The author of this article: Yuan Ying Yu Noya
Colorectal cancer is one of the most common malignancies. According to the latest data released by the National Cancer Center, it is estimated that there will be 517,100 new cases of colorectal cancer in mainland China in 2022, ranking second among malignant tumors, and 240,000 deaths due to colorectal cancer, ranking fourth among malignant tumors. About 20% of colorectal cancer patients develop metastases at the time of diagnosis, and more than 50% of colorectal cancer patients develop metastatic disease during the course of the disease. Most patients with metastatic colorectal cancer (mCRC) are incurable, except for a small number of patients with oligometastases who may be cured by a multidisciplinary approach, including surgery. For these patients with mCRC, chemotherapy and targeted therapy are the mainstays of prolonging survival.
Precise detection and precise treatment
Precision therapy based on molecular marker detection has gradually entered the clinical diagnosis and treatment practice of colorectal cancer, and the premise of precision treatment is accurate detection. How to standardize molecular testing and how to correctly interpret the test results to guide clinical treatment plans is one of the professional skills that clinicians urgently need to master.
The specimens used for the detection of molecular markers in colorectal cancer are mainly tumor tissue and blood/body fluid specimens of patients.
Tumor tissue specimens can truly reflect the mutation of genes in the tumor, but they cannot be used to diagnose genetic diseases (such as Lynch syndrome) because the source of the mutation cannot be distinguished. However, it is difficult to obtain samples due to the invasive procedures required to obtain tissue specimens. Molecular marker testing based on liquid specimens (blood, saliva, urine, ascites, pleural effusion, etc.) is an emerging method that is gradually partially replacing traditional tissue-based testing methods due to the convenience of sample acquisition and the convenience of reproducible sampling.
Among them, peripheral blood specimens include peripheral blood nucleated cells, blood cell-free fluid components, and circulating tumor cells (CTCs).
Genetic testing of blood nucleated cells can reflect the germline mutation of patients and can be used for the diagnosis of hereditary diseases. However, peripheral blood nucleated cells cannot reflect somatic mutations in tumor tissues, and cannot help clinically predict the therapeutic effect of related drugs.
CTC is a tumor cell that sheds from a primary or secondary tumor into the circulation, so it can reflect the somatic mutation of the tumor, but due to the relatively scarce distribution of tumor cells in the circulation, it needs to be isolated and enriched and identified by enriched cells, and its clinical application is still being explored.
Circulating cell-free DNA (cfDNA) in the cell-free fluid component of blood refers to extracellular DNA that is free in the blood, mainly derived from fragmented DNA released from senescent, apoptotic blood cells and tumor cells. Circulating tumor DNA (ctDNA) is one of the cfDNA, which is the free DNA that enters the bloodstream after apoptosis of tumor cells, and its detection of small residual lesions can help early diagnosis, or predict disease recurrence earlier than clinical imaging, and provide a reference for the evaluation of the efficacy of preoperative neoadjuvant therapy and postoperative adjuvant therapy.
A number of studies have shown that ctDNA can be used as a marker of postoperative recurrence risk in stage I~III. patients, and its dynamic monitoring can indicate tumor recurrence 3~6 months earlier than imaging, and may help stage II and III. patients make postoperative adjuvant treatment decisions and plan formulation. At present, ctDNA and minimal residual disease (MRD) concepts have been gradually accepted by clinicians, but they cannot be routinely carried out in clinical work because their detection methods and platforms still need to be standardized to ensure the reliability of the results.
Detection of molecular markers
Based on current guideline recommendations, testing for RAS gene (including KRAS and NRAS), BRAF V600E mutation, and microsatellite instability (MSI)/mismatch repair protein (MMR) status is strongly recommended for all patients with colorectal cancer, and HER2 amplification and NTRK gene fusion testing are recommended for patients who have failed standard therapy to guide clinical treatment.
RAS(KRAS/NRAS)
40%~50% of colorectal cancer patients have KRAS point mutations, and 3.8% of patients have NRAS gene point mutations. RAS point mutations can be detected using Sanger sequencing, PCR, and next-generation sequencing (NGS).
According to the existing guidelines and clinical practice, 5% is recommended as the cut-off value for mutation abundance in histological RAS gene detection. Several clinical studies have shown that patients with RAS mutant mCRC cannot benefit from anti-EGFR monoclonal antibody therapy, while patients with RAS wild type can benefit from anti-EGFR monoclonal antibody therapy, especially for patients with primary lesions in the left colon and rectum, and the median overall survival of patients receiving chemotherapy combined with anti-EGFR monoclonal antibody in the first line of palliative care can reach more than 55 months.
Therefore, it is recommended that patients with mCRC must clarify the status of RAS gene mutations before starting treatment, which can help patients develop a better individualized treatment plan.
FINE
In Asian colorectal cancer patients, the BRAF mutation rate is 5.4%~6.7%. Similar to the RAS gene, BRAF mutations can be detected using Sanger sequencing, PCR, NGS, and PCR is currently the most commonly used. Patients with BRAF V600E mutations have a worse prognosis and shorter survival than other patients. Both the NCCN guidelines and the CSCO guidelines recommend a combination of a BRAF inhibitor + cetuximab ± irinotecan, or cetuximab + BRAF inhibitor ± MEK inhibitor for the second-line treatment of patients with BRAF V600E mutant mCRC.
MSI status/MMR protein expression
MSI status/MMR protein expression is a predictor of the efficacy of immune checkpoint inhibitors in pan-tumor species, including colorectal cancer. For the detection of MSI, multiplex fluorescence PCR capillary electrophoresis technology is mainly used. MMR protein was detected by immunohistochemistry. In general, dMMR is equivalent to MSI-H and pMMR is equivalent to MSI-L or MSS. Patients with mCRC with dMMR/MSI-H respond well to immune checkpoint inhibitors, and PD-1/PD-L1 drugs represented by pembrolizumab have become the preferred treatment options for these patients. At the same time, MSI/MMR detection is also of great significance in the diagnosis of hereditary colorectal cancer, 95% of patients with Lynch syndrome present with MSI-H, and germline mutation of MMR gene is the gold standard for confirming Lynch syndrome.
HER2
The overall incidence of HER2 amplification/overexpression in colorectal cancer is approximately 5%. Patients with mCRC who have failed standard therapy can be tested for HER2 amplification/overexpression. However, there is currently no standard for the detection and interpretation of HER2 for colorectal cancer that has been certified by authoritative institutions. Both the NCCN and CSCO guidelines currently recommend that patients with HER2 amplification/overexpression can be treated with anti-HER2 targeted drugs, but the difference is that the CSCO guidelines recommend anti-HER2 therapy for patients with palliative first-line and beyond treatment, while the NCCN guidelines also recommend anti-HER2 therapy for patients with first-line palliative care who cannot tolerate high-intensity therapy.
NTRK
NTRK gene fusion is rare in colorectal cancer, with an incidence of 0.35%. Those who are positive for IHC screening need to be further validated with FISH, PCR, or NGS. Due to the extremely low incidence of NTRK gene fusions, testing is currently only recommended in patients who have failed standard therapy or in patients who have been screened for clinical studies. NTRK inhibitors are effective only in patients harboring NTRK fusions and not in patients with mutations.
Accurate detection is the premise of precision treatment! The detection time points and detection methods of molecular markers such as RAS, BRAF, MMR/MSI, HER-2, and NTRK can follow the consensus of the guidelines. Clinicians should fully understand the performance of each detection method when carrying out relevant molecular marker testing, and strengthen the attention and interpretation of sample types, detection methods, mutation types and other parts of the genetic testing report. NGS needs to be applied on the basis of certified platforms and technologies, strict quality control, and standardized operating procedures.
After-school research