KRAS Transitions and Villous Growth in Colorectal Adenomas

Colorectal cancer (CRC) is the second most common malignancy in the western world with a lifetime risk in the general population of about 5%. The genesis of CRC and its progression, mainly from pre-existing sporadic adenomas, are caused by somatic mutations of several genes including APC, KRAS, SMAD4, TP53, and β-catenin, by epigenetic events leading to gene activation and silencing, and by a large number of chromosomal aberrations leading to gene inactivation, amplification, and to more subtle gene dosage changes [7, 8,10,16,20]. The KRAS gene, in particular, one of the oldest oncogenes, is apparently having a second youth. The role of KRAS in the genesis and progression of CRC has been recently focused in a series of reviews linking the constitutive activation of this oncogene with a complex network of interactions leading to cell survival, apoptosis, angiogenesis, invasion and metastasis [17, 25,34]. An additional link of RAS mutations and chromosomal aberrations, a bona fide consequence of chromosomal instability (CIN), was suggested by a large series of in vitro and in vivo studies and during the human colorectal adenoma to carcinoma transition [5]. Though the mechanisms linking genetic and epigenetic events with CIN still remains unclear, it is commonly thought that CIN and aneuploidy represent early key genomic events in the genesis and progression of CRC and of other tumor types [4,8,9,16,21,24,26–30, 35–37]. Specific KRAS mutations induce different biological consequences by affecting differently the structural conformation and the function of the mutated protein [1,2,22]. In particular, CRC patient disease free survival and overall survival were shown to depend on the type of KRAS mutation [3,11]. In the latter study, in particular, it was shown that KRAS G → C and G → T transversions in CRC were related to a worse prognosis than G → A transitions. These findings are in general agreement with the literature data with a few exceptions [3,5]. An observation, however, that so far remained unexplained, is that KRAS G → A transitions in colorectal cancerized adenomas [16] and in adenocarcinomas [15] are more frequent than G → C/T transversions while in the precursor adenomas their incidences are about equal. More precisely, while KRAS G → C/T transversions range between 10 and 15% during the entire adenoma-carcinoma sequence, the G → A transitions frequency raises from about 10% in the adenomas to above 30% in the carcinomas. The present study indicates that KRAS G → A transitions strongly orientate DNA diploid adenoma morphogenesis toward the villous architecture, that in turn is known to be a reliable predictor of higher risk of cancer and may justify the relative increase of KRAS G→ A transitions in CRC. The study was conducted, after exclusion of serrated polyps [39] or polyps with sectors of serrated morphology, on 159 endoscopically removed and histologically proven colorectal polypoid adenomas (size range: 5– 80 mm; median 15 mm). The patients were 137 (60% men and 40% women aged 33–86; median 65 years) with a negative family history for colorectal neoplasia. Twenty-seven percent of adenomas were located in the proximal colon, 73% in the distal colon. Polyps were divided in two parts by a central mid-sagittal section. One part was fixed in 10% buffered formalin, embedded in paraffin and stained with H&E and Feulgen stain. The other part was immediately frozen in liquid nitrogen to provide multiple samples for DNA flow cytometry-sorting and KRAS analysis using a H&E stained cryostat section as a histotopographic reference. In accordance with WHO criteria [18], tubular adenomas were composed of branching neoplastic tubules in at least 80% of the tumour and villous adenomas were composed of leafor finger-like processes in at least 80% of the tumor; in tubulo-villous adenomas tubular and villous structures contributed to more than 20% of the tumour. Dysplasia was graded into low and high grade; the diagnostic grade was based on the most severely dysplastic area, independently on its extension.