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.
We used a novel method based on allele-specific quantitative polymerase chain reaction (Intplex) for the analysis of circulating cell.free DNA (ccfDNA) to compare total ccfDNA and KRAS- or BRAF-mutated ccfDNA concentrations in blood samples from mice xenografted with the human SW620 colorectal cancer (CRC) cell line and from patients with CRC. Intplex enables single-copy detection of variant alleles down to a sensitivity of ≥0.005 mutant to wild-type ratio. The proportion of mutant allele corresponding to the percentage of tumor-derived ccfDNA was elevated in xenografted mice with KRAS homozygous mutation and varied highly from 0.13% to 68.7% in samples from mutation-positive CRC patients (n = 38). Mutant ccfDNA alleles were quantified in the plasma of every patient at stages II/III and IV with a mean of 8.4% (median, 8.4%) and 21.8% (median, 12.4%), respectively. Twelve of 38 (31.6%) and 5 of 38 (13.2%) samples showed a mutation load higher than 25%and 50%, respectively. This suggests that an important part of ccfDNA may originate from tumor cells. In addition, we observed that tumor-derived (mutant) ccfDNA was more fragmented than ccfDNA from normal tissues. This observation suggests that the form of tumor-derived and normal ccfDNA could differ. Our approach revealed that allelic dilution is much less pronounced than previously stated, considerably facilitating the noninvasive molecular analysis of tumors.
We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non–small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference–mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance.
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.
Activating oncogenic mutations of BRAF have been described in patients with gastrointestinal stromal tumor (GIST), but treatment of GIST with BRAF inhibitors and mechanisms of mediating the emergence of resistance in GIST have not been reported. Dabrafenib is a potent ATP-competitive inhibitor of BRAF kinase and is highly selective for mutant BRAF in kinase panel screening, cell lines, and xenografts. We report prolonged antitumor activity in the first patient with V600E BRAF-mutated GIST who was treated with a BRAF inhibitor. Whole exome sequencing performed in tumor tissue obtained at the time of progressive disease demonstrated a somatic gain-of-function PIK3CA mutation (H1047R) as well as a CDKN2A aberration, which may have contributed to eventual resistance to treatment.
Gastrointestinal stromal tumor; Dabrafenib; GSK2118436; BRAF mutation; BRAF inhibition; V600E
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
The linear, double-stranded DNA genome of vaccinia virus contains covalently closed hairpin termini. These hairpin termini comprise a terminal loop and an A+T-rich duplex stem that has 12 extrahelical bases. DeMasi et al. have shown previously that proteins present in infected cells and in virions form distinct complexes with the telomeric hairpins and that these interactions require the extrahelical bases. The vaccinia virus I6 protein was identified as the protein showing the greatest specificity and affinity for interaction with the viral hairpins (J. DeMasi, S. Du, D. Lennon, and P. Traktman, J. Virol. 75:10090-10105, 2001). To gain insight into the role of I6 in vivo, we generated eight recombinant viruses bearing altered alleles of I6 in which clusters of charged amino acids were changed to alanine residues. One allele (temperature-sensitive I6-12 [tsI6-12]) conferred a tight ts phenotype and was used to examine the stage(s) of the viral life cycle that was affected at the nonpermissive temperature. Gene expression, DNA replication, and genome resolution proceeded normally in this mutant. However, proteolytic processing of structural proteins, which accompanies virus maturation, was incomplete. Electron microscopic studies confirmed a severe block in morphogenesis in which immature, but no mature, virions were observed. Instead, aberrant spherical virions and large crystalloids were seen. When purified, these aberrant virions were found to have normal protein content but to be devoid of viral DNA. We propose that the binding of I6 to viral telomeres directs genome encapsidation into the virus particle.
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 (BRAFV600E) are found in 3% of human non-small cell lung cancers. Contrary to KRAS mutant tumors, BRAFV600E-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 BRAFV600E oncogene is unknown. Our results describe a lung cell population in neonates mice where expression of BRAFV600E leads to lung adenoma development. Importantly, expression of BRAFV600E concomitant with the loss of only a single-copy of Lkb1, overcomes senencence–like features of BRAFV600E-mutant adenomas leading malignization to carcinomas. These results posit LKB1 haploinsufficiency as a risk factor for tumor progression of BRAFV600E mutated lung adenomas in human cancer patients.
The development of oral squamous cell carcinoma (OSCC) is a complex, multistep process. To date, numerous oncogenes and tumor-suppressor genes have been implicated in oral carcinogenesis. Of particular interest in this regard are genes involved in cell cycling and apoptosis, such BRAF, KRAS, and PIK3CA genes.
Mutations of BRAF, KRAS, and PIK3CA were evaluated by direct genomic sequencing of exons 1 of KRAS, 11 and 15 of BRAF, and 9 and 20 of PIK3CA in OSCC specimens.
Both BRAF and KRAS mutations were detected with a mutation frequency of 2% (1/42). PIK3CA mutations were detected at 3% (1/35).
This is the first report implicating BRAF mutation in OSCC. Our study supports that mutations in the BRAF, KRAS, and PIK3CA genes make at least a minor contribution to OSCC tumorigenesis, and pathway-specific therapies targeting these two pathways should be considered for OSCC in a subset of patients with these mutations.
BRAF; KRAS; PIK3CA; oncogene mutation; hot-spot mutation; oral squamous cell carcinoma; OSCC
A majority of malignant melanomas harbor an oncogenic mutation in either BRAF or NRAS. If BRAF and NRAS transform melanoma cells by a similar mechanism, then additional genetic aberrations would be similar (or random). Alternatively, distinct mutation-associated changes would suggest the existence of unique cooperating requirements for each mutation group. We first analyzed a panel of 52 melanoma cell lines (n= 35, 11, 6 for BRAF*, NRAS*, and BRAF/NRASwt/wt respectively) by array-based comparative genomic hybridization for unique alterations that associate with each mutation subgroup. Subsequently, those DNA copy number changes that correlated with a mutation subgroup were used to predict the mutation status of an independent panel of 43 tumors (n=17, 13, 13 for BRAF*, NRAS*, and BRAF/NRASwt/wt respectively). BRAF mutant tumors were classified with a high rate of success (74.4%, P = 0.002), while NRAS mutants were not significantly distinguished from wild types (26/43, P = 0.12). Copy number gains of 7q32.1-36.3, 5p15.31, 8q21.11 and 8q24.11 were most strongly associated with BRAF* tumors and cell lines, as were losses of 11q24.2-24.3. BRAF* melanomas appear to be associated with a specific profile of DNA copy number aberrations that is distinct from those found in NRAS* and BRAFNRASwt/wt tumors. These findings suggest that while both BRAF and NRAS appear to function along the same signal transduction pathway, each may have different requirements for cooperating oncogenic events. The genetic loci that make up this profile may harbor therapeutic targets specific for tumors with BRAF mutations.
DNA Copy number analysis; BRAF; melanoma; pharmacogenomics
The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), gefitinib and erlotinib, are reversible competitive inhibitors of the tyrosine kinase domain of EGFR that bind to its adenosine-5′ triphosphate-binding site. Somatic activating mutations of the EGFR gene, increased gene copy number and certain clinical and pathological features have been associated with dramatic tumor responses and favorable clinical outcomes with these agents in patients with non-small-cell lung cancer (NSCLC). The specific types of activating mutations that confer sensitivity to EGFR TKIs are present in the tyrosine kinase (TK) domain of the EGFR gene. Exon 19 deletion mutations and the single-point substitution mutation L858R in exon 21 are the most frequent in NSCLC and are termed ‘classical’ mutations. The NSCLC tumors insensitive to EGFR TKIs include those driven by the KRAS and MET oncogenes. Most patients who initially respond to gefitinib and erlotinib eventually become resistant and experience progressive disease. The point mutation T790M accounts for about one half of these cases of acquired resistance. Various second-generation EGFR TKIs are currently being evaluated and may have the potential to overcome T790M-mediated resistance by virtue of their irreversible inhibition of the receptor TK domain.
epidermal growth factor receptor; mutation; non-small-cell lung cancer; tyrosine kinase inhibitor; tyrosine kinase
New technologies as well as concerted brute-force approaches have increased the content (number of genes) that can be characterized for genomic DNA alterations. Recent advances include the detection of activating point mutations in key kinase genes (BRAF, EGFR, PIK3CA) in multiple cancer types; preliminary insight into the entire repertoire of genes that can be mutated in cancer; the discovery of new oncogenes by high-resolution profiling of DNA copy number alterations; and the bioinformatic-driven discovery of oncogenic gene fusions. High-content promoter methylation detection systems have been used to discover additional methylated genes and have provided evidence for a stem cell origin for certain tumors. Some of these advances have had significant impact on the development and clinical testing of new therapeutics.
BRAF is the main effector of KRAS in the RAS-RAF-MAPK axis, a signaling pathway downstream of EGFR. The activation of this cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules. Recent studies in metastatic colorectal cancer found that an oncogenic activation of BRAF by a point mutation in exon 15 (V600E) could bypass the EGFR-initiated signaling cascade with the effect that patients bearing the mutant BRAF allele are not likely to benefit from EGFR-targeted therapies. We designed an allele-specific PCR and screened 65 salivary gland carcinoma (SGC) of the main histopathological types for the BRAF V600E mutation. All 65 SGC in this cohort (100%) presented the BRAF wildtype. In a previous study, we found a KRAS wildtype in 98.5% of SGC.
These findings imply that SGC rarely acquires mutations that result in a constitutive activation of the signaling cascade downstream of EGFR and this pleads in favor of further therapeutic trials with EGFR-targeting monoclonal
Oncogenic mutations in PIK3CA, which encodes the phosphoinositide-3-kinase (PI3K) catalytic subunit p110α, occur in ∼25% of human breast cancers. In this study, we report the development of a knock-in mouse model for breast cancer where the endogenous Pik3ca allele was modified to allow tissue-specific conditional expression of a frequently found Pik3caH1047R (Pik3cae20H1047R) mutant allele. We found that activation of the latent Pik3caH1047R allele resulted in breast tumors with multiple histological types. Whole-exome analysis of the Pik3caH1047R-driven mammary tumors identified multiple mutations, including Trp53 mutations that appeared spontaneously during the development of adenocarinoma and spindle cell tumors. Further, we used this model to test the efficacy of GDC-0941, a PI3K inhibitor, in clinical development, and showed that the tumors respond to PI3K inhibition.
Pik3ca; H1047R; knock-in; mammary gland; Trp53; exome sequencing
KRAS mutations occur frequently in colorectal cancers (CRC) and predict lack of response to anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapy. CRC BRAF mutations, most commonly at V600E, occur less than 10% of the time, and occur usually in KRAS wild-type tumors, and more frequently in microsatellite instable tumors. Concomitant KRAS and BRAF mutant CRCs are rare (occurring in 0.001%); BRAF mutations should not be routinely tested in patients with KRAS mutant tumors, unless the patients is participating in a clinical trial enriching for the presence of a KRAS or BRAF tumor. Clinical trials treating patients with either KRAS or BRAF mutant tumors should address eligibility of patients with concomitant KRAS and BRAF mutations.
Concomitant KRAS; BRAF
The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.
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.
Epidermal growth factor receptor (EGFR)-targeting therapeutics have shown efficacy in the treatment of colorectal cancer patients. Clinical studies have revealed that activating mutations in the KRAS protooncogene predict resistance to EGFR-targeted therapy. However, the causality between mutant KRAS and resistance to EGFR inhibition has so far not been demonstrated. Here, we show that deletion of the oncogenic KRAS allele from colorectal tumor cells resensitizes those cells to EGFR inhibitors. Resensitization was accompanied by an acquired dependency on the EGFR for maintaining basal extracellular signal-regulated kinase (ERK) activity. Deletion of oncogenic KRAS not only resensitized tumor cells to EGFR inhibition but also promoted EGF-induced NRAS activation, ERK and AKT phosphorylation, and c-FOS transcription. The poor responsiveness of mutant KRAS tumor cells to EGFR inhibition and activation was accompanied by a reduced capacity of these cells to bind and internalize EGF and by a failure to retain EGFR at the plasma membrane. Of 16 human colorectal tumors with activating mutations in KRAS, 15 displayed loss of basolateral EGFR localization. Plasma membrane localization of the EGFR could be restored in vitro by suppressing receptor endocytosis through Rho kinase inhibition. This caused an EGFR-dependent increase in basal and EGF-stimulated ERK phosphorylation but failed to restore tumor cell sensitivity to EGFR inhibition. Our results demonstrate a causal role for oncogenic KRAS in desensitizing tumor cells not only to EGFR inhibitors but also to EGF itself.
Mutations of KRAS are known to occur in periampullary and ampullary adenomas and carcinomas. However, nothing is known about NRAS, HRAS, BRAF, and PIK3CA mutations in these tumors. While oncogenic BRAF contributes to the tumorigenesis of both pancreatic ductal adenocarcinoma and intraductal papillary mucinous neoplasms/carcinomas (IPMN/IPMC), PIK3CA mutations were only detected in IPMN/IPMC. This study aimed to elucidate possible roles of BRAF and PIK3CA in the development of ampullary and periampullary adenomas and carcinomas.
Mutations of BRAF, NRAS, HRAS, KRAS, and PIK3CA were evaluated in seven adenomas, seven adenomas with carcinoma in situ, and 21 adenocarcinomas of the periampullary duodenal region and the ampulla of Vater. Exons 1 of KRAS; 2 and 3 of NRAS and HRAS; 5, 11, and 15 of BRAF; and 9 and 20 of PIK3CA were examined by direct genomic sequencing.
In total, we identified ten (28.6%) KRAS mutations in exon 1 (nine in codon 12 and one in codon 13), two missense mutations of BRAF (6%), one within exon 11 (G469A), and one V600E hot spot mutation in exon 15 of BRAF. BRAF mutations were present in two of five periampullary tumors. All mutations appear to be somatic since the same alterations were not detected in the corresponding normal tissues.
Our data provide evidence that oncogenic properties of KRAS and BRAF but not NRAS, HRAS, and PIK3CA contribute to the tumorigenesis of periampullary and ampullary tumors; BRAF mutations occur more frequently in periampullary than ampullary neoplasms.
Ampullary cancer; Periampullary; KRAS; BRAF; PIK3CA
To assess prognostic roles of various KRAS oncogene mutations in colorectal cancer, BRAF mutation status must be controlled for because BRAF mutation is associated with poor prognosis, and almost all BRAF mutants are present among KRAS-wild-type tumors. Taking into account experimental data supporting a greater oncogenic effect of codon 12 mutations compared to codon 13 mutations, we hypothesized that KRAS codon 12 mutated colorectal cancers might behave more aggressively than KRAS-wild-type tumors and codon 13 mutants.
Utilizing molecular pathological epidemiology database of 1261 rectal and colon cancers, we examined clinical outcome and tumor biomarkers of KRAS codon 12 and 13 mutations in 1075 BRAF-wild-type cancers (i.e., controlling for BRAF status). Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for potential confounders, including stage, PIK3CA mutations, microsatellite instability, CpG island methylator phenotype, and LINE-1 methylation.
Compared to patients with KRAS-wild-type/BRAF-wild-type cancers (N=635), those with KRAS codon 12 mutations (N=332) experienced significantly higher colorectal cancer-specific mortality [log-rank P=0.0001; multivariate HR=1.30; 95% confidence interval (CI), 1.02–1.67; P=0.037], whereas KRAS codon 13 mutated cases (N=108) were not significantly associated with prognosis. Among the seven most common KRAS mutations, c.35G>T (p.G12V; N=93) was associated with significantly higher colorectal cancer-specific mortality (log-rank P=0.0007; multivariate HR=2.00, 95% CI, 1.38–2.90, P=0.0003) compared to KRAS-wild-type/BRAF-wild-type cases.
KRAS codon 12 mutations (in particular, c.35G>T), but not codon 13 mutations, are associated with inferior survival in BRAF-wild-type colorectal cancer. Our data highlight the importance of accurate molecular characterization in colorectal cancer.
colon cancer; genetics; oncogenic; molecular diagnostics; personalized medicine; RAF; RAS
Tumor progression is driven by genetic mutations, but little is known about the environmental conditions that select for these mutations. Studying the transcriptomes of paired colorectal cancer cell lines that differed only in the mutational status of their KRAS or BRAF genes, we found that GLUT1, encoding glucose transporter-1, was one of three genes consistently upregulated in cells with KRAS or BRAF mutations. The mutant cells exhibited enhanced glucose uptake and glycolysis and survived in low glucose conditions, phenotypes that all required GLUT1 expression. In contrast, when cells with wild-type KRAS alleles were subjected to a low glucose environment, very few cells survived. Most surviving cells expressed high levels of GLUT1 and 4% of these survivors had acquired new KRAS mutations. The glycolysis inhibitor, 3-bromopyruvate preferentially suppressed the growth of cells with KRAS or BRAF mutations. Together, these data suggest that glucose deprivation can drive the acquisition of KRAS pathway mutations in human tumors.
Vascular endothelial growth factor-2 (VEGFR-2 or KDR) is a known endothelial target also expressed in NSCLC tumor cells. We investigated the association between alterations in the KDR gene and clinical outcome in patients with resected NSCLC (n=248). KDR copy number gains (CNGs), measured by quantitative PCR and fluorescence in situ hybridization, were detected in 32% of tumors and associated with significantly higher KDR protein and higher microvessel density than tumors without CNGs. KDR CNGs were also associated with significantly increased risk of death (HR=5.16; P=0.003) in patients receiving adjuvant platinum-based chemotherapy, but no differences were observed in patients not receiving adjuvant therapy. To investigate potential mechanisms for these associations we assessed NSCLC cell lines and found that KDR CNGs were significantly associated with in vitro resistance to platinum chemotherapy as well as increased levels of nuclear HIF-1α in both NSCLC tumor specimens and cell lines. Furthermore, KDR knockdown experiments using small interfering RNA reduced platinum resistance, cell migration, and HIF-1α levels in cells bearing KDR CNGs, providing evidence for direct involvement of KDR. No KDR mutations were detected in exons 7, 11 and 21 by PCR-based sequencing; however, two variant SNP genotypes were associated with favorable overall survival in adenocarcinoma patients. Our findings suggest that tumor cell KDR CNGs may promote a more malignant phenotype including increased chemoresistance, angiogenesis, and HIF-1α levels, and that KDR CNGs may be a useful biomarker for identifying patients at high risk for recurrence after adjuvant therapy, a group that may benefit from VEGFR-2 blockade.
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.
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.
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).
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.
Documented sensitivity of melanoma cells to PLX4720, a selective BRAFV600E inhibitor, is based on the presence of mutant BRAFV600E alone, while wt-BRAF or mutated KRAS result in cell proliferation. In colon cancer appearance of oncogenic alterations is complex , since BRAF, like KRAS mutations, tend to co-exist with those in PIK3CA and mutated PI3K has been shown to interfere with the successful application of MEK inhibitors. When PLX4720 was used to treat colon tumours, results were not encouraging and herein we attempt to understand the cause of this recorded resistance and discover rational therapeutic combinations to resensitize oncogene driven tumours to apoptosis. Treatment of two genetically different BRAFV600E mutant colon cancer cell lines with PLX4720 conferred complete resistance to cell death. Even though p-MAPK/ ERK kinase (MEK) suppression was achieved, TRAIL, an apoptosis inducing agent, was used synergistically in order to achieve cell death by apoptosis in RKOBRAFV600E/PIK3CAH1047 cells. In contrast, for the same level of apoptosis in HT29BRAFV600E/PIK3CAP449T cells, TRAIL was combined with 17-AAG, an Hsp90 inhibitor. For cells where PLX4720 was completely ineffective, 17-AAG was alternatively used to target mutant BRAFV600E. TRAIL dependence on the constitutive activation of BRAFV600E is emphasised through the overexpression of BRAFV600E in the permissive genetic background of colon adenocarcinoma Caco-2 cells. Pharmacological suppression of the PI3K pathway further enhances the synergistic effect between TRAIL and PLX4720 in RKO cells, indicating the presence of PIK3CAMT as the inhibitory factor. Another rational combination includes 17-AAG synergism with TRAIL in a BRAFV600E mutant dependent manner to commit cells to apoptosis, through DR5 and the amplification of the apoptotic pathway. We have successfully utilised combinations of two chemically unrelated BRAFV600E inhibitors in combination with TRAIL in a BRAFV600E mutated background and provided insight for new anti-cancer strategies where the activated PI3KCA mutation oncogene should be suppressed.
CTEN/TNS4 is an oncogene in colorectal cancer (CRC) which enhances cell motility although the mechanism of Cten regulation is unknown. We found an association between high Cten expression and KRAS/BRAF mutation in a series of CRC cell lines (p = 0.03) and hypothesised that Kras may regulate Cten. To test this, Kras was knocked-down (using small interfering (si)RNA) in CRC cell lines SW620 and DLD1 (high Cten expressors and mutant for KRAS). In each cell line, Kras knockdown was mirrored by down-regulation of Cten Since Kras signals through Braf, we tested the effect of Kras knockdown in CRC cell line Colo205 (which shows high Cten expression and is mutant for BRAF but wild type for KRAS). Cten levels were unaffected by Kras knockdown whilst Braf knockdown resulted in reduced Cten expression suggesting that Kras signals via Braf to regulate Cten. Quantification of Cten mRNA and protein analysis following proteasome inhibition suggested that regulation was of Cten transcription. Kras knockdown inhibited cell motility. To test whether this could be mediated through Cten, SW620 cells were co-transfected with Kras specific siRNAs and a Cten expression vector. Restoring Cten expression was able to restore cell motility despite Kras knockdown (transwell migration and wounding assay, p<0.001 for both). Since KRAS is mutated in many cancers, we investigated whether this relationship could be demonstrated in other tumour models. The experiments were repeated in the pancreatic cancer cell lines Colo357 & PSN-1(both high Cten expressors and mutant for KRAS). In both cell lines, Kras was shown to regulate Cten and forced expression of Cten was able to rescue loss of cell motility following Kras knockdown in PSN-1 (transwell migration assay, p<0.001). We conclude that, in the colon and pancreas, Cten is a downstream target of Kras and may be a mechanism through which Kras regulates of cell motility.