Clinicopathologic features
A total of 166 cases of CRCs met the inclusion criteria for this study (Table 1). Briefly, female/male ratio is 1.30:1 and median age is 64 years (ranging from 32 to 91). The tumors were located in the cecum (38 or 22.9%), the ascending colon (34 or 20.5%), the transverse colon (18 or 10.8%), the descending colon (8 or 5.0%), the sigmoid colon (38 or 22.9%) and the rectum (30 or 18.0%). Across the entire cohort, 7 (4.2%) of them were classified as pathological stage T1, 10 (6.0%) as T2, 97 (58.4%) as T3, and 52 (31.3%) as T4. Immunohistochemical study for mismatch repair proteins (MLH1, MSH2, MSH6 and PMS-2) as surrogate markers for microsatellite instability was performed as part of routine assessment, and 42 (25.3%) of the cases were found to be MMR- deficient by immunohistochemistry. CRCs without mucinous component made up 70.5% of the cohort (n=117). 85 of 117 (72.6%) were morphologically classified as low grade (well and moderately differentiated) and the remaining 32 (27.4%) as high grade (poorly differentiated). At least 5% mucin by tumor volume was identified in 49 of 166 cases (29.5%). Among them, 16 cases (32.7%) had less than 30% mucinous component, 9 cases (18.4%) had 30%-50% mucinous component, and 24 cases (49.0%) had more than 50% mucinous component. No follow-up and survival data were available due to short period of time after surgical resections.
Frequency of gene mutations across colorectal carcinomas
The majority of CRCs (155 of 166, 93.4%) were found to have at least 1 mutation detected by NGS. The most commonly mutated gene was TP53 (77, 46.4%), followed by KRAS (76, 45.8%), APC (54, 32.5%), PIK3CA (43, 25.9%), BRAF (33, 19.9%), SMAD4 (18, 10.8%), FBXW7 (15, 9.0%) and PTEN (14, 8.4%). Mutations seldom associated with CRCs were KIT, GNAS, JAK2, RB1, RET, FGFR2, NOTCH, PIPN11, ERBB2, IDH2, ABL1, and KDR. In addition, multiple concurrent mutations were found to be common in our cohort. Two concurrent mutations were found in 61 CRCs (36.7%), 3 in 40 cases (24.1%), 4 in 23 cases (13.9%), 5 in 5 cases (3.0%), and 6 in 1 case (<1%).
Molecular and pathologic characteristics of CRCs with mucinous differentiation
The most commonly mutated genes in CRCs with at least 5% mucinous component were KRAS (28/49), BRAF (19/49), PIK3CA (16/49), followed by APC (12/49) and TP53 (11/49). In concordance with the results of study done by Gonsalves et al6, our study has shown that KRAS and BRAF mutations are almost mutually exclusive and only one case had both mutations in this group. The combination mutation rate of either KRAS or BRAF in this group of CRCs is 95.9% (47/49). Neither KRAS nor BRAF was identified in 2 of 49 cases this group. TP53 mutation was identified in one of these two tumors and the mutation of AKT1, APC, and PTEN were identified in other case. In the group of CRCs without mucin (or mucin component <5%), 61 of 117 cases had either KRAS (48/117, 41.0%) or BRAF (13/117, 11.1%) mutation, and no double mutations. The combination mutation rate of either KRAS or BRAF is 52.1% (61/117). TP53 mutation (66/117) was the most frequent mutation in this group, followed by KRAS (48/117, APC (42/117), PIK3CA (27/117) and BRAF (13/117).
These results showed that tumors with any amount of mucin (more than 5%) had a significantly higher likelihood arising from the serrated pathway as evidenced by BRAF mutation (38.7%, compared with 11.1% among conventional CRCs, p<0.001). Seventeen BRAF mutations detected were BRAFV600E and other BRAF mutations were BRAF W604L and BRAF G469A for each case. There was no class-3 BRAF mutation involving codon 594 detected in this study. All KRAS mutations detected in tumor with mucinous differentiation were within codon 12 (21 cases), 13 (3 cases), 61 (2 cases), 117 (1 case) and 146 (1 case). NRAS mutations were not identified in this study. Combined KRAS mutation, the EGFR signaling pathway in this group is near completely blocked due to either KRAS or BRAF mutation (figure 1, p<0.001 ). Our data also showed that the mutation distribution among three subgroups of CRCs with mucinous features (<30% mucin, 30-50% mucin, and >50% mucin) did not have significant difference, suggesting that they share the same or similar clinically relevant molecular genetics or biology (Table 2). Since the tumor with either KRAS or BRAF mutation has a poor response to anti-EGFR therapy, any mucinous component (more than 5%) may be an adverse indicator for poor responsiveness to anti-EGFR therapy.
Total 42 tumors were defined as MMR- deficient based on the results of immunochemical stain. 23 tumors had >5% mucinous component (23/49, 46.9%) and 19 tumors had no mucinous feature (19/117, 16.2%) (p < 0.0001). In both groups of CRCs with and without mucinous component, BRAF mutation was frequently identified in MMR- deficient tumors (15/23 vs. 4/26 and 6/19 vs. 7/98, p=0.001), while TP53 was more common in MMR- proficient tumors (10/26 vs. 1/23 and 61/98 vs. 519). However, KRAS mutation was frequently identified in MMR- proficient tumors with mucinous components (20/26 vs.8/23), while PTEN mutation was more common in MMR- deficient tumors with mucinous components (6/23 vs. 2/26). These findings were not shown in conventional tumors (Table 3).
High tumor grade is correlated with increased number of gene mutation in conventional CRCs.
In 117 conventional CRCs, 72 (61.5%) had <3 gene mutations and 45 (38.5%) had ≥ 3 gene mutations. In cancers with <3 gene mutations, 59 (81.9%) were low grade (well and moderately differentiated) and 13 were (18.1%) high grade (poorly differentiated) CRCs. In the cancers with ≥ 3 gene mutations, 26(57.8%) were diagnosed as low and 19(42.2%) as high-grade CRCs. The number of mutated genes is strongly associated with high tumor grade (p= 0.0010). High T stages (stage 4) does not correlate with mutation status in the cancers with <3 genes mutations (30/97) and with more than 3 gene mutations (22/69) (p>0.05). In addition, no correlation between the numbers of gene mutations and positive lymph nodes or MSI status was found in the cancers with <3 gene mutations (62/97 or 21/97) and with more gene mutations (37/69 or 21/69) (p>0.05). Since the grading of MAC is controversial and not recommended by WHO, the tumors with >5% of mucinous components were exclude when correlating tumor grade and the number of genes mutation.