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.

PD-L1 expression and microsatellite instability (MSI) in cancer of unknown primary site.

BACKGROUND: Cancer of unknown primary site (CUP) is a heterogeneous group of tumors for which the origin remains unknown. Clinical outcomes might be influenced by regulatory processes in its microenvironment. Microsatellite instability (MSI) is a predictive biomarker for cancer immunotherapy and its status, as well as co-occurrence with PD-L1 expression, is poorly evaluated. We aim to evaluate the expression of PD-L1 and the status of MSI in CUP and their possible associations with clinical-pathological features. METHODS: The combined positive score (CPS) PD-L1 expression was evaluated by immunohistochemistry. MSI status was assessed using a hexa-plex marker panel by polymerase chain reaction followed by fragment analysis. RESULTS: Among the 166 cases, MSI analysis was conclusive in 120, with two cases being MSI positive (1.6%). PD-L1 expression was positive in 18.3% of 109 feasible cases. PD-L1 expression was significantly associated with non-visceral metastasis and a dominance of nodal metastasis. The median overall survival (mOS) was 3.7 (95% CI 1.6-5.8) months and patients who expressed PD-L1 achieved a better mOS compared to those who did not express PD-L1 (18.7 versus 3.0 months, p-value: < .001). ECOG-PS equal to or more than two and PD-L1 expression were independent prognostic factors in multivariate analysis (2.37 and 0.42, respectively). CONCLUSION: PD-L1 is expressed in a subset (1/5) of patients with CUP and associated with improved overall survival, while MSI is a rare event. There is a need to explore better the tumor microenvironment as well as the role of immunotherapy to change such a bad clinical outcome.

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.

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.

Methylation profile of colon cancer genes in colorectal precursor lesions and tumor tissue: perspectives for screening.

AIMS: Epigenetic alterations of genes involved in colorectal carcinogenesis are likely to be informative biomarkers for early detection. We assessed the methylation profile of a panel of seven colon cancer-related genes comparing normal colon, colorectal cancer (CRC) precursor lesions and cancer tissues from a Brazilian cohort. METHODS: The cohort comprised 114 CRC patients, including 40 matched normal tissue, 47 patients with adenomas, 33 with serrated polyps and 8 with normal colonic biopsy. DNA methylation status of SEPT9, ALX4, NDRG4, BMP3, APC, p16 and MLH1 was determined by pyrosequencing and correlated with clinicopathological features. Sensitivity, specificity, positive predictive value and negative predictive value were calculated for all genes using cancer endpoint. RESULTS: The most frequently methylated genes in cancer and in precancer lesions were SEPT9, ALX4, NDRG4, and BMP3, ranging from 55.3 to 95% of the samples. Overall, the frequency of methylation of these four genes in normal colonic tissue was significantly lower as compared to cancer or precursor lesions both in adenoma-carcinoma (p < .001 and p < .050) and serrated (sessile-serrated lesion) (p < .001 and p < .050) pathways. Additionally, sensitivity for the cancer endpoint ranged from 65.6 to 91.8%, and specificity from 17.9 to 62.9% for SEPT9, ALX4, NDRG4, and BMP3 genes. Moreover, the comethylation of ≥4 genes was higher in sessile-serrated lesion (87.5%) and conventional adenomas (78.7%) than in hyperplastic polyps (43.7%) (p = .025) and was significantly associated with proximal cancers (p = .042). CONCLUSIONS: Our study suggests the DNA methylation can constitute potential biomarkers in CRC screening of Brazilian population.

Vemurafenib resistance increases melanoma invasiveness and modulates the tumor microenvironment by MMP-2 upregulation.

The BRAF(V600E) mutation confers constitutive kinase activity and accounts for >90% of BRAF mutations in melanoma. This genetic alteration is a current therapeutic target; however, the antitumorigenic effects of the BRAF(V600E) inhibitor vemurafenib are short-lived and the majority of patients present tumor relapse in a short period after treatment. Characterization of vemurafenib resistance has been essential to the efficacy of next generation therapeutic strategies. Herein, we found that acute BRAF inhibition induced a decrease in active MMP-2, MT1-MMP and MMP-9, but did not modulate the metalloproteinase inhibitors TIMP-2 or RECK in naïve melanoma cells. In vemurafenib-resistant melanoma cells, we observed a lower growth rate and an increase in EGFR phosphorylation followed by the recovery of active MMP-2 expression, a mediator of cancer metastasis. Furthermore, we found a different profile of MMP inhibitor expression, characterized by TIMP-2 downregulation and RECK upregulation. In a 3D spheroid model, the invasion index of vemurafenib-resistant melanoma cells was more evident than in its non-resistant counterpart. We confirmed this pattern in a matrigel invasion assay and demonstrated that use of a matrix metalloproteinase inhibitor reduced the invasion of vemurafenib resistant melanoma cells but not drug naïve cells. Moreover, we did not observe a delimited group of cells invading the dermis in vemurafenib-resistant melanoma cells present in a reconstructed skin model. The same MMP-2 and RECK upregulation profile was found in this 3D skin model containing vemurafenib-resistant melanoma cells. Acute vemurafenib treatment induces the disorganization of collagen fibers and consequently, extracellular matrix remodeling, with this pattern observed even after the acquisition of resistance. Altogether, our data suggest that resistance to vemurafenib induces significant changes in the tumor microenvironment mainly by MMP-2 upregulation, with a corresponding increase in cell invasiveness.

Immune-checkpoint gene expression and BCG response in non-muscle invasive bladder cancer.

METHODS: One-hundred-six patients diagnosed with non-muscle invasive bladder cancer and treated with intravesical BCG were included and divided into two groups, BCG-responsive (n = 47) and -unresponsive (n = 59). Immunohistochemistry was used to evaluate PD-L1 expression and MSI was assessed by a commercial multiplex PCR kit. The mRNA expression profile of 15 immune checkpoints was performed using the nCounter technology. For in silico validation, two distinct cohorts sourced from the Gene Expression Omnibus (GEO) database were used. RESULTS: Among the 106 patients, only one (<1 %) exhibited MSI instability. PD-L1 expression was present in 9.4 % of cases, and no association was found with BCG-responsive status. We found low gene expression of canonic actionable immune checkpoints PDCD1 (PD-1), CD274 (PD-L1), and CTLA4, while high expression was observed for CD276 (B7-H3), CD47, TNFRSF14, IDO1 and PVR (CD155) genes. High IDO1 expression levels was associated with worst overall survival. The PDCD1, CTLA4 and TNFRSF14 expression levels were associated with BCG responsiveness, whereas TIGIT and CD276 were associated with unresponsiveness. Finally, CD276 was validated in silico cohorts. CONCLUSION: In NMIBC, MSI is rare and PD-L1 expression is present in a small subset of cases. Expression levels of PDCD1, CTLA4, TNFRSF14, TIGIT and CD276 could constitute predictive biomarkers of BCG responsiveness.

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.

Association of microsatellite instability (MSI) status with the 5-year outcome and genetic ancestry in a large Brazilian cohort of colorectal cancer.

Colorectal cancer (CRC) has a high incidence and mortality worldwide. Microsatellite instability (MSI) is crucial in CRC, with distinct molecular and clinicopathological features in patients. Nowadays, it is a predictive marker for immunotherapy. We proposed to evaluate the 5-year outcome of MSI status in 1002 Brazilian CRC, and associate it with genetic ancestry, molecular and clinicopathological features. MSI evaluation was performed using molecular markers. MSI+ tumors were analyzed for alterations in 23 MSI-targeted genes. Genetic ancestry was evaluated using an Ancestry-Informative markers panel. MSI status was analyzed in relation to CRC specific survival and other clinical and genetic variables. MSI+ status was observed in 10.5% of cases. MSI+ status was significantly associated with the anatomic site right colon, mucinous histological type, clinical stage II, histological grade III/undifferentiated, no recurrence of disease, and live cases without cancer. No association of MSI status with genetic ancestry components was observed. MSI-targeted genes analyses showed the most frequently altered genes: ATM, EGFR, MRE11, ROCK1, and TGFBRII. There was a statistically significant difference in cancer-specific survival between cases according to MSI status. This study constitutes the most comprehensive analyses of the MSI impact on the Brazilian CRC. MSI+ frequency in Brazilian CRC agreed with the literature and was associated with several clinicopathological features related with less aggressive tumors, independently of their genetic ancestry.

Cancer immune profiling unveils biomarkers, immunological pathways, and cell type score associated with glioblastoma patients' survival.

Introduction: Glioblastoma (GBM), isocitrate dehydrogenase (IDH) wild-type (IDH wt), and grade 4 astrocytomas, IDH mutant (IDH mut), are the most common and aggressive primary malignant brain tumors in adults. A better understanding of the tumor immune microenvironment may provide new biomarkers and therapeutic opportunities. Objectives: We aimed to evaluate the expression profile of 730 immuno-oncology-related genes in patients with IDH wt GBM and IDH mut tumors and identify prognostic biomarkers and a gene signature associated with patient survival. Methods: RNA was isolated from formalin-fixed, paraffin-embedded sections of 99 tumor specimens from patients treated with standard therapy. Gene expression profile was assessed using the Pan-Cancer Immune Profiling Panel (Nanostring Technologies, Inc., Seattle, WA, USA). Data analysis was performed using nSolverSoftware and validated in The Cancer Genome Atlas. In addition, we developed a prognostic signature using the cox regression algorithm (Least Absolute Shrinkage and Selection Operator). Results: We found 88 upregulated genes, high immunological functions, and a high macrophage score in IDH wt GBM compared to IDH mut tumors. Regarding IDH wt GBM, we found 24 upregulated genes in short-term survivors (STS) and overexpression of CD274 (programmed death-ligand 1, PD-L1). Immune pathways, CD45, cytotoxic, and macrophage scores were upregulated in STS. Two different prognostic groups were found based on the 12-gene signature (CXCL14, PSEN2, TNFRSF13C, IL13RA1, MAP2K1, TNFSF14, THY1, CTSL, ITGAE, CHUK, CD207, and IFITM1). Conclusion: The elevated expression of immune-oncology-related genes was associated with worse outcome in IDH wt GBM patients. Increased immune functions, CD45, cytotoxic cells, and macrophage scores were associated with a more aggressive phenotype and may provide promising possibilities for therapy. Moreover, a 12 gene-based signature could predict patients' prognosis.

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.

Clinicopathological and molecular characterization of Brazilian families at risk for Lynch syndrome.

Lynch syndrome (LS), is the most common hereditary colorectal cancer syndrome. However, it is poorly characterized in Brazil. Therefore, we aimed to determine the spectrum of pathogenic variants in Mismatch Repair (MMR) genes and investigate the MLH1 promotor methylation role as a second hit in LS tumors. Tumor screening through microsatellite instability and immunohistochemistry for MMR proteins was performed in 323 cases who met clinical criteria. BRAF-V600E and MLH1 promoter methylation were analyzed for all MLH1-deficient tumors. Patients with MMR deficient tumor proceeded to germline genetic testing. MMR deficient tumors were detected in 41% of patients recruited. About half of patients carried a pathogenic germline variant. Two recurrent variants in MLH1 and three novel pathogenic variants were identified. Furthermore, pathogenic germline variants with concomitant somatic MLH1 hypermethylation were found in 6% of cases. Predictive genetic testing was offered to 387 relatives. Overall, 127 tumors were diagnosed in 100 LS patients, from 62 unrelated families. Our molecular data provide new information about the spectrum of MMR mutations, which contributes to a better characterization of LS in Brazil. Furthermore, we call attention to the possibility of failure in the diagnosis of germline MLH1 mutation carriers when somatic MLH1 hypermethylation is used to rule out LS.

A 4-Gene Signature Associated With Recurrence in Low- and Intermediate-Risk Endometrial Cancer.

BACKGROUND: The molecular profile of endometrial cancer has become an important tool in determining patient prognosis and their optimal adjuvant treatment. In addition to The Cancer Genome Atlas (TCGA), simpler tools have been developed, such as the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE). We attempted to determine a genetic signature to build a recurrence risk score in patients diagnosed with low- and intermediate-risk endometrial cancer. METHODS: A case-control study was conducted. The eligible patients were women diagnosed with recurrence low- and intermediate-risk endometrial cancer between January 2009 and December 2014 at a single institution; the recurrence patients were matched to two nonrecurrence patients with the same diagnosis by age and surgical staging. Following RNA isolation of 51 cases, 17 recurrence and 34 nonrecurrence patients, the expression profile was determined using the nCounter® PanCancer Pathways Panel, which contains 770 genes. RESULTS: The expression profile was successfully characterized in 49/51 (96.1%) cases. We identified 12 genes differentially expressed between the recurrence and nonrecurrence groups. The ROC curve for each gene was generated, and all had AUCs higher than 0.7. After backward stepwise logistic regression, four genes were highlighted: FN1, DUSP4, LEF1, and SMAD9. The recurrence risk score was calculated, leading to a ROC curve of the 4-gene model with an AUC of 0.93, sensitivity of 100%, and specificity of 72.7%. CONCLUSION: We identified a four-gene signature that may be associated with recurrence in patients with low- and intermediate-risk endometrial cancer. This finding suggests a new prognostic factor in this poorly explored group of patients with endometrial cancer.

Pyknon-Containing Transcripts Are Downregulated in Colorectal Cancer Tumors, and Loss of PYK44 Is Associated With Worse Patient Outcome.

Pyknons are specific human/primate-specific DNA motifs at least 16 nucleotides long that are repeated in blocks in intergenic and intronic regions of the genome and can be located in a new class of non-coding RNAs of variable length. Recent studies reported that pyknon deregulation could be involved in the carcinogenesis process, including colorectal cancer. We evaluated the expression profile of a set of 12 pyknons in a set of molecularly characterized colorectal cancer (CRC) patients. The pyknons (PYK10, PYK14, PYK17, PYK26, PYK27, PYK40, PYK41, PYK42, PYK43, PYK44, PYK83, and PYK90) expression was determined by qRT-PCR. A pilot analysis of 20 cases was performed, and consistent results were obtained for PYK10, PYK17, PYK42, PYK44, and PYK83. Further, the expression of the selected pyknons was evaluated in 73 CRC cases. Moreover, in 52 patients, we compared the expression profile in both tumor and normal tissues. All five pyknons analyzed showed significantly lower expression levels in the tumor compared to normal tissue. It was observed an association between expression of PYK10 with TP53 mutations (p = 0.029), PYK17 to histologic grade (p = 0.035), and PYK44 to clinical staging (p = 0.016). Moreover, levels of PYK44 were significantly associated with the patient's poor overall survival (p = 0.04). We reported the significant downregulation of pyknons motifs in tumor tissue compared with the normal counterpart, and the association of lower PYK44 expression with worse patient outcome. Further studies are needed to extend and validate these findings and determine the clinical-pathological impact.

Role of Genetic Ancestry in 1,002 Brazilian Colorectal Cancer Patients From Barretos Cancer Hospital.

Background: Colorectal cancer (CRC) is the third most frequent and the second deadliest cancer worldwide. The ethnic structure of the population has been gaining prominence as a cancer player. The purpose of this study was to determine the genetic ancestry of Brazilian CRC patients. Moreover, we intended to interrogate its impact on patients' clinicopathological features. Methods: Retrospective observational cohort study with 1,002 patients with CRC admitted from 2000 to 2014 at Barretos Cancer Hospital. Following tumor DNA isolation, genetic ancestry was assessed using a specific panel of 46 ancestry informative markers. Survival rates were obtained by the Kaplan-Meier method, and the log-rank test was used to compare the survival curves. Multivariable Cox proportional regression models were used to estimate hazard ratios (HRs). Results: We observed considerable admixture in the genetic composition, with the following average proportions: European 74.2%, African 12.7%, Asian 6.5%, and Amerindian 6.6%. The multivariate analysis for cancer-specific survival showed that clinical stage, lymphovascular invasion, and the presence of recurrence were associated with an increased relative risk of death from cancer (p < 0.05). High African proportion was associated with younger age at diagnosis, while high Amerindian proportion was associated with the mucinous histological subtype. Conclusions: This represents the larger assessment of genetic ancestry in a population of Brazilian patients with CRC. Brazilian CRC patients exhibited similar clinicopathological features as described in Western countries. Impact: Genetic ancestry components corroborated the significant admixture, and importantly, patients with high African proportion develop cancer at a younger age.

Mutation profiling of cancer drivers in Brazilian colorectal cancer.

The molecular basis of colorectal cancer (CRC) can guide patient prognosis and therapy. In Brazil, knowledge on the CRC mutation landscape is limited. Here, we investigated the mutation profile of 150 cancer-related genes by next-generation sequencing and associated with microsatellite instability (MSI) and genetic ancestry in a series of 91 Brazilian CRC patients. Driver mutations were found in the APC (71.4%), TP53 (56.0%), KRAS (52.7%), PIK3CA (15.4%) and FBXW7 (10.9%) genes. Overall, genes in the MAPK/ERK, PIK3/AKT, NOTCH and receptor tyrosine kinase signaling pathways were mutated in 68.0%, 23.1%, 16.5%, and 15.3% of patients, respectively. MSI was found in 13.3% of tumors, most of which were proximal (52.4%, P< 0.001) and had a high mutation burden. European genetic ancestry was predominant (median of 83.1%), followed by Native American (4.1%), Asian (3.4%) and African (3.2%). NF1 and BRAF mutations were associated with African ancestry, while TP53 and PIK3CA mutations were inversely correlated with Native American ancestry. Our study suggests that Brazilian CRC patients exhibit a mutation profile similar to other populations and identify the most frequently mutated genes, which could be useful in future target therapies and molecular cancer screening strategies.

Microbiota Profile and Impact of Fusobacterium nucleatum in Colorectal Cancer Patients of Barretos Cancer Hospital.

Microbial diversity has been pointed out as a major factor in the development and progression of colorectal cancer (CRC). We sought to explore the richness and abundance of the microbial community of a series of colorectal tumor samples treated at Barretos Cancer Hospital, Brazil, through 16S rRNA sequencing. The presence and the impact of Fusobacterium nucleatum (Fn) DNA in CRC prognosis was further evaluated by qPCR in a series of 152 CRC cases. An enrichment for potentially oncogenic bacteria in CRC was observed, with Fusobacterium being the most abundant genus in the tumor tissue. In the validation dataset, Fn was detected in 35/152 (23.0%) of fresh-frozen tumor samples and in 6/57 (10.5%) of paired normal adjacent tissue, with higher levels in the tumor (p = 0.0033). Fn DNA in the tumor tissue was significantly associated with proximal tumors (p = 0.001), higher depth of invasion (p = 0.014), higher clinical stages (p = 0.033), poor differentiation (p = 0.011), MSI-positive status (p < 0.0001), BRAF mutated tumors (p < 0.0001), and the loss of expression of mismatch-repair proteins MLH1 (p < 0.0001), MSH2 (p = 0.003), and PMS2 (p < 0.0001). Moreover, the presence of Fn DNA in CRC tissue was also associated with a worse patient cancer-specific survival (69.9 vs. 82.2% in 5 years; p = 0.028) and overall survival (63.5 vs. 76.5%; p = 0.037). Here we report, for the first time, the association of F. nucleatum presence with important clinical and molecular features in a Brazilian cohort of CRC patients. Tumor detection and classification based on the gut microbiome might provide a promising approach to improve the prediction of patient outcome.

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.

Advantage of HSP110 (T17) marker inclusion for microsatellite instability (MSI) detection in colorectal cancer patients.

Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Microsatellite instability (MSI) is a genetic pathway leading to CRC, associated with particular clinicopathological features, and recently a major biomarker of immunotherapy response. There is little information the frequency MSI among Brazilian CRC patients, and it is still debatable the ideal methodology for MSI screening in countries with limited resources. We proposed to evaluate MSI by molecular and immunohistochemistry (IHC) methods, to compare both methodologies and also to assess the inclusion of a novel microsatellite marker, HSP110 (T17). The molecular MSI evaluation was performed using a PCR-multiplex panel in a total of 1013 CRC patients. Mismatch repair (MMR) proteins (MLH1, MSH2, MSH6 and PMS2) expression were evaluated by IHC. HSP110 (T17) marker was analyzed by fragment analysis. Molecularly, 89.5% of cases were MSI-negative and 10.5% were MSI-positive. The IHC showed that 88.9% of cases exhibited MMR-proficient status, 10.2% were MMR-deficient and 0.9% was inconclusive. Genotyping of the HSP110 (T17) in 106 MSI-positive and 215 MSI-negative cases showed its alteration only among the MSI-positive cases. We observed agreement (0.956, Kappa Test) between both molecular and IHC methodologies, with only eight discordant results, and in this subset of cases the HSP110 (T17) corroborate the molecular findings. This study suggests the use of molecular assays over IHC for MSI analysis and proposes the inclusion HSP110 (T17) marker as a complementary analysis in discordant cases.