The Clinicopathological Correlations of hTERC Amplification with Esophageal Squamous Cell Precursor Lesions

The incidence of esophageal carcinoma has been increasing in recent years, making it the eighth most common malignancy and the sixth highest in terms of mortality worldwide [1]. There are approximately 400,000 newly diagnosed cases of esophageal carcinoma and approximately 300,000 related deaths worldwide each year, highlighting that this disease poses a serious threat to human health [2]. However, with ongoing advances in fiberoptic scopes to improve the detection rate of early-stage esophageal squamous cell carcinoma (ESCC), more cases of early-stage ESCC are being cured with surgery or radiotherapy; yet, most cases do not have obvious clinical symptoms, and most patients are diagnosed at an advanced stage of the disease, which is usually associated with a poor prognosis. Moreover, the prognosis of ESCC is still not as optimistic as other cancers of the digestive system, such as colorectal cancer. And the reported 5-year overall survival rate ranges from 15 to 40% [3, 4]. Therefore, early diagnosis and treatment of ESCC is an effective and critical approach to improve the patient survival rate.

ESCC undergoes a similar progression as squamous cell carcinoma in other tissue types, with a multistage, multifactorial, and progressive carcinogenic process. As an important stage in the progression of ESCC, esophageal squamous cell precursor lesions (ESPLs) have become the most controversial topics of the current research in terms of precise pathological grading, molecular mechanism, clinical management, and prognosis, and ESPLs are observed frequently in clinical and pathological practice. The histopathological manifestations of ESPLs are structural abnormalities (i.e., structural destruction of squamous epithelium and loss of normal cellular polarity) and cytological abnormalities (i.e., irregular size and shape of squamous cells, deeply stained nucleus, increased ratio of nucleus to cytoplasm, and more mitotic figures). Based on a 13.5-year follow-up study with 682 cases, Wang GQ et al. [5] found that the overall incidence of ESPLs was approximately 114/682 (16.7%), and ESPLs presented at various stages of squamous dysplasia, such as mild dysplasia (MID), moderate dysplasia (MOD), severe dysplasia (SD), and carcinoma in situ (CIS), with carcinogenesis rates of 2.9 (1.6–5.2)%, 9.8 (5.3–18.3)%, 28.3 (15.3–52.3)%, and 34.4 (16.6–71.4)%, respectively.

ESPLs are characterized by certain cytological and architectural changes in the esophageal epithelium. The World Health Organization (WHO) pathology and genetics classification system is the only one applied in China, and it is commonly used throughout the world. From the point of view of earlier studies, the squamous dysplasia mainly originates in the basal cells of squamous epithelium and undergoes histological and cytological microscopic changes from epithelial basal cells, progressing to the epidermal surface, until full-layer squamous dysplasia has occurred. Most pathologists are aware that the characteristic pathological morphology should be classified as MOD or SD, and most subjectively perceive that these histological features have good repeatability. However, there is no general agreement among multidisciplinary fields regarding the recognition, histological classification, management, and prognosis of these challenging ESPLs. Therefore, it is necessary to identify a molecular marker for the accurate histopathological classification and subsequent proper clinical management of ESPLs.

Telomeres are short, tandem repetitive DNA sequences located at the ends of chromosomes that contain double-stranded TTAGGG DNA sequences with a single-stranded 3′ telomeric overhang. Telomerase is a ribonucleoprotein responsible for maintaining telomere length. The telomerase core has two components: catalytic telomerase reverse transcriptase (TERT) and telomerase RNA (TERC). TERT utilizes the template region (3′-CAAUCCCAAUC-5′) of TERC to add TTAGGG DNA repeats and thereby extend single-stranded 3′ telomeric strands [6]. The human telomerase RNA (hTERC) gene is a cellular RNA-dependent DNA polymerase, a special type of reverse transcriptase. hTERC is the main component of telomerase, and it contains the template telomere DNA sequence and the telomerase protein-binding region. It also has the ability to reverse transcribe the human TTAGGG repeat sequence [7]. With the 3′ telomere as a primer, hTERC uses its own RNA as a template to synthesize telomeric DNA, whose function is to maintain telomere length at the end of chromosomes in eukaryotic cells, thereby protecting these chromosomal ends from being degraded or linked to other chromosomes [8]. In clinical studies, aggressive metastatic disease and poor prognosis have been correlated with high telomerase expression and activity in ovarian, breast, colorectal, prostate, and gastric cancer and melanoma [9], and some previous studies have shown the aberrant amplification of the hTERC gene in Barrett’s esophagus [10] and ESCC [11, 12]. In this study, we investigated the hTERC expression profile in formalin-fixed, paraffin-embedded (FFPE) ESPL tissues and retrospectively analyzed the relationship between hTERC expression and clinicopathological characteristics of ESPL patients using detailed follow-up data.

In the 2000 WHO classification [14], a two-tier system was initially introduced to replace the classical three-tier system of MID, MOD, and SD/CIS. The views of Japanese and Western pathologists have differed significantly regarding the pathological criteria of squamous dysplasia and ESCC. Before the term of intraepithelial neoplasia of the esophagus was introduced in the 2000 WHO classification [14], SD diagnosed according to the WHO classification was usually equivalent to CIS or noninvasive ESCC diagnosed by Japanese pathologists. Moreover, one particular situation is the presence of basal layer-type CIS, which is often underdiagnosed as low-/high-grade intraepithelial neoplasia by Western pathologists. In the 2010 WHO classification [12], the lower portion of MOD was grouped with MID to form the LGIEN group. The upper portion of MOD was grouped with SD/CIS to form the HGIEN group. To a large extent, these changes simplified the classification system for ESPLs and reduced the divergence among pathologists. While dealing with MOD, pathologists need to classify the lower and the upper portions into LGIEN and HGIEN according to the morphology. Then, there is inevitably subjective variability in the pathological classification. Additionally, because HGIEN ranges from some upper portion of MOD to CIS, both clinicians and pathologists still consider that the upper part of MOD should be treated differently from CIS, the latter of which is considered to require more aggressive treatment strategies. Therefore, in most Chinese hospitals, although the two-level classification criteria were adopted, most pathologists are still accustomed to adding an explicit suffix category, such as “MOD-SD” or “CIS,” as supplementary instructions to facilitate better guidance with proper management and prognosis, especially when dealing with grading of precursor oral, laryngeal, and esophageal lesions. Therefore, there are still some issues that need to be resolved in terms of the exact pathological classification of esophageal squamous epithelial lesions [15].

The most prominent clinical feature of ESPLs is the instability of bidirectional development: ESPLs can either progress into ESCC or remain unchanged at one stage for many years until they return to normal. Skacel et al. [16] analyzed the prognosis of esophageal low-grade intraepithelial lesions using 2-year and 6-month follow-up data of 25 cases and found that 60% naturally subsided, 12% remained at the original grade, and 28% progressed to high-grade intraepithelial lesions or ESCC. As the degree of dysplasia in the esophageal squamous epithelium increases, the risk of progression to ESCC also increases. Therefore, appropriate clinical treatment and management should be adopted according to different classifications of ESPLs. However, in clinical and pathological practice, the precise grading and histopathological diagnosis of ESPLs, especially for MOD or SD lesions, inevitably remains controversial. Wang GQ et al. [5] and the 2010 WHO Classification of Tumours of the Digestive System [10] both showed that SD and CIS have a similar risk of progression to ESCC and should be grouped together for clinical management. This finding may be due to the presence of residual squamous epithelium on the mucosal surface of the esophagus or incomplete histological structure of the squamous epithelium in the endoscopic biopsy tissue, which may confuse the pathological grading of ESPLs regarding whether squamous cell dysplasia exceeds one-half of the epithelium or covers the full thickness.

Telomeres have a capping function in which they protect the ends of chromosomes from degradation and fusion [17]. Telomerase activity is temporally regulated in accordance with organismal development; it is detected at the fetal and newborn stages but gradually declines to extremely low or absent levels thereafter, except in cells with higher regenerative proliferation, including hematopoietic, epidermal, and gastrointestinal cells [9, 18, 19]. Hsu et al. [11] found that the hTERT and hTERC genes were expressed in 64.9% and 79.7% of 74 specimens of ESCC, respectively. Even though telomerase expression is not related to tumor stages and prognosis, the cumulative 4-year survival rates of telomerase-positive and telomerase-negative cases were 35.9% and 31.2%, respectively (P = 0.84). In this study, hTERC gene amplification gradually increased with the severity of dysplasia in ESPLs and showed significant differences between the LGIEN (12.0%) and HGIEN (73.1%) groups in the two-tier classification, showing an approximately sixfold increase in hTERC amplification. These findings indicated that hTERC might be a valuable molecular marker for assisting with the pathological classification of ESPLs, especially for discriminating LGIEN and HGIEN. Moreover, during routine pathological practice, the pathologist could refer to hTERC expression to guide grading when encountering histological morphology with more divergence in indeterminate ESPLs between LGIEN and HGIEN. The existence of hTERC amplification may be indicative of high-risk lesions, and some pathologists would classify specimens with hTERC amplification as HGIEN; in contrast, no hTERC amplification may suggest low-risk lesions, and some specimens without hTERC amplification are more likely to be classified as LGIEN, thus enhancing the confidence of pathologists in the auxiliary classification of controversial cases of ESPLs and facilitating more appropriate and effective clinical treatment.

The altered expression of telomerase could illustrate its potential applications in diagnostic and prognostic assessments and clinical treatment, and it is expected to become a valuable molecular marker in clinicopathology. The distinct phenotypic disparity between different groups of ESPLs creates the potential for hTERC to serve as a universal cancer biomarker for disease diagnosis and prognosis. Previous studies have shown a potential correlation between high telomerase expression and poor prognosis and aggressive metastasis of some malignancies, including ovarian, breast, prostate, liver and colorectal cancer, and melanoma [9, 20‐23]. Moreover, there is a valuable correlation between telomerase expression and clinical factors related to drug resistance in breast [24] and gastric [25] cancer. Parallel in vitro studies have confirmed that TERT overexpression is associated with resistance to radiation and imatinib [26, 27]. However, studies concerning the clinicopathological relationship between hTERC dysregulation and clinicopathological features of ESPLs are rare. Combined with the exhaustive follow-up data on ESPLs, a comprehensive clinicopathological analysis explored the possible associations between hTERC expression and clinicopathological characteristics of ESPLs.

In our univariate analysis, two clinicopathological factors, pathological classification and ulcers, showed significant differences regarding hTERC amplification. As previously mentioned, hTERC amplification is correlated with both increased heteromorphism and the presence of ulcers in ESPLs. Subsequent logistic analysis further confirmed a statistically significant difference in hTERC amplification between LGIEN (12.0%) and HGIEN (73.1%); moreover, there was a higher rate of hTERC amplification in ESPLs with ulcers (68.2%) than in those without ulcers (31.8%). It is speculated that there might be some possible correlations between the factors of ulceration and hTERC amplification, which might stimulate the inflammatory factors and promote the progression of ESPLs into ESCC. Whether all of these speculations are reasonable or not, large sample studies are still necessary for further verification. In addition to these two parameters, hTERC amplification showed no statistical correlation with any other clinicopathological parameter of ESPLs. Although a difference in hTERC amplification between LGIEN and HGIEN was found in this study, we still did not find a clinicopathological correlation between hTERC and the progression of ESPLs. The reasons may be manifold: The sample size of the current study may be not sufficient to achieve a convincing conclusion, and the research designed here still needs further refinement, optimization, and improvement. However, a potential application value of the hTERC gene for classifying ESPLs was inferred.

Briefly, the current preliminary study potentially indicates that hTERC expression may be one of the factors related to the classification of pathological ulcers, which may be involved in the carcinogenic inflammatory mechanisms in ESPLs. In terms of the relationship between hTERC expression and ulcerative factors associated with ESPLs, the underlying mechanism of hTERC in this process remains unclear. There may be synergistic effects or interactions between inflammatory factors and the hTERC gene in the pathogenesis or progression of ESPLs, but these speculations require further in-depth experimental research to clarify. The relationship between hTERC and pathological ulcers may provide important clues and practical significance to perfect the pathological classification system used to guide clinical management and prognosis of ESPLs and to fundamentally reduce the incidence and progression of early ESCC. It is necessary to fully understand the role of hTERC in the molecular mechanism and characteristics of ESPL development for digestive system pathologists to reasonably assess the prognosis of ESPLs and to implement appropriate and effective treatment.