This is a retrospective study to investigate the survival outcome of metastatic CRC (mCRC) patients in Brunei Darussalam. The study was conducted at The Brunei Cancer Centre (TBCC), Pantai Jerudong Specialist Centre of Brunei Darussalam. We collected data of 76 mCRC patients diagnosed between 1 January 2013 and 31 December 2017 were collected and followed up until 30 April 2018. Age, gender, ethnicity, date of diagnosis, site of primary tumour, metastatic sites and tumour molecular analysis of KRAS mutation status (either KRAS mutated or KRAS wild-type) were recorded. KRAS mutation status was based on quantitative PCR results testing for missense mutations on codons 12 and 13 of KRAS from CAP accredited diagnostic laboratory in National University Hospital of Singapore. PCR testing for KRAS mutations is a validated method and a critical diagnostic tool for clinicians in prescription of anti-cancer drug type. KRAS testing includes testing for mutations in the following regions: both codons 12 and 13 of exon 2, codons 59 and 61 of exon 3 and codons 117 and 146 of exon 4. PCR testing is an inexpensive and simple method that provides good sensitivity, representing an excellent clinical molecular pathology diagnostic tool. A limitation of PCR methods is the restricted coverage of mutation hotspots defined by the designed primers. It should be noted that various PCR methods and tests may also have varying sensitivities [12]. The diagnostic date was the date of colonoscopy or sigmoidoscopy, or other diagnostic technique done. The metastatic sites were classified into either liver, lungs or others (inclusive of bone, brain, bladder and reproductive organs). Exclusion criteria were patients with known NRAS status, uncontactable patients and absence of metastasis. Known NRAS status was excluded to obtain a non-biased population of KRAS. Uncontactable patients refer to patients that were not followed up by the end of the follow up period. Fig. 1 illustrates the consort flow chart of sample patient categorisation.

Fig. 1.

Consort flow chart illustrating the numbers of Stage IV metastatic CRC patients and their known KRAS statuses.

Overall survival (OS) refers to total duration in months between patients’ date of diagnosis to the end of the follow up period or date of termination (date of death). The status of the patients (whether the patient was alive or not at the time of study), gender, age, site of primary tumour, metastasis location, RAS status and mutated codon were documented. Primary tumours located in the ileocecal valve, cecum, ascending colon to the transverse colon was classified as the right sided colon tumour (RCC), while the primary tumours found within the splenic flexure, descending colon, sigmoid and rectum were classified as left sided colon tumours (LCC). All duration of date are recorded in months. To correlate the survival impact of the metastatic site, we have categorised the patients in accordance to the two most common metastatic organs, that is, the liver and the lungs. In addition, the number of metastatic sites were taken into account and therefore, the OS based on either single or multiple metastatic site was studied. Survival analysis of mCRC with mutated KRAS was compared with that of wild-type KRAS. A further inspection on the survival outcome of different KRAS mutations, such as G12D, G12S, G12V and G12C in codon 12 and G13D in codon 13 was conducted.

Chi-squared test was used to compare the survival outcomes of mCRC patients, and Mann-Whitney U test was used to compare the median ages of both groups. Kaplan-Meier survival curves were drawn and logrank test was used to compare the survival outcome between two groups (namely between gender, age groups, location of primary tumour in the colon, and KRAS status). Statistical analysis was conducted using SPSS Statistics for Windows, version 17.0 (SPSS Inc., Chicago, III., USA) and R (ver.3.6.0, R Core Team, Vienna, Austria). A p-value of 0.05 is considered statistically significant.

During the study period, a total of 105 patients with Stage IV CRC were treated in TBCC. These patients were initially diagnosed by either sigmoidoscopy or colonoscopy histopathologic biopsy. They were classified as stage IV patients due to presence of metastatic tumour(s). Of the 105 cases, 29 were excluded from the study due to the following reasons: unknown KRAS status (n = 20), known NRAS status (n = 2), and inability to contact at the end of the follow-up period (n = 7). Hence, 76 metastatic CRC (mCRC) patients were included in this study, and were followed up for a total of 1951 person-months. Their median age was 58 years (range = 20–79 years) (Table 1). At the end of the study period, only 21 (61.8%) of the surviving mCRC patients were male and 13 (38.2%) were female. There was no significant difference between survival outcome and gender (p = 0.821) (Table 1). The median OS was 29 months (95% CI 23.7–34.3), with males and females having median survival of 27 months (95% CI 20.3–33.7) and 29 months (95% CI 23.7–34.3), respectively. No significant difference (p = 0.764) in terms of median survival between the two genders (Fig. 2 left panel, Table 2) and in terms of the median survival based on median age distribution of 58 years old was found (Fig. 2 middle panel).

Fig. 2.

Survival outcomes of mCRC patients (n = 76) based on gender, age and site of primary tumour in colon. (a) Survival outcomes of mCRC patients (n = 76) based on gender. p-value is derived from the log-rank test comparing the 2 Kaplan Meier curves for females (in yellow) and males (in blue). The shaded areas indicate the 95% confidence intervals for each group. (b) Survival outcomes of mCRC patients (n = 76) based on age distribution, with the median survival age of 58 years old as the cut-off point (58 years old in yellow; 58 years old and above in blue). (c) Survival analysis of mCRC patients (n = 76) based on the site of primary tumour in the colon (Left colon in yellow; Right colon in blue).

81.6% (n = 62) of mCRC patients were found to have the primary tumours on the left side of the colon (Table 1). Their median OS period was 26 months (95% CI 22.3–29.7) (Table 2, Fig. 2 right panel). Only 18.4% (n = 14) mCRC cases with primary tumours found in the right side of the colon (abbreviated here as RCC for right-sided colorectal cancer) were recorded, of which 6 died by the end of the study period, amounting to 14.3% of the deaths (Table 1). We were unable to calculate the median survival for mCRC patients with RCC because more than 50% of the patients were alive at the end of the study period (Table 2). Intriguingly, 19 of 26 (73.1%) mutant KRAS mCRC patients were found to have primary tumours on the left-side of the colon (abbreviated here as LCC to refer to as left-sided colorectal cancer), while 7 of 26 (26.9%) had RCC (Table 3). There were significantly more deaths among those with LCC, when compared to RCC (p-value = 0.035). 34.2% (n = 26) of the samples were mutant KRAS, generating a significant p-value of 0.024.

Additionally, the site of primary tumour was also classified either as in the colon, sigmoid colon or rectum. The primary tumours of most mCRC were found in the sigmoid colon (with n = 36) constituting 47.4%, followed by rectum (n = 21, 27.6%) and colon (n = 19, 25.0%). No significant difference (p-value = 0.289) with regards to the survival outcomes and sites of primary tumours was found (Table 1). The median survival of mCRC patients with primary tumours found in the rectum (34 months [95% CI: 28.4–39.6]) was significantly longer (p-value = 0.016) than mCRC patients with primary tumours in the sigmoid colon (25 months [95% CI: 20.8–29.2]). The median survival of mCRC patients with primary tumours in the colon was non-calculable (Table 2), as more than half of the mCRC patients with tumours in colon were alive at the end of follow-up period. Correspondingly, for mutant KRAS mCRC patients, there was no significant difference between these patients with primary tumours located in either colon, sigmoid colon or rectum (p-value = 0.080, Table 3). No significant difference was found in the median survival between the mutant KRAS mCRC patients with primary tumours in the colon, sigmoid colon or rectum (p-value = 0.348) (Table 4).

The median survival of mCRC patients with mutant and wild-type KRAS was 25 months (95% CI: 18.3–31.7) and 35 months (95% CI: 22.1–47.9), individually (Table 2, Fig. 3 left panel). Metastatic CRC patients with mutant KRAS were significantly more likely to have poorer median survival than mCRC patients with wild-type KRAS (p-value = 0.017, Table 2, Fig. 3 left panel). Further analysis was conducted specifically on the mCRC patients with mutated KRAS status (n = 26, Tables 3,4). The median survival of male and female mutant KRAS mCRC patients were 29 months (95% CI: 17.0–33.0) and 25 months (95% CI: 13.5–44.5), accordingly (Table 4). Among the mutant KRAS mCRC patients, 73.1% (n = 19) had LCC. Of these 19 patients, 84.2% (n = 16) died at the end of the study period (Table 3). The median survival of these 26 patients was 25 months (95% CI: 18.3–31.7) (Table 4, Fig. 3 right panel).

Fig. 3.

Survival outcomes of mCRC patients (n = 76) based on KRAS status and based on both KRAS status and primary tumour in the colon. (a) Survival outcomes of mCRC patients (n = 76) based on KRAS status (mutated in yellow; wild in blue). (b) Survival outcomes of mCRC patients with KRAS mutation (n = 26), based on the site of primary tumour in the colon (Left colon in yellow; Right colon in blue). The shaded areas indicate the 95% confidence intervals for each group. Metastatic sites of mCRC patients and their effects on survival periods.

We further investigated the number of metastatic sites detected in these mCRC patients. 30.3% (n = 23) of the study population had a single metastatic site detected (either liver, or lung or any other organs), while 69.7% (n = 53) of the 76 mCRC patients had two (double) or more metastatic sites (Table 1). mCRC patients with two or more metastatic sites were classified as multiple, regardless of the site of metastasis. There was a statistical significant difference between the number of metastatic sites and survival outcome (p-value = 0.004, Table 1). There was a statistically significance value of p = 0.047 between the survival outcomes of mCRC patients with a single metastatic site and mCRC patients with multiple metastatic sites (Table 2). At the end of the study period, 7 of the 23 single metastatic site CRC patients died while 35 of the 53 multiple (two or more) metastatic sites CRC patients died, yielding deaths rates of 30.4% and 66.0%, correspondingly (p-value = 0.004, Table 1).

Two metastatic target organs (liver and lungs) were also examined. 78.9% (n = 60) of the mCRC patients had liver metastasis, while 55.3% (n = 42) had metastasis to the lungs. It should be noted that a mCRC patient may have metastasis to both the liver and the lungs. The survival rate of mCRC patients with liver metastasis was 46.7% (n = 28), whilst the survival rate of mCRC patients with lung metastasis was 30.9% (n = 13). Chi-squared test showed no significant difference between the number of deaths and survival of mCRC patients due to liver metastasis (p-value of 0.512), but a significant difference with p-value of 0.007 was observed between the number of deaths and survival of mCRC patients due to lung metastasis (Table 1). The median survival for mCRC patients with liver metastasis and lung metastasis were at 29 months (95% CI: 20.1–37.9) and 25 (95% CI: 22.2–27.8), respectively. Survival analysis revealed a significant difference in the median survival times for mCRC patients with lung metastasis only (p = 0.011, Table 2).

In order to examine the survival outcomes of mutant KRAS mCRC patients (n = 26) with varying number of metastatic sites, mutant KRAS mCRC patients were categorised in accordance to the number of metastatic sites. Of the seven mutant KRAS mCRC patients who survived till the end of the study period, 4 (57.1%) have one metastatic site while 3 (42.9%) have two or more metastatic sites (Table 3). Mutant KRAS mCRC patients with multiple metastatic sites were significantly more likely to die by the end of the study period (p = 0.003, Table 3), as compared to mCRC patients with wild-type KRAS. The median survival of mutant KRAS mCRC patients with one metastatic site was non-calculable, however, the median survival for mutant mCRC patients with two or more metastatic sites was 25 (95% CI: 18.0–32.0) (Table 4). Survival analysis revealed a significant difference between these two mutant KRAS patient groups (p = 0.043).

Similarly, mutant KRAS mCRC patients were further analysed based on either liver or lung metastatic sites. Although majority of mutant KRAS mCRC patients with either metastatic site died, that is, 13 mutant KRAS mCRC patients with liver metastasis (68.4%) and 15 mutant KRAS mCRC patients with lung metastasis (78.9%) died at the end of the study period (Table 3).

The specific KRAS mutations detected in the mutant KRAS mCRC patients were studied. 69.2% (n = 18) and 30.8% (n = 8) of the mutant KRAS mCRC patients had mutations within codons 12 and 13, respectively (Tables 3 and 4). There was no significant difference between the survival outcomes of mutant KRAS patients with mutations in either codon 12 or 13 of the KRAS gene (Table 3). However, there is a significant difference in the median survival between the mutant KRAS mCRC patients with mutations in codon 12 and those with mutation in codon 13 of the KRAS gene (p-value = 0.003, Table 4). In our study population, the highest KRAS mutation was G12D (n = 10) (38.5%), followed by G13D (n = 8) (30.8%). There were 11.5% (n = 3) of G12S, followed by 7.7% each of G12V and G12C with 3.8% of G12A. Median survival for mutant KRAS mCRC patients possessing G12A, G12V and G12C missense mutations were non-calculable. Mutant KRAS mCRC patients with G12D, G12S and G13D had median survival of 23 months (95% CI 15.4–34.6), 25 months (95% CI 13.8–36.2) and 29 months (95% CI 24.4–33.6) (Table 4), correspondingly.