Colorectal cancer (CRC) patients in Indonesia are dominated by male (59%), more than 50 years old (59%), with well-differentiated (59%), stage 3B (40,9%), located in rectum (68%). Recently, incidence of CRC in young adults increased by 1,4% per year influenced by obesity and sedentary life style.13 High percentage of locally-advanced stage on hospital admission can be caused by low educational level about CRC risk factors and importance of screening especially in individuals with family history of malignancy. Intricate system of national health insurance also has role in slacking patients with unspecific complaints to see doctors before having clear disorder and getting worse. These are several of many reasons that cause delay in diagnosis and management of CRC.
The heterogeneous and dynamic nature of the CRC are related with its overlapping pathways of carcinogenesis. There are 4 principles of neoplasia in CRC, (1) colorectal tumors arise due to the activation of proto-oncogene mutations into oncogenes and inactivation of tumor suppressor genes14; (2) at least mutations in 4–5 genes are required for malignant formation; (3) accumulation of numbers is more important than sequence of mutations in determining tumor biologic behavior; (4) the mutated tumor suppressor gene continues to express the phenotype without loss of heterozygosity.2
The theory of colorectal neoplasia, namely adenoma-carcinoma sequence (ACS) states that the formation of colorectal carcinoma must be preceded by the presence of an adenoma.1,2 Changes in the normal intestinal mucosal epithelium to adenoma are triggered by mutations in the tumor suppressor gene, that is APC. APC can be detected in the aberrant crypt foci (ACF), which is a precursor lesion that occurred early in the beginning of the formation of adenomatous polyps and can only appear in dysplastic lesions.15
All subjects (100%) in this study had nonsynonymous mutations in APC. Only 2 patients had adenomas on colonoscopy. One of those had tubulous adenomas with mild dysplasia on colonoscopy and first-degree relative with CRC. Nonsense mutated APC was found at codons 879, 1095, 1123, which completely stopped glutamine production (Q). Meanwhile, in another patient with villous adenomas and well-differentiated adenocarcinoma, nonsense mutations were found at codons 876, 879, 1096, 1291, 1294 and 1517 that stopped the production of the amino acids glutamine (Q) and arginine (R). Mutations in APC are known to have high-penetrance that can reach 100% for FAP and CRC.16–19 In contrast to the Japanese population, whose APC mutations scattered at codons 142–1513, subjects in this study had APC mutations occur at codons 876–1517 with mutation cluster region (MCR) in exons 14–17.20,21
After the normal mucosal epithelium turned into an early adenoma, KRAS mutation occurred subsequently triggering the change of early into intermediate adenoma. However, in contrast to APC, KRAS can act on nondysplastic ACF precursor lesions.15
In this study, mutations in the KRAS gene occurred in 14 of 22 samples (63.6%) at 9 codons and were most commonly found in older age group, locally-advanced stage, well-differentiated/low grade, with positive lymphovascular invasion, and located at the rectum. There were differences of codon location in missense mutation between Jakarta (Indonesia) and United States population, i.e. codons 13, 14, 34, 58, 59, 146 VS 12, 13, 61, 146.22 In addition, nonsense mutations were also found at codon 22 which only occurred in 1 patient. This patient diagnosed with stage 2A (pT3N0M0) undergoing elective curative resection and was given 8 cycles of capecitabine adjuvant chemotherapy with complete response. Mutation located in codon 12 has more aggressive behavior than codon 13, because patients were commonly presented in advanced stage.22 Nevertheless, number of cases with metastases involving KRAS mutation in this study was found in 3 of 5 samples without involvement of codon 12.
KRAS mutation can occur concomitantly with APC mutation leading to increased accumulation of β-cathenin in the cytoplasm, by destroying its binding to E-cadherin, which has actually been increased due to loss of mutated APC degradation function. This causes the Wnt signal to become more active so that motility and cell invasion are more aggressive.15,18,21,23−26 In CRC, the combination of APC and KRAS mutations (co-occurring mutations) can occur up to 80%, whereas in this study only occurred in 63.6% of subjects.27
In this study, patients with APC, TP53 and KRAS mutations were predominantly ≥50 years old, with locally-advanced stage and positive lymphovascular invasion. Two shortest median life expectancy were found in patients with KRAS mutation (Fig. 1), in addition 50% of patients died within 6 months after therapy (Fig. 2).
Before turning into carcinoma, intermediate adenomas differentiate into late adenomas triggered by mutations in the SMAD4, CDC4, and DCC genes.2,7 In this study, we found SMAD4 nonsense and missense mutations in 18 of 22 patients (82%).
In ACS theory, late adenomas who developed into carcinomas have mutations in TP53, TGFBR2, BAX, and IGF2R. Mutated TP53 was found in all subjects in this study in the form of nonsense, missense, frameshift, splice-site, and silent mutation. Five most frequently occurred codon locations in this study were 237, 238, 127, G245S, and R248Q. Those are different compared to world database in The Cancer Genome Atlas Program (TCGA) portal which stated that the five codon positions with the highest frequency were 175, 282, 248, R273H, and R273C.28
In contrast to the UK population, in 64% (14 out of 22) subjects, TP53 and KRAS mutations co-occurred.18,21 In Indian population, these two combinations were only found in 13 of 112 cases, whereas the study by Timar can occur in up to ~ 40%.27,29 TP53 and KRAS activate different carcinogenesis pathways so that they rarely coexist.30
Similar to APC and TP53, PIK3CA mutations were found in all subjects (100%) with 9 SNVs. PIK3CA has no role in the aggressive behavior of CRC.31,32 Even so, when it occurs concurrently with KRAS mutations, it will show evident aggressive behavior, especially when occurs in exons 9 and 20. While in this study mutations occurred in exons 2, 3, and 4, aggressive behavior presenting as locally-advanced stage and positive lymphovascular invasion can be found.
Mutations in MLH1 can also occur in non-hereditary/sporadic CRC. The existence of microsatellite instability due to mutations in genes that play roles in the MMR system such as MLH1 actually provides a good prognosis with a higher survival rate.33 In this study, the group of cases with MLH1 mutations alone, had the highest median life expectancy and had a 30-month survival rate of up to 100%.
Referring to the colorectal neoplasia principle mentioned above, all subjects in this study indeed involved activation of oncogenes (PIK3CA and KRAS) and inactivation of tumor suppressor genes (APC, TP53 and MLH1) and also involved a range of 8–19 mutated genes per person. In this study, mutated APC and KRAS, which supposed to be occurred on early sequence of ACS, supports what Fearon stated about unimportance of mutational sequence in determining tumor biologic behavior.1,2
This is the first study fully describes the profile of pathogenic mutations of CRC in Indonesian population with its unique characteristics, compiled of various ethnicities with diverse diet and lifestyle which may have roles in contributing natures of Indonesian version of CRC presenting in locally-advanced stage with large tumor size and moderate-severe malnutrition status. This study is also the first in the world to examine the co-occurring mutations of APC, TP53, PIK3CA, KRAS, and MLH1.