Original contributionAnalysis of KRAS, NRAS, PIK3CA, and BRAF mutational profile in poorly differentiated clusters of KRAS-mutated colon cancer☆,☆☆
Introduction
Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer-related death worldwide [1]. At present, the pathological TNM (pTNM) stage assessed in accordance to the International Union Against Cancer TNM [2] and to the American Joint Committee on Cancer [3] represents the most relevant prognostic factor for CRC [4] and determines its postsurgical treatment. However, in some cases, pTNM stage has no correlation with the biological and clinical behavior of CRC [5], [6]. Hence, the identification of additional factors, which may predict the clinical course of CRC with more accuracy and regardless of pTNM stage, has been a main research focus over the years.
Recently, ours and other groups showed that a novel grading system based on the number of clusters composed of at least 5 cancer cells and lacking a gland-like structure—so-called poorly differentiated clusters (PDCs)—represents a significant prognostic factor in patients with CRC, independently of pTNM stage [6], [7], [8], [9], [10], [11], [12], [13]. In addition, the presence of a high number of PDCs was significantly associated with higher metastatic risk of CRC [7], [8], [9], [10], [11], [12]. This association may be explained by the relationship between PDC and epithelial-mesenchymal transition (EMT) [14], [15], a process by which neoplastic cells lose epithelial properties and acquire the mesenchymal cell potential to migrate through the extracellular matrix and to metastatize. Indeed, PDCs show several characteristics of EMT phenotype, such as reversed pattern of MUC1 expression, loss of or aberrant E-cadherin expression, up-regulation of WNT/β-catenin signaling pathway, and matrix metalloproteinase expression [15], [16], [17]. Thus, they have invasive properties similar to those seen in cells forming tumor budding foci [15]. However, because they are composed of at least 5 neoplastic cells [7], PDCs are more easily identified with conventional hematoxylin and eosin stain [18].
In a recent study, we found that CRCs with high counts of PDCs display mutations in KRAS gene with significantly higher frequency than CRCs with absent/low number of PDCs [19]. We speculated that this association may depend on the ability of RAS oncogenic mutations to induce dedifferentiation of CRC cells and EMT process [20], [21]. However, whether KRAS mutations actually exert a role in driving PDC formation is still to be determined. In addition, the molecular signature of PDCs has not been analyzed thus far.
On this premise, aims of the present study were as follows: (a) to investigate the mutational status of genes involved in the RAS/MAPK and PI3K-PTEN-AKT signaling pathways in the PDCs of a series of colonic carcinomas (CCs) showing KRAS mutations and (b) to compare the mutational status observed in PDCs with that revealed in the corresponding main tumor.
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Materials and methods
Twenty-five consecutive KRAS-mutated surgically resected CCs with more than 10 PDCs at the invasive front of growth (PDC G3 tumors) were taken from the files of the Unit of Pathological Anatomy of Modena, Italy. All cases were anonymously collected, and all procedures were performed in accordance with the Helsinki Declaration. All relevant issues were discussed with the local ethics committee that established that no further ethical approval was needed to revise histology or to perform
Results
Clinical and pathological features of CCs included in the study are summarized in Table 1.
We observed KRAS mutations in the main component of the CCs at the following sites: at codon 12 in 18 (72%) cases (G12A in 4 cases, G12C in 1, G12D in 11, G12R in 1, and G12S in 1), at codon 13 in 2 (8%) (G13C in 1 case and G13D in 1 case), at codon 61 (Q61H) in 2 (8%), at codon 59 in 1 (4%), at codon 146 (A146T) in 1 (4%), and at codon 117 in 1 (4%). In addition to KRAS mutations, the main tumor component
Discussion
Because of their ability to block downstream intracellular signaling of epidermal growth factor receptor (EGFR), monoclonal antibodies against EGFR are currently used as a therapy for CRC [22]. However, their therapeutic efficacy strictly depends on the integrity of genes involved in the pathways downstream to EGFR, that is, RAS/MAPK and PI3K-PTEN-AKT pathways. Indeed, mutations in KRAS, NRAS, or PIK3CA genes may activate the pathway in the absence of extracellular stimuli [23]. For this
Acknowledgments
We wish to thank Marzia Gozzoli and Rosa Zaramella for their technical support in the molecular analysis.
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2022, HeliyonCitation Excerpt :Together with the most frequent activating mutations E542K, E545K, H1047L, and H1047R, several additional mutations were selected for screening based on their frequency in previously published studies and confirmed functional significance [36]. Using the criteria mentioned above, mutations R88Q [37], N345K [37], C420R [38], Q546K [3], M1043I [39], H1047Y [38] (Table 3) were tested in the present study, as they were previously identified in samples from patients with CRC. A mutation E545A [40], characterized as activating, was discarded from the present study, as it was detected in the PIK3CA pseudogene [41].
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2018, Human PathologyCitation Excerpt :First, primary CRCs with high number of PDCs have higher probability of having nodal metastases [14] and even nodal micrometastases [31]. Second, when PDCs and main tumor mass have different biomolecular profile, nodal metastases may have the same genetic mutations as those observed in PDCs [32], which suggest that PDCs may represent the source of metastatic disease [32]. Interestingly, we found PDCs in at least 1 LM in most of our patients.
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Competing interests: We have no conflict of interest to declare.
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Funding/Support: This study was supported by “Programma di Ricerca Regione Università 2010-2012,” Regione Emilia Romagna, Italy, titled “Use of biomarkers and gene expression profiles to identify cancer patients with different prognosis and sensitivity to molecular targeted agents.”
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These authors contributed equally to this work.