Comparison of KRAS gene in circulating tumor DNA levels vs histological grading of colorectal cancer patients through liquid biopsy

Abstract

Background:

To determine KRAS gene in circulating tumor DNA in comparison with histological grading through liquid biopsy in colorectal cancer patients.

Methods:

This dual-centered cross-sectional study included 73 diagnosed patients of colorectal cancer at different grading levels [Grade I, well differentiated (n = 7, 9.5%); Grade II, moderately differentiated (n = 14,18.9%); and Grade III, poorly differentiated (n = 52, 70%)]. Blood was collected, and plasma was separated. ctDNA was extracted, using magnetic bead-based technique (MagMAX Cell-Free DNA kit). KRAS gene was quantified through qPCR. STRING database was used to find KRAS interactomes.

Results:

Mean threshold cycle (CT value) of KRAS gene in Grade III samples showed significantly higher (P = 0.001) levels of ctDNA (2.7 ± 1.14) compared with Grade II and Grade I (3.1 ± 0.68, 2.3 ± 0.60), respectively. Grading characterization showed that rectal cancer (n = 22, 42.3%) with Grade III (68.8%) was more prevalent than colon and sigmoid cancer (n = 19, 36.5%, n = 11, 21%, respectively). STRING database showed 10 functional genes interacting with KRAS expressed as gene/proteins.

Conclusion:

Liquid biopsy can be used to detect ctDNA in plasma of CRC patients and enabled to detect the KRAS gene by qPCR. The technique being less invasive and cost-effective is convenient for multiple biopsies in different cancers.

INTRODUCTION

Colorectal cancer (CRC) is a highly diversified, heterogeneous cancer with distinct histological cell types or genotypes.[1] Globally, it is causing high mortalities in both genders according to the GLOBACAN statistics 2020.[2] Most cases are diagnosed in advanced stage, since screening has not been helpful in proper detection. Distinctive genetic and epigenetic factors involved in the complexity of the inter- and intratumor genome framework as well as intricate treatment modalities could not be fully captured by the gold standard tissue biopsy.[3] Different treatment modalities currently being used are drugs (like panitumumab, cetuximab, and MoAbs along with anti-EGFR therapies) in wild-type RAS (KRAS and NRAS) mutations as a first-line monotherapy or in combination with chemotherapy. These mutations are also observed to confer resistance to anti-EGFR therapy; therefore, it is a prerequisite to test these mutations for anti-EGFR therapy.[4]

KRAS, a proto-oncogene, is an inhibitor of cancer growth, but if it converts into a carcinogen due to epigenetic factors, it transmutes into an oncogene, especially in colorectal cancer. The most commonly known locations involved in KRAS mutation are codons 12 and 13, which are present in 25-45% of colorectal cancers.[5] It plays a key role in the signaling of GTPase cycling between active GTP and (inactive) KRAS-GDP, resulting in uncontrolled cell growth and proliferation.

In Pakistan, like in other developing countries, although tissue biopsies are the gold standard, there are many inherent challenges associated with it such as potential surgical complications, clinical risks, discomfort, and medical expenditure. In this context, liquid biopsy provides several benefits over tissue biopsy including being less invasive, cost-effective, and easy accessibility, especially where multiple biopsies are required.[6] Circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and exosomes are all liquid biopsy specimens that can show a clear picture of a tumor’s microenvironment. Circulating tumor DNA (ctDNA) is extensively used to detect genetic changes, tumor progression, and therapeutic resistance and to assess the oncotherapy response.[7] This makes liquid biopsy a convenient diagnostic tool for the detection of gene mutations or genetic mapping of the disease in case of relapse and recurrence.[8] The aim of this study was to determine KRAS gene in circulating tumor DNA in comparison with histological grading through liquid biopsy in colorectal cancer patients.

PATIENTS AND METHODS

Patient population

A single institutional, cross-sectional study was performed with 73 diagnosed colorectal cancer (Grades I, II, or III) patients, recruited from the Department of Oncology, Dr. Ziauddin Hospital, between 2019 and 2020. This study was approved by the Ethics Review Committee (ERC) of Ziauddin University with the approach of nonprobability consecutive sampling. A written informed consent was obtained, and demographic data were taken from all the patients including family history, dietary habits, previous medical history, bowel habits, or presence of any type of tumor.

Methods

ctDNA Extraction: A total of 3-5 ml of blood was drawn, plasma was separated after centrifugation, and circulating tumor DNA (ctDNA) was extracted from plasma using MagMAX Cell Free DNA Kit Catalog number: A29319 (ThermoFisher, UK) by magnetic bead-based technique according to the manufacturer’s protocol, and then stored at -20°C.

Real-time PCR: Real-time PCR assay SLAN -48P Real-time PCR System (Shanghai, China) and the qPCR Master Mix (M-medical, Milan, Italy) were used to estimate the concentration of ctDNA in plasma samples.

The primers used for KRAS amplification were as follows:

Forward Primer: 5-CGATACACGTCTGCAGTCAAC-3

Reverse Primer: 5-ACCCTGACATACTCCCAAGGA-3,

The primers used for GAPDH, the housekeeping gene, were as follows:

Forward Primer: 5-ACCCACTCCTCCACCTTTGAC-3,

Reverse Primer 3-CTGTTGCTGTAGCCAAATTCG-5,

which was used as internal control. A three-step cycle was selected for the amplification after denaturation for 5 min at 95°C, and 42 cycles were carried out at 95°C, 60°C, and 25°C for 15 s, 1 min, and 10 s, respectively. Cycle threshold (CT) is the value where the PCR curve crosses the threshold, in the linear part of the curve. The higher the CT value (30-35), lower the ctDNA expression, as more cycles of amplification are required to detect the fluorescence. CT values were analyzed and interpreted accordingly. For data analysis, SPSS version 20 was used. One-way ANOVA and Chi-square tests were utilized. At 95% confidence level, a P value less than 0.05 was considered statistically significant.

RESULTS

The relative quantification of KRAS gene, investigated by real-time PCR, revealed that KRAS was highly expressed in Grade III (n = 52, poorly differentiated) compared with Grade II (n = 14, moderately differentiated) and Grade I (n = 7, well differentiated) patients. Almost 41.1% of Grade III CRC patients showed the lowest CT value, that is, 15.96, which means higher expression of ctDNA (P = 0.001). These results demonstrated that the concentration of ctDNA was higher in poorly differentiated cancer compared with well or moderately differentiated cancer. However, our results also showed a 1.25 fold change in KRAS gene expression in Grade III CRC patients, and significant differences of CT (P = 0.048) were observed, as shown in Table 1.

Table 1.

ΔCT Mean (KRAS), ΔΔCT, 2^-ΔΔCT, and P values for differentially expressed KRAS gene, among colorectal cancer grading

Grade	Grade I (Well Differentiated)	Grade II (Moderately Differentiated)	Grade III (Poorly Differentiated)	P
Minimum CT value	33.40	22.43	15.96	0.001**
Maximum CT value	35.32	30.1	23.57
ΔCT Mean (KRAS)	2.39	3.12	3.15	0.048*
ΔΔCT	-1.00	-0.41	-0.27	0.087
2-ΔΔCT	2.18	1.47	1.25	0.38

*Significant P<0.05, the mean ΔCT difference of KRAS gene is significant at the 0.05 level. **Highly significant value

Figure 1 illustrates threshold cycle (CT) spectrum of KRAS gene shown in different gradings of CRC. A total number of 42 cycles were run, and each sample was observed for its CT value as the fluorescence was detected by real-time PCR. Lower the CT value, higher the DNA expression.

Figure 1.

Showing respective CT values of KRAS, corresponding to Grade I to Grade III by qPCR

Moreover, the interactome network of KRAS in homo-sapiens was investigated using the STRING database analytical tool shown in Figure 2. By selecting LEGEND, a list of ten reputed KRAS interactors was shown, ranging from the highest confidence score (0.998) to the lowest confidence score (0.991). With a confidence score of 0.998, the gene phosphatidylinositol 4, 5-bisphosphate 3-kinase catalytic subunit alpha isoform and phosphoinositide-3-kinase (PI3K) confirm its maximal interaction with KRAS.

Figure 2.

KRAS interactome neighborhood in homo-sapiens, as obtained from the STRING database. The PPI-network discloses ten well-known KRAS interactors (first shell interactors). KRAS’s PPI data were mostly gathered from various databases, with some experiments and text mining thrown in for good measure

DISCUSSION

This is the first study that highlights liquid biopsy as a tool to investigate KRAS gene expression in plasma of CRC patients. Until now, the prognosis of CRC patients was largely determined by the staging and grading of the disease and this was considered as the gold standard. Luo et al.[9] investigated the potential value of serum-free circulating tumor DNA as a diagnostic marker to categorize patients with colon polyps and CRC. The mean index of ctDNA in CRC patients is roughly 50 times than that in the healthy individuals.[10]

In the current investigation, the lowest CT value of 15.96 was found in Grade III CRC (41.1%) patients, indicating higher expression of ctDNA compared with Grade I (well differentiated) CRC. These observations revealed that the concentration of ctDNA in poorly differentiated (Grade III) cancer cells is higher than in well-differentiated cancer cells, and the mean D CT was also significant in CRC from grade I (well differentiated) to grade III (poorly differentiated). Studies around the world have had similar results in prostate cancer or lymphomas, where in advanced metastatic conditions, liquid biopsy is a valuable source of CTC and ctDNA, since yields are significantly higher and it can be used as a practical tool to profile tumor dynamics for prognostic, predictive value.[11]

Thus, depending on the grade of cancer, ctDNA levels can vary widely in cancer patients, from 0.01 percent to more than 90 percent, elucidating the upregulation of the KRAS gene in ctDNA.[12] Though Healthy people also have ctDNA, but it is immediately phagocytized as soon it appears in the blood, whereas, in cancer patients, ctDNA variability is linked to the cellular turnover, vascularity, tumor load, and therapeutic response.[13] In a recent investigation, significantly higher concentrations of ctDNA were found in the early stages of cancer patients (TNM 0-II) compared with healthy individuals. They also discovered that primary CRC had higher ctDNA levels than intestinal polyps and healthy controls, and these levels were related to age, tumor stage, tumor grade, and histologic differentiation.[14] However, in this study, higher expression (lowest CT value) of KRAS was seen in Grade III cancer compared with Grades I and II. All these previous studies from developed countries reported the spectrum of KRAS mutation in relation to CRC, however, there is dearth of data available from developing countries. Therefore, it was important to examine KRAS gene expression in our study population.

Another hallmark of this research was to demonstrate the gene expression of KRAS in extracted ctDNA from CRC patients by qPCR. Numerous techniques have been used to test known mutations in ctDNA of CRC, the most well-known among them is the quantitative PCR or digital PCR,[15] as well as use of droplet digital PCR, which has been reported (ddPCR) by studies from developing countries for detecting mutation of genes through liquid biopsy (ctDNA).[16-18] However, only a few studies utilized ctDNA for detecting mutant gene expression by qPCR. Genetic variances can rise to interethnic differences, which can contribute to the development of certain malignancies or diseases. The incidence of rectal cancer, since 1974, is declining in older age groups, that is 55 years and above, compared to colon cancer.[19] These findings could be explained by the fact that in men, especially as they get older, their tendency to deposit visceral fat increases, which has a higher association with the risk of CRC. Unhealthy habits in men such as heavy smoking and intake of diet high in red or processed meat are some other risk factors.[20] There are several inherent disparities, undiscovered risk factors, and environmental factors within the colorectum, causing the initiation and progress in the development of cancer.

We evaluated known and predicted KRAS gene interactions by using the STRING database program. We have found functional association of KRAS with EGFR/MAPK and PI3K signaling pathway. Transcriptional silencing of tumor suppressor genes increases oncogenic events in colorectal cancer cells.

The EGFR signaling pathway

The EGFR is a signaling pathway implicated in a variety of biological activities, including proliferation, cell growth, and survival. EGFR pathway disruption affects cancer cell development, proliferation, survival, and its metastasis.[21] By attaching to phosphorylated tyrosine residues, this complex activates Ras-GTP. Following RAS activation, phosphorylation of “RAF, MEK, and ERK” initiates a cascade of activation of “RAF, MEK, and ERK.” When this cascade is deregulated, this leads to increased cell proliferation, prolonged survival, anti-apoptosis, angiogenesis, invasion, and metastasis, leading to malignant transformation and tumor growth.[22]

PI3K signaling pathway

PI3K/Akt is a crucial intracellular signaling pathway that regulates cell growth, proliferation, differentiation, and migration, among other activities.[23,24] EGFR signaling activates several pathways, including PI3K. There are two PI3K sub-units in this class, one regulatory (p85) and one catalytic (p110). The PI3K effects on tumor growth and progression are mediated by Akt, a “serine/threonine protein kinase” (Ser/Thr kinase). As a result, Akt is a PI3K downstream effector. In human CRCs, Akt phosphorylation has been related to cell growth and apoptosis suppression.[25] PI3K activation can also be activated by extracellular stimuli through receptor tyrosine kinases (RTK) or by stimulating Ras activation.[26,27] Phosphatidylinositol 4, 5 biphosphate (PIP2) is phosphorylated by activated PI3K, which forms “phosphatidylinositol 3, 4, 5 triphosphate” (PIP3).[28] The activation of AKT by PIP3 results in cell proliferation and survival. Overall, the PI3K signaling pathway has been shown to play an oncogenic role in the onset and progression of CRC.[24,29]

In conclusion, liquid biopsy is a less invasive, clinically relevant, and convenient method for biopsies in CRC patients. Expression of KRAS gene in CRC was efficiently detected in ctDNA through liquid biopsy. To further validate the clinical application of liquid biopsy (ctDNA), longer, prospective and large-scale studies are required for the diagnosis and prognosis of CRC.

Financial support and sponsorship

Nil.

Conflict of interest

There are no conflicts of interest.