Laia Agell

FGFR3 mutations in prostate cancer: association with low-grade tumors.

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.

KLF6 and TP53 mutations are a rare event in prostate cancer: distinguishing between Taq polymerase artifacts and true mutations.

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.

Molecular alterations of EGFR and PTEN in prostate cancer: association with high-grade and advanced-stage carcinomas

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.

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.

PI3K signaling pathway is activated by PIK3CA mRNA overexpression and copy gain in prostate tumors, but PIK3CA , BRAF , KRAS and AKT1 mutations are infrequent events

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.

A 12-Gene Expression Signature Is Associated with Aggressive Histological in Prostate Cancer: SEC14L1 and TCEB1 Genes Are Potential Markers of Progression

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.

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.

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.