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

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

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.

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

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

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

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

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

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

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

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

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.

EGF+61 A>G polymorphism does not predict response to first-generation EGFR tyrosine kinase inhibitors in lung cancer patients.

Epidermal growth factor (EGF) and its receptor (EGFR) play a paramount role in lung carcinogenesis. The polymorphism in the EGF promoter region EGF+61A>G (rs4444903) has been associated with cancer susceptibility, but its role in lung cancer patients treated with tyrosine kinase inhibitors (TKIs) remains unknown. Here, we aimed to evaluate the predictive and prognostic role of EGF+61A>G SNP in lung cancer from Brazilian EGFR-mutated TKI-treated patients. Herein, patients carrying EGFR-sensitizing mutations submitted to TKI treatment (gefitinib/erlotinib) were analyzed (n = 111) for EGF+61A>G genotype by TaqMan genotyping assay. TKI treatment was classified as partial response (PR), stable disease (SD), and disease progression (DP), according to RECIST1.1. Association analysis was assessed by chi-square and Fisher's test (univariate) and multinomial model (multivariate) and survival analysis by Kaplan-Meier method and log-rank test. The EGF+61A>G genotype frequencies observed were: AA = 31.5% (n = 35), AG = 49.6% (n = 55) and GG = 18.9% (n = 21). The allelic frequencies were 56.3% for A, and 43.7% for G and the population was in Hardy-Weinberg equilibrium (P = 0.94). EGF+61A>G codominant model (AA vs. AG vs. GG) was associated with a response to TKIs (P = 0.046), as well as a recessive model (AA vs. AG + GG; P = 0.023). The multinomial regression showed an association between the codominant model (AG) and recessive model (AG + GG) with SD compared with DP (P = 0.01;OR = 0.08; 95% CI = 0.01-0.60 and P = 0.02;OR = 0.12; 95% CI = 0.20-0.72, respectively). No association between genotypes and progression-free or overall survival was observed. In conclusion, the EGF+61 polymorphism (AG and AG + GG) was independently associated with stable disease in lung cancer patients although it was not associated with the overall response rate to first-generation TKIs or patient outcome.

Lack of KBTBD4 Mutations in Molecularly Classified Brazilian Medulloblastomas.

Medulloblastoma is the most frequent malignant brain tumor in children, representing 20% of all childhood brain tumors. Currently, medulloblastomas are molecularly classified in 4 subgroups that are associated with distinctive clinicopathological features. KBTBD4 mutations were recently described in a subset of MBGRP3 and MBGRP4 medulloblastomas subgroups. However, no other studies reported KBTBD4 mutations in medulloblastomas. Thus, our aim was to investigate KBTBD4 mutations in a Brazilian series of medulloblastoma. We evaluated 128 medulloblastoma patients molecularly classified from 4 Brazilian reference centers. DNA from formalin-fixed, paraffin-embedded samples was screened for KBTBD4 hotspot mutations by Sanger sequencing. Most of the patients were male, average age was 16.5 years old and average overall survival was 55.9 months. The predominant histological subtype was the classic subtype, followed by nodular/desmoplastic, and the predominant medulloblastoma molecular subtype was the MBSHH subgroup (46%), followed by MBGRP3 and MBGRP4 (19%/each), and MBWNT (16%). Among the 128 samples, 111 were successfully sequenced. No KBTBD4 mutations were identified in 111 samples. Our findings suggest that KBTBD4 mutations are uncommon in Brazilian MBGRP3 and MBGRP4 medulloblastomas subgroups. Further studies in a larger series of MBGRP3 and MBGRP4 medulloblastomas are warranted to better assess role of KBTBD4 mutations.