The Role of KRAS in Endometrial Cancer: A Mini-Review

Current Staging

EC staging consensus keeps with the 2009 International Federation of Gynaecology and Obstetrics (FIGO) revised classification. Revised FIGO staging defines four stages (I-IV) following radical surgical resection (7). Stage I refers to a uterus-confined tumour, stage II to involvement of the cervix, stage III to adnexal or lymph node involvement, and stage IV to the presence of any metastatic deposits outside the pelvis (7).

Treatment Stratification

To-date, the stratification of treatment options relies on the disease stage; this includes certain histopathological features which are integrated into the FIGO staging. The cornerstone of EC treatment is to offer (radical) excision of the tumour; this includes total hysterectomy with/without bilateral salpingo-oophorectomy, and if indicated, systematic pelvic/para-aortic lymphadenectomy. Besides its role in treatment, radical surgery is also the basis for staging and stratifying patients for further adjuvant treatment options (8-10). Gupta et al. suggest an EC risk group classification; this is primarily based on disease staging after primary resection and stratifies the need for further adjuvant treatment depending on the potential for disease recurrence (11). Adjuvant treatment options include chemo-radiotherapy, pelvic external beam radiation therapy or vaginal cuff brachytherapy. Based on this model, surgery is the only treatment in early, low-risk EC, whereas intermediate high-risk EC would require additional adjuvant treatment (11).

Molecular Staging

Although FIGO remains the gold standard in EC staging, there is an increasing need to identify novel molecular biomarkers in order to achieve individualised treatment options. Several efforts have been described in the literature, however, consensus is yet to be reached. All efforts aim to provide a more accurate framework which can predict both prognosis as well as response to treatment and the need for additional adjuvant therapy schemes.

A classic example is The Cancer Genome Atlas (TCGA) classification. TCGA provides a molecular taxonomy for EC based on an integrated multi-platform incorporating genomic, transcriptomic and proteomic profiling (1, 12). TCGA classifies EC into four groups, each of which is based on different histopathology or molecular sub-type, as well as prognostic potential. Group 1 includes tumours with a hypermutant profile and mainly DNA polymerase epsilon, catalytic subunit (POLE) exonuclease inactivation mutations, which have a favourable overall prognosis. Group 2 refers to EC which is associated with microsatellite instability (MSI), and more specifically with hypermethylation of the promoter region of the mutL homolog 1 (MLH1) gene; the latter has been found to be the primary MSI-associated mechanism of carcinogenesis in sporadic colorectal cancer (CRC) (13). Group 3 includes tumours with low somatic copy number alterations (SCNA); the latter refers to various segmental aneuploidies, focal events, and whole-chromosome aneuploidies (14). SCNA are strongly associated with chromosomal instability; these mechanisms explain why cancer cells can potentially deviate from a diploid karyotype and can also be the fundamental cause for a degree of general heterogeneity within an individual tumour. Groups 2 and 3 have similar prognosis. Group 4 represents a high SCNA group, which mostly incorporates TP53 mutation, and includes serous-like EC, indicating a poor overall prognosis.

Further to stratifying prognosis, the incorporation of molecular features into the classification of EC aims to optimise personalised treatment options and to predict potential responses to (neo)adjuvant treatment (12). Recent studies have shown that in the case of Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutant EC, a combination therapy of mitogen-activated extracellular kinase (MEK) inhibitors plus anti-oestrogen agents may alter oestrogen signalling and thus improve the response rate (15).

KRAS: A Marker in Cancer Molecular Biology

KRAS is a proto-oncogene (Gene ID: 3845) located at chromosome 12 (12p12.1) and is primarily involved in the cellular response to extracellular signals. It is strongly associated with down-regulation of mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene (PI3K/AKT) pathways (16-18). KRAS encodes a 21-kDa signalling protein which connects activated membrane receptors to MAPK and PI3K/AKT pathways (5). Mutant KRAS promotes down-regulation of MAPK or PI3K/AKT, which further results in excessive cell proliferation and subsequently carcinogenesis (5). KRAS mutations refer to a frequent alteration of guanine to adenine (G>A), a mutation most frequently found in codon 12. Codons 12 and 13 in exon 2 constitute 90% of all KRAS mutations (19). In total, there have been 85 different mutations reported, many of which are pathway-specific (20).

KRAS is tethered to several cell membrane receptors and acts as a signalling transducer molecule. A classic example of such receptors are the surface tyrosine kinase receptors, including the epidermal growth factor receptor (EGFR) across the cell membrane of colonic and rectal epithelial cells. There is a large family of anti-EGFR chemotherapy agents, for example cetuximab and panitumumab, that target these receptors. KRAS mutations can cause resistance to EGFR inhibitors (20). Hence, especially in the case of CRC, KRAS-mutant status is directly associated with chemotherapy resistance. Therefore, KRAS is currently being used in clinical practice as a predictive biomarker for response to anti-EGFR chemotherapy agents (21-25).

KRAS Mutations as Biomarkers of Early-stage Type I EC

Type I and II EC are thought to be associated with different distinct mutations (4, 5). KRAS mutations have been mostly associated with type I oestrogen-related EC and their frequency is estimated at around 10-30% (4, 5).

In their review of mechanisms of EC development, Banno et al. supported the assumption that KRAS mutations occur at the early stages of the EC pathway (5). Further to this, KRAS mutations are present in 6-16% of endometrial hyperplasia specimens (26). Similar notions have been discussed in the case of CRC, where in the classic adenoma–carcinoma pathway, KRAS mutations seem to appear early in the neoplastic route. Several studies concluded that KRAS may play a significant role in early stage CRC (27-29). In 1988, Vogelstein et al. stated that early mutations of adenomatous polyposis coli gene result in deregulation of the wingless-related integration site (WNT) pathway (30); KRAS mutations follow deregulation of the WNT pathway and certainly take place prior to TP53 gene inactivation. Similarly, in the case of EC, KRAS mutations appear to be a stage ahead of TP53 involvement, and TP53 signifies the transition from low-grade to high-grade type I EC (5).

Further to this, Tsuda et al. stress the role of KRAS in predicting invasive proliferation of well-differentiated (grade I) tumours (31). Therefore, the role of KRAS in both an early checkpoint of transition from hyperplasia to EC, as well as a marker of the invasive potential in the case of grade I tumours, is clear.

Another interesting feature is the association of KRAS mutations with MSI-positive EC. Microsatellites are short repetitive DNA sequences which are involved in the DNA repair system. The vast majority of MSI involvement is either via direct base substitutions (point mutations), or via hypermethylation of promoters of involved genes (epigenetic changes). In the case of Lynch syndrome, the whole series of mismatch repair (MMR) genes, including MLH1, mutS homolog 2 (MSH2), mutS homolog 6 (MSH6) and post-meiotic segregation increased 2 (PMS2), is affected (13). Nevertheless, in sporadic CRC and type I EC, MMR defects are primarily a consequence of hypermethylation of gene promoters, and this primarily affects the MLH1 (4, 13). A classic example was shown by Muraki et al., who reported hypermethylation of MLH1 promoter in 40% of type I ECs (32). KRAS promoter can equally be affected by hypermethylation and this can explain its concurrent presence with defective expression of MMR genes (in MSI-positive EC). Both hypermethylation changes, as reflected by reduced presence of DNA repair proteins (MMR), and KRAS mutations, are generally thought to occur early in the EC pathway.

Translation of KRAS Mutation Status into Clinical Information for Type I EC

A narrative review of the literature was performed to summarize the current views on the prognostic and predictive value of KRAS mutations in EC. PubMed database was searched using any (AND, OR) combination of keywords “KRAS” and “Endometrial Cancer”. Any original study which involved KRAS mutation in EC was identified and critically commented on.

Most studies focus on explaining the role of KRAS in type I oestrogen-related EC. van der Putten et al. supported the view that KRAS mutations are found adjacent to hyperplastic endometrial tissue (33). Based on this, KRAS status was used as a prognostic marker to describe a possible transition from hyperplastic tissue to malignancy; 27% of their type I EC specimens were positive for KRAS mutation, most of which were next to hyperplastic endometrium. In 5% of cases, hyperplastic (non-malignant) endometrial specimens were also positive for KRAS mutation. Further to this study, Zauber et al. supported the involvement of KRAS early in the carcinogenesis pathway, suggesting that biopsies confirming endometrial hyperplasia should be analysed for KRAS status, along with MSI status (34). Similarly, Berg et al. concluded that KRAS involvement happens early, and that molecular alterations related to KRAS mutations and inflammation are more common in obese patients (35). Similarly, Duggan et al. concluded that there is early-stage involvement of KRAS gene in type I EC, prior to clonal expansion (36).

Many studies correlated KRAS status with certain histopathological features; Xiong et al. supported the role of KRAS mutations in the formation of superficial epithelial changes in endometrioid EC, which has been further associated with focal mucinous differentiation (37). A similar association between KRAS and mucinous differentiation was reported by He et al. (38). Another interesting study by Steward et al. identified KRAS mutations in 12 out of 42 endometriosis-associated endometrioid adenocarcinomas (39).

As discussed previously, current literature concludes that KRAS mutations are primarily found in type I oestrogen-related EC (5). An interesting question would be to explore the relationship between the KRAS gene and oestrogen receptors (ER), as there is extremely limited evidence for this. Tu et al. supported the assumption that the transcriptional activity of the ER is up-regulated by KRAS mutation (40). In other words, ER expression may be seen as a regulator of the RAS signalling pathway which directly affects directly the tumorigenesis of EC.

Several studies support the role of KRAS as a potential prognostic marker, both in terms of transition from pre-malignant to malignant cell status, as well as progression from early to more advanced invasive cancer. Ninomiya et al. identified K- and NRAS-mediated signalling pathways as potential inducers of cell apoptosis (41). Birkeland et al. noted an increase in KRAS amplification and KRAS mRNA expression during transition from primary to metastatic disease (42). Alexander-Sefre et al. suggests a molecular assessment of the depth of myometrial invasion of EC based on KRAS (43). Mizuuchi et al. correlated the presence of KRAS mutation (codon 12 or 13) with poor prognosis (44). Ito et al. attributed KRAS mutations as being responsible for more aggressive clinical behaviour of EC in postmenopausal women (type II EC) (45). On the other hand, Varras et al. (46) and Trowbridge et al. (47) did not find correlation of KRAS status with any clinicopathological features. From the aforementioned evidence, it is apparent that a consensus on the exact way that KRAS overall affects EC prognosis is yet to be achieved.

Another interesting question is the association of KRAS status following tamoxifen exposure after breast cancer. Wallen et al. supported the existence of a link between tamoxifen use and KRAS codon 12 mutation (48). Nagy et al. noted a higher trend in KRAS mutation following exposure to tamoxifen (49).

Finally, although KRAS has an established predictive value in CRC, there are extremely limited data on this aspect in the case of EC. Byron et al. state that KRAS and fibroblast growth factor receptor 2 (FGFR2) mutations may alter the effectiveness of anti-FGR or anti-MEK biological therapies (50).

A promising study by Alomari et al. showed that KRAS mutation had a positive predictive value of 88% in diagnosing complex atypical hyperplasia (51). Based on the previous discussion, this could be an extremely important finding which may alter the current management of endometrial hyperplasia. Current practice in the United Kingdom, as defined by the Green-top guideline (No. 67), suggests first line management of endometrial hyperplasia with atypia in premenopausal women who wish to maintain fertility, by offering the Levonorgestrel Intrauterine System (LNG-IUS) and second-line by administering oral progestogen supplements or combination of both for at least 6 months (52). Identifying novel biomarkers which can predict the course of lesions and their progression to EC would be useful to optimise care provision and reduce anxiety from both the patient's and clinician's point of view.

Limitations

We recognise a series of limitations in this narrative mini-review. Although PubMed was searched systematically using certain key words, only the studies that were thought to be relevant and of high quality were considered in raising the discussion points. Secondly, most of the included cohorts were small with several limitations. Furthermore, existing evidence was conflicting. Lastly, a single biomarker was searched, which may provide a biased view as it may be optimistic to explain a complex carcinogenesis progression using a single gene.