Clinicopathological features and BRAFV600E mutation analysis in colorectal cancer

INTRODUCTION

Colorectal cancer (CRC) is the third most common cancer worldwide, causing considerable cancer-related deaths in both sexes.^[1] The prevalence of colorectal cancer varies globally due to factors such as demographics, ethnicity, sedentary lifestyle, culture, genetics, and intake of high carbohydrate and low fiber diets. Over the last few years, the global incidence of CRC is about 151030; affecting 53.4% of men and 46.57% of women.^[2] There is variation in the incidence rate of CRC within Asia, being uniformly low in all South Asian countries and high in all developed Asian countries.^[3] In India, the incidence rates of CRC in males and females are 4.3 and 3.4 per 100,000, respectively, but recent studies have shown an increasing trend in the Indian population.^[4]

CRC is a divergent disease resulting from multifarious genomic and epigenomic aberrations involving several signaling pathways. Based on the genetic mutation, CRC can be categorized as inherited, familial, and sporadic type, of which sporadic cases form a major share.^[5] Three main molecular pathways due to several mechanisms are identified in the pathogenesis of CRC. 1. Chromosomal instability (CIN). 2.Microsatellite instability (MSI). 3. CpG island methylator phenotype (CIMP) pathway.^[6,7] The BRAF (Rat sarcoma virus RAF and B-Raf proto-oncogene) gene is a valuable biomarker among the various genetic mutations.^[8] BRAF-mutated CRC is associated with the worst overall (OS) survival and is considered a negative prognostic marker of CRC compared to wild-type BRAF.^[9] According to the English literature on CRC among Asian countries, India has the highest BRAF prevalence rate of 11.1%, and the lowest number of 1.1% is reported from Taiwan. In contrast, China has a heterogeneous distribution of genetic mutations.^[10] As per the location, the colon has the highest rate of BRAF mutations at 67.9% and a higher incidence of 59.9% in an advanced stage of cancer.^[10] The BRAF oncogene is found on chromosome arm 7q34, which has 18 exons and codes for a 766 amino acid peptide with a molecular weight of 84 kDa. It produces a serine/ threonine kinase, a vital part of the RAS/RAF/MEK/ERK signaling cascade that encourages cell growth.^[11,12] Numerous mutations have been identified in BRAF, the most prevalent of which is the Thymine to Adenine transversion (c.1799T>A) inside codon 600, which results in the amino acid level substitution of valine by glutamate (p.V600E). BRAFV600E mutation is seen in nearly one in ten patients with advanced colorectal cancer and approximately accounts for 5 to 9 % of the total cases. Some studies have reported that the V600E substitution is linked to specific clinicopathological characteristics such as proximal localization, microsatellite instability, mucinous histology, and chemotherapy resistance.^[7,13,14] Non-V600E BRAF mutations are seen in younger CRC patients with a better prognosis.^[15] In India, a literature survey has shown few reports from North India and no reports from South India.^[15-19] In the present study, we analyzed the prevalence of BRAF mutations in CRC and correlated them with clinicopathological characteristics.

MATERIAL AND METHODS

The present study was conducted in the Department of Pathology, in a Tertiary Care Institution in South India, for eighteen months after approval from the Institutional Ethics Committee (IEC Ref No: 72/19/IEC/JMMC&RI). Informed consent was obtained from all the patients included in the study whose specimens were received in the department, and the Declaration of Helsinki procedures were followed. Formalin-fixed paraffin-embedded (FFPE) samples (85) of primary CRC were selected. All other non-epithelial malignant tumors of the colon and patients who received neoadjuvant radiotherapy/chemotherapy were excluded.

RESULTS

Histopathological evaluation and molecular analysis

The size distribution of tumors was 0-5 cm in 45 (52.9%), 6-10 cm in 36 (42.4%), and 11-15cm in 4 (4.7%) cases. In our study majority of tumors were located in the sigmoid colon (31.8%), followed by the rectum (16.5%), ascending colon (14.1%), hepatic flexure (10.6%), caecum (7.1%), transverse colon (5.9%), descending colon (4.7%), and splenic flexure (1.2%). Regarding the tumor grade, 78 (91.8%) were low-grade tumors, and 7 (8.2%) were high-grade. The p-value for tumor grade and size was not statistically significant (P=0.638). In this study, pT3-43 (50.6%) was the most frequent tumor pT stage among the 85 study participants, while pT1- 2 (2.4%) was scarce. pN0- 43 (50.6%) was the most common nodal status, while pN2- 2 (2.4%) was infrequent. BRAF mutation analysis was performed in all cases. In our study, only 2.4% (2 patients) were positive for the BRAFV600E mutation, and the remaining were negative. [Figures 1 and 2]

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Figure 1:

Representative ethidium bromide-stained agarose gel showing wild type (97bp) and control (200bp) bands. Marker (M) is of 50bp ladder mixed with 2X gel loading buffer. Patient samples: Lanes1,2,3 and 4.

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Figure 2:

Representative ethidium bromide-stained agarose gel showing BRAF mutation (144bp). Marker (M) is of 100bp ladder mixed with 2X gel loading buffer. Lane 1: Positive case. Lanes 2 – 6: Negative cases.

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DISCUSSION

CRC is the third most common cancer leading to cancer-related mortality globally. Early diagnosis of colorectal cancers, surgical treatment, and response to adjuvant therapy have contributed to better outcomes in patients. The revelation of different signaling pathways in CRC has revolutionized the therapeutic perspective due to the use of combined targeted therapy, thereby prolonging the overall survival period.^[21] Mainly, three diverse molecular pathways: the chromosomal instability pathway (mutations in P53 and KRAS), the mutator pathway (loss of function of DNA mismatch repair proteins), and the serrated pathway (BRAFV600E mutation) lead to CRC. Among these, the BRAFV600E mutation is usually observed in CRC with microsatellite instability (MSI). Screening is essential as patients harboring the BRAF mutation have an aggressive outcome and are unlikely to have Lynch syndrome.^[22]

In our study, patients older than 65 years (55.3%) were the predominant age group involved. Gender-wise, CRC was greater in males (63.1%) than in females (36.5%), which is identical to the earlier reports from India and worldwide.^[10,22-24] In this study, the age and gender of the patients did not show any statistically significant correlation. This disparity could be attributed to the varying sample sizes employed in different individual studies. The difference in male-to-female incidence in CRC is pertinent in prognostication, as males have a poorer outcome.^[10] As the genetic pathways of CRC in the proximal and distal colon are divergent while some mutations are mutually exclusive, the location of the tumors is crucial.^[25] In our cohort, the sigmoid colon (31.8%) was the preponderant site, followed by the rectum (16.5%), which is comparable to previous reports from Asia, including India.^[22] There are isolated Indian reports of a higher number of right-sided colonic cancer and rectal cancer in younger individuals.^[26,27] According to the ethnicity of the study samples, the worldwide BRAF mutation rate ranges from 7- 10%.^[8]

In this study, BRAFV600E mutation analysis was performed on all 85 cases, of which two cases were positive (2.4%).^[16] This is the same as the Indian study report by Lashkar et al on rectal cancers.^[15] Other Indian reports showed varying numbers of BRAF positivity; the highest number of 21% of BRAF mutation in CRC is reported by R. Eachkoti et al.^[17-19,23] The Asian population shows a lower frequency of BRAFV600E mutation compared to the Caucasian population.^[28] However, one report from Iran recorded a complete lack of BRAF mutation in CRC.^[29] Our results of low BRAF positivity concur with overall low incidence in Asia compared to the West. In this study, we found BRAF mutation in each male and female patient with low-grade tumors located in the right and left colon, respectively, in contrast to the previous report of BRAF mutation commonly found in females >70 years and the right colon.^[30] Patil et al.^[31] found a correlation of BRAF with low-grade carcinomas.^[31] As T tumor stage (BRAFV600E mutation frequency is substantially greater in stage II/III than in stage IV) is still debatable in terms of how well it predicts outcomes in early disease, and more research is needed.^[30] The majority (50.6%) of the cases in our study had pT3 tumors, and the least cases (2.4%) had T4 tumors, which should alert the treating physician to BRAF mutational testing. A survey by Lie et al.^[32] reported a strong association of BRAF mutation with clinicopathological parameters like age, gender, location, histological type, and TNM staging, which was conspicuously absent in our study similar to the previous Indian data.^[23]

The role of BRAF mutation in the prognosis of CRC is well recognized and is typically poorer.^[12] Due to the increasing significance of this mutation, the National Comprehensive Cancer Network Guidelines now recommend BRAF mutation testing in patients with metastatic disease.^[33] Moreover, BRAF mutant metastatic CRCs present a unique metastatic pattern, showing high rates of peritoneal metastases and distant lymph node metastases.^[34] In the present study, there was no metastasis recorded in both BRAF-positive cases. Additionally, BRAF mutant metastatic CRC patients frequently show an early resistance to targeted therapy, and only about half of these patients respond to second-line chemotherapy, suggesting that more aggressive and individualized combined therapies may be effective in selected patients’ cohorts.^[34] Recent studies have proved the conjunctive efficacy of targeted therapy using EGFR and BRAF inhibitors in metastatic CRC.^[35] There is only one documented Indian report of Non-BRAF V600E mutation.^[17]

On follow-up with our BRAF-positive patients, the first female patient, an elderly person, did not receive chemotherapy and succumbed to colonic cancer during the pandemic, five months after the right hemicolectomy. The second male patient on colostomy following sigmoid resection was deferred chemotherapy due to comorbidities, such as uncontrolled diabetes mellitus, hypothyroidism, cirrhosis liver, and renal failure. Four years later, he died following liver metastasis.

The limitation of this study was the small sample size. A collaborative study of molecular tests in a large Indian population is necessary for explicit mutational data in CRC.

CONCLUSION

This is the first South Indian cohort study of CRC on BRAF mutation. The small sample size, demographic disparities in the study population, and pre-analytical factors could explain the low positivity number. In the future, routine molecular testing for various biomarkers, including BRAF may become inevitable in targeted therapy of CRC for practicing precision medicine.