The Spectrum, Tendency and Predictive Value of PIK3CA Mutation in Chinese Colorectal Cancer Patients

Background PIK3CA is a high-frequency mutation gene in colorectal cancer, while its prognostic value remains unclear. This study evaluated the mutation tendency, spectrum, prognosis power and predictive power in cetuximab treatment of PIK3CA in Chinese CRC cohort. Methods The PIK3CA exon 9 and 20 status of 5763 CRC patients was detected with Sanger sequencing and a high-resolution melting test. Clinicopathological characteristics of 5733 patients were analyzed. Kaplan-Meier method and nomogram were used to evaluate the overall survival curve and disease recurrence, respectively. Results Fifty-eight types of mutations in 13.4% (771/5733) of the patients were detected. From 2014 to 2018, the mutation rate of PIK3CA increased from 11.0% to 13.5%. At stage IV, exon 20 mutated patients suffered shorter overall survival time than wild-type patients (multivariate COX regression analysis, HR = 2.72, 95% CIs = 1.47-5.09; p-value = 0.012). At stage III, PIK3CA mutated patients were more likely to relapse (multivariate Logistic regression analysis, exon 9: OR = 2.54, 95% CI = 1.34-4.73, p = 0.003; exon 20: OR = 3.89, 95% CI = 1.66-9.10, p = 0.002). The concordance index of the nomogram for predicting the recurrence risk of stage III patients was 0.685. After cetuximab treatment, the median PFS of PIK3CA exon 9 wild-type patients (n = 9) and mutant patients (n = 5) did not reach a significant difference (3.6 months vs. 2.3 months, Log-rank test, p-value = 0.513). Conclusions We found that PIK3CA mutation was an adverse predictive marker for the overall survival of stage IV patients and recurrence of stage III patients, respectively. Further more, we suggested that PIK3CA exon 9 mutations are not negative predictors of cetuximab treatment in KRAS, NRAS, and BRAF wild-type mCRC patients.


INTRODUCTION
Colorectal cancer (CRC) is the third most prevalent malignancy worldwide, which leads to more than 860,000 deaths every year (1). In China, CRC has become the third most common cancer and the fifth leading cause of cancer-related mortality (2), while the incidence is predicted to be growing (3).
To date, the most effective treatment of CRC is adjuvant chemotherapy after surgical resection. Recently, the significant predictive value of some genetic mutation status has been reported by various clinical studies. KRAS mutation status was proved to be a robust predictive biomarker for the efficacy of anti-epidermal growth factor receptor (anti-EGFR) therapies (4). BRAF mutation has been wildly recognized as a reliable indicator of poor prognosis (5,6). PIK3CA is one of the most frequently mutated oncogenes in CRC. It was reported that about 15-20% of CRC patients carried PIK3CA mutation (7), 80% of which was found in exon 9 and exon 20 (8). Previous studies indicated that patients with PIK3CA mutation could benefit from regular Aspirin treatment (9,10), while the prognostic impact of PIK3CA mutation has far been controversial (11)(12)(13)(14)(15). Besides, the relationship between PIK3CA mutant mCRC tumor and resistance of anti-EGFR agents, cetuximab, is lack of investigation and did not reach consistency (16)(17)(18)(19)(20).
In this retrospective study, we analyzed PIK3CA mutations in 5763 CRC patients, using Sanger sequencing and high-resolution melting (HRM) test, and evaluated the associations between PIK3CA mutations and the clinicopathological characteristics. We also conducted a survival analysis to investigate the prognostic value of PIK3CA mutations, and evaluated the role of PIK3CA mutations in first-line chemotherapy. A nomogram was then constructed to predict the recurrence risk of CRC patients at stage III. Further, the predictive effect of PIK3CA exon 9 mutation in cetuximab treatment was investigated with wildtype KRAS, NRAS, BRAF mCRC patients.

Subjects
This study was approved by the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University (L2017ZSLYEC-003). All patients underwent an informed consent process approved by the Hospital Institutional Review Board.
Our study included 5763 CRC patients diagnosed at the Sixth Affiliated Hospital of Sun Yat-sen University from January 2014 to December 2018. In this study, colorectal tumor specimens were fixed in formalin, embedded in paraffin after surgery, and confirmed histologically. The clinicopathologic features of these patients were collected from their medical records. The definition of the rectum is 12 cm or less from the anal verge.
Among the 5763 CRC patients, a sub-cohort composed of 1946 patients with available follow-up records was used to evaluate the prognosis value of PIK3CA exon 9 and 20 mutations. Another sub-cohort composed of 377 stage III patients who had received at least six courses of 5-fluorouracil based chemotherapy treatment after surgical excision was used to evaluate the associations between PIK3CA mutations and CRC recurrence.

Mutation Detections
In this study, 5763 patients' primary intestinal tumors were collected for genetic testing. Genomic DNA extraction and Sanger sequencing were followed by the procedure of our previous study (25).
Besides, high-resolution melting (HRM) test was also applied to detect the mutation status of PIK3CA. In brief, the PCR reagent used in the HRM test was LightCycler 480 High-Resolution Melting Master Reagent Kit (Cat no: 04909631001, Roche Diagnostics Indianapolis, USA). The reaction system of the HRM test was: 50-100ng genomic DNA, 10ml 2×Master mix, 2.8ml MgCl 2 , and 1ml 10µM of each primer for exon 9 test, or 0.8ml 10µM of each primer for exon 20 test, and the mixtures were added up to 15ml with H 2 O. The primers of the exon 9 HRM test were: 5'-GAGACAATGAATTAAGGGAAAATG-3' and 5'-CACTTACCTGTGACTCCATAG-3'; The primers of the exon 20 HRM test were: 5'-ACCCTAGCCTTAGATAAAA CTGAGC-3' and 5'-TCCATTTTTGTTGTCCAGCCACCAT-3'. The reaction conditions of cycling and melting for exon 9 HRM test were: 95°C for 10 min; 45 cycles of 95°C for 20s, 60°C for 20s and 72°C for 20s; 95°C; for 30s and 37°C for 30s; followed by a highresolution melt of 74°C to 87°C with 45 acquisitions/°C 40°C for 10s. The exon 20 HRM test's reaction condition was the same as that of exon 9 except that the annealing temperature was 62°C.
The PIK3CA exon 9 and 20 HRM tests' detection limits were evaluated with spiked plasmid samples containing wild-type copies and mutant-type copies at various percentages. In detail, the exon 9 mutation type was c.1633G>A, and the initial concentration of mutant and wild-type plasmids were both 9.2×10 5 copies/ml. The exon 20 mutation type was c.3140A>T, and the initial concentration of mutant and wild-type plasmids were both 4.9×10 5 copies/ml. PIK3CA mutant plasmid was mixed with its corresponding wild type plasmid at various dilutions, at ratio of 50%, 40%, 30%, 20%, 10%, 8%, 6%, 5%. The minimum detectable dilution serves as the detection limit in this study.
Sanger sequencing was wildly considered a golden standard method for mutation detection. According to the Sanger sequencing results, sensitivities and specificities were calculated for PIK3CA exon 9 and 20 by HRM tests. Only the samples that obtained consistent results from the two methods were included for this study's subsequent analysis.
For association analysis, the mutation information of KRAS exon 2 and BRAF codon 600 in our cohort was also obtained by the previously described detection method (26).

Statistic Analysis
Chi-square test, Mann-Whitney U test, and Fisher's exact test were applied to analyze the association of PIK3CA exon 9 and 20 mutation status with clinical characteristics, including the mutation status of KRAS exon 2 and BRAF V600E . The analyses were initially evaluated with continuous variables, categories data analysis, and further accessed with logistic regression models to evaluate the association based on estimating the odds ratios (OR) and their 95% confidence intervals (CIs). The significance test was two-sided, and a p-value < 0.05 was considered as statistically significant. All the statistical analyses were performed with SPSS 20.0 packages (SPSS, Chicago, IL, USA).
The annual mutation rates of PIK3CA exon 9 and 20 from 2014 to 2018 were calculated separately, and the mutation rates tendency was analyzed with the joinpoint regression model (Joinpoint 4.6.0.0., Calverton, MD, USA).
Kaplan-Meier survival curves for overall survival (OS) of 1946 patients with available follow-up records were performed with GraphPad Prism 5 (Graph Pad Software Inc., San Diego, CA, USA). The Log-rank test was used to assess the significance, and the p-value less than 0.05 was considered statistically significant. Besides, the logistic regression was used to screen other clinical characteristics related to the recurrence of patients at stage III. These variables were then enrolled to create a nomogram with rms package (version 5.1-4, https://cran.r-project.org/web/ packages/rms/) in R statistical software (version 3.4.3).

Demographic and Clinical Characteristics
The study cohort involved 5733 patients (Figure 1), getting the general profiles of CRC patients in China. Some basic clinical features of the cohort were summarized in Table 1. Briefly, there were more male patients (60.9%) in this cohort. The age of the first diagnosis ranged from 17 to 96 years old. We assigned patients into four groups, younger than 45 years old, between 45 to 49 years old, between 50 to 75 years old, and older than 75 years old, with 13.8%, 9.4%, 67.2%, and 9.6% of the study cohort respectively. For all CRC patients' tumor sites, 47.4% of tumors were located in the rectum, 30.9% of tumors were located in the left colon, and 21.6% of tumors were located in the right colon. The vast majority of patients included in this study were tubular adenocarcinoma, of which 17.6%, 74.6%, and 7.8% were well, moderately, and poorly differentiated, respectively. Among these 5733 patients, only 3153 patients' TNM stages information was available, in which 10.9%, 37.6%, 34.9%, and 16.5% of patients were assigned to stage I, stage II, stage III, and stage IV.

Associations of PIK3CA Exon 9 and 20 Mutations With Clinicopathologic Characteristics
The associations of PIK3CA exon 9 and 20 mutations and clinicopathologic characteristics were shown in Table 1 and Table S1. In exon 9, mutations were more likely to occur at right colon (13.4%) than at other sides (6.8% at rectum, 9.1% at left side; p-value < 0.001), while less likely to present at poor differentiation tumors (4.8%) than at well and moderate differentiation tumors (9.5% and 8.9% respectively; p-value = 0.012). In TNM stage analysis, the exon 9 mutation frequency was higher in stage II (18.3%) than the other three stages (12.8% at stage I, 12.4% at stage III, and 15.5% at stage IV, p-value = 0.001, Table S1). In exon 20, mutations were more likely to occur in younger patients (7.2% in patients younger than 45 years old and 6.1% in patients between 45 to 49 years old) than those older than 50 years old (4.1% in patients between 50 to 75 years old and 3.8% at older than 75 years old, p-value < 0.001), as well as more likely to occur at the right colon (9.0%) than other sites (both 3.5% at the rectum and left colon, p-value < 0.001). In TNM stage analysis, exon 20 mutations were significantly more common at stage II (11.4%) than in other stages (5.5% at stage I, 5.9% at stage III, and 6.5% at stage IV; p-value < 0.001, Table S1).
Among all the 5733 patients, only ten patients mutated in both exons simultaneously, which indicated that exon 9 and exon 20 of PIK3CA are mutually exclusive in CRC ( Table 1). KRAS exon 2 mutations were associated with PIK3CA exon 9 and exon 20 mutations (p-value < 0.001 for both exons), while BRAF V600E mutation did not show any association with both PIK3CA exons.
Features which were significantly associated with PIK3CA mutations in Table 1 or considered to have important effects in The Trends of the Mutation Rate of PIK3CA Mutations The mutation rates of PIK3CA among Chinese CRC patients from 2014 to 2018 were shown in Figure S1 and Table S3. The mutation rate of PIK3CA exon 9 shows a slightly ascending tendency from 6.

Mutation Types and Classification of PIK3CA Gene in CRC Patients
PIK3CA exon 9 and 20 mutations were tested in all 5763 samples with Sanger sequencing and the HRM test ( Figure 1). Sanger sequencing failed to generate analyzable results from 16 samples, which can be detected by the HRM test. In the rest of 5747 samples, 14 had inconsistent results. The detection limits of PIK3CA exon 9 and exon 20 HRM tests were both 5% (Supplementary Figures S2 and S3), which is more sensitive than Sanger sequencing (20%). The specificities of PIK3CA exon 9 and exon 20 HRM tests were 99.77% and 99.96%, respectively, and the sensitivities of these two detecting methods were both 100% (Chinese patent, NO. 201610524420.9). Among the final cohort with 5733 samples, 13.4% (771/5733) patients mutated in PIK3CA with 58 types of mutations ( Table  2). Among these 771 mutated patients, 501 mutated at exon 9, 260 mutated at exon 20, and only 10 mutated at both exons simultaneously.
Among 58 mutation types, only 12 types of mutations could be detected by ARMS-based commercial PIK3CA mutation detection kits, which comprised 78.6% (606/771) of mutated patients.
In this study, we also annotated these somatic mutations following the instruction of ACMG guides on the interpretation of sequence variants (45). There were 4, 30, 21, and 3 mutation types in PIK3CA that were classified as Tier I, II, III, and IV variants in colorectal cancer, respectively ( Table 2).

Survival Analysis
In order to evaluate the prognosis value of PIK3CA mutation in CRC patients, 1946 patients with available follow-up information were collected for survival analysis (clinicopathologic characteristics were shown in Table S4). The follow-up started from the day of surgery and ended on August 30, 2019. The median follow-up for the 1946 cohort is 16 months (0-64 months). Their overall survival rates were analyzed with the Kaplan-Meier method. In this follow-up cohort, 447 patients carried exon 9 mutations, 238 patients carried exon 20 mutations, and the remaining 1270 patients had wild-type PIK3CA gene. During the following-up, 178 patients died, among which 138 patients died from colorectal cancer or related diseases. No significant difference was detected between patients with and without PIK3CA mutation (Log-rank test, p-value = 0.289; Figure 2A), exon 9 mutation (Log-rank test, p-value = 0.241; Figure 2B), and exon 20 mutation (Log-rank test, p-value = 0.772; Figure 2C), respectively. For further analyses, patients were divided into four subgroups according to their TNM stages at the first diagnosis and conducted the survival analysis separately (Figures 2D-F). We found that only patients at stage IV who carried exon 20 mutations experienced significantly shorter OS than wild-type patients (median followup: 14 months; median survival for PIK3CA wild-typed and exon 20 mutated patients: 40 months vs. 23 months; Log-rank test, pvalue = 0.002; Figure 2F). To assess the PIK3CA exon 20 mutations' influence on the survival of stage IV patients, the COX regression model was applied. In comparison with patients carried PIK3CA exon 20 wild-type tumors, those with PIK3CA exon 20 mutated stage IV patients showed a decrease in OS (univariate HR = 2.52, 95% CIs = 1.38-4.59; p-value = 0.003, Table 3). In the multivariate COX regression model, PIK3CA exon 20 mutation was associated with a significant decrease in OS of stage IV CRC patients (HR = 2.72, 95% CIs = 1.47-5.09; p-value =0.012, Table 3).

Recurrence Nomogram of Patients With Stage III CRC
In this study, the impact of PIK3CA mutations on 5-fluorouracil based chemotherapy treatment was evaluated in stage III patients who had received consistent chemotherapy in our hospital (clinicopathologic characteristics were shown in Table S5). In total, 377 patients were included in this sub-cohort, and 76 (20.2%) were observed to have disease progression (median follow-up: 13 months; median time-to relapse: 13 months). Univariate and multivariate logistic analyses were applied to screen independent factors relative to disease recurrence. In univariate and multivariate analysis, PIK3CA exon 9, and tumor sites were related to stage III patients' disease relapse (p-value < 0.05 for all, Table 4), indicating that these variants were independent predictive markers in recurrence. Besides, PIK3CA exon 20 was found to be on the cusp of conventional  Table 4).
A nomogram incorporated these five variables of stage III patients' disease recurrence was established ( Figure 3A), and the concordance index of this nomogram was 0.685, which serves as a reasonable accuracy for prediction. The calibration curves also showed high coherence between the observed and predicted disease relapse in the nomogram ( Figure 3B).

PIK3CA Exon 9 Mutations Is Not A Negative Biomarker for Wild-Type KRAS, NRAS, and BRAF mCRC Patients in Cetuximab Treatment
We collected a cohort consist of 14 stage IV patients who received cetuximab treatment ( Table 5) from the 5733 patients. All of these 14 patients carried wild-typed KRAS, NRAS, and BRAF. Five patients carried PIK3CA exon 9 mutations, and the rest nine patients carried wild-typed PIK3CA. For wild-typed PIK3CA patients, most of them (7/9) were evaluated as stable disease (SD), one patient was evaluated as partial response (PR), and one patient was evaluated as progressive disease (PD). For PIK3CA exon 9 mutant patients, four patients were evaluated as SD, and one was evaluated as PR. The disease control rate (DCR) was 88.9% (8/9) in wild-typed patients and 100% (5/5)

DISCUSSION
In this study, we reported 58 types of PIK3CA mutation in Chinese CRC patients with two detecting methods. Most of the commercial kits for PIK3CA mutation detection were based on the ARMS-PCR technique, which can only detect five types of PIK3CA mutations (E542K, E545K, E545D, H1047L, and H1047R). In this case, there were only 12 mutation types in our real-world data that could be detected by such commercial kits, while 21.4% of mutated patients will be missed. Since we found that PIK3CA mutation was a valuable predictive biomarker in survival and disease recurrence, it is essential to achieve precise results in clinical testing. Here we proved that the HRM test was a more sensitive detection method than Sanger sequencing at a lower cost, which can also cover all the 58 mutation types we detected. For these reasons, it can be used as an auxiliary detection method. Moreover, our study indicated that PIK3CA mutation was a neutral biomarker when considered patients at all stages (n = 1946). However, PIK3CA exon 20 was an adverse prognostic factor for CRC patients at stage IV (n = 213). We also found that PIK3CA mutation was a potential molecular biomarker for predicting resistance to 5-fluorouracil based chemotherapy regimens in stage III CRC patients. These findings may provide instructive information in clinical practice.
Mutations at PIK3CA could activate the PI3K/AKT signaling pathway, which is an essential factor that leads to the occurrence of various human malignancies (46)(47)(48). High mutation frequency was observed in multiple cancers, including breast cancer, bladder cancer, and colorectal cancer (8). Previous researches reported that the mutation rates of PIK3CA in CRC are 10-20% (7). In this study, we found that about 13.4% of Chinese CRC patients carried PIK3CA mutation, 8.7% were at exon 9, 4.5% were at exon 20, and 0.2% were found at both exons. The mutation rates of PIK3CA in Chinese CRC patients were varied from different studies, which might be due to the size of the cohort and detection technique (49,50). Our research collected a large cohort of Chinese CRC patients and detected PIK3CA mutations with two different methods, which ensured that the mutation rates reported in this study are representative. Though the mutation rate was relatively high, the prognostic effect of PIK3CA in CRC remains controversial. Some studies showed that PIK3CA mutation was associated with a shorter OS. For example, Ogino et al. found that PIK3CA mutations were an adverse prognostic factor in CRC patients at stage I-III, while this effect was only restricted to patients with wild-type KRAS (11).   Besides, some studies indicated that mutations at different exons carried different prognostic effects. Research conducted by Farina et al. found that patients with PIK3CA exon 20 mutations conferred a more reduced disease-free survival (DFS) than patients with wildtype PIK3CA in stage III, and such an adverse effect was not observed in patients with PIK3CA exon 9 mutations (13). More studies, however, found that PIK3CA is a neutral prognostic factor that did not show any prognostic effects in CRC patients (14,15). The reasons that lead to inconsistency may arise from different mutation sites, patient ethnicity, and the cohorts' size. PIK3CA exon 9 and exon 20 were located in the helical and kinase domain separately. Mutations at these two domains show a gain of enzymatic function by activating the AKT signaling pathway to induce oncogenic transformation (51). A previous in vitro study suspected that the oncogenic mechanisms of these two domains were different. Briefly, mutations in the helical and kinase domain are both required to bind to p85 to induce the gain offunction, while the former is required to interact with RAS-GTP simultaneously (52). The different mechanisms could be the reason that leads to the different prognostic effects of PIK3CA exon 9 and exon 20. Chemotherapeutic treatment after surgical resection is a mainstream treatment in stage III CRC patients. 5-fluorouracil is the most widely used drug and is usually combined with other drugs as regimens, such as FOLFOX and XELOX. Recently, more and more CRC patients showed resistance to first-line chemotherapy (21)(22)(23). It is urgent to search for biomarkers that could predict chemotherapy resistance in CRC. A few studies suspected that PIK3CA mutation is a potential predictive marker in chemotherapy resistance, while the study cohorts were limited and the effect did not focus on stage III CRC patients (24), while patients at stage III with PIK3CA mutation were more likely to have disease recurrence or progression.
Nomogram is an effective method to visualize the regression model with various factors, which is valuable in clinical application. Most of the studies focus on constructing nomograms for survival prediction in CRC, including risk factors like TNM stage, age, and tumor location (53,54). Our study is one of few that tried to construct a nomogram for predicting the recurrence risk of stage III CRC patients (55), and it is the first nomogram that involved PIK3CA mutation as a risk factor. The concordance index and the calibration curve indicated that the nomogram we constructed in this study served a well predictive function in clinical practice.
Previous studies indicated that mutation in PIK3CA exon 9 did not influence the outcome of anti-EGFR target therapy, while exon 20 mutation might suffer worse outcomes (16). Other studies, however, suspected that PIK3CA gene as a whole was a negative biomarker to cetuximab treatment in mCRC patients. The reason that leads to this inconsistency might be that these researches did not exclude KRAS, NRAS, and BRAF mutations during the analysis (17)(18)(19)(20). In clinical treatment, bevacizumab treatment is more wildly used for patients with PIK3CA mutations. So in our study, only five mCRC patients with PIK3CA exon 9 mutations were treated with cetuximab. We found that the DCR in PIK3CA exon 9 mutation patients was higher than wild-type patients (100% vs. 88.9%), though the Kaplan-Meier analysis found that the PFS between these two subgroups did not differ significantly (Log-rank test, p-value =0.513). With these results, we suggested that PIK3CA exon 9 mutation did not affect cetuximab treatment among patients with wild-type KRAS, NRAS, and BRAF. For the low mutation rate, we could not obtain data of exon 20 mutation to cetuximab response. A larger cohort is required to clarify the role of PIK3CA in cetuximab resistance in the future.
There are some shortcomings in our study. First of all, though we collected a substantial cohort, patients involved in this study came from a single hospital, so there is a risk of bias in the cohort's characteristics. Secondly, the concordance index in this nomogram was not very high (0.685). Additional researches were required to build a more reliable predictive model.

CONCLUSION
In this study, we uncover the PIK3CA mutation profile with a 5763 cohort. We found that 13.4% of Chinese CRC patients carried PIK3CA mutation with 58 types of mutants. And 21.4% of mutated patients could be missed if tested with commercial detection kits. We also found that the mutation trend of PIK3CA in our cohort remains steady from 2014 to 2018. In survival analysis, we found that PIK3CA exon 20 was an adverse biomarker in stage IV CRC patients, and Stage III patients with PIK3CA mutation were more likely to have disease recurrence than those with wild-type PIK3CA. We also found that PIK3CA exon 9 mutations is not a negative biomarker for wild-type KRAS, NRAS, and BRAF mCRC patients in cetuximab treatment.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the ethics committee of the Sixth Affiliated Hospital of Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study.