PIK3CA gene encoding a catalytic subunit of the phosphatidylinositol-3-kinase (PI3K) is mutated and/or amplified in various neoplasia, including lung cancer. Here we investigated PIK3CA gene alterations, the expression of core components of PI3K pathway, and evaluated their clinical importance in non-small cell lung cancer (NSCLC).
Materials and methods
Oncogenic mutations/rearrangements in PIK3CA, EGFR, KRAS, HER2, BRAF, AKT1 and ALK genes were detected in tumors from 1117 patients with NSCLC. PIK3CA gene copy number was examined by fluorescent in situ hybridization and the expression of PI3K p110 subunit alpha (PI3K p110α), p-Akt, mTOR, PTEN was determined by immunohistochemistry in PIK3CA mutant cases and 108 patients without PIK3CA mutation.
PIK3CA mutation was found in 3.9% of squamous cell carcinoma and 2.7% of adenocarcinoma. Among 34 PIK3CA mutant cases, 17 tumors harbored concurrent EGFR mutations and 4 had KRAS mutations. PIK3CA mutation was significantly associated with high expression of PI3K p110α (p<0.0001), p-Akt (p = 0.024) and mTOR (p = 0.001), but not correlated with PIK3CA amplification (p = 0.463). Patients with single PIK3CA mutation had shorter overall survival than those with PIK3CA-EGFR/KRAS co-mutation or wildtype PIK3CA (p = 0.004). A significantly worse survival was also found in patients with PIK3CA mutations than those without PIK3CA mutations in the EGFR/KRAS wildtype subgroup (p = 0.043)
PIK3CA mutations frequently coexist with EGFR/KRAS mutations. The poor prognosis of patients with single PIK3CA mutation in NSCLC and the prognostic value of PIK3CA mutation in EGFR/KRAS wildtype subgroup suggest the distinct mutation status of PIK3CA gene should be determined for individual therapeutic strategies in NSCLC.
Phosphatidylinositol 3-kinases (PI3Ks) are a group of lipid kinases that regulate signaling pathways involved in cell proliferation, adhesion, survival, and motility. Even though PIK3CA amplification and somatic mutation have been reported previously in various kinds of human cancers, the genetic change in PIK3CA in human breast cancer has not been clearly identified.
Fifteen breast cancer cell lines and 92 primary breast tumors (33 with matched normal tissue) were used to check somatic mutation and gene copy number of PIK3CA. For the somatic mutation study, we specifically checked exons 1, 9, and 20, which have been reported to be hot spots in colon cancer. For the analysis of the gene copy number, we used quantitative real-time PCR and fluorescence in situ hybridization. We also treated several breast cancer cells with the PIK3CA inhibitor LY294002 and compared the apoptosis status in cells with and without PIK3CA mutation.
We identified a 20.6% (19 of 92) and 33.3% (5 of 15) PIK3CA somatic mutation frequency in primary breast tumors and cell lines, respectively. We also found that 8.7% (8 of 92) of the tumors harbored a gain of PIK3CA gene copy number. Only four cases in this study contained both an increase in the gene copy number and a somatic mutation. In addition, mutation of PIK3CA correlated with the status of Akt phosphorylation in some breast cancer cells and inhibition of PIK3CA-induced increased apoptosis in breast cancer cells with PIK3CA mutation.
Somatic mutation rather than a gain of gene copy number of PIK3CA is the frequent genetic alteration that contributes to human breast cancer progression. The frequent and clustered mutations within PIK3CA make it an attractive molecular marker for early detection and a promising therapeutic target in breast cancer.
Despite considerable recent progress in the treatment of lung adenocarcinoma, there has been little progress in the development of efficacious molecular targeted therapies for squamous cell lung cancer. In addition to the recent comprehensive genome-wide characterization of squamous cell lung cancer, it is also important to genotype this form of cancer. We therefore conducted the Shizuoka Lung Cancer Mutation Study to analyze driver mutations in patients with thoracic malignancies. Here we report the results of genotyping in patients with squamous cell lung cancer.
Based on the biobanking system, in conjunction with the clinic and pathology lab, we developed a genotyping panel designed to assess 24 mutations in 10 genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN, HER2 and DDR2), EGFR, MET, PIK3CA, FGFR1 and FGFR2 copy numbers, and EML4-ALK and ROS1 translocations, using pyrosequencing plus capillary electrophoresis, quantitative polymerase chain reaction (PCR) and reverse-transcription PCR, respectively.
A total of 129 patients with squamous cell lung cancer and adenosquamous carcinoma were enrolled in this study between July 2011 and November 2012. We detected genetic alterations in 40% of all cases. Gene alterations included: EGFR mutations, 6%; KRAS mutations, 4%; PIK3CA mutations, 13%; NRAS mutations, 1%; KIF5b-RET fusion gene, 1%; EGFR copy number gain, 5%; PIK3CA copy number gain, 15%; and FGFR1 copy number gain, 5%. Twelve patients (9%) harbored simultaneous genetic alterations. Genetic alterations were detected more frequently in surgically-resected, snap-frozen samples than in formalin-fixed, paraffin-embedded samples (50% vs. 29%). In addition, patients aged ≤70 years old and never-smokers showed high frequencies of genetic alterations.
This study represents one of the largest prospective tumor-genotyping studies to be performed in Asian patients with squamous cell lung cancer. These results suggest that incorporation of genetic profiling into lung cancer clinical practice may facilitate the administration of personalized cancer treatments in patients with squamous cell lung cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-786) contains supplementary material, which is available to authorized users.
Lung cancer; Squamous cell carcinoma; Adenosquamos carcinoma; Genetic profiling; Driver mutation; PIK3CA mutation; FGFR1 copy number gain
Deregulation of the EGFR signaling pathway is one of the most frequently observed genetic abnormalities that drives cancer development. Although mutations in the downstream components of the EGFR signaling pathway, including KRAS, BRAF and PIK3CA, have been reported in numerous cancers, extensive mutation and copy number analysis of these genes in clinical samples has not been performed for head and neck squamous cell carcinoma (HNSCC).
We examined the mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients.
We used DNA sequencing to detect mutations and the copy number changes were evaluated by qPCR and array comparative genomic hybridization (CGH) analysis.
We examined the mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients. We identified 3 mutations (2.6%) in K-RAS and 3 mutations (2.6%) in PIK3CA. Copy number amplification was found in 37 cases (32.2%) for PIK3CA, 10 cases (8.7%) for K-RAS and 2 cases (1.7%) for BRAF. Kaplan-Meier survival analysis revealed that copy-number amplification of PIK3CA was markedly associated with cancer relapse in patients without lymph node metastasis. (Log-rank test, p = 0.026)
Copy number amplification of the PIK3CA gene is associated with poor prognosis in HNSCC patients without lymph node metastasis. The PIK3CA copy number status will serve as a marker of poor prognosis in patients with HNSCC.
PIK3CA; KRAS; BRAF; Copy number analysis; Prognostic Factor
Cetuximab is a monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). Although approved for use in EGFR over-expressing advanced colorectal cancer, recent studies have demonstrated a lack of association between EGFR over-expression and cetuximab response, requiring the identification of novel biomarkers predictive of response to this agent. To do so, 22 colon cancer cell lines were screened for cetuximab response in-vitro and sensitive and resistant lines identified. In sensitive cell lines cetuximab induced a G0/G1 arrest without inducing apoptosis. Notably, cetuximab sensitive but not resistant cell lines were preferentially responsive to EGF-stimulated growth. While neither EGFR protein/mRNA expression nor gene copy number correlated with cetuximab response, examination of the mutation status of signaling components downstream of EGFR demonstrated that cells lines with activating PIK3CA mutations or loss of PTEN expression (PTEN null) were more resistant to cetuximab than PIK3CA wild type/PTEN expressing cell lines (14±5.0% versus 38.5±6.4% growth inhibition, mean ± SEM, p=0.008). Consistently, PIK3CA mutant isogenic HCT116 cells showed increased resistance to cetuximab compared to PIK3CA wild type controls. Furthermore, cell lines that were PIK3CA mutant/PTEN null and Ras/BRAF mutant were highly resistant to cetuximab compared to those without dual mutations / PTEN loss (10.8±4.3% versus 38.8±5.9% growth inhibition, respectively, p=0.002), indicating constitutive and simultaneous activation of the Ras and PIK3CA pathways confers maximal resistance to this agent. A priori screening of colon tumors for PTEN expression status and PIK3CA and Ras/BRAF mutation status could help stratify patients likely to benefit from this therapy.
cetuximab; colon cancer cell lines; EGFR expression; EGFR copy number; PIK3CA; PTEN; Ras; BRAF
We performed this analysis to improve the understanding of the clinicopathological characteristics and clinical outcome of non-small cell lung cancer (NSCLC) patients harboring the primary epidermal growth factor receptor (EGFR) T790M mutation along with activating EGFR mutation.
Resected tumors from 1903 NSCLC patients were analyzed for mutation in EGFR, as well as KRAS (Kirsten rat sarcoma viral oncogene homolog), BRAF (v-raf murine sarcoma viral oncogene homolog B), HER2 (human epidermal growth factor 2), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha), and EML4 (echinoderm microtubule associated protein like 4)–ALK (anaplastic lymphoma receptor tyrosine kinase) fusion. Fluorescence in situ hybridization was performed to define EGFR and c-MET (met proto-oncogene gene amplification. Expression of PIK3CA and p-Akt (phosphorylated protein kinase B) were tested using immunohistochemistry. Clinical and pathological data, including sex, age at diagnosis, stage, tumor differentiation, smoking history, histological subtype, relapse-free and overall survival, were further analyzed.
In all, 16 NSCLC patients were found to harbor primary EGFR T790M mutation, including 14 adenocarcinomas and two adenosquamous carcinomas, accounting for 2.04% of all the EGFR mutant cases and 0.84% of the total. No c-MET amplification was found to coexist with primary EGFR T790M. Fewer EGFR copy-number variations were found in samples harboring EGFR T790M mutations compared with those in patients with exon 19 deletions and L858R. Overall survival was significantly shorter for patients harboring EGFR T790M mutation than it was for patients with exon 19 deletions (logrank P=0.008). When taking patients harboring EGFR L858R or exon 19 deletions as one group, the overall survival was also significantly longer than that in patients with T790M mutation (logrank P=0.012). There was no significant difference in relapse-free survival among three subgroups of patients.
Our study described the clinicopathological and molecular characteristics of NSCLC patients harboring primary EGFR T790M mutations. Its value of being a predictor for worse prognosis was established. Primary EGFR T790M mutation is a rare event in NSCLC cases, but the therapeutic strategies for this subtype of patients should be precisely considered.
driver mutation; survival; clinicopathological profile; EGFR tyrosine kinase inhibitor; acquired resistance
Cancer cells expressing constitutively active phosphatidylinositol-3 kinase (PI3K) are proliferative regardless of the absence of insulin, and they form dietary restriction (DR)-resistant tumors in vivo. Because the binding of insulin to its receptors activates the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling cascade, activating mutations in the PIK3CA oncogene may determine tumor response to DR-like pharmacological strategies targeting the insulin and mTOR pathways. The anti-diabetic drug metformin is a stereotypical DR mimetic that exerts its anti-cancer activity through a dual mechanism involving insulin-related (systemic) and mTOR-related (cell-autonomous) effects. However, it remains unclear whether PIK3CA-activating mutations might preclude the anti-cancer activity of metformin in vivo. To model the oncogenic PIK3CA-driven early stages of cancer, we used the clonal breast cancer cell line MCF10DCIS.com, which harbors the gain-of-function H1047R hot-spot mutation in the catalytic domain of the PI3KCA gene and has been shown to form DR-refractory xenotumors. To model PIK3CA-activating mutations in late stages of cancer, we took advantage of the isogenic conversion of a PIK3CA-wild-type tumor into a PIK3CA H1047R-mutated tumor using the highly metastatic colorectal cancer cell line SW48. MCF10DCIS.com xenotumors, although only modestly affected by treatment with oral metformin (approximately 40% tumor growth inhibition), were highly sensitive to the intraperitoneal (i.p.) administration of metformin, the anti-cancer activity of which increased in a time-dependent manner and reached >80% tumor growth inhibition by the end of the treatment. Metformin treatment via the i.p. route significantly reduced the proliferation factor mitotic activity index (MAI) and decreased tumor cellularity in MCF10DCIS.com cancer tissues. Whereas SW48-wild-type (PIK3CA+/+) cells rapidly formed metformin-refractory xenotumors in mice, ad libitum access to water containing metformin significantly reduced the growth of SW48-mutated (PIK3CAH1047R/+) xenotumors by approximately 50%. Thus, metformin can no longer be considered as a bona fide DR mimetic, at least in terms of anti-cancer activity, because tumors harboring the insulin-unresponsive, DR-resistant, PIK3CA-activating mutation H1047R remain sensitive to the anti-tumoral effects of the drug. Given the high prevalence of PIK3CA mutations in human carcinomas and the emerging role of PIK3CA mutation status in the treatment selection process, these findings might have a significant impact on the design of future trials evaluating the potential of combining metformin with targeted therapy.
Metformin; cancer; PI3K; PIK3CA mutations; dietary restriction; calorie restriction; rapamycin
Deregulation of EGFR signaling is common in non-small cell lung cancers (NSCLC) and this finding led to the development of tyrosine kinase inhibitors (TKIs) that are highly effective in a subset of NSCLC. Mutations of EGFR (mEGFR) and copy number gains (CNGs) of EGFR (gEGFR) and HER2 (gHER2) have been reported to predict for TKI response. Mutations in KRAS (mKRAS) are associated with primary resistance to TKIs.
We investigated the relationship between mutations, CNGs and response to TKIs in a large panel of NSCLC cell lines. Genes studied were EGFR, HER2, HER3 HER4, KRAS, BRAF and PIK3CA. Mutations were detected by sequencing, while CNGs were determined by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH). IC50 values for the TKIs gefitinib (Iressa) and erlotinib (Tarceva) were determined by MTS assay. For any of the seven genes tested, mutations (39/77, 50.6%), copy number gains (50/77, 64.9%) or either (65/77, 84.4%) were frequent in NSCLC lines. Mutations of EGFR (13%) and KRAS (24.7%) were frequent, while they were less frequent for the other genes. The three techniques for determining CNG were well correlated, and qPCR data were used for further analyses. CNGs were relatively frequent for EGFR and KRAS in adenocarcinomas. While mutations were largely mutually exclusive, CNGs were not. EGFR and KRAS mutant lines frequently demonstrated mutant allele specific imbalance i.e. the mutant form was usually in great excess compared to the wild type form. On a molar basis, sensitivity to gefitinib and erlotinib were highly correlated. Multivariate analyses led to the following results:
1. mEGFR and gEGFR and gHER2 were independent factors related to gefitinib sensitivity, in descending order of importance.
2. mKRAS was associated with increased in vitro resistance to gefitinib.
Our in vitro studies confirm and extend clinical observations and demonstrate the relative importance of both EGFR mutations and CNGs and HER2 CNGs in the sensitivity to TKIs.
Oral tongue squamous cell carcinomas (TSCC) are a unique subset of head and neck cancers with a distinct demographic profile, where up to half of the cases are never smokers. A small proportion of patients with OSCC are known to respond to EGFR TKI. We used a high-sensitivity mass spectrometry-based mutation profiling platform to determine the EGFR mutation status, as well as other actionable alterations in a series of Asian TSCC.
66 TSCC patients treated between 1998-2009 with complete clinico-pathologic data were included in this study. Somatic mutation profiling was performed using Sequenom LungCarta v1.0, and correlated with clinical parameters.
Mutations were identified in 20/66(30.3%) of samples and involved TP53, STK11, MET, PIK3CA, BRAF and NRF2. No activating EGFR mutations or KRAS mutations were discovered in our series, where just over a third were never smokers. The most common mutations were in p53 (10.6%; n = 7) and MET (10.6%, n = 11) followed by STK11 (9.1%, n = 6) and PIK3CA (4.5%, n = 3). BRAF and NRF2 mutations, which are novel in TSCC, were demonstrated in one sample each. There was no significant correlation between overall mutation status and smoking history (p = 0.967) or age (p = 0.360). Positive MET alteration was associated with poorer loco-regional recurrence free survival (LRFS) of 11 months [vs 90 months in MET-negative group (p = 0.008)]. None of the other mutations were significantly correlated with LRFS or overall survival. Four of these tumors were propagated as immortalized cell lines and demonstrated the same mutations as the original tumor.
Using the Sequenom multiplexed LungCarta panel, we identified mutations in 6 genes, TP53, STK11, MET, PIK3CA, BRAF and NRF2, with the notable absence of EGFR and HER2 mutations in our series of Asian OSCC. Primary cell line models recapitulated the mutation profiles of the original primary tumours and provide an invaluable resource for experimental cancer therapeutics.
Druggable; Therapeutic; Squamous cell carcinoma; Sequenom; Oral cancers; HNSCC
Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.
We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival.
MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.
As molecular targets continue to be identified and more targeted inhibitors are developed for personalized treatment of non-small cell lung cancer (NSCLC), multigene mutation determination will be needed for routine oncology practice and for clinical trials. In this study, we evaluated the sensitivity and specificity of multigene mutation testing by using the Snapshot assay in NSCLC. We retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which epidermal growth factor receptor (EGFR) mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected for EGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase 1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cell line DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies of EGFR, KRAS, PIK3CA, PTEN, and MEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in the HER2, NRAS, or BRAF genes. Three of the 51 mutant samples harbored double mutations: two PIK3CA mutations coexisted with KRAS or EGFR mutations, and another KRAS mutation coexisted with a PTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens; the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay is a sensitive and easily customized assay for multigene mutation testing in clinical practice.
Non-small cell lung cancer; multigene mutation; Snapshot assay; Sanger sequencing
Genomic testing to identify driver mutations that enable targeted therapy is emerging for patients with non-small cell lung cancer (NSCLC). We report the implementation of systematic prospective genotyping for somatic alterations in BRAF, PIK3CA, HER2, and ALK, in addition to EGFR and KRAS, in NSCLC patients at the Dana-Farber Cancer Institute.
Patients with NSCLC were prospectively referred by their providers for clinical genotyping. Formalin-fixed, paraffin embedded tumor samples were analyzed by Sanger sequencing for mutations in selected exons of EGFR, KRAS, BRAF, PIK3CA, and HER2. ALK rearrangements were detected by FISH or immunohistochemistry.
Between 7/1/2009 and 8/1/2010, 427 specimens from 419 patients were referred for genomic characterization; 344 (81%) specimens were successfully genotyped with a median turnaround time of 31 days (range, 9-155). Of the 344 specimens, 185 (54%) had at least one identifiable somatic alteration (KRAS: 24%, EGFR: 17%, ALK: 5%, BRAF: 5%, HER2: 4%, PIK3CA: 2%). As of 8/1/2011, 63/288 (22%) advanced NSCLC patients had received molecularly targeted therapy based on their genotypic results, including 34/42 (81%) patients with EGFR mutations, 12/15 (80%) with ALK rearrangements, and 17/95 (18%) with KRAS, BRAF or HER2 mutations.
Large scale testing for somatic alterations in EGFR, KRAS, BRAF, PIK3CA, HER2 and ALK is feasible and impacts therapeutic decisions. As the repertoire for personalized therapies expands in lung cancer and other malignancies, there is a need to develop new genomics technologies that can generate a comprehensive genetic profile of tumor specimens in a time and cost effective manner.
Lung cancer; cancer genomics; molecular targeted therapy
Mutations in PIK3CA (the gene encoding the p110α catalytic subunit of phosphatidylinositide-3-kinase, PI3K) play an important role in colorectal carcinogenesis. Experimental evidence suggests that PIK3CA exon 9 and exon 20 mutations trigger different biological effects, and that concomitant mutations in both exons 9 and 20 synergistically enhance tumorigenic effects. Thus, we hypothesized that PIK3CA exon 9 and exon 20 mutations might have differential effects on clinical outcome in colorectal cancer, and that concomitant PIK3CA exon 9 and 20 mutations might confer aggressive tumor behavior.
We sequenced PIK3CA by pyrosequencing in 1170 rectal and colon cancers in two prospective cohort studies, and found 189 (16%) PIK3CA-mutated tumors. Mortality hazard ratio (HR) according to PIK3CA status was computed using Cox proportional hazards model, adjusting for clinical and molecular features including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and BRAF and KRAS mutations.
Compared to PIK3CA wild-type cases, patients with concomitant PIK3CA mutations in exons 9 and 20 experienced significantly worse cancer-specific survival [log-rank P=0.031; multivariate HR=3.51; 95% confidence interval (CI), 1.28–9.62] and overall survival (log-rank P=0.0008 ; multivariate HR=2.68; 95% CI, 1.24–5.77). PIK3CA mutation in either exon 9 or 20 alone was not significantly associated with patient survival. No significant interaction of PIK3CA mutation with BRAF or KRAS mutation was observed in survival analysis.
Co-existence of PIK3CA (the PI3K p110α subunit) exon 9 and 20 mutations, but not PIK3CA mutation in either exon 9 or 20 alone, is associated with poor prognosis of colorectal cancer patients.
colon cancer; PI3K; RAF; RAS; biomarker
Bronchopulmonary neuroendocrine tumours (BP-NETs) comprise a large spectrum of tumours including typical carcinoids (TCs), atypical carcinoids (ACs), large-cell neuroendocrine carcinomas (LCNECs) and small-cell lung carcinomas (SCLCs) that exhibit considerably different biological aggressiveness and clinical behaviours. The phosphatidylinositol-3-kinase α catalytic subunit (PIK3CA) gene is known to be involved in the pathogenesis of several types of human cancers through gene amplification, deletions or somatic missense mutations within the helical and catalytic domains. However, the PIK3CA gene status in BP-NETs has yet to be explored. This study aimed to investigate the PIK3CA gene status in a large series of BP-NETs by direct gene sequencing and to analyse its correlation with the main clinicopathological parameters. To the best of our knowledge, we demonstrated for the first time a high frequency of somatic missense mutations (23.2%) in the PIK3CA gene in a series of 190 BP-NETs, including 75 TCs, 23 ACs, 17 LCNECs and 75 SCLCs. The frequency of the PIK3CA gene mutation in the kinase domain was higher (17.9%) than that in the helical domain (5.3%). When the mutational status of the PIK3CA gene was compared with the main clinical and pathological characteristics of the BP-NET patients, we found a significant association between PIK3CA gene mutations and BP-NET histology (P=0.011). Interestingly, the frequency of PIK3CA gene mutations increased with the biological aggressiveness of all BP-NETs, except LCNECs. In conclusion, our results suggest that PIK3CA gene mutations may play a key role in tumourigenesis and aggressiveness of BP-NETs. The PIK3CA gene may represent a favourable candidate for an effective therapeutic strategy in the treatment of patients with BP-NETs.
bronchopulmonary neuroendocrine tumours; PI3K/Akt/mTOR pathway; phosphatidylinositol-3-kinase α catalytic subunit mutations; biomarkers
Fifty percent of lung adenocarcinomas harbor somatic mutations in six genes that encode proteins in the EGFR signaling pathway, i.e., EGFR, HER2/ERBB2, HER4/ERBB4, PIK3CA, BRAF, and KRAS. We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this signaling pathway that could contribute to lung tumorigenesis.
We analyzed genomic DNA from a total of 261 resected, clinically annotated non-small cell lung cancer (NSCLC) specimens. The coding sequences of 39 genes were screened for somatic mutations via high-throughput dideoxynucleotide sequencing of PCR-amplified gene products. Mutations were considered to be somatic only if they were found in an independent tumor-derived PCR product but not in matched normal tissue. Sequencing of 9MB of tumor sequence identified 239 putative genetic variants. We further examined 22 variants found in RAS family genes and 135 variants localized to exons encoding the kinase domain of respective proteins. We identified a total of 37 non-synonymous somatic mutations; 36 were found collectively in EGFR, KRAS, BRAF, and PIK3CA. One somatic mutation was a previously unreported mutation in the kinase domain (exon 16) of FGFR4 (Glu681Lys), identified in 1 of 158 tumors. The FGFR4 mutation is analogous to a reported tumor-specific somatic mutation in ERBB2 and is located in the same exon as a previously reported kinase domain mutation in FGFR4 (Pro712Thr) in a lung adenocarcinoma cell line.
This study is one of the first comprehensive mutational analyses of major genes in a specific signaling pathway in a sizeable cohort of lung adenocarcinomas. Our results suggest the majority of gain-of-function mutations within kinase genes in the EGFR signaling pathway have already been identified. Our findings also implicate FGFR4 in the pathogenesis of a subset of lung adenocarcinomas.
Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessed PIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as these have not been previously reported.
Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutations within the C2, helical and kinase domain "hot spots" where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations to assess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice.
We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studied to date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model.
These data suggest that activating mutations in the Ras/Raf-MAPK/PI3K signaling cascades occur infrequently in neuroblastoma. Further, despite compelling evidence for MYC and RAS cooperation in vitro and in vivo to promote tumourigenesis, activation of RAS signal transduction does not constitute a preferred secondary pathway in neuroblastomas with MYCN deregulation in either human tumors or murine models.
Phosphatidylinositol-3-kinase (PI3K) activation involves the epidermal growth factor receptor (EGFR) and plays an important role in cell survival signaling in pancreaticobiliary cancer. EGFR gene mutations have been correlated with clinical response to EGFR inhibitors in patients with advanced non-small cell lung cancer. This study examined the prevalence of PIK3CA and EGFR mutations in pancreaticobiliary cancer where erlotinib, an EGFR inhibitor, is approved for therapy.
Thirty patients who underwent pancreatectomy for pancreaticobiliary carcinoma were identified. Genomic DNA was extracted from formalin fixed paraffin embedded tumor and adjacent normal tissue, and exons 9 and 20 (for the PIK3CA gene) and exons 18-21 (for the EGFR gene) were amplified by PCR and sequenced. Literature review on EGFR and/or PIK3CA mutations in pancreaticobiliary adenocarcinomas was conducted.
No mutations in either PIK3CA or EGFR genes were identified. The study identified one synonymous single nucleotide polymorphism (SNP) (rs1050171) in the coding region of EGFR. A previously unreported change, suspected to be a SNP, was observed in intron 18 of EGFR (IVS18+15, C>T). Review of the literature showed EGFR mutation rate of 2% and 10.5% in pancreatic and biliary tract carcinomas, respectively. PIK3CA mutations were found in 3.6% and 11.7% of pancreatic and biliary tract carcinomas, respectively.
A low prevalence of EGFR or PIK3CA mutations exists in pancreatic cancer (<5%), indicating that mutation screening may not be as useful in determining prognosis or response to targeted inhibition.
Phosphatidylinositol-3-kinase catalytic subunit (PIK3CA); epidermal growth factor receptor (EGFR); mutations; pancreas; biliary; cancer
Somatic mutations in PIK3CA (phosphatidylinositol-4,5-bisphosphonate 3-kinase [PI3K], catalytic subunit alpha gene) activate the PI3K-AKT signaling pathway and contribute to pathogenesis of various malignancies, including colorectal cancer.
We examined associations of PIK3CA oncogene mutation with relapse, survival, and treatment efficacy in 627 stage III colon carcinoma case subjects within a randomized adjuvant chemotherapy trial (5-fluorouracil and leucovorin [FU/LV] vs irinotecan [CPT11], fluorouracil and leucovorin [IFL]; Cancer and Leukemia Group B 89803 [Alliance]). We detected PIK3CA mutation in exons 9 and 20 by polymerase chain reaction and pyrosequencing. Cox proportional hazards model was used to assess prognostic and predictive role of PIK3CA mutation, adjusting for clinical features and status of routine standard molecular pathology features, including KRAS and BRAF mutations and microsatellite instability (mismatch repair deficiency). All statistical tests were two-sided.
Compared with PIK3CA wild-type cases, overall status of PIK3CA mutation positivity or the presence of PIK3CA mutation in either exon 9 or 20 alone was not statistically significantly associated with recurrence-free, disease-free, or overall survival (log-rank P > .70; P > .40 in multivariable regression models). There was no statistically significant interaction between PIK3CA and KRAS (or BRAF) mutation status in survival analysis (P
interaction > .18). PIK3CA mutation status did not appear to predict better or worse response to IFL therapy compared with FU/LV therapy (P
interaction > .16).
Overall tumor PIK3CA mutation status is not associated with stage III colon cancer prognosis. PIK3CA mutation does not appear to serve as a predictive tumor molecular biomarker for response to irinotecan-based adjuvant chemotherapy.
Background and aims
Recent studies have reported high frequencies of somatic mutations in
the phosphoinositide-3-kinase catalytic-α (PIK3CA)
gene in various human tumors. Three hot-spot mutations in the exons 9 and 20
have been proven to activate the Akt signalling pathway. The Raf/MEK/ERK
(mitogen-activated protein kinase) signal transduction is an important
mediator of a number of cellular fates including growth, proliferation, and
survival. The BRAF gene is activated by oncogenic RAS, leading to
cooperative effects in cells responding to growth factor signals. Here we
evaluate the mutational status of PIK3CA, KRAS, and BRAF in intraductal
papillary mucinous neoplasm/carcinoma (IPMN/IPMNC) of the pancreas.
Materials and methods
Exons 1, 4, 5, 6, 7, 9, 12, 18, and 20 of PIK3CA,
exons 1 of KRAS, and exons 5, 11, and 15 of
BRAF were analyzed in 36 IPMN/IPMC and two mucinous
cystadenoma specimens by direct genomic DNA sequencing.
We identified four somatic missense mutations of PIK3CA within the 36
IPMN/IPMC specimens (11%). One of the four mutations, H1047R, has
been previously reported to be a hot-spot mutation. Furthermore, we found 17
(47%) KRAS mutations in exon 1 and one missense mutation
(2.7%) in exon 15 of BRAF.
This data is the first report of PIK3CA mutation in
pancreatic cancer and it appears to be the first oncogene to be mutated in
IPMN/IPMC but not in conventional ductal adenocarcinoma of the pancreas. Our
data provide evidence that PIK3CA and BRAF
contribute to the tumorigenesis of IPMN/IPMC, but at a lower frequency than
IPMN; IPMC; Pancreas; PIK3CA; KRAS; BRAF; Mutation
The phosphatidylinositol-3 kinase (PI3K) pathway regulates a number of cellular processes, including cell survival, cell growth, and cell cycle progression. Consequently, this pathway is commonly deregulated in cancer. In particular, mutations in the gene PIK3CA that encodes the p110α catalytic subunit of the PI3K enzymes result in cell proliferation and resistance to apoptosis in vitro and induce breast tumors in transgenic mice. These data underscore the role of this pathway during oncogenesis. Thus, an ongoing, large-scale effort is underway to develop clinically active drugs that target elements of the PI3K pathway. However, conflicting data suggest that gain-of-function PIK3CA mutations may be associated with either a favorable or a poor clinical outcome, compared with the wild-type PIK3CA gene. In the current study, we performed a systematic review of breast cancer clinical studies. Upon evaluation of 2587 breast cancer cases from 12 independent studies, we showed that patients with tumors harboring a PIK3CA mutation have a better clinical outcome than those with a wild-type PIK3CA gene. Importantly, this improved prognosis may pertain only to patients with mutations in the kinase domain of p110α and to postmenopausal women with estrogen receptor-positive breast cancer. We propose three potential explanations for this paradoxical observation. First, PIK3CA mutations may interfere with the metastasis process or may induce senescence, which results in a better outcome for patients with mutated tumors. Secondly, we speculate that PIK3CA mutations may increase early tumor diagnosis by modification of the actin cytoskeleton in tumor cells. Lastly, we propose that PIK3CA mutations may be a favorable predictive factor for response to hormonal therapy, giving a therapeutic advantage to these patients. Ultimately, an improved understanding of the clinical impact of PIK3CA mutations is critical for the development of optimally personalized therapeutics against breast cancer and other solid tumors. This effort will be important to prevent or explain therapeutic failures and select patients who are most likely to respond to new therapies that inhibit the PI3K pathway.
PI3K; PIK3CA gene; mutation; breast cancer
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.
Approximately 10–30 % of colorectal cancers exhibit somatic mutations in the phosphoinositide-3-kinase, catalytic, alpha polypeptide gene (PIK3CA). We evaluated the relationship between PIK3CA mutation status and demographic factors, lifestyle factors, and other tumor characteristics and the relationship between PIK3CA mutation status and colorectal cancer survival.
The population-based study included postmenopausal women with invasive colorectal cancer diagnosed between 1998 and 2002 in Western Washington State. Participants were interviewed, and tumor specimens were tested for PIK3CA mutations in exons 9 and 20 hotspots, KRAS exon 2 mutations, BRAF p.V600E mutation, and microsatellite instability. We used Cox regression to evaluate the association between PIK3CA mutation status and disease-specific and overall survival. Stratified analyses were conducted by KRAS mutation status.
PIK3CA mutations were evident in approximately 13 % of cases (N=35). Women with PIK3CA-mutated colorectal cancer were significantly more likely than those with PIK3CA wild-type disease to be non-white, to have proximal colon cancer, and to have KRAS-mutated tumors (p<0.05). In Cox proportional hazards regression analyses, overall survival was poorer, although not statistically significantly so, for women with PIK3CA-mutated versus wild-type colorectal cancer (hazard ratio=1.74, 95 % confidence interval 0.86–3.50). This association between PIK3CA mutation status and survival was evident only when analyses were restricted to cases without somatic KRAS mutations (hazard ratio=2.94, 95 % confidence interval 1.12–7.73).
PIK3CA-mutated colorectal cancer appears to have a distinct epidemiologic profile that is of clinical significance. Women with PIK3CA-mutated colorectal cancer experience a poorer prognosis than those with PIK3CA wild-type disease.
Colorectal cancer; PIK3CA; Survival; KRAS
Mutations of the PIK3CA gene may predict response to phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) inhibitors. Concomitant mutations in the mitogen-activated protein kinase (MAPK) pathway may mediate resistance.
Patients and Methods
Tumors from patients with breast, cervical, endometrial, and ovarian cancer referred to the Clinical Center for Targeted Therapy (Phase I Program) were analyzed for PIK3CA, KRAS, NRAS, and BRAF mutations. Patients with PIK3CA mutations were treated, whenever feasible, with agents targeting the PI3K/AKT/mTOR pathway.
Of 140 patients analyzed, 25 (18%) had PIK3CA mutations, including five of 14 patients with squamous cell cervical, seven of 29 patients with endometrial, six of 29 patients with breast, and seven of 60 patients with ovarian cancers. Of the 25 patients with PIK3CA mutations, 23 (median of two prior therapies) were treated on a protocol that included a PI3K/AKT/mTOR pathway inhibitor. Two (9%) of 23 patients had stable disease for more than 6 months, and seven patients (30%) had a partial response. In comparison, only seven (10%) of 70 patients with the same disease types but with wild-type PIK3CA treated on the same protocols responded (P = .04). Seven patients (30%) with PIK3CA mutations had coexisting MAPK pathway (KRAS, NRAS, BRAF) mutations (ovarian cancer, n = 5; endometrial cancer, n = 2), and two of these patients (ovarian cancer) achieved a response.
PIK3CA mutations were detected in 18% of tested patients. Patients with PIK3CA mutations treated with PI3K/AKT/mTOR inhibitors demonstrated a higher response rate than patients without mutations. A subset of patients with ovarian cancer with simultaneous PIK3CA and MAPK mutations responded to PI3K/AKT/mTOR inhibitors, suggesting that not all patients demonstrate resistance when the MAPK pathway is concomitantly activated.
PI3K/AKTsignaling pathway plays an important role in cell growth, proliferation, and tumorgenesis of various malignancies. This signaling pathway has been shown to be frequently altered in several human cancers including ovarian cancers. However the role of this oncogenic signaling pathway has not been explored in the Middle Eastern epithelial ovarian cancer (EOC). Therefore, we investigated PI3K/AKT genetic alterations such as PIK3CA amplification, PIK3CA mutation, PTEN protein loss and their relationships with various clinicopathological characteristics in 156 EOCs.
Fluorescence in situ hybridization (FISH) technique and DNA sequencing were used to analyze PIK3CA amplification and mutation respectively. Expression of PIK3CA protein expression (p110 α), PTEN, p-AKT and Ki-67 was analyzed by immunohistochemistry. PIK3CA amplification was seen in 54 of 152 (35.5%) EOC cases analyzed; PIK3CA gene mutations in 6/153 EOC (3.9%); KRAS mutations in 3/154 EOC (1.9%), BRAF mutations in 3/156 EOC (1.9%), p53 mutation in 50/154 EOC (32.5%), and loss of PTEN protein expression in 33/144 EOC (22.9%). p110 α overexpression was associated with increased phosphorylation of AKT-Ser 473 and with the proliferation marker Ki-67.
Our data showed mutual exclusivity between the molecular event of PIK3CA amplification and mutations in PIK3CA, KRAS, BRAF genes, which suggests that each of these alterations may individually be sufficient to drive ovarian tumor pathogenesis independently. High prevalence of genetic alterations in PI3K/AKT pathway in a Middle Eastern ovarian carcinoma provides genetic evidence supporting the notion that dysregulated PI3K/AKT pathways play an important role in the pathogenesis of ovarian cancers.
Activated PI3K-AKT pathway may contribute to decrease sensitivity to inhibitors of key pathogenetic effectors (mutated BRAF, active NRAS or MEK) in melanoma. Functional alterations are deeply involved in PI3K-AKT activation, with a minimal role reported for mutations in PIK3CA, the catalytic subunit of the PI3K gene. We here assessed the prevalence of the coexistence of BRAF/NRAS and PIK3CA mutations in a series of melanoma samples.
A total of 245 tumor specimens (212 primary melanomas and 33 melanoma cell lines) was screened for mutations in BRAF, NRAS, and PIK3CA genes by automated direct sequencing.
Overall, 110 (44.9%) samples carried mutations in BRAF, 26 (10.6%) in NRAS, and 24 (9.8%) in PIK3CA. All identified PIK3CA mutations have been reported to induce PI3K activation; those detected in cultured melanomas were investigated for their interference with the antiproliferative activity of the BRAF-mutant inhibitor vemurafenib. A reduced suppression in cell growth was observed in treated cells carrying both BRAF and PIK3CA mutations as compared with those presenting a mutated BRAF only. Among the analysed melanomas, 12/245 (4.9%) samples presented the coexistence of PIK3CA and BRAF/NRAS mutations.
Our study further suggests that PIK3CA mutations account for a small fraction of PI3K pathway activation and have a limited impact in interfering with the BRAF/NRAS-driven growth in melanoma.
Melanoma; Mutation analysis; PIK3CA gene; Resistance to BRAF/MEK inhibitors