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We evaluated primary tumors from two cohorts, Spain (N = 111) and Greece (N = 102), for patients who were treated with platinum‐based chemotherapy. Patients were tested for HER2 status (IHC score of 3+ or FISH ratio of ≥2.2) by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), DNA copy number, mRNA expression, and mutation status in patients with metastatic urothelial carcinoma (UC), and its impact on survival. ERBB2 mutation was determined by hotspot sequencing. mRNA expression was assessed using NanoString counting. Association of overall survival (OS) and HER2 status was assessed by a Cox regression model. NIH‐3T3 cells containing HER2 V777L were assessed for growth, invasion, and HER2 kinase activation. In all, 22% of Spanish and 4% of Greek cohorts had 3+ HER2 staining by IHC. FISH amplification was identified in 20% of Spanish and 4% of Greek cohorts. Kappa coefficient between FISH and IHC was 0.47. HER2 status was not associated with OS in univariate (Spanish P = 0.34; Greek P = 0.11) or multivariate (Spanish P = 0.49; Greek P = 0.12) analysis. HER2‐positive tumors expressed higher levels of HER2 mRNA than HER2‐negative tumors (P < 0.001). HER2 mutations (V777L and L755S) were identified in two (2%) patients. In vitro analysis of V777L results in transformation of NIH‐3T3 cells, leading to increased growth, invasion on soft agar, and HER2 kinase constitutive activation. In summary, HER2 overexpression or amplification in the primary tumor did not predict OS in patients with metastatic UC. HER2 positivity rates can differ between different populations. Further trials in genomically screened patients are needed to assess HER2‐targeted therapies in UC. The association between HER2 status and overall survival (OS) in UC remains unclear. HER2 positivity may not only be a biomarker for more aggressive disease but also a potential therapeutic target. We report that HER2 overexpression or amplification in the primary tumor did not predict OS in patients with metastatic UC. Further trials in genomically screened patients are needed to assess HER2‐targeted therapies in UC.

Krüppel-like factor 6 ( KLF6 ) has been reported to act as a tumor suppressor gene involved in the regulation of the cell cycle by activating p21 in a p53-independent manner. Many studies suggest that KLF6 is inactivated by allelic loss and somatic mutation. However, there is a high variability in the reported frequency of mutations (from 1 to 55%). TP53 also regulates the cell cycle through the activation of p21. In prostate cancer, the reported frequency of TP53 mutations ranges from 3 to 42%. In all these reports, there is a considerable degree of methodological heterogeneity. Our aim was to determine the frequency of KLF6 and TP53 mutations in a well-defined group of prostate tumors with different stages and Gleason grades. The four exons of KLF6 and exons 4–9 of TP53 were studied in 103 cases, including 90 formalin-fixed, paraffin-embedded (FFPE) and 13 frozen samples. All tumors were analyzed through PCR and direct sequencing. All changes found were confirmed by a second independent PCR and sequencing reaction. For KLF6 , mutation (E227G) was only detected in one tumor (1%) and for TP53 , three different mutations (L130H, H214R, and Y234C) were detected in five tumors (5%). This low mutation index is in keeping with recent papers on the subject. Our study strongly supports the notion that KLF6 and TP53 mutations are not frequent events in prostate cancer. When using FFPE tissues, it is mandatory to perform at least two independent rounds of PCR and sequencing to confirm mutations and exclude Taq polymerase-induced artifacts.

Anaplastic lymphoma kinase (ALK) genomic alterations have emerged as a potent predictor of benefit from treatment with ALK inhibitors in several cancers. Currently, there is no information about ALK gene alterations in urothelial carcinoma (UC) and its correlation with clinical or pathologic features and outcome. Samples from patients with advanced UC and correlative clinical data were collected. Genomic imbalances were investigated by array comparative genomic hybridization (aCGH). ALK gene status was evaluated by fluorescence in situ hybridization (FISH). ALK expression was assessed by immunohistochemistry (IHC) and high-throughput mutation analysis with Oncomap 3 platform. Next generation sequencing was performed using Illumina Genome Analyzer IIx, and Illumina HiSeq 2000 in the FISH positive case. 70 of 96 patients had tissue available for all the tests performed. Arm level copy number gains at chromosome 2 were identified in 17 (24%) patients. Minor copy number alterations (CNAs) in the proximity of ALK locus were found in 3 patients by aCGH. By FISH analysis, one of these samples had a deletion of the 5'ALK. Whole genome next generation sequencing was inconclusive to confirm the deletion at the level of the ALK gene at the coverage level used. We did not observe an association between ALK CNA and overall survival, ECOG PS, or development of visceral disease. ALK genomic alterations are rare and probably without prognostic implications in UC. The potential for testing ALK inhibitors in UC merits further investigation but might be restricted to the identification of an enriched population.

Prostate cancer is the second cause of cancer-related death in men of the Western world. The potential prognostic role of the combined alterations in EGFR and PTEN in prostate cancer is not well established. It was the aim of the study to investigate this role. Prevalence of EGFR and PTEN somatic mutations, EGFR amplification and EGFR protein expression were investigated in a series of prostate adenocarcinomas, classified according to the current Gleason grading system. Mutational analysis revealed eight EGFR and three PTEN mutations in 98 (8%) and 92 (3%) prostate adenocarcinomas, respectively. The combined prevalence of EGFR–PTEN mutations was 11%. EGFR overexpression was present in 31% of adenocarcinomas, with a marginally significant difference ( P =0.068) between Gleason grade ≤7 adenocarcinomas and Gleason grade ≥8 and metastatic adenocarcinomas. Four cases (4 of 31; 13%) had an EGFR gene gain due to chromosome 7 polysomy. In 35% of adenocarcinomas we found some type of EGFR–PTEN alteration, with a tendency to be associated with advanced-stage prostate adenocarcinomas ( P =0.04). The IVS18+19 polymorphism was also associated with more advanced prostate adenocarcinomas. This is the first study reporting mutations of EGFR and PTEN in the same series of prostate adenocarcinomas. Protein overexpression is the most frequent EGFR abnormality. Mutations in EGFR and PTEN genes are a minor event, although prostate cancer represents the third neoplasm in which the EGFR gene mutations are more prevalent. Alterations in the EGFR–PTEN signaling pathway are present in a third of prostate adenocarcinomas, particularly affecting the more advanced cases.

The phosphatidylinositol 3-kinase (PI3K)–AKT and RAS–MAPK pathways are deregulated in a wide range of human cancers by gain or loss of function in several of their components. Our purpose has been to identify genetic alterations in members of these pathways in prostate cancer. A total of 102 prostate tumors, 79 from prostate cancer alone (group G1) and 23 from bladder and prostate cancer patients (G2), are the subject of this study. In 20 of these 23, the bladder tumors were also analyzed. PIK3CA, KRAS, BRAF and AKT1 mutations were analyzed by direct sequencing, and BRAF also by pyrosequencing. PIK3CA quantitative mRNA expression and fluorescence in situ hybridization (FISH) gains were tested in 25 and 32 prostate tumors from both groups (G1 and G2), respectively. Immunohistochemistry for pAKT was performed in 55 prostate tumors. Of 25 prostate tumors, 10 (40%) had PIK3CA mRNA overexpression that was statistically associated with Gleason score ≥7 ( P =0.018). PIK3CA copy gain was detected in 9 of 32 (28%) prostate tumors. Of 20 bladder tumors, 3 (15%) displayed mutations in PIK3CA , KRAS and AKT1 , the corresponding prostate tumors being wt. We also detected a previously not reported PIK3CA polymorphism (IVS9+91) in two prostate tumors. In all, 56% of prostate tumors overexpressed pAKT. There is a statistical association ( P <0.0001) of strong pAKT immunostaining with high Gleason score, and with PIK3CA alterations (mRNA overexpression and/or FISH gains). PIK3CA gene is deregulated by mRNA overexpression and DNA gain in ∼40 and 28% of prostate tumors, respectively. High-grade prostate tumors are associated with PIK3CA mRNA overexpression, but not with FISH status. PIK3CA , BRAF , KRAS and AKT1 mutations are very infrequent events in prostate tumors. However, PI3K signaling pathway is activated by PIK3CA FISH gain and/or mRNA overexpression, leading to an increased pAKT protein expression.

Prostate cancer is the second cause of cancer-related death in men of the Western World. The role of FGFR3 and its abnormalities in prostate cancer are not known. FGFR3 mutations have been reported in some human tumors. Few studies have analyzed the mutations of FGFR3 in prostate tumors, and no mutations have been previously reported. Prevalence of FGFR3 somatic mutations was investigated in a series of prostate tumors. The presence of other tumors in these patients, including urothelial, skin, colon, and lung neoplasms, was recorded. Mutational analysis of exons 7, 10, and 15 of FGFR3 revealed 9 mutations in the 112 prostate tumors studied (8%). Most of them consisted of the missense change S249C. The prevalence of mutations in tumors with combined Gleason score=6 is 18% (8/45) compared to 3% (1/36) for tumors with grade=7, and 0% (0/31) for those with grade ≥8 and metastases ( P =0.007). The frequency of FGFR3 mutations in autopsy and biopsy samples was 6 and 9%, respectively. The prevalence of FGFR3 mutations in prostate tumors from patients with only prostate cancer was 2% compared to 23% in prostate tumors from patients with other associated neoplasms ( P =0.001). This is the first report of molecular changes of FGFR3 in prostate cancer. This gene does not seem to be central to the pathogenesis of prostate cancer, but it is significantly associated with a subgroup of low-grade prostate tumors, and with the finding of other tumors, mainly arising in bladder and skin.

Background Anaplastic lymphoma kinase (ALK) genomic alterations have emerged as a potent predictor of benefit from treatment with ALK inhibitors in several cancers. Currently, there is no information about ALK gene alterations in urothelial carcinoma (UC) and its correlation with clinical or pathologic features and outcome. Methods Samples from patients with advanced UC and correlative clinical data were collected. Genomic imbalances were investigated by array comparative genomic hybridization (aCGH). ALK gene status was evaluated by fluorescence in situ hybridization (FISH). ALK expression was assessed by immunohistochemistry (IHC) and high-throughput mutation analysis with Oncomap 3 platform. Next generation sequencing was performed using Illumina Genome Analyzer IIx, and Illumina HiSeq 2000 in the FISH positive case. Results 70 of 96 patients had tissue available for all the tests performed. Arm level copy number gains at chromosome 2 were identified in 17 (24%) patients. Minor copy number alterations (CNAs) in the proximity of ALK locus were found in 3 patients by aCGH. By FISH analysis, one of these samples had a deletion of the 5'ALK. Whole genome next generation sequencing was inconclusive to confirm the deletion at the level of the ALK gene at the coverage level used. We did not observe an association between ALK CNA and overall survival, ECOG PS, or development of visceral disease. Conclusions ALK genomic alterations are rare and probably without prognostic implications in UC. The potential for testing ALK inhibitors in UC merits further investigation but might be restricted to the identification of an enriched population.

Amblyopia is the leading cause of vision impairment in children, and its early detection can avoid irreversible consequences for a child’s visual ability. In this prospective study, to detect amblyopia risk factors, we examined patients aged 18 to 30 months in primary care settings. Patients were referred to an ophthalmologist for confirmation. The main aims were to detect amblyogenic risk factors and to assess the usefulness of a photoscreener in such settings. Out of 453 patients, 42 (9.3%) presented visual alterations according to the photoscreener, with astigmatism being the most common. The instrument had good sensitivity (89%) and specificity (91%), with a positive predictive value of 76% and a negative predictive value of 96%. Overall, 38% of the patients required follow-up, and 47% needed glasses. The automated screening device allowed these children to be diagnosed at an early stage.Conclusions: The use of a photoscreener to screen 2-year-old children in primary care settings was helpful and accurate.What is Known:• Early detection of amblyopic risk factors is important to avoid vision defects. However, it is very difficult to measure visual acuity using visual charts in children younger than 4 years old.What is New:• Instrument-based screening in children aged 18 to 30 months allows excellent detection of early amblyopia risk factors in primary care settings.