PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers

Background

Oncogenic mutations of PIK3CA, RAS (KRAS, NRAS), and BRAF have been identified in various malignancies, and activate the PI3K/AKT/mTOR and RAS/RAF/MEK pathways, respectively. Both pathways are critical drivers of tumorigenesis.

Methods

Tumor tissues from 504 patients with diverse cancers referred to the Clinical Center for Targeted Therapy at MD Anderson Cancer Center starting in October 2008 were analyzed for PIK3CA, RAS (KRAS, NRAS), and BRAF mutations using polymerase chain reaction-based DNA sequencing.

Results

PIK3CA mutations were found in 54 (11%) of 504 patients tested; KRAS in 69 (19%) of 367; NRAS in 19 (8%) of 225; and BRAF in 31 (9%) of 361 patients. PIK3CA mutations were most frequent in squamous cervical (5/14, 36%), uterine (7/28, 25%), breast (6/29, 21%), and colorectal cancers (18/105, 17%); KRAS in pancreatic (5/9, 56%), colorectal (49/97, 51%), and uterine cancers (3/20, 15%); NRAS in melanoma (12/40, 30%), and uterine cancer (2/11, 18%); BRAF in melanoma (23/52, 44%), and colorectal cancer (5/88, 6%). Regardless of histology, KRAS mutations were found in 38% of patients with PIK3CA mutations compared to 16% of patients with wild-type (wt)PIK3CA (p = 0.001). In total, RAS (KRAS, NRAS) or BRAF mutations were found in 47% of patients with PIK3CA mutations vs. 24% of patients wtPIK3CA (p = 0.001). PIK3CA mutations were found in 28% of patients with KRAS mutations compared to 10% with wtKRAS (p = 0.001) and in 20% of patients with RAS (KRAS, NRAS) or BRAF mutations compared to 8% with wtRAS (KRAS, NRAS) or wtBRAF (p = 0.001).

Conclusions

PIK3CA, RAS (KRAS, NRAS), and BRAF mutations are frequent in diverse tumors. In a wide variety of tumors, PIK3CA mutations coexist with RAS (KRAS, NRAS) and BRAF mutations.     

PIK3CA Activating Mutation in Colorectal Carcinoma: Associations with Molecular Features and Survival

Mutations in PIK3CA are present in 10 to 15% of colorectal carcinomas. We aimed to examine how PIK3CA mutations relate to other molecular alterations in colorectal carcinoma, to pathologic phenotype and survival. PIK3CA mutation testing was carried out using direct sequencing on 757 incident tumors from the Melbourne Collaborative Cohort Study. The status of O-6-methylguanine-DNA methyltransferase (MGMT) was assessed using both immunohistochemistry and methyLight techniques. Microsatellite instability, CpG island phenotype (CIMP), KRAS and BRAF V600E mutation status, and pathology review features were derived from previous reports. PIK3CA mutation was observed in 105 of 757 (14%) of carcinomas, characterized by location in the proximal colon (54% vs. 34%; P<0.001) and an increased frequency of KRAS mutation (48% vs. 25%; P<0.001). High-levels of CIMP were more frequently found in PIK3CA-mutated tumors compared with PIK3CA wild-type tumors (22% vs. 11%; P = 0.004). There was no difference in the prevalence of BRAF V600E mutation between these two tumor groups. PIK3CA-mutated tumors were associated with loss of MGMT expression (35% vs. 20%; P = 0.001) and the presence of tumor mucinous differentiation (54% vs. 32%; P<0.001). In patients with wild-type BRAF tumors, PIK3CA mutation was associated with poor survival (HR 1.51 95% CI 1.04–2.19, P = 0.03). In summary, PIK3CA-mutated colorectal carcinomas are more likely to develop in the proximal colon, to demonstrate high levels of CIMP, KRAS mutation and loss of MGMT expression. PIK3CA mutation also contributes to significantly decreased survival for patients with wild-type BRAF tumors.     

BRAF,PIK3CA,and HER2 Oncogenic Alterations According to KRAS Mutation Status in Advanced Colorectal Cancers with Distant Metastasis

Background

Anti-EGFR antibody–based treatment is an important therapeutic strategy for advanced colorectal cancer (CRC); despite this, several mutations—including KRAS, BRAF, and PIK3CA mutations, and HER2 amplification—are associated with the mechanisms underlying the development of resistance to anti-EGFR therapy. The aim of our study was to investigate the frequencies and clinical implications of these genetic alterations in advanced CRC.

Methods

KRAS, BRAF, and PIK3CA mutations were determined by Cobas real-time polymerase chain reaction (PCR) in 191 advanced CRC patients with distant metastasis. Microsatellite instability (MSI) status was determined by a fragmentation assay and HER2 amplification was assessed by silver in situ hybridization. In addition, KRAS mutations were investigated by the Sanger sequencing method in 97 of 191 CRC cases.

Results

Mutations in KRAS, BRAF, and PIK3CA were found in 104 (54.5%), 6 (3.1%), and 25 (13.1%) cases of advanced CRC, respectively. MSI-high status and HER2 amplification were observed in 3 (1.6%) and 16 (8.4%) cases, respectively. PIK3CA mutations were more frequently found in KRAS mutant type (18.3%) than KRAS wild type (6.9%) (P = 0.020). In contrast, HER2 amplifications and BRAF mutations were associated with KRAS wild type with borderline significance (P = 0.052 and 0.094, respectively). In combined analyses with KRAS, BRAF and HER2 status, BRAF mutations or HER2 amplifications were associated with the worst prognosis in the wild type KRAS group (P = 0.004). When comparing the efficacy of detection methods, the results of real time PCR analysis revealed 56 of 97 (57.7%) CRC cases with KRAS mutations, whereas Sanger sequencing revealed 49 cases (50.5%).

Conclusions

KRAS mutations were found in 54.5% of advanced CRC patients. Our results support that subgrouping using PIK3CA and BRAF mutation or HER2 amplification status, in addition to KRAS mutation status, is helpful for managing advanced CRC patients.     

Effectors of Epidermal Growth Factor Receptor Pathway: The Genetic Profiling of KRAS,BRAF, PIK3CA,NRAS Mutations in Colorectal Cancer Characteristics and Personalized Medicine

Mutations in KRAS oncogene are recognized biomarkers that predict lack of response to anti- epidermal growth factor receptor (EGFR) antibody therapies. However, some patients with KRAS wild-type tumors still do not respond, so other downstream mutations in BRAF, PIK3CA and NRAS should be investigated. Herein we used direct sequencing to analyze mutation status for 676 patients in KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons12, 13 and 61). Clinicopathological characteristics associations were analyzed together with overall survival (OS) of metastatic colorectal cancer patients (mCRC). We found 35.9% (242/674) tumors harbored a KRAS mutation, 6.96% (47/675) harbored a BRAF mutation, 9.9% (62/625) harbored a PIK3CA mutation and 4.19% (26/621) harbored a NRAS mutation. KRAS mutation coexisted with BRAF, PIK3CA and NRAS mutation, PIK3CA exon9 mutation appeared more frequently in KRAS mutant tumors (P = 0.027) while NRAS mutation almost existed in KRAS wild-types (P<0.001). Female patients and older group harbored a higher KRAS mutation (P = 0.018 and P = 0.031, respectively); BRAF (V600E) mutation showed a higher frequency in colon cancer and poor differentiation tumors (P = 0.020 and P = 0.030, respectively); proximal tumors appeared a higher PIK3CA mutation (P<0.001) and distant metastatic tumors shared a higher NRAS mutation (P = 0.010). However, in this study no significant result was found between OS and gene mutation in mCRC group. To our knowledge, the first large-scale retrospective study on comprehensive genetic profile which associated with anti-EGFR MoAbs treatment selection in East Asian CRC population, appeared a specific genotype distribution picture, and the results provided a better understanding between clinicopathological characteristics and gene mutations in CRC patients.     

The Homeobox Gene MEIS1 Is Methylated in BRAF p.V600E Mutated Colon Tumors

Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAF ^p.V600E mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAF ^p.V600E mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAF ^p.V600E with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log₂ fold change: 0.89, false discovery rate-adjusted P-value 2.8*10^-9). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAF ^p.V600E and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1 _D27, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAF ^p.V600E tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAF ^p.V600E stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAF ^p.V600E colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression.     

The Prognostic Value of Microsatellite Instability,KRAS, BRAF and PIK3CA Mutations in Stage II Colon Cancer Patients

In the era of personalized cancer medicine, identifying mutations within patient tumors plays an important role in defining high-risk stage II colon cancer patients. The prognostic role of BRAF V600E mutation, microsatellite instability (MSI) status, KRAS mutation and PIK3CA mutation in stage II colon cancer patients is not settled. We retrospectively analyzed 186 patients with stage II colon cancer who underwent an oncological resection but were not treated with adjuvant chemotherapy. KRAS mutations, PIK3CA mutation, V600E BRAF mutation and MSI status were determined. Survival analyses were performed. Mutations were found in the patients with each mutation in the following percentages: 23% (MSI), 35% (KRAS), 19% (BRAF) and 11% (PIK3CA). A trend toward worse overall survival (OS) was seen in patients with an MSI (5-year OS 74% versus 82%, adjusted hazard ratio [HR] 1.8, 95% confidence interval [CI] 0.6–4.9) and a KRAS-mutated tumor (5-year OS 77% versus 82%, adjusted HR 1.7, 95% CI 0.8–3.5). MSI and BRAF-mutated tumors tended to correlate with poorer disease-free survival (DFS) (5-year DFS 60% versus 78%, adjusted HR 1.6, 95% CI 0.5–2.1 and 5-year DFS 57% versus 77%, adjusted HR 1.1, 95% CI 0.4–2.6 respectively). In stage II colon cancer patients not treated with adjuvant chemotherapy, BRAF mutation and MSI status both tended to have a negative prognostic effect on disease-free survival. KRAS and MSI status also tended to be correlated with worse overall survival.     

DNA sequence profiles of the colorectal cancer critical gene set KRAS-BRAF-PIK3CA-PTEN-TP53 related to age at disease onset

The incidence of colorectal cancer (CRC) increases with age and early onset indicates an increased likelihood for genetic predisposition for this disease. The somatic genetics of tumor development in relation to patient age remains mostly unknown. We have examined the mutation status of five known cancer critical genes in relation to age at diagnosis, and compared the genomic complexity of tumors from young patients without known CRC syndromes with those from elderly patients. Among 181 CRC patients, stratified by microsatellite instability status, DNA sequence changes were identified in KRAS (32%), BRAF (16%), PIK3CA (4%), PTEN (14%) and TP53 (51%). In patients younger than 50 years (n = 45), PIK3CA mutations were not observed and TP53 mutations were more frequent than in the older age groups. The total gene mutation index was lowest in tumors from the youngest patients. In contrast, the genome complexity, assessed as copy number aberrations, was highest in tumors from the youngest patients. A comparable number of tumors from young (<50 years) and old patients (>70 years) was quadruple negative for the four predictive gene markers (KRAS-BRAF-PIK3CA-PTEN); however, 16% of young versus only 1% of the old patients had tumor mutations in PTEN/PIK3CA exclusively. This implies that mutation testing for prediction of EGFR treatment response may be restricted to KRAS and BRAF in elderly (>70 years) patients. Distinct genetic differences found in tumors from young and elderly patients, whom are comparable for known clinical and pathological variables, indicate that young patients have a different genetic risk profile for CRC development than older patients.     

The Relationship of BrafV600E Mutation Status to FDG PET/CT Avidity in Thyroid Cancer: A Review and Meta-Analysis

Objective: Papillary thyroid cancer (PTC) harboring a BRAF^V600E gene mutation has been shown to exhibit aggressive tumor behavior and carries higher risks of recurrence and disease-specific death. In this systematic review and meta-analysis, we examined published evidence related to the accuracy of fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) in detection of residual disease in patients with BRAF^V600E mutated thyroid cancer.Methods: We extracted data from PUBMED/MEDLINE and EMBASE published between January 1995 and March 2017. We included studies that compared FDG PET standardized uptake values (SUVs) between BRAF^V600E-positive and BRAF^V600E-negative subjects, as well as those that evaluated the odds of having FDG avidity between BRAF^V600E-positive and -negative patients with thyroid cancer.Results: There were a total of 12 studies in the systematic review. Seven studies qualified for the analysis for calculating the pooled odds ratio (OR). The pooled cohort with binary data had 1,144 patients out of which 843 were BRAF^V600E positive and 301 were BRAF^V600E negative. Those with a BRAF^V600E mutation had a significantly greater likelihood of having FDG-avid lesions. The pooled OR was 2.12 (confidence interval &lsqb;CI] 1.53–3.00, P<.01). The pooled mean SUV (cohort of 315 patients) was significantly higher in BRAF^V600E-positive compared to BRAF^V600E negative patients, with a pooled mean difference of 5.1 (CI 4.3–5.8).Conclusion: Our meta-analysis shows that presence of BRAF^V600E mutation in PTC confers a higher likelihood of FDG PET avidity and is associated with higher SUV uptake values compared to BRAF^V600E-mutation negative status.Abbreviations: BRAF = B-Raf proto-oncogene, serine/threonine kinase; CI = confidence interval; CT = computed tomography; DTC = differentiated thyroid cancer; FDG = fluorodeoxyglucose; PET = positron emission tomography; PTC = papillary thyroid cancer; SUV = standardized uptake value     

Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.PIK3CA¹, the gene encoding the p110α catalytic subunit of phosphatidylinositide-3 kinase (PI3K), is one of the two most frequently mutated genes in breast cancer. Approximately 80% of these mutations occur in two hot spots in the helical domain (E545K, E542K) and in the catalytic domain (H1047R). PIK3CA activating mutations occur in ∼40% of luminal and HER2-enriched breast cancer subtypes and ∼10% of basal-like breast cancer (BLBC) (1). In this last tumor subtype, mutations in PIK3CA are the most frequent activating kinase mutation. Thus, understanding of how PIK3CA mutations operate in BLBC is important for identifying therapeutic targets in this subtype of the disease, which lacks approved targeted therapies.To elucidate mechanisms by which mutant PIK3CA transforms MECs, we used immortalized, nontumorigenic MCF10A cells, which exhibit basal-like gene expression. Although MCF10A cells require growth factors for proliferation (2), heterozygous knock-in of E545K or H1047R PIK3CA mutation allows growth factor-independent proliferation (3). These knock-in PIK3CA mutant MECs provide a robust model in which to study the impact of these mutations without the effects of random insertion and overexpression associated with ectopic gene transduction. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of these cells identified 72 proteins concordantly altered by both PIK3CA mutations. A significant fraction of these were secreted proteins, cell surface receptors or ECM interacting molecules, suggesting PIK3CA mutations induce changes involving communication with the tumor microenvironment. This analysis identified a PI3K-induced amphiregulin (AREG)-EGFR-ERK signaling pathway that was required for growth of PIK3CA-mutant cells as well as adjacent PIK3CA-WT cells. In addition, these protein changes correlated with poor clinical outcome in BLBC. EGFR antagonists inhibited growth of PIK3CA mutant BLBC tumors, suggesting a potential therapeutic strategy for patients with this molecular subtype of breast cancer.     

Network Analysis Reveals A Signaling Regulatory Loop in the PIK3CA-mutated Breast Cancer Predicting Survival Outcome

Mutated genes are rarely common even in the same pathological type between cancer patients and as such, it has been very challenging to interpret genome sequencing data and difficult to predict clinical outcomes. PIK3CA is one of a few genes whose mutations are relatively popular in tumors. For example, more than 46.6% of luminal-A breast cancer samples have PIK3CA mutated, whereas only 35.5% of all breast cancer samples contain PIK3CA mutations. To understand the function of PIK3CA mutations in luminal A breast cancer, we applied our recently-proposed Cancer Hallmark Network Framework to investigate the network motifs in the PIK3CA-mutated luminal A tumors. We found that more than 70% of the PIK3CA-mutated luminal A tumors contain a positive regulatory loop where a master regulator (PDGF-D), a second regulator (FLT1) and an output node (SHC1) work together. Importantly, we found the luminal A breast cancer patients harboring the PIK3CA mutation and this positive regulatory loop in their tumors have significantly longer survival than those harboring PIK3CA mutation only in their tumors. These findings suggest that the underlying molecular mechanism of PIK3CA mutations in luminal A patients can participate in a positive regulatory loop, and furthermore the positive regulatory loop (PDGF-D/FLT1/SHC1) has a predictive power for the survival of the PIK3CA-mutated luminal A patients.     

A systematic study of gene mutations in urothelial carcinoma; inactivating mutations in TSC2 and PIK3R1

Background

Urothelial carcinoma (UC) is characterized by frequent gene mutations of which activating mutations in FGFR3 are the most frequent. Several downstream targets of FGFR3 are also mutated in UC, e.g., PIK3CA, AKT1, and RAS. Most mutation studies of UCs have been focused on single or a few genes at the time or been performed on small sample series. This has limited the possibility to investigate co-occurrence of mutations.

Methodology/Principal Findings

We performed mutation analyses of 16 genes, FGFR3, PIK3CA, PIK3R1 PTEN, AKT1, KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, TSC1, TSC2, APC, CTNNB1, and TP53, in 145 cases of UC. We show that FGFR3 and PIK3CA mutations are positively associated. In addition, we identified PIK3R1 as a target for mutations. We demonstrate a negative association at borderline significance between FGFR3 and RAS mutations, and show that these mutations are not strictly mutually exclusive. We show that mutations in BRAF, ARAF, RAF1 rarely occurs in UC. Our data emphasize the possible importance of APC signaling as 6% of the investigated tumors either showed inactivating APC or activating CTNNB1 mutations. TSC1, as well as TSC2, that constitute the mTOR regulatory tuberous sclerosis complex were found to be mutated at a combined frequency of 15%.

Conclusions/Significance

Our data demonstrate a significant association between FGFR3 and PIK3CA mutations in UC. Moreover, the identification of mutations in PIK3R1 further emphasizes the importance of the PI3-kinase pathway in UC. The presence of TSC2 mutations, in addition to TSC1 mutations, underlines the involvement of mTOR signaling in UC.     

BRAF and PIK3CA genes are somatically mutated in hepatocellular carcinoma among patients from South Italy

Poor data have been previously reported about the mutation rates in K-RAS, BRAF, and PIK3CA genes among patients with hepatocellular carcinoma (HCC). Here we further elucidated the role of these genes in pathogenesis of primary hepatic malignancies. Archival tumour tissue from 65 HCC patients originating from South Italy were screened for mutations in these candidate genes by direct sequencing. Overall, oncogenic mutations were detected in 15 (23%) patients for BRAF gene, 18 (28%) for PIK3CA gene, and 1 (2%) for K-RAS gene. Using statistical analysis, BRAF mutations were significantly correlated with the presence of either multiple HCC nodules (P=0.021) or higher proliferation rates (P=0.034). Although further extensive screenings are awaited in HCC patients among different populations, our findings clearly indicated that mutational activation of both BRAF and PIK3CA genes does contribute to hepatocellular tumorigenesis at somatic level in Southern Italian population.     

Multi-Determinants Analysis of Molecular Alterations for Predicting Clinical Benefit to EGFR-Targeted Monoclonal Antibodies in Colorectal Cancer

Background

KRAS mutations occur in 35–45% of metastatic colorectal cancers (mCRC) and preclude responsiveness to EGFR-targeted therapy with cetuximab or panitumumab. However, less than 20% patients displaying wild-type KRAS tumors achieve objective response. Alterations in other effectors downstream of the EGFR, such as BRAF, and deregulation of the PIK3CA/PTEN pathway have independently been found to give rise to resistance. We present a comprehensive analysis of KRAS, BRAF, PIK3CA mutations, and PTEN expression in mCRC patients treated with cetuximab or panitumumab, with the aim of clarifying the relative contribution of these molecular alterations to resistance.

Methodology/Principal Findings

We retrospectively analyzed objective tumor response, progression-free (PFS) and overall survival (OS) together with the mutational status of KRAS, BRAF, PIK3CA and expression of PTEN in 132 tumors from cetuximab or panitumumab treated mCRC patients. Among the 106 non-responsive patients, 74 (70%) had tumors with at least one molecular alteration in the four markers. The probability of response was 51% (22/43) among patients with no alterations, 4% (2/47) among patients with 1 alteration, and 0% (0/24) for patients with ≥2 alterations (p<0.0001). Accordingly, PFS and OS were increasingly worse for patients with tumors harboring none, 1, or ≥2 molecular alteration(s) (p<0.001).

Conclusions/Significance

When expression of PTEN and mutations of KRAS, BRAF and PIK3CA are concomitantly ascertained, up to 70% of mCRC patients unlikely to respond to anti-EGFR therapies can be identified. We propose to define as ‘quadruple negative’, the CRCs lacking alterations in KRAS, BRAF, PTEN and PIK3CA. Comprehensive molecular dissection of the EGFR signaling pathways should be considered to select mCRC patients for cetuximab- or panitumumab-based therapies.     

<Emphasis Type="Italic">PIK3CA</Emphasis> mutations in <Emphasis Type="Italic">KRAS</Emphasis> and <Emphasis Type="Italic">BRAF</Emphasis> wild type colorectal cancer patients. A study of Spanish population

The objective of the work was to study PIK3CA mutations in wild type KRAS and BRAF colorectal cancer. Clinicopathological data and paraffin-embedded specimens were collected on 73 patients who underwent colorectal resections at General Yagüe Hospital in Burgos. KRAS, BRAF and PIK3CA status were analyzed by real-time PCR in all patients. PIK3CA mutations were present in 8.22% of wild type KRAS and BRAF colorectal cancers. The most frequent mutation is E545K/D in exon 9 which represents 83.3% of all mutations. By contrast, we did not found any tumour harbouring H1047R mutation in exon 20. Among the patients who undergo a curative resection of colorectal cancer, PIK3CA mutation is present in an important percentage of KRAS and BRAF wild type tumours. PIK3CA mutation may be considered as it could be a hypothetic reason to be not responder to anti-EGFR antibodies.     

Tumor Homogeneity between Primary and Metastatic Sites for BRAF Status in Metastatic Melanoma Determined by Immunohistochemical and Molecular Testing

BRAF inhibitors have demonstrated improvement of overall survival in patients with metastatic melanoma and BRAF^V600 mutations. In order to evaluate BRAF tumor heterogeneity between primary and metastatic site, we have evaluated the performance of immunohistochemistry (IHC) with an anti-BRAF^V600E antibody in both localization by comparison with high resolution melting analysis followed by Sanger sequencing in a parallel blinded study. A total of 230 samples distributed as primary melanoma (n = 88) and different types of metastatic samples (n = 142) were studied in 99 patients with advanced or metastatic melanoma (stage III or IV). The prevalence of each BRAF mutation was c.1799T>A, BRAF^V600E (45.2%), c.1799_1800TG>AA, BRAF^V600E2 (3.0%), c.1798_1799GT>AA, BRAF^V600K (3.0%), c.1801 A>G, BRAF^K601E (1.3%), c.1789_1790CT>TC, BRAF^L597S (0.4%), c.1780G>A, BRAF^D594N (0.9%) respectively. IHC was positive in 109/112 samples harboring BRAF^V600E/E2 mutations and negative in other cases. The cytoplasmic staining was either strongly positive in tumor cells of BRAF^V600E mutated cases. It appeared strong brown, different from the vesicular grey cytoplasmic pigmentation of melanophages. Concordance between the two techniques was 96.4%. Sensitivity of IHC for detecting the BRAF^V600E/E2 mutations was 97.3%, while specificity was 100%. Both our IHC and molecular study demonstrated homogeneity between primary and metastatic sites for BRAF status in melanoma. This study also provides evidence that IHC may be a cost-effective first-line method for BRAF^V600E detection. Thereafter, molecular techniques should be used in negative, ambiguous or non-contributive cases.     

FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy

Background

Fifty percent of patients with muscle–invasive bladder cancer (MI-BC) die from their disease and current chemotherapy treatment only marginally increases survival. Novel therapies targeting receptor tyrosine kinases or activated oncogenes may improve outcome. Hence, it is necessary to stratify patients based on mutations in relevant oncogenes. Patients with non-muscle-invasive bladder cancer (NMI-BC) have excellent survival, however two-thirds develop recurrences. Tumor specific mutations can be used to detect recurrences in urine assays, presenting a more patient-friendly diagnostic procedure than cystoscopy.

Methodology/Principal Findings

To address these issues, we developed a mutation assay for the simultaneous detection of 19 possible mutations in the HRAS, KRAS, and NRAS genes. With this assay and mutation assays for the FGFR3 and PIK3CA oncogenes, we screened primary bladder tumors of 257 patients and 184 recurrences from 54 patients. Additionally, in primary tumors p53 expression was obtained by immunohistochemistry. Of primary tumors 64% were mutant for FGFR3, 11% for RAS, 24% for PIK3CA, and 26% for p53. FGFR3 mutations were mutually exclusive with RAS mutations (p = 0.001) and co-occurred with PIK3CA mutations (p = 0.016). P53 overexpression was mutually exclusive with PIK3CA and FGFR3 mutations (p≤0.029). Mutations in the RAS and PIK3CA genes were not predictors for recurrence-free, progression-free and disease-specific survival. In patients presenting with NMI-BC grade 3 and MI-BC, 33 and 36% of the primary tumors were mutant. In patients with low-grade NMI-BC, 88% of the primary tumors carried a mutation and 88% of the recurrences were mutant.

Conclusions/Significance

The mutation assays present a companion diagnostic to define patients for targeted therapies. In addition, the assays are a potential biomarker to detect recurrences during surveillance. We showed that 88% of patients presenting with low-grade NMI-BC are eligible for such a follow-up. This may contribute to a reduction in the number of cystoscopical examinations.     

The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer

Purpose

To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC).

Experimental Design

PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM) model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined.

Results

In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC₅₀ = 9.0–14.3 nM). Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01) vs. a 43% decrease (p = 0.008) in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003), no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013).

Conclusions

These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC.     

Lkb1 Loss Promotes Tumor Progression of BRAFV600E-Induced Lung Adenomas

Aberrant activation of MAP kinase signaling pathway and loss of tumor suppressor LKB1 have been implicated in lung cancer development and progression. Although oncogenic KRAS mutations are frequent, BRAF mutations (BRAF^V600E) are found in 3% of human non-small cell lung cancers. Contrary to KRAS mutant tumors, BRAF^V600E-induced tumors are benign adenomas that fail to progess. Interestingly, loss of tumor supressor LKB1 coexists with KRAS oncogenic mutations and synergizes in tumor formation and progression, however, its cooperation with BRAF^V600E oncogene is unknown. Our results describe a lung cell population in neonates mice where expression of BRAF^V600E leads to lung adenoma development. Importantly, expression of BRAF^V600E concomitant with the loss of only a single-copy of Lkb1, overcomes senencence–like features of BRAF^V600E-mutant adenomas leading malignization to carcinomas. These results posit LKB1 haploinsufficiency as a risk factor for tumor progression of BRAF^V600E mutated lung adenomas in human cancer patients.