ReviewTargeting KRAS for diagnosis, prognosis, and treatment of pancreatic cancer: Hopes and realities
Introduction
Pancreatic cancer remains one of the most deadly types of cancer: the 5-year survival rate after diagnosis is <3.5% [1]. The sole curative treatment is surgical resection, which is, unfortunately, applicable in no more than 15% of cases. Single-agent gemcitabine, FOLFIRINOX, and nab-paclitaxel–gemcitabine protocols, although not dramatically improving survival beyond 11 months, have demonstrated significant clinical benefits and have become the standard chemotherapy for advanced and metastatic pancreatic ductal adenocarcinoma (PDAC) [1], [2], [3], [4], [5]. Our understanding of pancreatic carcinoma has increased greatly from experimental models of genetic/epigenetic alterations and molecular expression, from analyses of pre-cancerous and cancerous tissues, from the use of molecular amplification, and from large-scale transcriptome analyses.
INK4a/ARF, TP53, DPC4/Smad4 tumour-suppressor pathways are genetically inactivated in the majority of pancreatic carcinomas (associated with losses of heterozygosity of, respectively, 9p21, 17p, and 18q), whereas oncogenic KRAS is activated [6], [7], [8]. At a late stage of tumour development, there is an increase in telomerase activity, over-expression of growth factors and/or their receptors (Epidermal growth factor [EGF], nerve growth factor, gastrin), and of pro-angiogenic factors (Vascular endothelial growth factor [VEGF], Fibroblast growth factor [FGF], Platelet derived growth factor [PDGF]), and increased invasive factors (metalloproteinases, tissue plasminogen activators). The microenvironment plays also a key role in the invasive and metastatic process of pancreatic carcinoma, with a strong relationship between cancerous cells and pancreatic stellate cells, as well as the extracellular matrix. This microenvironment strongly participates in tumour fibrosis, hypoxia, and hypovascularisation, resulting in inaccessibility of drugs [8], [9]. However, the activating point mutation of the KRAS oncogene on codon 12 (Exon 2) remains the major event (70–95% of PDAC cases: 71% of pancreatic cancer specimens in the COSMICS database harbour KRAS mutations) [10].
This mutation occurs early in pancreatic carcinogenesis as attested by its presence in common pre-neoplastic lesions, such as PanIN (pancreatic intraductal neoplasia) and intraductal mucinous papillary pancreatic neoplasia [8], [12]. The single-nucleotide mutation induces a replacement of the GGT sequence (encoding for glycine) by the GAT sequence (aspartic acid – G12D – c35 G > A), GTT (valine – G12V – c35 G > T), CGT (arginine – G12R – C34 G > C), or GCT (alanine – G12A – c35 G > C). A point mutation can also occur, but less frequently, on codon 13 (G13D) or 61 (Q61L or Q61H) [7], [8], [10].
The KRAS gene encodes for the protein P21 RAS, which is a small Guanosine triphosphatase (GTPase) that acts as a molecular switch by coupling cell-membrane growth factor receptors to intracellular signalling pathways and transcription factors to control various cellular processes. P21 RAS is localised in the inner surface cell-membrane and interacts with more than 20 effector proteins. The RAS protein requires membrane association for its biological activity. This membrane association results in modification of lipids by a farnesyl isoprenoid. The point mutation of KRAS impairs intrinsic GTPase activity of RAS and prevents GTPase activating proteins to promote conversion of Guanosine triphosphate (GTP) (active) to Guanosine diphosphate (GDP) (inactive). P21 RAS is thus permanently bound to GTP and activates downstream signalling pathways, such as phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) or RAF/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinases (independently of upstream growth factor receptor activation) [11]. Following this activation, nuclear transcription factors are also activated (such as ELK/JUN/MYC) with stimulation of cell proliferation, transformation, adhesion, and survival (posttranslational regulation and signalling of RAS are summarised in Fig. 1) [11], [12].
Studying KRAS mutations has improved our understanding of the processes involved in transformation, uncontrolled proliferation, and invasion of pancreatic cancer cells, as well as the development of transgenic animal models. Nowadays, these models are at the centre of all pathophysiological studies, especially since the creation of the KC KRASG12D mouse model [13]. These mice reliably reproduce the human pathology with the presence of various PanIN lesions in 100% of animals. This confirms the initiating role of a KRAS mutation in pancreatic carcinogenesis, but also the importance of multistep genetic mutations in pancreatic carcinogenesis. Although KC mice develop PDAC in only 10% of cases, when inactivation of TP53, Smad4 and Ink4A/Arf is added after crossbreeding of transgenic mice, significant cancerous lesions are obtained [14], [15], [16], [17]. By using these models, the role of the signalling pathway, stroma, and microenvironment can be studied, as well as the co-factors, such as inflammation [18], [19]. These murine models offer also a wide range of applications in the domain of diagnosis and therapy. Biomarkers can be tested in blood and imaging such as positron emission tomography scan, computed tomography or ultrasound can be applied [13], [20], [21], [22], [23], [24]. These models displayed not only primary tumours with an organised tumour microenvironment but also metastasis. All these lesions cannot be reproduced by means of transplantable models. In consequence, pre-clinical test and proof of concept can be conducted for future antitumour agents [22], [23].
Activation of the RAS pathway is also important in the carcinogenesis of various types of tumours [25], [26], [27], [28]. Because the RAS-mediated signalling pathway links downstream growth factors (especially epidermal growth factor receptor [EGFR]), studies conducted in colon and lung cancer have found that the KRAS mutation and EGFR expression can be indicators for a poor prognosis [25], [26], [27]. Moreover, the absence of a KRAS mutation is correlated with the therapeutic response of colon carcinoma to anti-EGFR antibodies and the therapeutic response of lung cancer to anti-EGFR/HER molecules, with or without chemotherapy [28], [29], [30]. Taking into account the high frequency of KRAS mutations in PDAC and the major role of this mutation in proliferation and progression of pancreatic cancer, numerous studies have been conducted over the past 20 years to investigate if targeting the KRAS mutation can be applied clinically to either diagnose or provide a prognosis or treatment for PDAC. The aim of this review was to synthesise all the published ‘positive’ and ‘negative’ studies in this field to gain a straightforward picture of the main issues in terms of the possible applications of targeting the KRAS mutation in current clinical practice.
Section snippets
KRAS mutation assay to improve positive and differential diagnosis of PDAC
Nowadays due to its invasiveness, ERCP is focused on therapeutic approaches of biliary and pancreatic disease. More than 15 years of endoscopic ultrasound (EUS) experience now allows safe guided fine-needle aspiration biopsies (FNA) of solid pancreatic lesions for cytopathological analysis [31]. EUS-guided FNA (EUS-FNA) is, thus, now an effective technique to diagnose and assess the staging of PDAC [31]. However, its accuracy to diagnose malignancy varies widely, with a sensitivity ranging from
KRAS mutation assay to assess a prognosis for PDAC
Several groups, including ours, have investigated whether the presence or not of a KRAS mutation can influence the prognosis of PDAC, especially in advanced tumours that are only investigated using EUS-FNA. All studies that have included ≥50 patients (range: 50–272) are summarised in Table 2. The numbers of KRAS mutations found in samples (biopsies or resected specimens) varied between 41 and 75%, and last, populations included either resected or non-resectable (locally advanced and/or
Therapeutic approaches to targeting KRAS
Several strategies have been proposed for targeting the RAS protein. The main strategies and molecular targets are shown in Fig. 1. The RAS-membrane association is induced by farnesyl transferase, which attaches a C15 farnesyl isoprenoid lipid to the cysteine of the RAS-terminal CAXX motif; however, the use of farnesyl transferase inhibitors (such as tipifarnib or lonafarnib) showed no clinical benefit [70], [71], [72]. This is because several RAS isoforms, such as NRAS, do not rely on
Conclusion
The oncogene KRAS point mutation is the major molecular event in PDAC. Nowadays, qPCR technology enables reliable assessment of KRAS mutations, both from tissues and from FNA biopsies. Numerous studies report that the cytopathology and KRAS mutation assay used on EUS-FNA material can improve the positive and differential diagnosis of PDAC. In this way, benign versus malignant solid masses can be more easily distinguished, thus avoiding unnecessary pancreatectomies. Conversely, in cases of
Conflict of interest statement
The authors have no conflict of interest to declare.
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