KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker.

Our aim was to characterize and validate that the location and age of onset of the tumor are both important criteria to classify colorectal cancer (CRC). We analyzed clinical and molecular characteristics of early-onset CRC (EOCRC) and late-onset CRC (LOCRC), and we compared each tumor location between both ages-of-onset. In right-sided colon tumors, early-onset cases showed extensive Lynch syndrome (LS) features, with a relatively low frequency of chromosomal instability (CIN), but a high CpG island methylation phenotype. Nevertheless, late-onset cases showed predominantly sporadic features and microsatellite instability cases due to BRAF mutations. In left colon cancers, the most reliable clinical features were the tendency to develop polyps as well as multiple primary CRC associated with the late-onset subset. Apart from the higher degree of CIN in left-sided early-onset cancers, differential copy number alterations were also observed. Differences among rectal cancers showed that early-onset rectal cancers were diagnosed at later stages, had less association with polyps, and more than half of them were associated with a familial LS component. Stratifying CRC according to both location and age-of-onset criteria is meaningful, not only because it correlates the resulting categories with certain molecular bases, but with the confirmation across larger studies, new therapeutical algorithms could be defined according to this subclassification.

Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR

Early-onset colorectal cancer (EOCRC) is an increasing and worrisome entity. The aim of this study was to analyze its association with polyps concerning prognosis and surveillance. EOCRC cases were compared regarding the presence or absence of associated polyps (clinical and molecular features), during a minimum of 7 years of follow-up. Of 119 cases, 56 (47%) did not develop polyps (NP group), while 63 (53%) did (P group). The NP group showed a predominant location of the CRC in the rectum (50%), of sporadic cases (54%), and diagnosis at advanced stages: Only P53 and SMARCB1 mutations were statistically linked to this group. The P group, including mainly early-diagnosed tumors, was linked with the most frequent and differential altered chromosomal regions in the array comparative genomic hybridization. The two most frequent groups according to the follow-up were the NP group (40%), and patients developing polyps in the first 5 years of follow-up (P < 5FU) (34%) (these last groups predominantly diagnosed at the earliest stage and with adenomatous polyps (45%)). EOCRC with polyps that developed during the entire follow-up (PDFU group) were mainly located in the right colon (53%), diagnosed in earlier stages, and 75% had a familial history of CRC. Patients developing polyps after the first 5 years (P > 5FU) showed a mucinous component (50%). Our results show that the absence or presence of polyps in EOCRC is an important prognostic factor with differential phenotypes. The development of polyps during surveillance shows that it is necessary to extend the follow-up time, also in those cases with microsatellite-stable EOCRC.