Silvia de Muga

Association of ERG and TMPRSS2‐ERG with grade, stage, and prognosis of prostate cancer is dependent on their expression levels

There is controversy in the literature on the role of the fusion TMPRSS2‐ERG in the pathogenesis and progression of prostate cancer. The quantitative differences in TMPRSS2‐ERG fusion expression have received very limited attention in the literature.

HER2 as a target in invasive urothelial carcinoma.

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.

A 12-gene expression signature is associated with aggressive histological in prostate cancer: SEC14L1 and TCEB1 genes are potential markers of progression. American journal of pathology

The main challenge for clinical management of prostate cancer is to distinguish tumors that will progress faster and will show a higher tendency to recur from the more indolent ones. We have compared expression profiles of 18 prostate cancer samples (seven with a Gleason score of 6, eight with a Gleason score of 7, and three with a Gleason score of ≥8) and five nonneoplastic prostate samples, using the Affymetrix Human Array GeneChip Exon 1.0 ST. Microarray analysis revealed 99 genes showing statistically significant differences among tumors with Gleason scores of 6, 7, and ≥8. In addition, mRNA expression of 29 selected genes was analyzed by real-time quantitative RT-PCR with microfluidic cards in an extended series of 30 prostate tumors. Of the 29 genes, 18 (62%) were independently confirmed in the extended series by quantitative RT-PCR: 14 were up-regulated and 4 were down-regulated in tumors with a higher Gleason score. Twelve of these genes were differentially expressed in tumors with a Gleason score of 6 to 7 versus ≥8. Finally, IHC validation of the protein levels of two genes from the 12-gene signature (SEC14L1 and TCEB1) showed strong protein expression levels of both genes, which were statistically associated with a high combined Gleason score, advanced stage, and prostate-specific antigen progression. This set of genes may contribute to a better understanding of the molecular basis of prostate cancer. TCEB1 and SELC14L1 are good candidate markers for predicting prognosis and progression of prostate cancer.

CXCR4 mRNA overexpression in high grade prostate tumors: Lack of association with TMPRSS2-ERG rearrangement

The TMPRSS2-ERG fusion has been reported in 42 to 78% of prostate tumors. More than 90% of ERG-overexpressing tumors harbor the fusion. The relationship between the TMPRSS2-ERG fusion and prognosis is controversial. Different studies have suggested an association between CXCR4 and ERG overexpression resulting from the TMPRSS2-ERG rearrangement. The aim of this study was to investigate the relationship between CXCR4 expression, TMPRSS2-ERG fusion and Gleason grade in prostate cancer. TMPRSS2-ERG rearrangement was investigated by FISH (n=44), ERG protein by IHC (n=84), and CXCR4 by quantitative RT-PCR (n=44). TMPRSS2-ERG rearrangement and ERG protein expression were present in almost 50% of the cases, without statistical differences between the different Gleason score groups. There was a very high concordance between FISH and IHC techniques (Kappa Index=0.954). Seventy percent of Gleason ⩾ 8 prostate tumors overexpressed CXCR4 mRNA, and the difference in CXCR4 expression with Gleason < 8 cases was statistically significant (p=0.009). There was no association between ERG protein and CXCR4 mRNA expression. In conclusion, our results reveal for the first time that CXCR4 overexpression is associated with high Gleason score prostate tumors, but that it is independent of the TMPRSS2-ERG rearrangement.

Identification of ALK Gene Alterations in Urothelial Carcinoma

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.

Assessment of ALK Status by FISH on 1000 Spanish Non-Small Cell Lung Cancer Patients

Introduction: Patients with non-small cell lung cancer (NSCLC) harboring anaplastic lymphoma kinase (ALK) rearrangement selectively respond to ALK inhibitors. Thus, identification of ALK rearrangements has become a standard diagnostic test in advanced NSCLC patients. Our institution has been a referral center in Spain for ALK determination by Fluorescent in situ hybridization (FISH). The aim of our study was to assess the feasibility and the FISH patterns of the ALK gene and to evaluate the clinical and pathological features of patients with ALK alterations. Methods: Between 2010 and 2014, 1092 samples were evaluated for ALK using FISH technique (927 histological samples, 165 cytological samples). Correlation with available clinical-pathological information was assessed. Results: ALK rearrangement was found in 35 patients (3.2%). Cytological samples (using either direct smears or cell blocks), were more frequently non-assessable than histological samples (69% versus 89%, respectively) (p < 0.001). Within the ALK-rearranged cases the majority were female, non-smokers, and stage IV. Conclusions: Although assessable in cytological samples, biopsies are preferred when available for ALK evaluation by FISH. The ALK translocation prevalence and the associated clinico-pathological features in Spanish NSCLC patients are similar to those previously reported.

Concurrent TMPRSS2‐ERG and SLC45A3‐ERG rearrangements plus PTEN loss are not found in low grade prostate cancer and define an aggressive tumor subset

SLC45A3 is the second most common ERG partner in prostate cancer (PrCa). Coexisting TMPRSS2 and SLC45A3 rearrangements are found in a subset of cases, but the meaning is still unknown.

FOXO1 down-regulation is associated with worse outcome in bladder cancer and adds significant prognostic information to p53 overexpression

Nuclear FOXOs mediate cell cycle arrest and promote apoptosis. FOXOs and p53 could have similar effects as tumor suppressor genes. In spite of extensive literature, little is known about the role of FOXO1 and its relationship with p53 status in bladder cancer. Expression of FOXO1 and p53 were analyzed by immunohistochemistry in 162 urothelial carcinomas (UC). Decreased FOXO1 expression, p53 overexpression and the combination FOXO1 down-regulation/p53 overexpression were strongly associated with high grade (P=.030; P=.017; P=.004, respectively), high stage (P=.0001; P<.0001; P<.0001, respectively) or both (P=.0004; P<.0001; P<.0001, respectively). In the overall series of cases, p53 overexpression was associated with tumor progression (hazard ratio [HR]=3.18, 95% confidence interval [CI] 1.19-8.48, P=.02), but this association was even stronger if having any alteration in any of the 2 genes was considered (HR=3.51, 95% CI 1.34-9.21, P=.01). Having both FOXO1 down-regulation and p53 overexpression was associated with disease recurrence (HR=2.75, 95% CI 1.06-7.13, P=.03). In the analysis of the different subgroups, having any alteration in any of the 2 genes was associated with progression in low-grade (P=.005) and pTa (P=.006) tumors. Finally, the combined FOXO1 down-regulation/p53 overexpression was associated with disease recurrence specifically in high-grade (P=.04) and in pT1 stage tumors (P=.007). Adding FOXO1 expression to the immunohistochemical analysis of p53 can provide relevant prognostic information on progression and recurrence of bladder cancer. It may be particularly informative on the risk of progression in the more indolent and on the risk of recurrence in the more aggressive tumors.

ERG overexpression plus SLC45A3 (prostein) and PTEN expression loss: Strong association of the triple hit phenotype with an aggressive pathway of prostate cancer progression

TMPRSS2 and SLC45A3 rearrangements may coexist in the same tumor. ERG rearrangements and PTEN loss are concomitant events in prostate cancer (PrCa), and can cooperate in progression. We have reported that mRNA expression of TMPRSS2-ERG and SLC45A3-ERG rearrangements plus PTEN loss define an aggressive tumor subset. The aim of this study has been to validate these results by immunohistochemistry in a large cohort of tumors. ERG, SLC45A3 and PTEN immunostaining and their association with pathological features and PSA progression-free survival were analyzed in 220 PrCa (PSMAR-Biobank, Barcelona, Spain). ERG protein expression was found in 46.8% and SLC45A3 and PTEN loss in 30% and 34% tumors, respectively. Single ERG positive immunostaining was associated with GS = 6 tumors (p = 0.016), double ERG+/PTEN loss with GS = 7 (p = 0.008) and Grade Group 2 (GG) or GG3 cases (p = 0.042), ERG+/SLC45A3 loss/PTEN loss (“triple hit”) with GS ≥ 8 (p < 0.0001) and GG4 or GG5 tumors (p = 0.0003). None of GS = 6 nor = GG1 cases showed this combination. In the GS ≥ 8 group, ERG+ (p = 0.002), PTEN loss (p = 0.009) and “triple hit” (p = 0.003) were associated with Gleason pattern 3 component, and single SLC45A3 loss (p = 0.036) with GS ≥ 8 without pattern 3. The number of aberrant events and the triple hit were strongly associated with shorter PSA progression-free survival. In GS = 6 PrCa, single ERG+ was also associated with progression. ERG+ identifies a distinct pathway of PrCa. Additional assessment of PTEN and SLC45A3 adds relevant prognostic information. The triple hit phenotype (ERG+/SLC45A3 loss/PTEN loss) is associated with progression and could be used for patient stratification, treatment and follow-up.

Abstract 1168: Urothelial cell carcinomas harbor very frequent mutations in FGFR3-PI3K-AKT pathway and highest prevalence of mutations is related to low-grade tumors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC More than 90% of bladder cancers are urothelial cell carcinomas. Superficial/papillary and invasive tumors progress via different molecular pathways. FGFR3 and PI3KCA mutations are more commonly found in superficial/papillary tumors. The PI3K-AKT pathway emerges as an alternative pathogenic mechanism linked to bladder cancer. We have analyzed the prevalence of mutations in FGFR3, PI3KCA, KRAS, BRAF and AKT1 genes in a set of 61 bladder tumors classified as TaG1 (n=10), TaG2 (n=19), TaG3 (n=8), T1G2 (n= 3), T1G3 (n= 11), T2G2 (n=1) and T2G3 (n=10). We focused on analyzing the hot spot mutation codons and surrounding regions. Mutation analysis of DNA from paraffin-embedded tumors was performed by direct sequencing of PCR products. Thirty-six (60%) tumors presented mutations, 22 cases with only one mutated gene, whereas 14 of 61 cases had mutations in two genes. The most frequently mutated gene was FGFR3 in 25 tumors, followed by PI3KCA in 16 tumors. The distribution and combinations of mutations was: FGFR3 (n= 16), PI3KCA (n= 3), AKT1 (n= 2), KRAS (n= 1), FGFR3-PI3KCA (n= 9), FGFR3-AKT1 (n= 1), and PI3KCA-KRAS (n= 4). No tumor showed BRAF mutations. According to tumor stage, 26 of 37 (70%) Ta tumors, 3 of 13 (23%) T1 tumors and 7 of 11 (63%) of T2 tumors harbored mutations. On the other hand, according to tumor grade, the distribution of mutations was: grade 1, 9 of 11 (82%); grade 2, 15 of 22 (68%); and grade 3, 12 of 28 (43%), with an overall rate of 27 mutations in 50 high grade tumors (54%). In grade 1, 100% of mutations were found in FGFR3 and/or PI3KCA, with FGFR3 mutations in 9 of 10 tumors. In grade 2, 11 of 15 mutated tumors (73%) showed alterations in FGFR3 and/or PI3KCA, and 4 tumors displayed mutations in AKT1 and KRAS (3 of them in combination with FGFR3 or PI3KCA). In grade 3, 8 of 12 mutated tumors (66%) showed mutations in FGFR3 and/or PI3KCA, and 4 tumors harbored mutations in AKT1 and KRAS (2 of them in combination with PI3KCA). Mutations in AKT1 and KRAS were found in grade 2 and 3, but not in grade 1 tumors. Percentage of mutations in the FGFR3-PI3K-AKT1 pathway is inversely proportional to grade, and it is more often associated with low grade tumors. Mutations in FGFR3 are often found alone in bladder tumors, whereas PI3KCA mutations are more frequent in combination with other mutated genes, such as FGFR3 or KRAS. FGFR3 and KRAS mutations seem to be mutually exclusive, but not PI3KCA and KRAS, nor FGFR3 and AKT1 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1168.