KRAS mutations in tongue squamous cell carcinoma

Background: p16^INK4a (p16) expression in tongue cancer (TC) is reportedly not associated with human papilloma virus (HPV). Mutations of KRAS in cancer cells are most frequently observed within codon 12. However, few reports have investigated the association between KRAS mutations and p16 status in TC.

Objectives: This study aimed to evaluate the influence of KRAS mutations on TC.

Methods: Clinical records and surgically resected specimens of 85?TC patients were analyzed. Tumor samples were analyzed for mutations of KRAS located within codons 12 and 13. p16 staining was performed and considered positive in cases with moderate to strong nuclear and cytoplasmic staining.

Results: Positive p16 staining was observed in 10 cases (11.8%). A KRAS mutation was detected in one case (1.2%). The case with KRAS mutation showed negative p16 staining. Despite being at an early stage, the patient died of lung metastasis at 43 months from initial treatment.

Conclusions and Significance: KRAS mutations are not associated with p16 expression in TC and may predict poor prognosis in TC patients. Further analysis of mutations in regions other than codons 12 and 13 of KRAS will be necessary to determine the relationship between KRAS mutations and prognosis of this disease.     

Early detection and treatment of ovarian cancer: shifting from early stage to minimal volume of disease based on a new model of carcinogenesis

The goal of ovarian cancer screening is to detect disease when confined to the ovary (stage I) and thereby prolong survival. We believe this is an elusive goal because most ovarian cancer, at its earliest recognizable stage, is probably not confined to the ovary. We propose a new model of ovarian carcinogenesis based on clinical, pathological, and molecular genetic studies that may enable more targeted screening and therapeutic intervention to be developed. The model divides ovarian cancer into 2 groups designated type I and type II. Type I tumors are slow growing, generally confined to the ovary at diagnosis and develop from well-established precursor lesions so-called borderline tumors. Type I tumors include low-grade micropapillary serous carcinoma, mucinous, endometrioid, and clear cell carcinomas. They are genetically stable and are characterized by mutations in a number of different genes including KRAS, BRAF, PTEN, and beta-catenin. Type II tumors are rapidly growing, highly aggressive neoplasms that lack well-defined precursor lesions; most are advanced stage at, or soon after, their inception. These include high-grade serous carcinoma, malignant mixed mesodermal tumors (carcinosarcomas), and undifferentiated carcinomas. The type II tumors are characterized by mutation of TP53 and a high level of genetic instability. Screening tests that focus on stage I disease may detect low-grade type I neoplasms but miss the more aggressive type II tumors, which account for most ovarian cancers. A more rational approach to early detection of ovarian cancer should focus on low volume rather than low stage of disease.     

Components of the phosphatidylserine endoplasmic reticulum to plasma membrane transport mechanism as targets for KRAS inhibition in pancreatic cancer

KRAS is mutated in 90% of human pancreatic ductal adenocarcinomas (PDACs). To function, KRAS must localize to the plasma membrane (PM) via a C-terminal membrane anchor that specifically engages phosphatidylserine (PtdSer). This anchor-binding specificity renders KRAS–PM localization and signaling capacity critically dependent on PM PtdSer content. We now show that the PtdSer lipid transport proteins, ORP5 and ORP8, which are essential for maintaining PM PtdSer levels and hence KRAS PM localization, are required for KRAS oncogenesis. Knockdown of either protein, separately or simultaneously, abrogated growth of KRAS-mutant but not KRAS–wild-type pancreatic cancer cell xenografts. ORP5 or ORP8 knockout also abrogated tumor growth in an immune-competent orthotopic pancreatic cancer mouse model. Analysis of human datasets revealed that all components of this PtdSer transport mechanism, including the PM-localized EFR3A-PI4KIIIα complex that generates phosphatidylinositol-4-phosphate (PI4P), and endoplasmic reticulum (ER)–localized SAC1 phosphatase that hydrolyzes counter transported PI4P, are significantly up-regulated in pancreatic tumors compared to normal tissue. Taken together, these results support targeting PI4KIIIα in KRAS-mutant cancers to deplete the PM-to-ER PI4P gradient, reducing PM PtdSer content. We therefore repurposed the US Food and Drug Administration–approved hepatitis C antiviral agent, simeprevir, as a PI4KIIIα inhibitor In a PDAC setting. Simeprevir potently mislocalized KRAS from the PM, reduced the clonogenic potential of pancreatic cancer cell lines in vitro, and abrogated the growth of KRAS-dependent tumors in vivo with enhanced efficacy when combined with MAPK and PI3K inhibitors. We conclude that the cellular ER-to-PM PtdSer transport mechanism is essential for KRAS PM localization and oncogenesis and is accessible to therapeutic intervention.

RAS proteins are small GTPases that switch between active GTP-bound and inactive GDP-bound states, regulating cell proliferation, differentiation, and apoptosis. RAS is regulated by guanine nucleotide exchange factors that promote GDP–GTP exchange, thereby activating RAS, and GTPase-activating proteins (GAPs), which stimulate intrinsic RAS GTPase activity to return it to its inactive state. Approximately 20% of human cancers express oncogenic RAS with mutations at residues 12, 13, or 61 (1), which prevent RASGAPs from stimulating GTP hydrolysis, rendering RAS constitutively active. The RAS isoforms, HRAS, NRAS, KRAS4A, and KRAS4B (hereafter referred to as KRAS), have near-identical G-domains, which are implicated in guanine nucleotide binding and effector interaction. However, they have different C termini and membrane anchors, which contribute to their differential signaling outputs (2). KRAS is the most-frequently mutated isoform in cancer and hence represents the major clinical concern, especially in pancreatic, colon, and non–small cell lung cancers (NSCLCs) in which mutant KRAS is expressed in ∼90%, ∼50%, and ∼25% of cases, respectively (3).RAS proteins must localize to the plasma membrane (PM) and organize into nanoclusters for biological activity (4–8), whereby RAS.GTP recruits its effectors to PM nanoclusters, leading to downstream pathway activation. KRAS interacts with the PM via its C-terminal membrane anchor that comprises a farnesyl-cysteine-methyl-ester and a polybasic domain (PBD) of six contiguous lysines (9–11). Together, the KRAS PBD sequence and prenyl group define a combinatorial code for lipid binding, resulting in a membrane anchor that specifically interacts with asymmetric species of phosphatidylserine (PtdSer) that contain one saturated and one desaturated acyl chain (8, 12–14). Since PtdSer binding specificity is hardwired into its anchor structure, KRAS–PM interactions are PtdSer dependent. KRAS that partitions into the cytosol following endocytosis is captured by PDEδ, which, upon interacting with ARL2, is released to the recycling endosome (RE) for forward transport back to the PM (15). Capture of KRAS by the RE is again PtdSer dependent; therefore, abrogating PtdSer delivery to the PM will reduce PM and RE PtdSer content, abrogating both KRAS PM binding and KRAS recycling back to the PM. In sum, KRAS–PM localization, nanoclustering, and signaling capacity are all exquisitely dependent on PM PtdSer levels.Previous attempts at preventing KRAS–PM localization to inhibit its function include the development of farnesyltransferase inhibitors (FTIs), which inhibit the first posttranslational processing step that generates the KRAS membrane anchor. FTIs were clinically unsuccessful since KRAS can alternatively be geranylgeranylated by geranylgeranyl transferase1 when cells are treated with FTIs, allowing for continued PM localization (2, 16, 17). We recently leveraged the dependence of KRAS on PM PtdSer to inhibit KRAS signaling by targeting the cellular machinery that actively maintains PM PtdSer levels (18). Genetic knockdown (KD) of ORP5 or ORP8, two lipid transporters that function at endoplasmic reticulum (ER)–PM membrane contact sites to transport PtdSer to the PM (Fig. 1), mislocalized KRAS from the PM and reduced nanoclustering of any remaining KRAS. Consequently, ORP5/8 KD decreased proliferation and anchorage-independent growth of multiple KRAS-dependent pancreatic cancer cell lines. In this study, we examine the effects of ORP5/8 genetic KD and knockout (KO) on tumor growth in vivo and provide compelling evidence that these proteins are essential for tumor maintenance in KRAS-dependent pancreatic cancer. ORP5/8 function by exchanging phosphoinositide-4-phosphate (PI4P) synthesized on the PM by PI4KIIIα for PtdSer synthesized in the ER (19, 20). We demonstrate both in vitro and in vivo that PI4KIIIα inhibitors can potently inhibit oncogenic KRAS function. One such inhibitor is simeprevir, a US Food and Drug Administration (FDA)–approved antiviral agent used for the treatment of hepatitis C, that may have potential for repurposing as a therapeutic for mutant KRAS-driven cancers.Open in a separate windowFig. 1.ORP5 and ORP8 transport PtdSer to the PM. ORP5 and ORP8 exchange ER PtdSer with PM PI4P. This is driven by a PI4P concentration gradient whereby PM PI4P levels are kept high by PI4KIIIα and low at the ER by SAC1P, which hydrolyzes PI4P. ORP, oxysterol-binding protein-related protein; PI4KIIIα, class III PI4 kinase alpha; and SAC1P, SAC1-like phosphatidylinositide phosphatase.     

Increased sensitivity of KRAS mutation detection by high‐resolution melting analysis of COLD‐PCR products

Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low‐level tumor specific KRAS mutations. Coamplification at lower denaturation temperature‐PCR (COLD‐PCR) is a new form of PCR that selectively amplifies mutation‐containing templates based on the lower melting temperature of mutant homoduplexes versus wild‐type homoduplexes. We have developed a fast COLD‐PCR and high‐resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD‐PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen^® KRAS mutation kit. The sensitivity was increased by 5‐ to 100‐fold for melting temperature decreasing mutations when using COLD‐PCR compared to standard PCR. Mutations, undetectable by the TheraScreen^® kit in clinical samples, were detected by COLD‐PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD‐PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

PTEN loss and KRAS activation leads to the formation of serrated adenomas and metastatic carcinoma in the mouse intestine

Mutation or loss of the genes PTEN and KRAS have been implicated in human colorectal cancer (CRC), and have been shown to co‐occur despite both playing a role in the PI3' kinase (PI3'K) pathway. We investigated the role of these genes in intestinal tumour progression in vivo, using genetically engineered mouse models, with the aim of generating more representative models of human CRC. Intestinal‐specific deletion of Pten and activation of an oncogenic allele of Kras was induced in wild‐type (WT) mice and mice with a predisposition to adenoma development (Apc^fl/+). The animals were euthanized when they became symptomatic of a high tumour burden. Histopathological examination of the tissues was carried out, and immunohistochemistry used to characterize signalling pathway activation. Mutation of Pten and Kras resulted in a significant life‐span reduction of mice predisposed to adenomas. Invasive adenocarcinoma was observed in these animals, with evidence of activation of the PI3'K pathway but no metastasis. However, mutation of Pten and Kras in WT animals not predisposed to adenomas led to perturbed homeostasis of the intestinal epithelium and the development of hyperplastic polyps, dysplastic sessile serrated adenomas and metastasizing adenocarcinomas with serrated features. These studies demonstrate synergism between Pten and Kras mutations in intestinal tumour progression, in an autochthonous and immunocompetent murine model, with potential application to preclinical drug testing. In particular, they show that Pten and Kras mutations alone predispose mice to the spectrum of serrated lesions that reflect the serrated pathway of CRC progression in humans. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk     

Prognostic significance of alterations in KRAS isoforms KRAS‐4A/4B and KRAS mutations in colorectal carcinoma

Somatic KRAS mutation is an early well‐known event in colorectal carcinogenesis but a complete understanding of RAS function and dysfunction in colorectal cancer is still to come. Our aim was to study the incidence of KRAS mutation; KRAS splice variants: KRAS4A and KRAS4B; and their relationships with various clinico‐pathological characteristics in colorectal cancer (CRC).In this study, 285 CRC cases were analysed for KRAS mutation by direct DNA sequencing followed by immunohistochemical analysis after validation with real‐time PCR assay, to study the protein expression of KRAS4A and ‐4B isoforms. KRAS gene mutations were seen in 80/285 CRCs (28.1%) and of the mutated cases, the majority of the mutations were seen in codon 12 (81.2%) as opposed to codon 13 (18.8%). CRCs with KRAS mutations were associated with a poor overall survival (p = 0 . 0009). Furthermore, KRAS mutations at codon 12 were associated with a poor overall survival of 64.4% at 5 years compared with a 5‐year overall survival of 75.8% and 78.2% with codon 13 mutation and absence of KRAS mutations, respectively (p = 0 . 0025). KRAS4A protein expression was predominantly seen in the cytoplasm, while KRAS4B protein was nuclear. KRAS4A overexpression was significantly associated with left colon, histology subtype of adenocarcinoma, p27kip1, and cleaved caspase3 expression. Interestingly, KRAS4A overexpression was associated with a better overall survival (p = 0 . 0053). On the other hand, KRAS4B overexpression (33.2%) was significantly associated with larger tumour size (p = 0 . 0234) and inversely correlated with p27kip1 protein (p = 0 . 0159). Both KRAS mutation and KRAS4A were independent prognostic markers in a multivariate analysis with age, gender, stage, differentiation, and MSI status. Our results highlight the differential role of KRAS isoforms in CRC, their utility as a prognostic biomarker, and underline the importance of KRAS alterations as a potential therapeutic target for CRC. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Specific mosaic KRAS mutations affecting codon 146 cause oculoectodermal syndrome and encephalocraniocutaneous lipomatosis

Oculoectodermal syndrome (OES) and encephalocraniocutaneous lipomatosis (ECCL) are rare disorders that share many common features, such as epibulbar dermoids, aplasia cutis congenita, pigmentary changes following Blaschko lines, bony tumor‐like lesions, and others. About 20 cases with OES and more than 50 patients with ECCL have been reported. Both diseases were proposed to represent mosaic disorders, but only very recently whole‐genome sequencing has led to the identification of somatic KRAS mutations, p.Leu19Phe and p.Gly13Asp, in affected tissue from two individuals with OES. Here we report the results of molecular genetic studies in three patients with OES and one with ECCL. In all four cases, Sanger sequencing of the KRAS gene in DNA from lesional tissue detected mutations affecting codon 146 (p.Ala146Val, p.Ala146Thr) at variable levels of mosaicism. Our findings thus corroborate the evidence of OES being a mosaic RASopathy and confirm the common etiology of OES and ECCL. KRAS codon 146 mutations, as well as the previously reported OES‐associated alterations, are known oncogenic KRAS mutations with distinct functional consequences. Considering the phenotype and genotype spectrum of mosaic RASopathies, these findings suggest that the wide phenotypic variability does not only depend on the tissue distribution but also on the specific genotype.     

Consistent mutation status within histologically heterogeneous lung cancer lesions

Mattsson J S M, Imgenberg‐Kreuz J, Edlund K, Botling J & Micke P
(2012) Histopathology  61, 744–748 Consistent mutation status within histologically heterogeneous lung cancer lesions Aims: Activating epidermal growth factor receptor (EGFR) and KRAS mutations characterize molecular subgroups of non‐small‐cell lung cancer (NSCLC) with a strong predictive value for response to EGFR inhibitor therapy. However, the temporal occurrence and clonal stability of these mutations during the course of cancer progression are debated. The aim of this study was to characterize the presence of EGFR and KRAS mutations in histologically different areas of primary NSCLC lesions. Methods and results: Formalin‐fixed paraffin‐embedded cancer specimens from six cases with EGFR mutations and five cases with KRAS mutations were selected from a pool of primary resected NSCLC patients. From each tumour, three morphologically distinct areas were manually microdissected and analysed for the presence of mutations. The results demonstrated consistent EGFR and KRAS mutation status in the different histological areas of all primary tumours. Conclusions: The results support the concept that activating EGFR and KRAS mutations are oncogenic events that are consistently present throughout the primary tumour independently of histological heterogeneity. Thus, for molecular diagnostics, any part of the tumour is likely to be representative for EGFR and KRAS mutation testing.     

Disproportionate representation of KRAS gene mutation in atypical adenomatous hyperplasia, but even distribution of EGFR gene mutation from preinvasive to invasive adenocarcinomas

In the resected lung, additional small lesions are occasionally found incidentally, and include the full spectrum of preinvasive to invasive lesions under the current putative schema of the sequential development of lung cancer. In this study, we examined EGFR and KRAS gene mutations in 119 synchronous pulmonary lesions, including 40 precursor lesions (atypical adenomatous hyperplasia, AAH), 26 carcinomas in situ (non-mucinous bronchioloalveolar carcinoma, BAC), 14 minimally invasive adenocarcinomas, 34 overt invasive adenocarcinomas, and five of other subtypes of cancer. Although the mutually exclusive nature of KRAS and EGFR gene mutations was maintained even in preinvasive lesions, the incidences of the lesions along the putative progression schema were quite different. The KRAS gene was mutated in 33% of AAH, 12% of carcinomas in situ, 8% of minimally invasive adenocarcinomas and 0% of well-differentiated adenocarcinomas, whereas the frequencies of EGFR mutation did not fluctuate greatly, at 25%, 51%, 36%, 86% and 67%, respectively. These results are consistent with the findings of a published gene-targeted mouse model; the mice expressing oncogenic KRAS developed AAH but not invasive adenocarcinoma, whereas a spectrum of preinvasive to invasive adenocarcinomas was observed in the mice expressing mutant EGFR. Taking these factors together, it is suggested that AAH could develop by either KRAS or EGFR gene mutation, but AAH harbouring a KRAS gene mutation might not progress further to an invasive cancer.