Esophageal cancer (EC) is a fatal upper gastrointestinal malignant tumor. Approximately, there were 604,100 new EC cases and 544,076 deaths worldwide in 2020, ranking 7th and 6th in all cancer, respectively (1). The concealment of EC occurrence led to a poor clinical outcome of EC that associated with late diagnosis at advanced stage and therapy resistance, resulting in a poor 5-year survival rate of 30.3% (2). It is crucial to facilitate the EC precise diagnosis and therapy.

Recently, studies have characterized that the CpG island of the P16/CDKN2A promoter is highly methylated in EC and might be a promising biomarker for EC personalized treatment and prognosis prediction (3–5). Since only a single sample was obtained to represent the methylation status of the whole tumor in present studies generally (6, 7), a wide variation of P16 methylated rate was noticed in different studies that based on EC tissues, 40.0%–90.0%, which might be caused by ITH (intratumoral heterogeneity) that hindered its clinical application (8–14). Many studies uncovered the adverse effects of ITH on the cancer precise molecular classification, biomarker screening, and individualized precise treatment (15–17). Researches revealed that ITH can significantly affect the DNA methylation status of malignant tumors including breast cancer and lymphoma (15, 18). Since few studies focused on the impact of ITH on P16 methylation in EC (6, 19), how much it impacts on P16 methylation is still unclear.

In this study, we measured the P16 methylation level in EC and evaluated the impact of ITH on P16 methylation quantitatively to provide novel insights into the relationship between P16 methylation and EC.

Due to the unfavorable prognosis of advanced EC, a number of studies hoped to seek biomarkers that have a diagnostic value in the early stage of EC (19). P16 methylation was found prevalent in EC tissues and demonstrated a promising diagnostic, therapeutic, and prognostic value (3, 5, 22–24). However, many studies have shown large variations in detecting P16 methylation with a positive rate ranging from 45% to 88%, even in the same race and same sites (25–27). Researchers had already recognized the variation in EC and speculated that it might be influenced by ITH on tumor suppressor methylation (6, 7, 28).

In this study, we designed a systematic sampling method to measure the ITH quantitatively and the representativeness of tumor biopsy on pathological diagnosis and P16 methylation. We found a positive rate of 63.0% on P16 methylation in the tumor tissues that were obtained by the GSM, which is at the same level as others’ reports (27, 29). The positive rate of P16 methylation in nonposition-specific and center tumor tissue was 85.0% and 100.0%, respectively, which was significantly higher than that in the GSM samples. This suggests that the tumor tissue from the center position was the most representative of the whole tumor lesion compared to other positions. Thus, the ITH might influence the positive rate of P16 methylation if only one piece of tumor tissue was collected randomly (19, 28, 30). Our current study recommends that tumor tissue should be obtained at the center of the EC tumor rather than other position in future studies. Other researchers also revealed it in EC and other malignant tumor including breast cancer, liver cancer, colorectal cancer, and Ewing sarcoma (15–18).

We also noticed P16 methylation prevalent in the paired surgical margin obtained from EC patients, which was consistent with others’ studies (31, 32), revealing that P16 methylation has already occurred and developed in the “normal” tissue, and the surgical margin is not the most appropriate blank control in EC biomarker screening (5).

The tumor biopsy specimen was commonly analyzed in a number of studies, especially in studies of EC screening and early diagnosis, to represent whole individual cases (33, 34). However, researchers found that one single biopsy did not represent tumor or lesions adequately. On the aspect of pathological diagnosis, our results revealed that more than a third (35.6%) of biopsies obtained from the EC tumor surface were diagnosed as non-EC. A significant pathological difference between noncenter and center biopsies was found (58.3% vs. 88.9%). There were two patients that could be pathologically diagnosed as EC by using four biopsies from different position at least, suggesting that inadequate diagnosis (false negative) could occur easily when pathological diagnosis was made based on only one biopsy. For P16 methylation, a lower positive rate of 68.9% was found in tumor biopsies compared to tumor tissues (85.0%). Although a P16 methylation positive rate between nonspecific position, clock position, and center position was insignificantly different, a positive correlation was found between the P16 methylation rate and the severity of dysplasia lesions, which was consistent with other researchers’ reports (35). Since one-third of the biopsies were P16 methylation negative even from the center position, we considered that the position of tumor biopsy scarcely influenced the detection of P16 methylation. Nonetheless, a smaller number of biopsies might result in a higher false negative.

Among the 40 pairs of tumor biopsies and corresponding tissues, 25.0% were inconsistent on P16 methylation results in total, and with a kappa value of 0.31, had shown a lowly concordant between the biopsy and surgically resected specimens from the same patients. Taking the P16 methylation result for tumor tissues as the golden standard, a false negative rate of 23.5% and a false positive rate of 33.3% were noticed in tumor biopsies. However, Ken Hatogai et al. found that highly concordant between endoscopic biopsy and surgically resected specimens from the same EC patients in detecting PIK3CA mutation status with an overall concordance rate of 98.3% (178/181) (36), which is inconsistent with our results.

The esophageal squamous epithelium region between the tumor area and the surgical margin was considered as autologous blank control in many studies usually. However, some researchers discovered that the P16 methylation might be positive even in a histologically normal sample of EC patients, with positive rates of 36.8% (32) and 38.3% (37), which is consistent with the rate of 33.3% in our study. A lower positive rate, ranging from 0.0% to 21.3%, was also found by other researchers (26, 38). Our results suggested that this controversial finding might be induced by the length of the esophageal squamous epithelium region and the normal tissue position. It is worth noting that the positive rate of P16 methylation increased to approximately 60.0% suddenly if the biopsy position moved forward just 1 cm to the surgical margin, and it remains at a lower positive rate of P16 methylation only when it reaches over 3 cm from the tumor lesion. This might result in an uncanny false positive rate and confuse other researchers. Therefore, when an autologous blank control is essential, a normal tissue should be obtained at a position 3 cm far from the tumor lesion if conditions permit.

There were several limitations in this study. Primarily, the sample size is small, and the EC patients were heterogenous, which reduced the reliability of our findings and caused a limited extend power. This is a pilot study on the representativity of pathological diagnosis and DNA methylation of tumor suppressors applied on EC screening. We will validate the results in a larger cohort in the future. Secondly, we tested P16 methylation only, and the heterogeneity status of other tumor suppressors is still not clear yet.

Significant impacts of ITH on pathological diagnosis and P16 methylation of EC and the concordance between tumor tissue and biopsy samples were observed in our SSM analyses Several suggestions about proper sampling to improve the performance of endoscope examination/biopsy were recommended. More studies with a larger sample size, more biomarkers, and high-resolution methylation testing are needed to be implemented in a larger cohort to evaluate the sampling method in the future.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Institutional Review Board of the Cancer Hospital of Chinese Academy of Medical Sciences. The patients/participants provided their written informed consent to participate in this study.

YQ, WW, and DD contributed to the conception and design of the study and wrote the manuscript. YQ, JZ, WW, and DD interpreted the results, literature search, and construction of tables and figures. DL reviewed the pathological slides. YQ, JG, and XL contributed to questionnaire information and specimen collection. All authors contributed to the article and approved the submitted version.

This study is supported by the National Key R&D Program of China (2016YFC0901400, 2016YFC0901404) and the National Natural Science Foundation of China (81974493).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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