Somatic mutational profiling and clinical impact of driver genes in Latin-Iberian medulloblastomas: Towards precision medicine.

Medulloblastoma (MB) is the most prevalent malignant brain tumor in children, known for its heterogeneity and treatment-associated toxicity, and there is a critical need for new therapeutic targets. We analyzed the somatic mutation profile of 15 driver genes in 69 Latin-Iberian molecularly characterized medulloblastomas using the Illumina TruSight Tumor 15 panel. We classified the variants based on their clinical impact and oncogenicity. Among the patients, 66.7% were MBSHH, 13.0% MBWNT, 7.3% MBGrp3, and 13.0% MBGrp4. Among the 63 variants found, 54% were classified as Tier I/II and 31.7% as oncogenic/likely oncogenic. We observed 33.3% of cases harboring at least one mutation. TP53 (23.2%, 16/69) was the most mutated gene, followed by PIK3CA (5.8%, 4/69), KIT (4.3%, 3/69), PDGFRA (2.9%, 2/69), EGFR (1.4%, 1/69), ERBB2 (1.4%, 1/69), and NRAS (1.4%, 1/69). Approximately 41% of MBSHH tumors exhibited mutations, TP53 (32.6%) being the most frequently mutated gene. Tier I/II and oncogenic/likely oncogenic TP53 variants were associated with relapse, progression, and lower survival rates. Potentially actionable variants in the PIK3CA and KIT genes were identified. Latin-Iberian medulloblastomas, particularly the MBSHH, exhibit higher mutation frequencies than other populations. We corroborate the TP53 mutation status as an important prognostic factor, while PIK3CA and KIT are potential therapeutic targets.

Detection of NTRK fusions by RNA-based nCounter is a feasible diagnostic methodology in a real-world scenario for non-small cell lung cancer assessment.

NTRK1, 2, and 3 fusions are important therapeutic targets for NSCLC patients, but their prevalence in South American admixed populations needs to be better explored. NTRK fusion detection in small biopsies is a challenge, and distinct methodologies are used, such as RNA-based next-generation sequencing (NGS), immunohistochemistry, and RNA-based nCounter. This study aimed to evaluate the frequency and concordance of positive samples for NTRK fusions using a custom nCounter assay in a real-world scenario of a single institution in Brazil. Out of 147 NSCLC patients, 12 (8.2%) cases depicted pan-NTRK positivity by IHC. Due to the absence of biological material, RNA-based NGS and/or nCounter could be performed in six of the 12 IHC-positive cases (50%). We found one case exhibiting an NTRK1 fusion and another an NTRK3 gene fusion by both RNA-based NGS and nCounter techniques. Both NTRK fusions were detected in patients diagnosed with lung adenocarcinoma, with no history of tobacco consumption. Moreover, no concomitant EGFR, KRAS, and ALK gene alterations were detected in NTRK-positive patients. The concordance rate between IHC and RNA-based NGS was 33.4%, and between immunohistochemistry and nCounter was 40%. Our findings indicate that NTRK fusions in Brazilian NSCLC patients are relatively rare (1.3%), and RNA-based nCounter methodology is a suitable approach for NRTK fusion identification in small biopsies.

High frequency of WNT-activated medulloblastomas with CTNNB1 wild type suggests a higher proportion of hereditary cases in a Latin-Iberian population.

Purpose: Medulloblastomas are the most common primary malignant brain tumors in children. They are divided into molecular subgroups: WNT-activated, SHH-Activated, TP53 mutant or wild type, and non-WNT/non-SHH (Groups 3 and 4). WNT-activated medulloblastomas are usually caused by mutations in the CTNNB1 gene (85%-90%), and most remaining cases of CTNNB1 wild type are thought to be caused by germline mutations in APC. So far, the frequencies of CTNNB1 have been reported mainly in North American and European populations. The aim of this study was to report the frequency of CTNNB1 mutations in WNT-activated medulloblastomas in a Latin-Iberian population and correlate with their clinicopathological characteristics. Methods: A total of 266 medulloblastomas from seven different institutions from Brazil (n=211), Portugal (n=38), and Argentina (n=17) were evaluated. Following RNA and DNA isolation from formalin-fixed, paraffin-embedded (FFPE) tumor tissues, the molecular classification and CTNNB1 mutation analysis were performed by nCounter and Sanger sequencing, respectively. Results: WNT-activated medulloblastomas accounted for 15% (40/266) of the series. We observed that 73% of WNT-activated medulloblastomas harbored CTNNB1 mutations. CTNNB1 wild-type cases (27%) were more prevalent in female individuals and suggested to be associated with a worse outcome. Among the CTNNB1 wild-type cases, the available analysis of family history revealed two cases with familiar adenomatous polyposis, harboring APC germline variants. Conclusion: We observed a lower incidence of CTNNB1 mutations in WNT-activated medulloblastomas in our Latin-Iberian cohort compared to frequencies previously described in other populations. Considering that CTNNB1 wild-type cases may exhibit APC germline mutations, our study suggests a higher incidence (~30%) of hereditary WNT-activated medulloblastomas in the Latin-Iberian population.

Genomic profile of two Brazilian choroid plexus tumors by whole-exome sequencing.

Choroid plexus tumors (CPTs) are rare intracranial neoplasms, representing <1% of all brain tumors, yet they represent 20% of first-year pediatric brain tumors. Although these tumors have been linked to TP53 germline mutations in the context of Li-Fraumeni syndrome, their somatic driver alterations remain poorly understood. In this study, we report two cases of lateral ventricle tumors: 3-yr-old male diagnosed with an atypical choroid plexus papilloma (aCPP), and a 6-mo-old female diagnosed with a choroid plexus carcinoma (CPC). We performed whole-exome sequencing of paired blood and tumor tissue in both patients, categorized somatic variants, and determined copy-number alterations. Our analysis revealed a tier II variant (Association for Molecular Pathology [AMP] criteria) in BRD1, a H3 and TP53 acetylation agent, in the aCPP. In addition, we detected copy-number gains on Chromosomes 12, 18, and 20 and copy-number losses on Chromosomes 13q and 22q (BRD1 locus) in this tumor. The CPC tumor had only a pathogenic germline TP53 variant, based on American College of Medical Genetics (ACMG) criteria, with a clinical and familiar history of Li-Fraumeni syndrome. The CPC patient presented loss of heterozygosity (LoH) of TP53 loci and hyperdiploid genome. Both tumors were microsatellite-stable. This is the first study performing whole-exome sequencing in Brazilian choroid plexus tumors, and in line with the literature, we corroborate the absence of recurrent somatic mutations in these tumors. Further studies with larger sample sizes are necessary to confirm our findings and better understand the underlying biology of these tumors.

Digital expression profile of immune checkpoint genes in medulloblastomas identifies CD24 and CD276 as putative immunotherapy targets.

Introduction: Medulloblastoma is the most common and lethal pediatric malignant brain tumor. It comprises four main molecular subgroups: WNT-activated, SHH-activated, Group 3, and Group 4. Medulloblastoma treatment is surgical resection, craniospinal radiation, and chemotherapy. However, many patients do not respond to therapy, and most suffer severe side effects. Cancer immunotherapy targeting immune checkpoints (IC) (PD-1, PD-L1, and CTLA4) has been getting disappointing outcomes in brain tumors. Nevertheless, other less explored immune checkpoints may be promising candidates for medulloblastoma therapy. Objectives: In the present study, we aimed to characterize the expression profile of 19 immune checkpoints in medulloblastoma. Methods: We analyzed 88 formalin-fixed paraffin-embedded medulloblastomas previously classified for each molecular subgroup and three non-tumoral brain tissue. mRNA levels of 19 immune checkpoint-related genes were quantified using the nCounter (PanCancer Immune Profiling Panel) assay. Further in silico analysis was performed in two larger public microarray datasets, one of which enabled comparisons between tumoral and non-tumoral tissues. Immunohistochemistry of PD-L1 was performed in a subset of cases. Microsatellite instability was also molecularly analyzed. Results: We observed an absence of expression of the canonic ICs, namely PDCD1 (PD-1), CD274 (PD-L1), and CTLA4, as well as CD80, CD86, BTLA, IDO1, CD48, TNFSF14, CD160, CEACAM1, and CD244. PD-L1 protein expression was also practically absent. We found higher mRNA levels of CD24, CD47, CD276 (B7-H3), and PVR, and lower mRNA levels of HAVCR2, LAG3, and TIGIT genes, with significant differences across the four molecular subgroups. Compared to the non-tumor tissues, the expression levels of CD276 in all subgroups and CD24 in SHH, Group 3, and Group 4 subgroups are significantly higher. The in silico analysis confirmed the expression profile found in the Brazilian cohort, including the lower/absent expression of the canonic ICs. Moreover, it confirmed the overexpression of CD24 and CD276 in medulloblastomas compared with the non-tumor tissue. Additionally, CD276 and CD24 high levels were associated with worse survival. Conclusion: These results highlight the low or absence of mRNA levels of the canonic targetable ICs in medulloblastomas. Importantly, the analysis revealed overexpression of CD24 and CD276, which can constitute prognostic biomarkers and attractive immunotherapy targets for medulloblastomas.

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.

Single nCounter assay for prediction of MYCN amplification and molecular classification of medulloblastomas: a multicentric study.

PURPOSE: Medulloblastoma is the most frequent pediatric malignant brain tumor, and is divided into four main subgroups: WNT, SHH, group 3, and group 4. MYCN amplification is an important medulloblastoma prognostic biomarker. We aimed to molecular classify and predict MYCN amplification in a single assay. METHODS: It was included 209 medulloblastomas from 205 patients (Brazil, Argentina, and Portugal), divided into training (n = 50) and validation (n = 159) sets. A nCounter assay was carried out using a custom panel for molecular classification, with additional genes, including MYCN. nSolver 4.0 software and the R environment were used for profiling and MYCN mRNA analysis. MYCN amplification by FISH was performed in 64 cases. RESULTS: The 205 medulloblastomas were classified in SHH (44.9%), WNT (15.6%), group 3 (18.1%) and group 4 (21.4%). In the training set, MYCN amplification was detected in three SHH medulloblastomas by FISH, which showed significantly higher MYCN mRNA counts than non-FISH amplified cases, and a cutoff for MYCN amplification was established ([Formula: see text] + 4σ = 11,124.3). Applying this threshold value in the validation set, we identified MYCN mRNA counts above the cutoff in three cases, which were FISH validated. CONCLUSION: We successfully stratified medulloblastoma molecular subgroups and predicted MYCN amplification using a single nCounter assay without the requirement of additional biological tissue, costs, or bench time.

A Unique Case Report of Infant-Type Hemispheric Glioma (Gliosarcoma Subtype) with TPR-NTRK1 Fusion Treated with Larotrectinib.

Herein, we present a rare case of a nine-month-old boy diagnosed with infant-type hemispheric glioma (gliosarcoma subtype) at the left frontal lobe. Following subtotal resection, the patient started chemotherapy with the BABY POG protocol. We describe the clinical diagnosis, histological characteristics, radiological features, molecular aspects, and management of this tumor. A comprehensive molecular analysis on the tumor tissue showed a TPR-NTRK1 gene fusion. The patient was treated with a TRK inhibitor, larotrectinib, and exhibited a stable disease with residual lesion following 8 months of target therapy. The present study is the first report of an infantile gliosarcoma harboring NTRK1 rearrangement treated with larotrectinib.

A PCR-Based Approach for Driver Mutation Analysis of EGFR, KRAS, and BRAF Genes in Lung Cancer Tissue Sections.

Driver mutations in non-small cell lung cancer (NSCLC) have a relevant significance for clinical management. EGFR mutations are the most important predictive biomarkers for NSCLC, although KRAS and BRAF mutations can also be prognostic and predictive biomarkers, respectively. PCR-based approaches followed by sequencing are useful for EGFR, KRAS, and BRAF mutational analysis. Herein, all steps for a PCR-based technique, from DNA isolation from tumor tissue sections to DNA sequencing for genetic analysis of EGFR, KRAS, and BRAF hotspot regions are described.

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.

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.

EGF+61 A>G polymorphism is not associated with lung cancer risk in the Brazilian population.

Epidermal growth factor (EGF) and its receptor (EGFR) play an important role in lung carcinogenesis. A functional single nucleotide polymorphism (SNP) in EGF promoter region (EGF+61 A>G-rs4444903) has been associated with cancer susceptibility. Yet, in lung cancer, the EGF+61 A>G role is unclear. The aim of this study was to evaluate the risk of lung cancer associated with EGF+61 A>G SNP in the Brazilian population. For that, 669 lung cancer patients and 1104 controls were analyzed. EGF+61 A>G genotype was assessed by PCR-RFLP and TaqMan genotyping assay. Both patients and controls were in Hardy-Weinberg equilibrium. As expected, uni- and multivariate analyses showed that tobacco consumption and age were significant risk factors for lung cancer. The genotype frequencies in lung cancer patients were 27.3% of AA, 47.4% of AG and 25.3% of GG, and for controls were 25.3% of AA, 51.6% of AG and 23.1% of GG. The allele frequencies were 51.1% of A and 48.9% of G for both cases and controls. No significant differences for the three genotypes (AA, AG and GG-codominant model) were observed between cases and controls. We then grouped AG and GG (recessive model) genotypes, as well as AA and AG (dominant model), and again, no significant differences were also found. This is the largest study to explore EGF+61 A>G polymorphism association with lung cancer risk and suggests that this SNP is not a risk factor for lung cancer in the Brazilian population.

Syncytia Induction by Clinical Isolates of Human Respiratory Syncytial Virus A.

OBJECTIVE: Syncytia formation is the hallmark of the cytopathic effect caused by human respiratory syncytial virus (HRSV), which is the most important viral respiratory pathogen in children. This article reports methodological improvements in primary HRSV isolation and the importance of syncytia formation and mRNA levels of F protein for the progeny yield, using clinical isolates of HRSV. METHODS: The A and B strains of HRSV were isolated in HEp-2 cell cultures from fresh and frozen nasopharyngeal aspirates. The formation of syncytia was evaluated using 2 different assays. Levels of F protein mRNA were quantified by real-time PCR while HRSV progeny titration was done by plaque assay. RESULTS: HRSV was primarily isolated from 238 of 312 (90.7%) samples, and 13 of these (12 HRSV-A and 1 HRSV-B) were continuously passaged in vitro. The quantity and size of syncytia formed by 6 pure HRSV-A clinical isolates were different, as were the levels of F protein mRNA. CONCLUSION: There is a direct correlation of quantities of syncytia and inoculum size, but not with mRNA levels of HRSV-A F protein. Importantly, levels of F protein mRNA were directly related to progeny production.

Cytotoxicity of allitinib, an irreversible anti-EGFR agent, in a large panel of human cancer-derived cell lines: KRAS mutation status as a predictive biomarker.

BACKGROUND: The epidermal growth factor receptor (EGFR) is a member of the HER family of growth factors that activates several intracellular signaling pathways promoting proliferation and survival. EGFR over-expression is frequently associated with gene mutation or amplification, thereby constituting a major target for molecular therapies. Recently, a new generation of EGFR inhibitors has been developed with pan-HER properties and irreversible actions. Allitinib® (AST1306) is an orally active, highly selective irreversible inhibitor of the HER family of receptor tyrosine kinases with promising efficacies. In the present study we aimed to investigate the cytotoxicity of allitinib in a large panel of human cancer-derived cell lines and to correlate its efficacy to the mutational status of the EGFR, KRAS, BRAF, PI3KCA and PTEN genes. In addition, we aimed to evaluate the functional role of KRAS mutations in the response to this new inhibitor. RESULTS: In total 76 different cancer-derived cell lines, representing 11 distinct histological types, were analyzed and classified into three groups: highly sensitive (HS), moderately sensitive (MS) and resistant (R). We found that 28 (36.8 %) cancer-derived cell lines exhibited a HS phenotype, 19 (25.0 %) a MS phenotype and 29 (38.1 %) a R phenotype. Allitinib showed a stronger cytotoxicity in head and neck, esophageal, melanoma and lung cancer-derived cell lines. We found that KRAS mutations were significantly associated with the R phenotype. To substantiate these results, an allitinib-sensitive lung cancer-derived cell line (H292) was transfected with plasmids carrying the two most common activating KRAS mutations (p.G12D and p.G12S). We found that both mutations reverted the allitinib-sensitive phenotype in these cells. CONCLUSIONS: The current study represents the largest in vitro assessment of allitinib cytotoxicity performed to date. Through this study, we identified cancer types that could potentially benefit from this drug. Additionally, our findings suggest that prevalent KRAS mutations constitute potential predictive biomarkers for allitinib response.

Severe lower respiratory tract infection in infants and toddlers from a non-affluent population: viral etiology and co-detection as risk factors.

BACKGROUND: Lower respiratory tract infection (LRTI) is a major cause of pediatric morbidity and mortality, especially among non-affluent communities. In this study we determine the impact of respiratory viruses and how viral co-detections/infections can affect clinical LRTI severity in children in a hospital setting. METHODS: Patients younger than 3 years of age admitted to a tertiary hospital in Brazil during the months of high prevalence of respiratory viruses had samples collected from nasopharyngeal aspiration. These samples were tested for 13 different respiratory viruses through real-time PCR (rt-PCR). Patients were followed during hospitalization, and clinical data and population characteristics were collected during that period and at discharge to evaluate severity markers, especially length of hospital stay and oxygen use. Univariate regression analyses identified potential risk factors and multivariate logistic regressions were used to determine the impact of specific viral detections as well as viral co-detections in relation to clinical outcomes. RESULTS: We analyzed 260 episodes of LRTI with a viral detection rate of 85% (n = 222). Co-detection was observed in 65% of all virus-positive episodes. The most prevalent virus was Respiratory Syncytial Virus (RSV) (54%), followed by Human Metapneumovirus (hMPV) (32%) and Human Rhinovirus (HRV) (21%). In the multivariate models, infants with co-detection of HRV + RSV stayed 4.5 extra days (p = 0.004), when compared to infants without the co-detection. The same trends were observed for the outcome of days of supplemental oxygen use. CONCLUSIONS: Although RSV remains as the main cause of LRTI in infants our study indicates an increase in the length of hospital stay and oxygen use in infants with HRV detected by RT-PCR compared to those without HRV. Moreover, one can speculate that when HRV is detected simultaneously with RSV there is an additive effect that may be reflected in more severe clinical outcome. Also, our study identified a significant number of children infected by recently identified viruses, such as hMPV and Human Bocavirus (HBov), and this is a novel finding for poor communities from developing countries.