Methylated BCAT1/IKZF1 DNA: a breakthrough in colorectal cancer diagnosis?

Dear Editor,

Colorectal cancer (CRC) is one of the most common cancers in the world and the second-leading cause of cancer-related deaths. There have been 52 580 deaths estimated in the United States in 2022, and almost 1 million deaths were recorded. Remarkably, CRC cases in developed nations have been decreasing in recent years as accomplished by large-scale screening, timely diagnosis, and treatment¹. On the other hand, low- and low-middle-income countries (LLMICs) are reporting increased incidences of CRC and associated deaths, sadly due to lack of screening and late diagnosis. Developed nations have raised their standards and practices in CRC diagnosis with the advancement of sophisticated molecular genetic techniques; nevertheless, LLMICs have been relying on conventional carcinoembryonic antigen (CEA), histological and radiological findings for both diagnostic and prognostic markers. The pitfall of relying on plasma CEA levels in establishing CRC diagnosis is its aberrant false-positive elevation and inadequacy in detecting most potentially curable CRC recurrences. Furthermore, since the cut-offs of CEA vary according to laboratories, oncologists do face confusion in interpreting the results, which makes the need for a sensitive and reliable diagnostic tool evident.

Undeniably, molecular genetic testing has benefitted CRC management through precise screening, timely diagnosis, identifying risk, monitoring, and foreseeing treatment outcomes by means of analyzing the presence of specific genetic alterations related to CRC. Novel approaches to the detection of circulating tumor DNA (ctDNA) in CRC are being recently emphasized. ctDNAs are released from the tumor cells into the bloodstream and their detection has been linked to tumor diagnosis, progression, recurrence, and even recovery^2,3. In the case of CRC, research has put forward impressive diagnostic performance using a PCR assay for the detection of epigenetic markers in CRC. Hypermethylated DNA of BCAT1/IKZF1 genes has been found in over 95% of CRCs. A study has found that 95% of CRC cases have hypermethylated regions in both of these genes, and 99% of cases have that in any one of the two genes^4,5. Detecting the hypermethylated BCAT1/IKZF1 has shown higher sensitivity and specificity in detecting CRC recurrence than plasma CEA estimation. The mechanism underlies the fact that ctDNAs decline following tumor resection and elevate as metastases develop.

Symonds et al.² compared the sensitivity and specificity of CEA and methylated BCAT1/IKZF1 genes in CRC recurrence. The investigators found a lower sensitivity and specificity of the conventional CEA in contrast to quantitative ctDNA analysis (sensitivity, specificity: 31.9%, 96.4% and 66.0%, 97.9%, respectively). Also, the odds of developing recurrence were significantly higher with a positive quantitative BCAT1/IKZF1 methylated ctDNA (12.7 for CEA and 89.3 for ctDNA). BCAT1/IKZF1 methylation testing has further been indicated as an independent prognostic indicator in CRC. Pedersen et al.⁶ found significantly higher 3-year recurrence-free survival (RFS) in patients without detectable methylation.

Testing for BCAT1/IKZF1 hypermethylated genes is a moderately easy-to-perform assay and is in compliance with the patient’s comfort and well-being since it follows the least invasive procedure, phlebotomy, and blood samples are obtained. The procedure is often referred to as liquid biopsy in a bundle of literature⁷. DNAs are extracted and PCR assay is then employed, targeting the methylated regions near to the promoter sequence of these genes. A real-time PCR is carried out in a thermal cycler with 50 cycles, and the result is expressed as positive if methylated BCAT1/IKZF1 is detected in any of the replicates⁵.

This two-gene PCR assay is commercially available as COLVERA, which is supplied with validated primers and reagents⁸. The utilization of COLVERA testing for CRC diagnosis and prognostication has been rising in the United States. This molecular testing holds the potential to uplift the standards in CRC diagnosis and patient management via its highly sensitive and CRC-specific performance in detecting methylated BCAT1/IKZF1 ctDNA. Winter et al.⁹ compared the methylation analysis in different tumors and found the assay to be sensitive and specific only to CRC, while that for breast and prostate cancers were statistically insignificant. These evidences further advocate prospects of the testing to CRC-specific screening. However, the appropriateness on its worldwide routine implementation is questionable until strong validation studies are performed. There lie few pitfalls on standardization on specific risk cut-offs since the methylation profiles can vary based on age, sex, and the biopsy sampling. Also, variations in the methylation levels were reported between biopsies sampled from luminal surfaces and the central bulk of cancer.

Turning to another flip, the epigenetic landscape in cancer is intricate, and methylation of multiple genes along with other epigenetic alternations, contributes to the disease¹⁰. Furthermore, correlation with mutations on the mismatch repair genes is highly important for its stringent association with CRC¹¹. Therefore, it is essential to consider the broader genetic and epigenetic context when validating screening tests. The field of epigenetic markers in cancer is continually evolving, and the relevance and significance of BCAT1 and IKZF1 methylation in CRC may change as more research is conducted. There indeed lies the requirement for further research and conclusions so as to implement these methylation markers into routine CRC screening.

Ethical approval

Ethics approval was not required for this editorial article.

Consent

Informed consent was not required for this editorial article.

Sources of funding

No funding was available for the study.

Author contribution

A.B. and S.S: wrote the original manuscript, reviewed, and edited the manuscript; P.K., R.S., S.S., and H.A.S.: reviewed and edited the manuscript.

Conflicts of interest disclosure

Authors have no conflicts of interest to declare.

Research registration unique identifying number (UIN)

Not required.

Guarantor

Sangam Shah.

Data availability statement

All the required information are in the manuscript itself.

Provenance and peer review

Not commissioned, externally peer-reviewed.