Biomarker driven anti-cancer therapy underwent a paradigm shift when the USFDA and ASCO recommended that the use of anti EGFR mAb in mCRC be restricted to patients with a WT KRAS gene status, because of a lack of benefit in those with a mutation [47
]. In fact, there appears to be a negative outcome when patients with a KRAS mutation are treated with the anti-EGFR drugs [50
]. Furthermore, there is emerging data that not all KRAS mutations are alike, and one particular mutation, G13D, may not be a valid biomarker of exclusion after all [52
]. In this retrospective study, we performed a comprehensive analysis of mutations in the PI3K/PTEN-AKT-mTOR pathway to identify further determinants of anti-EGFR treatment benefit. While we failed to identify mutations in AKT or PIK3R1 we observed that loss of expression of PTEN expression and mutations in PIK3CA, both of which hyper activate the PI3K/PTEN-AKT-mTOR pathway, are further predictors of anti-EGFR treatment response.
Consistent with our published pre-clinical observation [24
], we observe that collective consideration of PIK3CA mutations and PTEN loss predict for an superior clinical outcome when treated with anti-EGFR therapy, with an improvement in OS and a strong trend in PFS, in the KRAS WT patients. A clinically meaningful approach is likely to be an evaluation for KRAS mutations, followed by PIK3CA mutation assessment and PTEN IHC staining in the KRAS WT tumors; offering anti-EGFR therapy for the KRAS WT/PIK3CA WT/PTEN positive patients. This approach could potentially wean out 60–70% of patients from therapy with these drugs, thereby markedly improving the clinical benefit rate. Thus, PTEN preservation is a potential biomarker of inclusion, while KRAS and PIK3CA are markers of exclusion, of therapy with anti-EGFR drugs. This identification of PTEN/PIK3CA status as a putative biomarker is consistent with prior reports [24
]. Interestingly enough, one group has reported its findings on the possibility of using gene expression profiles to characterize KRAS, BRAF or PIK3CA activated like tumors, as a further refinement of clinical therapy with anti-EGFR agents [56
We observed an interesting overlap of mutations within the same tumor. One sample harbored mutations in both BRAF (V600E) and KRAS codon 12, and another with mutations in KRAS codons 12 and 13. Such simultaneous mutations have been reported previously, as in BRAF and KRAS, KRAS codons 12 and 61, and in KRAS codons 12 and 13 [52
]. It appears that this is the first report of simultaneous mutations in codons 12 and 13. Furthermore, we found one tumor sample that harbored mutations in both exons 9 (E545K) and 20 (H1047R) of the PIK3CA gene. It is likely that as more tumor samples are analyzed for multiple somatic mutations, similar overlapping simultaneous mutations will continue to be identified. The co appearance of mutations in the MAPK and PI3K pathway has been reported extensively in the literature [24
Another important observation is that simultaneous consideration of PTEN methylation and truncating mutation is significantly associated with loss of PTEN expression by IHC. Moreover, careful study of the matched pairs elucidated that there was an increase in methylation in the metastases as compared to the primary. We therefore propose a hypothesize that PTEN loss requires a dual “hit”, either by homozygous mutation, or an initial allelic loss by a truncating mutation, followed by a second allelic loss by promoter methylation, that takes place between the initial diagnosis and development of metastases. This methylation event is a potential mediator of PTEN loss in the metastases and is clearly worthy of further investigations in large scale tissue studies.
The lack of an association of PTEN methylation alone with IHC expression may be explained by the fact that we interrogated four sites by the MSP and may have missed other potential important sites of methylation. Broadening the panel of primers to interrogate more CpG islands may further improve the yield of methylation detection. A second possibility is that methylation alone is insufficient to downregulate PTEN expression. For example, in a recent publication, in patients with Cowden syndrome, methylation in the PTEN promoter did not result in transcriptional repression of gene expression [58
]. As regards PTEN mutations, we were unable to establish their association with overall outcome or response data. However, considering the ease of mutation testing, its role as a simple potential biomarker needs further exploration. Taken together, despite its limitations, PTEN IHC appears to remain the most robust assay to determine its expression.
The most significant finding in our study is clearly the confirmation that the loss of expression of PTEN (by IHC), and mutations in the PIK3CA gene are potential biomarkers of resistance to therapy with the anti-EGFR agents, particularly in patients with a WT KRAS in their cancer. These findings need to be further explored in large prospective randomized studies. Further identification of novel biomarkers will lead to small incremental steps towards truly individualizing therapy for patients with mCRC, thereby improving outcomes, reducing healthcare costs, and most importantly minimizing toxicity by the selective use of the most effective, and minimizing the use of drugs unlikely to benefit the patient.
CLINICAL PRACTICE POINTS
Recent recognition that patients with KRAS mutant metastatic colorectal cancer do not experience clinical benefit from the anti EGFR monoclonal antibodies cetuximab and panitumumab, and the subsequent recommendation that they be excluded from this therapy marked a paradigm shift in the way we manage patients and heralded the era of personalized therapy in cancer. However, not all KRAS WT patients derive benefit, emphasizing the urgent need for development of better predictive biomarkers. BRAF, an oncogene downstream of KRAS is purported to be a prognostic rather than a predictive biomarker. We collected tumor rich tissue from FFPE blocks from patients who were treated with cetuximab or panitumumab at our institution, and investigated for perturbations in the alternate signaling pathway, the PI3K-mTOR-AKT-PTEN pathway that transfers signal from the cell membrane to the nucleus. Similar to our pre-clinical in vivo findings, we find that collective consideration of PIK3CA activating mutations and loss of PTEN expression are predictive for lack of benefit from these drugs. If adopted in clinical practice, excluding patients with KRAS and/or PIK3CA mutations or those who lack PTEN expression could narrowly define the 30–40% of patients with metastatic colorectal cancer most likely to benefit (and exclude close to 60–70% of patients) from the use of anti-EGFR therapy. These novel potent biomarkers will lead to further refinement of therapy for these patients, facilitating better outcome, a better tolerable toxicity profile, and lower cost of health care delivery.