Surgical treatment of 186 sinonasal inverted papillomas and analysis of the immunohistochemical and molecular features associated with recurrences.

INTRODUCTION: Inverted papillomas (IP) are benign epithelial tumors with a tendency to be locally invasive and with disposition to recur. The aim of our study is to present the results of IP treatment, considering pathological, immunohistochemical and molecular features of recurrence. MATERIAL AND METHODS: From 1978 to 2020, 186 sinonasal IPs surgeries corresponding to 152 patients were treated in our center. We performed a pathology evaluation of all the recurrent cases reviewing the histological diagnosis, the presence of mixed component other than IP, the koilocytic changes, the p16 over expression and HPV-DNA detection. RESULTS: Overall recurrence rate was 19 % (35/186). The 35 IP recurrences correspond to 22 patients, 9 of whom presented a single recurrence (single recurrence group) while 13 of them presented more than one recurrence (multi-recurrent group). Immunohistochemical analysis showed a higher percentage of p16 overexpression (54 % vs 33 % p = 0.415) and HPV-DNA presence (23 % vs 0 % p = 0.240) in the multi-recurrent group compared with single recurrence group. In addition, the revision showed more IP with exophytic papilloma focus (38 vs 22 % p = 0.648) and a higher proportion of IP with koilocytotic changes (61 % vs 22 % p = 0.099) in the multirecurrent group. There is no significant difference between groups in our results. CONCLUSION: The analysis of our patients may differentiate between two groups with recurrent papillomas. A single recurrence group where the cause of recurrence is probably an anatomical problem related to an incomplete resection, and a second pattern, the multi-recurrence group, where HPV infection may be the main cause of recurrence.

Small Non-Coding RNAs and Their Role in Locoregional Metastasis and Outcomes in Early-Stage Breast Cancer Patients.

Deregulation of small non-coding RNAs (sncRNAs) has been associated with the onset of metastasis. We evaluated the expression of sncRNAs in patients with early-stage breast cancer, performing RNA sequencing in 60 patients for whom tumor and sentinel lymph node (SLN) samples were available, and conducting differential expression, gene ontology, enrichment and survival analyses. Sequencing annotation classified most of the sncRNAs into small nucleolar RNA (snoRNAs, 70%) and small nuclear RNA (snRNA, 13%). Our results showed no significant differences in sncRNA expression between tumor or SLNs obtained from the same patient. Differential expression analysis showed down-regulation (n = 21) sncRNAs and up-regulation (n = 2) sncRNAs in patients with locoregional metastasis. The expression of SNHG5, SNORD90, SCARNA2 and SNORD78 differentiated luminal A from luminal B tumors, whereas SNORD124 up-regulation was associated with luminal B HER2+ tumors. Discriminating analysis and receiver-operating curve analysis revealed a signature of six snoRNAs (SNORD93, SNORA16A, SNORD113-6, SNORA7A, SNORA57 and SNORA18A) that distinguished patients with locoregional metastasis and predicted patient outcome. Gene ontology and Reactome pathway analysis showed an enrichment of biological processes associated with translation initiation, protein targeting to specific cell locations, and positive regulation of Wnt and NOTCH signaling pathways, commonly involved in the promotion of metastases. Our results point to the potential of several sncRNAs as surrogate markers of lymph node metastases and patient outcome in early-stage breast cancer patients. Further preclinical and clinical studies are required to understand the biological significance of the most significant sncRNAs and to validate our results in a larger cohort of patients.

Circulating miRNA Expression Is Inversely Correlated with Tumor Tissue or Sentinel Lymph Nodes in Estrogen Receptor-Positive Early Breast Cancer Patients.

The deregulation of microRNAs (miRNAs) is associated with the various steps of the metastatic process. In addition, circulating miRNAs are remarkably stable in peripheral blood, making them ideal noninvasive biomarkers for disease diagnosis. Here, we performed a proof-of-principle study to determine whether tumor-tissue-derived miRNAs are traceable to plasma in ER-positive early breast cancer patients. We performed RNA-sequencing on 30 patients for whom plasma, sentinel lymph nodes (SLNs) and tumor tissue were available. We carried out differential expression, gene ontology and enrichment analyses. Our results show that circulating miRNAs are inversely expressed compared with tumor tissue or SLNs obtained from the same patients. Our differential expression analysis shows the overall downregulation of circulating miRNAs. However, the expression of miR-643a-3p and miR-223 was up-regulated in patients with positive SLNs. Furthermore, gene ontology analysis showed the significant enrichment of biological processes associated with the regulation of epithelial cell proliferation and transcriptional regulation commonly involved in the promotion of metastases. Our results suggest the potential role of several circulating miRNAs as surrogate markers of lymph node metastases in early breast cancer patients. Further preclinical and clinical studies are required to understand the biological significance of the most significant miRNAs and to validate our results in a larger cohort of patients.

Intratumor cholesteryl ester accumulation is associated with human breast cancer proliferation and aggressive potential: a molecular and clinicopathological study.

BACKGROUND: The metabolic effect of intratumor cholesteryl ester (CE) in breast cancer remains poorly understood. The objective was to analyze the relationship between intratumor CE content and clinicopathological variables in human breast carcinomas. METHODS: We classified 30 breast carcinoma samples into three subgroups: 10 luminal-A tumors (ER+/PR+/Her2-), 10 Her-2 tumors (ER-/PR-/Her2+), and 10 triple negative (TN) tumors (ER-/PR-/Her2-). We analyzed intratumor neutral CE, free cholesterol (FC) and triglyceride (TG) content by thin layer chromatography after lipid extraction. RNA and protein levels of lipid metabolism and invasion mediators were analyzed by real time PCR and Western blot analysis. RESULTS: Group-wise comparisons, linear regression and logistic regression models showed a close association between CE-rich tumors and higher histologic grade, Ki-67 and tumor necrosis. CE-rich tumors displayed higher mRNA and protein levels of low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SCARB1). An increased expression of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in CE-rich tumors was also reported. CONCLUSIONS: Intratumor CE accumulation is intimately linked to proliferation and aggressive potential of breast cancer tumors. Our data support the link between intratumor CE content and poor clinical outcome and open the door to new antitumor interventions.

ERCC1 and ERCC2 Polymorphisms Predict the Efficacy and Toxicity of Platinum-Based Chemotherapy in Small Cell Lung Cancer.

Standard first-line chemotherapy in small cell lung cancer (SCLC) is based on the platinum plus etoposide combination. Despite a high objective response rate, responses are not durable and chemotherapy-induced toxicity may compromise treatment. Genetic variants in genes involved in the DNA-repair pathways and in etoposide metabolization could predict treatment efficacy and safety and help personalize platinum-based chemotherapy. Germline polymorphisms in XRCC1, ERCC1, ERCC2, ABCB1, ABCC3, UGT1A1 and GSTP1 genes were investigated in 145 patients with SCLC. The tumor expression of ERCC1 was determined using immunohistochemistry, and the tumor expression of ERCC1-XPF was determined via a proximity ligation assay. Survival analyses showed a statistically significant association between the ERCC1 rs11615 variant and median progression-free survival (PFS) in patients with limited-stage (LS) SCLC (multivariate: hazard ratio 3.25, [95% CI 1.38-7.70]; p = 0.007). Furthermore, we observed differences between the ERCC1-XPF complex and median PFS in LS-SCLC, although statistical significance was not reached (univariate: positive expression 10.8 [95% CI 4.09-17.55] months versus negative expression 13.3 [95% CI 7.32-19.31] months; p = 0.06). Safety analyses showed that the ERCC2 rs1799793 variant was significantly associated with the risk of grade ≥ 3 thrombocytopenia in the total cohort (multivariate: odds ratio 3.15, [95% CI 1.08-9.17]; p = 0.04). Our results provide evidence that ERCC1 and ERCC2 variants may predict the efficacy and safety of platinum-based chemotherapy in SCLC patients. LS-SCLC patients may benefit most from ERCC1 determination, but prospective studies are needed.

RET Fusion Testing in Patients With NSCLC: The RETING Study.

Introduction: RET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC. Methods: This situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion-positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay. Results: The most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively). Conclusions: In-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.

Differences in tumor thickness between hematoxylin and eosin and Melan-A immunohistochemically stained primary cutaneous melanomas.

Tumor thickness (Breslow thickness) represents the main prognostic factor in primary melanoma. Potential differences in melanoma tumor thickness measurements between conventional hematoxylin and eosin (H&E) and Melan-A immunohistochemical staining were evaluated. Ninety-nine excisional biopsies were included in the study. From each sample, 2 consecutive histological sections were stained with H&E and Melan-A, respectively. Tumor thickness was measured from both sections by 2 independent observers. In 59 biopsy specimens (59.6%), higher tumor thickness measurements were recorded in Melan-A-stained than in H&E-stained sections. In 42.4% of such cases (25 biopsies), the observed differences were ≥0.2 mm. After Melan-A evaluation, 33% of in situ melanoma cases were reclassified as invasive melanoma, with thickness measurements ranging from 0.15 to 0.35 mm. In 23 biopsies, identical values were recorded with both techniques, whereas in 17 cases, measurements obtained with H&E staining were slightly higher (from 0.01 to 0.18 mm) than those obtained with Melan-A staining. A high rate of interobserver agreement was noted, and significant intertechnique measurement differences were detected. Significant discrepancies (≥0.2 mm) in thickness measurements between the 2 techniques were mainly attributed to the presence of individual or small clusters of melanocytic cells in the papillary dermis. These melanocytic cells could be easily overlooked in H&E-stained sections, especially in sections showing dense lymphohistiocytic inflammatory infiltrates, numerous melanin-containing histiocytic cells in the upper dermis, or extensive fibrotic changes or regression phenomena. This study confirms the practical interest of immunohistochemical staining with Melan-A in evaluating primary melanoma and, specifically, in situ melanoma cases.

Reduced Number of Thymoma CTLA4-Positive Cells Is Associated With a Higher Probability of Developing Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Myasthenia gravis (MG) is an autoimmune disease associated with comorbid thymoma in 10%-15% of cases. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) expressed by T cells downregulates T-cell-mediated immune response. Polymorphisms in the CTLA4 gene have been associated with the development of MG. In this context, we aimed to determine whether CTLA4 expression in the thymoma differs between patients with and without MG and whether CTLA4 gene polymorphisms are associated with these differences. METHODS: This is a retrospective study of all patients, with and without MG, surgically treated at our institution for thymoma between January 2010 and December 2020. Ten samples were obtained from normal thymuses as controls. The number of CTLA4-positive cells in paraffin-embedded thymoma samples was determined by immunohistochemistry. The presence of follicular-center and regulatory T-cell lymphocytes was determined by immunohistochemistry (B-cell lymphoma [BCL]-6 expression) and double immunofluorescence-based staining of CD4-FOXP3, respectively. We evaluated the association between thymic expression of CTLA4 and the development of MG. We also determined the association between CTLA4 expression and various clinical and prognostic characteristics of MG. We sequenced the CTLA4 gene and evaluated possible associations between CTLA4 polymorphisms and thymic CTLA4 expression. Finally, we assessed the potential association between these polymorphisms and the risk of MG. RESULTS: Forty-one patients with thymoma were included. Of them, 23 had comorbid MG (56.1%). On average, patients with MG had fewer CTLA4-positive cells in the thymoma than non-MG patients: 69.3 cells/mm2 (95% CIs: 39.6-99.1) vs 674.4 (276.0-1,024.0) cells/mm2; p = 0.001 and vs controls (200.74 [57.9-343.6] cells/mm2; p = 0.02). No between-group differences (MG vs non-MG) were observed in the number of cells positive for BCL6 or CD4-FOXP3. CTLA4 expression was not associated with differences in MG outcome or treatment refractoriness. Two polymorphisms were detected in the CTLA4 gene, rs231770 (n = 30 patients) and rs231775 (n = 17). MG was present in a similar proportion of patients for all genotypes. However, a nonsignificant trend toward a lower CTLA4-positive cell count was observed among carriers of the rs231775 polymorphism vs noncarriers: 77.9 cells/mm2 (95% CI: -51.5 to 207.5) vs 343.3 cells/mm2 (95% CI: 126.2-560.4). DISCUSSION: Reduced CTLA4 expression in thymoma may predispose to a higher risk of developing MG.

Validation of a Pathological Score for the Assessment of Bronchial Biopsies in Severe Uncontrolled Asthma: Beyond Blood Eosinophils.

BACKGROUND: Blood eosinophil count (BEC) is currently used as a surrogate marker of T2 inflammation in severe asthma but its relationship with tissue T2-related changes is elusive. Bronchial biopsy could add reliable information but lacks standardization. OBJECTIVES: To validate a systematic assessment of the bronchial biopsy for the evaluation of severe uncontrolled asthma (SUA) by standardizing a pathological score. METHODS: A systematic assessment of submucosal inflammation, tissue eosinophilic count/field (TEC), goblet cells hyperplasia, epithelial changes, basement membrane thickening, prominent airway smooth muscle and submucosal mucous glands was initially agreed and validated in representative bronchial biopsies of 12 patients with SUA by 8 independent pathologists. In a second phase, 62 patients with SUA who were divided according to BEC≥300cells/mm3 or less underwent bronchoscopy with bronchial biopsies and the correlations between the pathological findings and the clinical characteristics were investigated. RESULTS: The score yielded good agreement among pathologists regarding submucosal eosinophilia, TEC, goblet cells hyperplasia and mucosal glands (ICC=0.85, 0.81, 0.85 and 0.87 respectively). There was a statistically significant correlation between BEC and TEC (r=0.393, p=0.005) that disappeared after correction by oral corticosteroids (OCS) use (r=0.170, p=0.307). However, there was statistically significant correlation between FeNO and TEC (r=0.481, p=0.006) that was maintained after correction to OCS use (r=0.419, p=0.021). 82.4% of low-BEC had submucosal eosinophilia, 50% of them moderate to severe. CONCLUSION: A standardized assessment of endobronchial biopsy is feasible and could be useful for a better phenotyping of SUA especially in those receiving OCS.

Early hospital readmission increases short and long - term mortality in patients with interstitial lung disease.

OBJECTIVE: To investigate the prognostic impact of early readmission (30 days) on hospitalized patients with Interstitial Lung Disease (ILD). METHODS: Observational study analysing a cohort of patients hospitalized in a respiratory ward at a University Hospital. Demographic, clinical data and survival status were collected from patients' records. Early readmission was defined as hospitalization within 30 days after patient's discharge. The primary outcome was 90-day and 1-year all-cause mortality. RESULTS: Between 2013 to 2016, a total of 2.238 patients were admitted to the respiratory ward and 98 (%) had a diagnosis of ILD. Among them, 74 patients were discharged (25% in-hospital mortality). Early readmission was observed in 15 cases (20.2%). Early readmitted patients were more frequently current smokers (20% vs. 2%, p=0.02). After a multivariate analysis, early readmission was found to be independently associated with 90-day and 1 year mortality (Odds Ratio (OR) 17.6, 95% Confidence Interval (CI) 4.5-69-2, p=0.001 and OR 4.5; 95CI 1.3-15.2, p=0.01, respectively). CONCLUSION: In patients with ILD, early readmission after hospitalization increases both short-term and long term mortality. Thus, preventing early readmission after discharge from hospital admission may have an impact in the clinical course of ILD patients. Further studies are required to identify factors contributing to early readmission.

Circulating microRNAs in Early Breast Cancer Patients and Its Association With Lymph Node Metastases.

MicroRNAs have emerged as important regulators of the metastatic process. In addition, circulating miRNAs appear to be surprisingly stable in peripheral blood making them ideal noninvasive biomarkers for disease diagnosis. Here, we performed a proof-of-principle study to investigate the expression profile of circulating miRNAs and their association with the metastatic lymph node status in early breast cancer patients. Sentinel lymph node status was detected by one-step nucleic acid (OSNA) analysis. We performed RNA-sequencing in 16 plasma samples and validated the results by qPCR. Gene Ontology term enrichment and KEGG pathway analyses were carried out using DAVID tools. We found16 differentially expressed miRNAs (q < 0.01) in patients with positive SLNs. Fourteen miRNAs were down-regulated (miR-339-5p, miR-133a-3p, miR-326, miR-331-3p, miR-369-3p, miR-328-3p, miR-26a-3p, miR-139-3p, miR-493-3p, miR-664a-5p, miR-146a-5p, miR-323b-3p, miR-1307-3p and miR-423-3p) and 2 were up-regulated (miR-101-3pand miR-144-3p). Hierarchical clustering using differentially expressed miRNAs clearly distinguished patients according to their lymph node status. Gene ontology analysis showed a significant enrichment of biological processes associated with the regulation of the epithelial mesenchymal transition, cell proliferation and transcriptional regulation. Our results suggest the potential role of several circulating miRNAs as surrogate markers of lymph node metastases in early breast cancer patients. Further validation in a larger cohort of patients will be necessary to confirm our results.

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very-high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies.

MicroRNA-1291 Is Associated With Locoregional Metastases in Patients With Early-Stage Breast Cancer.

Evidence that microRNAs (miRNAs) regulate the various steps of metastasis is increasing. Several studies have looked at the miRNA expression profile in primary breast tumors but few have compared primary tumor and sentinel lymph node (SLN) metastasis. We correlated the expression of miRNAs with the SLN status and the outcome of axillary lymph node dissection (ALND) in 60 patients with early breast cancer. We profiled the expression of miRNAs in paired breast tumor samples and SLNs using the NextSeq500 Illumina platform and key findings were validated by qPCR. MultiMiR Bioconductor and Reactome pathways analysis were performed to identify target genes and signaling pathways affected by altered expressed miRNAs. Our results show that nine miRNAs were differentially expressed in tumor tissues (q ≤ 0.05). In tumor samples, a 13.5-fold up-regulation of miR-7641-2 (q < 0.001) and a 2.9-fold down-regulation of miR-1291 (q < 0.001) were associated with tumors with positive SLNs. However, only down-regulation of miR-1291 (q = 0.048) remained significant in paired SLNs samples. Interestingly, a 10.5 up-regulation of miR-1291 in SLNs samples was associated with additional axillary lymph node involvement (q < 0.001). The enrichment analyses showed that canonical and non-canonical WNT pathways and negative regulation of various receptor tyrosine kinases signaling pathways were targets of miR-1291 and supports the role of miR-1291 as a tumor suppressor gene (TSG). Further studies are warranted to investigate the use of miR-1291 as a surrogate biomarker of SLN node metastasis in patients with early-stage breast cancer.

ApoA-I mimetic administration, but not increased apoA-I-containing HDL, inhibits tumour growth in a mouse model of inherited breast cancer.

Low levels of high-density lipoprotein cholesterol (HDLc) have been associated with breast cancer risk, but several epidemiologic studies have reported contradictory results with regard to the relationship between apolipoprotein (apo) A-I and breast cancer. We aimed to determine the effects of human apoA-I overexpression and administration of specific apoA-I mimetic peptide (D-4F) on tumour progression by using mammary tumour virus-polyoma middle T-antigen transgenic (PyMT) mice as a model of inherited breast cancer. Expression of human apoA-I in the mice did not affect tumour onset and growth in PyMT transgenic mice, despite an increase in the HDLc level. In contrast, D-4F treatment significantly increased tumour latency and inhibited the development of tumours. The effects of D-4F on tumour development were independent of 27-hydroxycholesterol. However, D-4F treatment reduced the plasma oxidized low-density lipoprotein (oxLDL) levels in mice and prevented oxLDL-mediated proliferative response in human breast adenocarcinoma MCF-7 cells. In conclusion, our study shows that D-4F, but not apoA-I-containing HDL, hinders tumour growth in mice with inherited breast cancer in association with a higher protection against LDL oxidative modification.

Chromosome 17 centromere duplication and responsiveness to anthracycline-based neoadjuvant chemotherapy in breast cancer.

Human epidermal growth factor receptor 2 (HER2) and topoisomerase II alpha (TOP2A) genes have been proposed as predictive biomarkers of sensitivity to anthracycline chemotherapy. Recently, chromosome 17 centromere enumeration probe (CEP17) duplication has also been associated with increased responsiveness to anthracyclines. However, reports are conflicting and none of these tumor markers can yet be considered a clinically reliable predictor of response to anthracyclines. We studied the association of TOP2A gene alterations, HER2 gene amplification, and CEP17 duplication with response to anthracycline-based neoadjuvant chemotherapy in 140 patients with operable or locally advanced breast cancer. HER2 was tested by fluorescence in situ hybridization and TOP2A and CEP17 by chromogenic in situ hybridization. Thirteen patients (9.3%) achieved pathologic complete response (pCR). HER2 amplification was present in 24 (17.5%) of the tumors. TOP2A amplification occurred in seven tumors (5.1%). CEP17 duplication was detected in 13 patients (9.5%). CEP17 duplication correlated with a higher rate of pCR [odds ratio (OR) 6.55, 95% confidence interval (95% CI) 1.25-34.29, P = .026], and analysis of TOP2A amplification showed a trend bordering on statistical significance (OR 6.97, 95% CI 0.96-50.12, P = .054). TOP2A amplification and CEP17 duplication combined were strongly associated with pCR (OR 6.71, 95% CI 1.66-27.01, P = .007). HER2 amplification did not correlate with pCR. Our results suggest that CEP17 duplication predicts pCR to primary anthracycline-based chemotherapy. CEP17 duplication, TOP2A amplifications, and HER2 amplifications were not associated with prognosis.

Mutations in FGFR3 and PIK3CA, singly or combined with RAS and AKT1, are associated with AKT but not with MAPK pathway activation in urothelial bladder cancer.

Different members of the phosphoinositide 3 kinase--serine threonine protein kinase (PI3K-AKT) pathway are altered in bladder cancer. Fibroblast growth factor receptor 3 (FGFR3) mutations characterize the low-grade tumors, and RAS genes are mutated in approximately 13% of all bladder tumors. Interestingly, a percentage of bladder tumors have alterations in more than 1 PI3K-AKT or rat sarcoma viral oncogene homolog-RAF mitogen activated protein kinase (RAS-MAPK) pathway gene or their upstream regulators, but some combinations are mutually exclusive. We analyzed mutations in FGFR3, phosphoinositide 3 kinase catalytic alpha polypeptide (PIK3CA), v-akt murine thymoma viral oncogene homolog 1 (AKT1), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) in 88 urothelial cell carcinomas and the immunohistochemical expression of phospho-v-akt murine thymoma viral oncogene homolog (AKT) and mitogen-activated protein kinase 1 and 2 (pERK1/2) in 80 and 77 urothelial cell carcinomas, respectively. Approximately 43% and 20.5% of tumors presented 1 and 2 mutated genes, respectively. FGFR3 mutations were more frequent alone, whereas PIK3CA mutations were associated with another mutated gene (FGFR3 and KRAS). Overall, mutated FGFR3 (FGFR3(mut)) and mutated FGFR3 (FGFR3(mut))-mutated PIK3CA (PIK3CA(mut)) genotypes were associated with low-grade bladder tumors and mutated PIK3CA (PIK3CA(mut))-mutated KRAS (KRAS(mut)) and mutated AKT1 (AKT1(mut)) were only present in high-grade tumors. There are no mutated FGFR3 (FGFR3(mut))-mutated RAS (RAS(mut)) nor mutated PIK3CA (PIK3CA(mut))-mutated AKT1 (AKT1(mut)) combinations. Fifty percent and 56% of tumors showed high levels of pAKT and pERK1/2, respectively. High levels of pAKT were associated with total mutations, FGFR3(mut), and PIK3CA(mut) tumors but not with tumor grade or stage. Wild-type tumors presented significantly higher pERK1/2 expression. Mutations in FGFR3 and FGFR3-PIK3CA but not single PIK3CA mutations characterize low-grade bladder tumors. Single FGFR3 or PIK3CA mutations and the different mutation combinations FGFR3-PIK3CA/AKT1 and PIK3CA-RAS can activate the AKT but not the MAPK pathway. Other genes different from FGFR3 may be related with the pERK activation in bladder tumors.