytes producing IL-6 ≤ 68.5%, AUC = 0.905) or in T3 (classical monocytes MFI of HLA-DR ≤ 2587.5, AUC = 0.675; and frequency of monocytes producing TNF-α ≤ 93.5%, AUC = 0.833), a variable to define immunoparalysis was obtained (100% sensitivity, 81.5% specificity). Forty per cent of patients were assigned to the immunoparalysis group. In this: a higher frequency of nosocomial infection (p = 0.011), vasoactive inotropic score (p = 0.014) and length of hospital stay (p = 0.036) was observed. In the subgroup with the diagnosis of sepsis/septic shock (n = 5), patients showed higher percentages of non-classical monocytes (p = 0.004). No mortality was recorded. A reduction in classical monocytes HLA-DR expression with lower frequencies of monocytes producing TNF-α and IL-6 during the first week of critical illness, appears to be a good marker of immunoparalysis; these findings relate to an increased risk of nosocomial infection and deleterious outcomes. The increased frequency of non-classical monocytes in patients with sepsis/septic shock is suggestive of a better prognosis.

Perceptions of Siblings of People with Trisomy 21 on Family Relationships / Percepções de Irmãos de Pessoas com Trissomia 21 sobre as Relações Familiares

ABSTRACT: The birth of a child with a disability brings implications, changes in the environment and family dynamics. The family goes through a process of overcoming until the child acceptance with disability and the establishment of a suitable family environment that includes and meets his/her needs. Trisomy 21 (T21) is the most frequent genetic anomaly among those diagnosed. This study aimed to identify the perception of a sibling group of people with T21 regarding their family and social relationships. The sample consisted of 18 adolescents aged between 11 and 16 years, siblings of people with T21, and 17 guardians. Data collection occurred through semi-structured interviews conducted by video calls, with an average duration of 10 to 15 minutes. The interviews were recorded, transcribed and analyzed qualitatively based on the creation of thematic nuclei and categories. The results reinforce the importance of family relationships as a security factor and acceptance of the siblings' needs.

RESUMO: O nascimento de uma criança com deficiência traz implicações, mudanças no ambiente e na dinâmica familiar como um todo. A família passa por um processo de superação até a aceitação da criança com deficiência e a instalação de um ambiente familiar propício que a inclua e atenda às suas necessidades. A trissomia 21 (T21) é a anomalia genética mais frequente dentre as diagnosticadas. Este estudo teve como objetivo conhecer a percepção de um grupo de irmãos de pessoas com T21 quanto às suas relações familiares e sociais. A amostra foi composta por 18 adolescentes com idades entre 11 e 16 anos, irmãos de pessoas com T21 e 17 responsáveis. A coleta de dados ocorreu por meio de entrevista semiestruturada realizadas por meio de chamada de vídeo, com duração média de dez a 15 minutos. As entrevistas foram gravadas, transcritas e analisadas qualitativamente a partir da criação de núcleos temáticos e categorias. Os resultados reforçam a importância das relações familiares como um fator segurança e acolhimento das necessidades dos irmãos.

Parathyroid Hormone-Related Peptide (PTHrP): Evaluation of Pediatric, Covariate-Stratified Reference Intervals.

Parathyroid hormone-related peptide (PTHrP) is expressed at a wide range of sites in the body and performs different functions including vasodilation, relaxation of smooth muscle cells, and regulation of bone development. PTHrP also mediates hypercalcemia related to neoplastic diseases. However, reference ranges specific method and age were not evaluated. We establish PTHrP reference ranges in apparently healthy, normocalcemic, normophosphatemic pediatric individuals. In this observational prospective, study we measured PTHrP in serum from 178 samples (55.06% male 44.94% female) from apparently healthy pediatric subjects [median age 10 years (range 1-18)] subunit ELISA method The statistical analysis performed provided for the calculation of the 95% reference interval, right-sided, with a non-parametric percentile method (CLSI C28-A3). Upper reference limits (URL) for PTHrP was 2.89 ng/mL (2.60 to 3.18; 90% CI). No significant differences were found between the median PTHrP concentrations in males vs females and in the age range categories selected. Comprehensive normal values for PTHrP are indispensable to the assessment of calcium phosphorus dysfunction in children. Severe hypercalcemia is a rare, but clinically significant condition, in infancy and childhood. PTHrP values higher than the reference value may help to distinguish the hypercalcemic product of a malignancy, paraneoplastic syndromes mediated by PTHrP, from other causes.

Síndrome de Turner / Turner's Syndrome / Sindrome de Turner

Introducción: el síndrome de Turner es una enfermedad genética caracterizada por la pérdida total o parcial de un cromosoma X, siendo sus características fundamentales la talla baja, la disgenesia gonadal y hallazgos fenotípicos característicos. Tiene una amplia variabilidad en su forma de presentación. Grandes estudios epidemiológicos muestran que la morbilidad aumenta en mujeres con este síndrome, debido a una amplia gama de enfermedades asociadas, sobre todo cardiovasculares, que eleva la mortalidad de manera significativa. Objetivo: realizar una revisión de la literatura, en base a la presentación de un caso clínico, para recabar información sobre las ultimas pautas de manejo y presentar los nuevos objetivos de tratamiento. Conclusiones: el diagnóstico temprano es fundamental, y tiene características propias y criterios de sospecha según la etapa en la que se efectúa, el reto actual en el manejo de estas pacientes consiste en la formación de un equipo médico multidisciplinario, conformado por una amplia gama de especialistas para el adecuado seguimiento, con el fin de disminuir las complicaciones y ayudar a que la paciente alcance sus objetivos para una vida plena. Se presenta el caso de una paciente con síndrome de Turner vista por el equipo médico en el Hospital Pediátrico del Centro Hospitalario Pereira Rossell, Montevideo-Uruguay.

Introduction: Turner's syndrome is a genetic disease characterized by total or partial loss of an X chromosome, its main features being low height, gonadal dysgenesis and characteristic phenotypic findings. It has a wide variability in its form of presentation. Large epidemiological studies show that morbidity increases in women with this syndrome, due to a wide range of associated diseases, especially cardiovascular disease, which significantly raises mortality. Objectives: to carry out a review of the literature, based on a clinical case in order to gather information regarding the latest treatment guidelines and present the new treatment goals. Conclusions: early diagnosis is essential, and has its own characteristics and suspicion criteria according to the stage in which it is carried out. The present challenge regarding the management of these patients consists of the training of a multidisciplinary medical team made up of a wide range of specialists able to carry out proper follow-up, in order to reduce complications and help the patient live a full life. We present a case of a patient with Turner's syndrome assisted at the Pereira Rossell Hospital Center in Montevideo-Uruguay.

Introdução: a síndrome de Turner é uma doença genética caracterizada pela perda total ou parcial de um cromossomo X, sendo suas características fundamentais de baixa estatura, disgenesia gonadal e achados fenotípicos característicos. Tem uma ampla variabilidade em sua forma de apresentação. Consideráveis (grandes, amplos, extensos) estudos epidemiológicos mostram que a morbidade aumenta em mulheres com essa síndrome, devido a uma ampla gama de doenças associadas, especialmente cardiovasculares, o que aumenta significativamente a mortalidade. Objetivos: realizar uma revisão da literatura, a partir da apresentação de um caso clínico, reunir informações sobre as últimas diretrizes de tratamento e apresentar os novos objetivos do tratamento. Conclusões: o diagnóstico precoce é fundamental, e possui características próprias e critérios de suspeita de acordo com a etapa em que é realizado, o desafio atual na gestão desses pacientes consiste na formação de uma equipe médica multidisciplinar, formada por uma ampla gama de especialistas para o acompanhamento adequado, a fim de reduzir complicações e ajudar a paciente a alcançar uma vida plena. Apresentamos o caso de uma paciente com síndrome de Turner atendido pela equipe médica do Hospital Pediátrico do Centro Hospitalar Pereira Rossell, Montevidéu-Uruguai.

KRAS and NRAS mutational analysis in plasma ctDNA from patients with metastatic colorectal cancer by real-time PCR and digital PCR.

PURPOSE: Mutations in the KRAS and NRAS (RAS) genes are negative predictors of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC). The detection of mutations in circulating tumor DNA (ctDNA) has emerged as a less invasive strategy to assess the molecular profile of mCRC patients. We aimed to perform RAS mutational analysis in ctDNA from mCRC patients using BEAMing Digital PCR (OncoBEAM) and Idylla ctDNA qPCR and evaluate the concordance rate with RAS mutational status in tumor tissue and between these two methodologies with different limits of detection. METHODS: Blood samples were collected from 47 mCRC patients previously tested for RAS mutations in tumor tissue. DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit, and RAS mutation analysis was conducted using OncoBEAM RAS CRC and Idylla ctRAS assays. RESULTS: The overall agreement between tumor tissue and ctDNA analyses was 83% and 78.7% using the OncoBEAM and Idylla assays, respectively, with the concordance being 96.2% and 88.5% in naive treatment patients. The overall agreement between OncoBEAM and Idylla ctDNA analyses was 91.7%. CONCLUSIONS: Analysis of ctDNA is a viable strategy for clinical management of mCRC patients. Although the OncoBEAM assay sensitivity is somewhat higher, the fully automated Idylla platform also has good performance, while being cheaper and much less labor-intensive, for the detection of RAS mutations in plasma, either at diagnosis or after progression when considering anti-EGFR treatment rechallenge.

Expression Profiling in Ovarian Cancer Reveals Coordinated Regulation of BRCA1/2 and Homologous Recombination Genes.

Predictive biomarkers are crucial in clarifying the best strategy to use poly(ADP-ribose) polymerase inhibitors (PARPi) for the greatest benefit to ovarian cancer patients. PARPi are specifically lethal to cancer cells that cannot repair DNA damage by homologous recombination (HR), and HR deficiency is frequently associated with BRCA1/2 mutations. Genetic tests for BRCA1/2 mutations are currently used in the clinic, but results can be inconclusive due to the high prevalence of rare DNA sequence variants of unknown significance. Most tests also fail to detect epigenetic modifications and mutations located deep within introns that may alter the mRNA. The aim of this study was to investigate whether quantitation of BRCA1/2 mRNAs in ovarian cancer can provide information beyond the DNA tests. Using the nCounter assay from NanoString Technologies, we analyzed RNA isolated from 38 ovarian cancer specimens and 11 normal fallopian tube samples. We found that BRCA1/2 expression was highly variable among tumors. We further observed that tumors with lower levels of BRCA1/2 mRNA showed downregulated expression of 12 additional HR genes. Analysis of 299 ovarian cancer samples from The Cancer Genome Atlas (TCGA) confirmed the coordinated expression of BRCA1/2 and HR genes. To facilitate the routine analysis of BRCA1/2 mRNA in the clinical setting, we developed a targeted droplet digital PCR approach that can be used with FFPE samples. In conclusion, this study underscores the potential clinical benefit of measuring mRNA levels in tumors when BRCA1/2 DNA tests are negative or inconclusive.

Corrigendum: Generation of Two Paclitaxel-Resistant High-Grade Serous Carcinoma Cell Lines With Increased Expression of P-Glycoprotein.

[This corrects the article DOI: 10.3389/fonc.2021.752127.].

Identifying the Steps Required to Effectively Implement Next-Generation Sequencing in Oncology at a National Level in Europe.

Next-generation sequencing (NGS) may enable more focused and highly personalized cancer treatment, with the National Comprehensive Cancer Network and European Society for Medical Oncology guidelines now recommending NGS for daily clinical practice for several tumor types. However, NGS implementation, and therefore patient access, varies across Europe; a multi-stakeholder collaboration is needed to establish the conditions required to improve this discrepancy. In that regard, we set up European Alliance for Personalised Medicine (EAPM)-led expert panels during the first half of 2021, including key stakeholders from across 10 European countries covering medical, economic, patient, industry, and governmental expertise. We describe the outcomes of these panels in order to define and explore the necessary conditions for NGS implementation into routine clinical care to enable patient access, identify specific challenges in achieving them, and make short- and long-term recommendations. The main challenges identified relate to the demand for NGS tests (governance, clinical standardization, and awareness and education) and supply of tests (equitable reimbursement, infrastructure for conducting and validating tests, and testing access driven by evidence generation). Recommendations made to resolve each of these challenges should aid multi-stakeholder collaboration between national and European initiatives, to complement, support, and mutually reinforce efforts to improve patient care.

Generation of Two Paclitaxel-Resistant High-Grade Serous Carcinoma Cell Lines With Increased Expression of P-Glycoprotein.

Debulking surgery followed by chemotherapy are the standard of care for high-grade serous carcinoma. After an initial good response to treatment, the majority of patients relapse with a chemoresistant profile, leading to a poor overall survival. Chemotherapy regimens used in high-grade serous carcinomas are based in a combination of classical chemotherapeutic drugs, namely, Carboplatin and Paclitaxel. The mechanisms underlying drug resistance and new drug discovery are crucial to improve patients' survival. To uncover the molecular mechanisms of chemoresistance and test drugs capable of overcoming this resistant profile, it is fundamental to use good cellular models capable of mimicking the chemoresistant disease. Herein, we established two high-grade serous carcinoma cell lines with intrinsic resistance to Carboplatin and induced Paclitaxel resistance (OVCAR8 PTX R C and OVCAR8 PTX R P) derived from the OVCAR8 cell line. These two chemoresistant cell line variants acquired an enhanced resistance to Paclitaxel-induced cell death by increasing the drug efflux capacity, and this resistance was stable in long-term culture and following freeze/thaw cycles. The mechanism underlying Paclitaxel resistance resides in a significant increase in P-glycoprotein expression and, when this drug efflux pump was blocked with Verapamil, cells re-acquired Paclitaxel sensitivity. We generated two high-grade serous carcinoma cell lines, with a double-chemoresistant (Carboplatin and Paclitaxel) phenotype that mimics the majority of tumor recurrences in ovarian cancer context. This robust tool is suitable for preliminary drug testing towards the development of therapeutic strategies to overcome chemoresistance.

Next Generation Sequencing of Tumor and Matched Plasma Samples: Identification of Somatic Variants in ctDNA From Ovarian Cancer Patients.

Genetic testing to detect somatic alterations is usually performed on formalin-fixed paraffin-embedded tumor samples. However, tumor molecular profiling through ctDNA analysis may be particularly interesting with the emergence of targeted therapies for ovarian cancer (OC), mainly when tumor is not available and biopsy is not viable, also allowing representation of multiple neoplastic subclones. Using a custom panel of 27 genes, next-generation sequencing (NGS) was performed on tumor and matched plasma samples from 96 OC patients, which were combined in two groups (treatment naive and post-treatment). Overall, at least one somatic variant present in the tumor sample was also detected in the matched plasma sample in 35.6% of the patients, a percentage that increased to 69.6% of the treatment naive patients and 83.3% of those with stage IV disease, showing the potential of ctDNA analysis as an alternative to identify somatic variants in these patients, namely those that have predictive value for targeted therapy. In fact, of the two treatment-naive patients with somatic BRCA1 variants identified in tumor samples, in one of them we detected in ctDNA a BRCA1 somatic variant that was present in the tumor with a VAF of 53%, but not in the one that had a VAF of 5.4%. We also showed that ctDNA analysis has a complementary role to molecular unraveling of inter- and intra-tumor heterogeneity, as exemplified by one patient diagnosed with bilateral OC in which different somatic variants from both tumors were detected in ctDNA. Interestingly, as these bilateral tumors shared a rare combination of two of the three variants identified in ctDNA, we could conclude that these morphologically different tumors were clonally related and not synchronous independent neoplasias. Moreover, in the post-treatment group of patients with plasma samples collected after surgery, those with detectable somatic variants had poor prognosis when compared with patients with no detectable somatic variants, highlighting the potential of ctDNA analysis to identify patients at higher risk of recurrence. Concluding, this study demonstrated that somatic variants can be detected in plasma samples of a significant proportion of OC patients, supporting the use of NGS-based ctDNA testing for noninvasive tumor molecular profiling and to stratify patients according to prognosis.

Secondary Hepatic Injury in Pediatric Intensive Care: Risk Factors and Prognostic Impact.

OBJECTIVES: The aim of this study was to assess the profile of secondary hepatic injury (SHI), to determine risk factors and to evaluate its impact on prognosis of pediatric intensive care patients. METHODS: An exploratory observational and retrospective study was conducted in a Pediatric Intensive Care Unit. Two groups were defined: with SHI [alanine aminotransferase (ALT) ≥100 IU/L or gamma glutamyl transpeptidase (GGT)≥100 IU/L or direct bilirubin ≥30 µmol/L] and without. SHI was divided into 3 patterns: cytolysis, cholestasis, and mixed. RESULTS: SHI occurred in 16.5%, cytolysis in 5%, cholestasis in 4%, and mixed pattern in 7%. Independent risk factors for SHI were: organ dysfunction score PELOD-2 in D1 in cytolysis (n = 28); total parenteral nutrition and Pediatric Index of Mortality 3 (PIM3) in cholestasis (n = 23); sepsis, oncologic comorbidities, PIM3, and respiratory dysfunction in mixed pattern (n = 37). The ALT was an independent risk factor and a good predictor of mortality (AUC = 0.865) with a cut-off of 137 IU/L. CONCLUSIONS: SHI was associated with worst prognostic. ALT may be useful for detecting patients at increased risk of death, probably being a surrogate marker of the illness severity, reflecting a secondary injury.