KRAS mutation and immunohistochemical profile in intraductal papillary neoplasm of the intrahepatic bile ducts

Intraductal papillary neoplasm of bile duct (IPNB) is characterized by a spectrum of diseases ranging from low-grade intraepithelial neoplasia to invasive carcinoma. In the present study, we aimed to investigate immunophenotypic features and KRAS mutations in relation to pathological subtypes and grades in Chinese patients with IPNBs. A total of 46 patients with IPNBs and 11 invasive adenocarcinomas arising in IPNBs (invasive IPNBs) were enrolled and clinicopathological data were analyzed. It was found that CK7 was expressed in 42 of the 46 neoplastic lesions. HepPar1 was expressed in 11 of the 46 noninvasive IPNBs, but not in invasive IPNBs. Additionally, CK19 was frequently expressed in both noninvasive IPNBs and invasive IPNBs. The intestinal-type IPNBs had a significantly higher percentage of MUC2 expression relative to the pancreaticobiliary (P = 0.015) and gastric-type IPNBs (P < 0.001). High-grade IPNBs and invasive IPNBs showed increased expression of cyclin D1, Ki-67, p53, mCEA, and CA19-9. The rate of KRAS mutation was significantly higher in high-grade IPNBs (P = 0.001) and invasive IPNBs (P = 0.006) than that in low- to intermediate-grade IPNBs. Additionally, KRAS mutation was significantly associated with tumor size, and Ki-67 expression. In conclusion, the expression of cyclin D, Ki-67, p53, mCEA and CA19-9 and KRAS mutation status are significantly correlated with histological grades of IPNBs.     

KRASP1于KRAS基因突变检测中的潜在影响

目的 论证KRAS假基因对KRAS基因突变检测结果存在潜在性影响.方法 检测5个确证结直肠癌的肿瘤组织序列,并以序列比对的方式对KRASP1与KRAS基因进行同源性比较与分析.结果 KRASP1与KRAS存在高度同源性.在KRAS基因突变检测的实验中,KRASP1的存在对扩增类反应产生一定的不良影响,影响程度与检测方法的类型以及实验的设计有关.结论 KRASP1的产生与其原始序列可能是来自于同期的KRAS基因的一个转录本,并随着时间的推移逐渐差异化,但仍保留着大部分的相似性,并有进一步的分析所证实.KRASP1对KRAS基因突变检测结果存在潜在影响,如何避免同源性干扰将取决于分子诊断方法的实验设计.     

新疆维吾尔族、汉族结直肠癌患者中KRAS及BRAF基因突变差异及其与预后的关系研究

目的探讨新疆维吾尔族(维族)、汉族结直肠癌伴肝转移患者中KRAS基因及BRAF基因突变差异及其与临床病理特征和预后的关系。方法选择新疆医科大学第一附属医院2005年1月-2010年12月收治的100例新疆维、汉族结直肠癌伴肝转移患者,应用PCR及Pyrosequencing测序法检测分析KRAS基因及BRAF基因突变情况,分析其与临床病理特征和预后的关系。结果维、汉族结直肠癌伴肝转移患者的KRAS与BRAF基因突变率分别为28%(28/100)与5%(5/100)。50例汉族患者中KRAS基因突变18例,BRAF基因突变2例;50例维吾尔族患者中KRAS基因突变10例,BRAF基因突变3例。原发肿瘤组织学分级、区域淋巴结转移、脉管瘤栓、肝外侵犯或转移、KRAS和BRAF为结直肠癌的预后影响因素(P<0.05);多因素分析显示,脉管瘤栓(P=0.035)、KRAS基因(P=0.001)为结直肠癌的独立的预后因素。结论在新疆维、汉族结直肠癌伴肝转移患者中,脉管瘤栓及KRAS基因具有主要的预后价值。积极检测KRAS基因,可望延长患者生存期。     

KRAS (but not BRAF) mutations in ovarian serous borderline tumour are associated with recurrent low‐grade serous carcinoma

BRAF and KRAS mutations in ovarian serous borderline tumours (OSBTs) and ovarian low‐grade serous carcinomas (LGSCs) have been previously described. However, whether those OSBTs would progress to LGSCs or whether those LGSCs were developed from OSBT precursors in previous studies is unknown. Therefore, we assessed KRAS and BRAF mutations in tumour samples from 23 recurrent LGSC patients with a known initial diagnosis of OSBT. Paraffin blocks from both OSBT and LGSC samples were available for five patients, and either OSBTs or LGSCs were available for another 18 patients. Tumour cells from paraffin‐embedded tissues were dissected out for mutation analysis by conventional polymerase chain reaction (PCR) and Sanger sequencing. Tumours that appeared to have wild‐type KRAS by conventional PCR–Sanger sequencing were further analysed by full COLD (co‐amplification at lower denaturation temperature)‐PCR and deep sequencing. Full COLD‐PCR was able to enrich the amplification of mutated alleles. Deep sequencing was performed with the Ion Torrent personal genome machine (PGM). By conventional PCR–Sanger sequencing, BRAF mutation was detected only in one patient and KRAS mutations were detected in ten patients. Full COLD‐PCR deep sequencing detected low‐abundance KRAS mutations in eight additional patients. Three of the five patients with both OSBT and LGSC samples available had the same KRAS mutations detected in both OSBT and LGSC samples. The remaining two patients had only KRAS mutations detected in their LGSC samples. For patients with either OSBT or LGSC samples available, KRAS mutations were detected in seven OSBT samples and six LGSC samples. Surprisingly, patients with the KRAS G12V mutation have shorter survival times. In summary, KRAS mutations are very common in recurrent LGSC, while BRAF mutations are rare. The findings indicate that recurrent LGSC can arise from proliferation of OSBT tumour cells with or without detectable KRAS mutations. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Activating HRAS mutation in a case of inverted ductal papilloma of the salivary gland

Inverted ductal papilloma (IDP) is one of the least common benign papillary/cystic neoplasms of the salivary duct system, being characterized histologically by florid hyperplasia of duct-type epithelial cells into a cystic lumen near the orifice with occasional endophytic growth of the surface squamous epithelium along the terminus of the affected excretory duct. Given its rarity, the exact etiology of IDP remains to be defined. We herein present the first evidence of oncogenic HRAS mutation in a case of oral IDP. This finding, together with the frequent and specific BRAF mutations in sialadenoma papilliferum reported in the recent literature, supports an active role of the MAP-kinase cascade in the pathogenesis of benign papillary neoplasms of terminal duct origin.     

Sensitive Detection of KRAS Mutations Using Enhanced‐ice‐COLD‐PCR Mutation Enrichment and Direct Sequence Identification

A number of methods allowing the detection of low levels of KRAS mutations have been developed in the last years. However, although these methods have become increasingly sensitive, they can rarely identify the mutated base directly without prior knowledge on the mutated base and are often incompatible with a sequencing‐based read‐out desirable in clinical practice. Here, we present a modified version of the ice‐COLD‐PCR assay called Enhanced‐ice‐COLD‐PCR (E‐ice‐COLD‐PCR) for KRAS mutation detection and identification, which allows the enrichment of the six most frequent KRAS mutations. The method is based on a nonextendable chemically modified blocker sequence, complementary to the wild‐type (WT) sequence leading to the enrichment of mutated sequences. This assay permits the reliable detection of down to 0.1% mutated sequences in a WT background. A single genotyping assay of the amplification product by pyrosequencing directly following the E‐ice‐COLD‐PCR is performed to identify the mutated base. This developed two‐step method is rapid and cost‐effective, and requires only a small amount of starting material permitting the sensitive detection and sequence identification of KRAS mutations within 3 hr. This method is applied in the current study to clinical colorectal cancer samples and enables detection of mutations in samples, which appear as WT using standard detection technologies.     

Oncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4B^G12D), and nontransforming cytosolic double mutant (BirA-KRAS4B^G12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRAS^G12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.

A total of 60,430 new cases of pancreatic cancer were estimated for 2021, and the 5-y relative survival rate has consistently remained below 11% (1). About 85% of these pancreatic cancer tumors are pancreatic ductal adenocarcinoma (PDAC) (2). Poor outcomes of PDAC cases result from late diagnoses leading to unresectable and heterogeneous tumors as well as ineffective therapies, which only prolong survival on the order of months (3–5). Mutations in the KRAS proto-oncogene are present in over 90% of PDAC cases and are associated with a poor prognosis (6). Furthermore, mice expressing mutant KRAS in the pancreas develop precursor lesions, which sporadically progress into frank PDAC. This progression is accelerated when combined with other mutations or deletion of tumor suppressor genes (7–11). Additionally, independent studies have shown that the maintenance of murine PDAC cells require KRAS (12–14).As a RAS GTPase, KRAS acts as a molecular switch at the plasma membrane that relays growth factor signaling from receptor tyrosine kinases to downstream pathways such as RAF/MEK and PI3K/AKT (15). GTP binding alters the conformation of the KRAS G domain, thereby creating binding sites for downstream effectors to trigger enzymatic cascades that promote cell transformation (16–19). Intrinsically, KRAS slowly hydrolyzes GTP into GDP to halt signaling; however, GTPase activating proteins (GAPs) such as neurofibromin 1(NF1) catalyze this process (20). In contrast, guanine nucleotide exchange factors, such as son of sevenless homolog 1 (SOS1), catalyze the exchange of GTP for bound GDP. In most PDAC cases, KRAS is mutated at the 12th residue located in the G domain from glycine to either a valine (G12V), or more commonly, aspartate (G12D). These mutations sterically prevent the “arginine finger domain” of GAPs from entering the GTPase site, thereby blocking extrinsic allosteric GTPase activation and stabilizing RAS-GTP (21, 22). Activating mutations in KRAS constitutively trigger RAF/MEK and PI3K/AKT pathways leading to increased cell proliferation as well as other prooncogenic behaviors (15). KRAS signaling not only relies on the G domain but also the C-terminal hypervariable domain (HVR), which is required to stabilize KRAS on membranes where signaling is most efficient (23–26). Independent studies suggest that specific biochemical and cellular consequences of KRAS activation are attributed to the unique properties of the HVR of the predominant splice form KRAS4B, namely the polybasic domain and the lipid anchor (27–30). Localization of RAS proteins to the plasma membrane requires the prenylation of the CAAX motif (23). Additionally, for KRAS4B, the hypervariable region contains a highly polybasic domain consisting of several consecutive lysines, which can interact with the negative charges on the polar heads of phospholipids and stabilize protein interactions (31). Structural and biochemical characterization of the HVR and G domain has contributed to a better understanding of the signaling outputs of KRAS and led to KRAS-targeting strategies.Various approaches to inhibit KRAS include direct inhibition, expression interference, mislocalization, and targeting of downstream effectors (32). Thus far, direct inhibitors against KRAS have only successfully targeted the G12C mutant, which comprises 2.9% of KRAS mutant PDAC (21, 33). For other KRAS mutants, targeting downstream effectors of KRAS in pancreatic cancer remains an alternative approach. Unfortunately, dual inhibition of MEK and AKT pathways was ineffective in PDAC patients (34). Difficulty in targeting KRAS due to adaptive resistance and feedback regulation motivates a better understanding of KRAS biology (35). For example, although PDAC typically features a mutant KRAS, there may be a role for its wild-type (WT) counterpart as well as WT RAS paralogs (HRAS and NRAS), which are GAP sensitive and subject to signaling feedback. While oncogenic KRAS has been shown to activate WT HRAS and NRAS via allosteric stimulation of SOS1 (36), WT KRAS has been proposed to be a tumor suppressor in some KRAS mutant cancers based on the commonly observed mutant-specific allele imbalance that occurs throughout tumor progression (37). Additionally, the reintroduction of WT KRAS abolished tumor T cell acute lymphoblastic leukemia development and impaired tumor growth in KRAS mutant lung cancer cells in vivo (37–39). The discovery of novel KRAS protein interactors involved in downstream signaling or feedback and compensatory pathways may elucidate why inhibition of downstream pathways have had limited clinical impact in PDAC. Here, we perform proximity labeling experiments by expressing a fusion of BirA^R118G biotin ligase and KRAS in PDAC cells, which, in the presence of high concentrations of biotin, generates reactive biotinoyl-AMP that labels lysines of nearby proteins, such as interactors of its fusion partner KRAS (40–42). The biotinylated interactor proteins can be isolated by streptavidin pulldown and analyzed by proteomics to identify novel protein interactors (43–45). Because covalent labeling occurs in living cells, enzymatic labeling may potentially identify transient interactors and protein complexes.Two recent studies used proximity-dependent biotin identification (BioID) labeling methods to identify KRAS interactors in 293T and colon cancer cells (46, 47). These studies uncovered and validated the functional relevance of PIP5KA1 and mTORC2 in PDAC cells. However, BirA-KRAS screens in PDAC models have not yet been performed. Since the tumor context may determine protein expression and relevant interactions, we sought to perform a BirA-KRAS screen in PDAC cells. We hypothesize that proximity labeling with BioID presents a means for identifying new mutant KRAS-specific interactions in PDAC, which may unveil new insights into therapeutic design for this malignancy.     

MiR-30c通过靶向KRAS抑制肝癌细胞系生长侵袭能力的体外研究

目的 探讨MiR-30c对肝癌细胞生长和侵袭的影响.方法 采用脂质体Lipofectamine 2000将MiR-30c模拟物组和MiR-30c模拟物组阴性对照转染至肝癌细胞Huh7,qRT-PCR检测转染效果,细胞免疫荧光、Western blot检测转染后目的蛋白的表达,Transwell、划痕实验检测细胞迁移侵袭能力、MTT法检测细胞增殖活性,流式细胞术检测细胞周期分布的改变.结果 成功构建稳定过表达MiR-30c的肝癌细胞亚系Huh7-MiR30c,荧光定量PCR结果显示MiR-30c表达量明显上调,过表达MiR-30c后Huh7细胞的侵袭和增殖能力均受到明显抑制;抑制MiR-30c能明显抑制KRAS蛋白和减低p-ERK蛋白表达水平,对ERK蛋白水平无明显影响.结论 抑制MiR-30c表达可能通过调控RAS/MAPK/ERK通路抑制肝癌细胞Huh7增殖和侵袭.     

Correlations of morphology and molecular alterations in traditional serrated adenoma

Traditional serrated adenoma was first reported by Longacre and Fenoglio-Presier in 1990. Their initial study described main features of this lesion, but the consensus diagnostic criteria were not widely adopted until recently. Traditional serrated adenoma presents with grossly protuberant configuration and pinecone-like appearance upon endoscopy. Histologically, it is characterized by ectopic crypt formation, slit-like serration, eosinophilic cytoplasm and pencillate nuclei. Although much is now known about the morphology and molecular changes, the mechanisms underlying the morphological alterations are still not fully understood. Furthermore, the origin of traditional serrated adenoma is not completely known. We review recent studies of the traditional serrated adenoma and provide an overview on current understanding of this rare entity.