Abstract
Background/Aim: The prognostic significance of proliferating cell nuclear antigen (PCNA) has not yet been defined in either colorectal adenoma or carcinoma. This study aimed to compare the differences in PCNA expression between pathologically altered tissue (polyp, adenoma, carcinoma) and the surrounding tissue (termed PCNA expression difference, PCNA-ED) as a potential prognostic marker in colorectal tumor progression.
Patients and Methods: Tissue specimens used for this study were obtained from 63 non-neoplastic epithelial polyps, 211 colorectal adenomas, and 156 colorectal adenocarcinomas, as well as adjacent normal mucosa.
Results: PCNA-ED was absent in non-neoplastic polyps, present in 13.7% of adenomas, and significantly more frequent in adenocarcinomas (33.3%). Higher PCNA-ED correlated with increasing adenoma size, grade of dysplasia, depth of invasion, and Dukes staging. High PCNA-ED was significantly associated with early recurrence, lymphovascular invasion, liver metastases, and reduced survival. Among patients with Astler-Coller B2 tumors, high PCNA-ED suggested a need for adjuvant chemotherapy.
Conclusion: PCNA-ED is strongly associated with malignant transformation, recurrence, and adverse prognosis in colorectal neoplasms. Its measurement may offer valuable prognostic insight and inform postoperative management strategies.
Introduction
One of the most interesting models of human carcinogenesis is the so-called adenoma-carcinoma sequence in the colon, namely, the development of a cancer from a dysplastic, polypoid focal precursor: the adenoma (1-3). Although most colorectal carcinomas are thought to originate in colorectal adenomas, most adenomas, which are quite common, do not progress into invasive carcinomas, and only 1-10% of people with resected adenomas later develop invasive cancer (1, 4). Given the high prevalence of adenomas (4), in contrast to the rarity of colorectal cancer, identifying risk factors for dysplastic lesions may facilitate the development of strategies to prevent the process leading to colorectal cancer. Because disruptions in cell growth regulation occur early and accumulate throughout carcinogenesis, assessing proliferative activity across stages of the adenoma–carcinoma sequence can provide valuable insights into the biology of colorectal cancer (5).
The prognostic significance of proliferating cell nuclear antigen (PCNA) in colorectal adenoma or carcinoma has not yet been clarified; the relationship between PCNA expression and clinical outcome is unclear. According to previous studies, PCNA is significantly increased in adenomas with high-grade dysplasia (6) and may be a useful marker for the morphological progression of invasive colonic polyps (6-8). However, other researchers have found no predictive value of PCNA expression in the state of adenomas (5, 9, 10). In case of colorectal adenocarcinoma, several authors have found a correlation between PCNA and improved survival (11), whilst others have concluded that PCNA expression is not related to tumor progression (12).
In this study, we aimed to determine the difference in PCNA expression between pathologically altered tissue (polyp, adenoma, carcinoma) and the surrounding normal mucosa in the same patient- a parameter we defined as PCNA expression difference (PCNA-ED). We then examined the correlation between PCNA-ED and clinical and pathological variables of colorectal polyps, adenomas, and carcinomas. Particular attention was placed on evaluating whether PCNA-ED could be a potential prognostic marker in human colorectal carcinogenesis and tumor progression.
Patients and Methods
Tissue specimens. Tissue specimens used for this study were obtained from 63 non-neoplastic epithelial polyps, 211 colorectal adenomas, and 156 colorectal adenocarcinomas as well as adjacent normal mucosa (Table I, Table II, Table III, Table IV), which were either obtained by endoscopic polypectomy or resected surgically at the 1st Department of Surgery, Semmelweis University, Budapest, Hungary from 2002 to 2017. Corresponding adjacent normal mucosa were obtained either from adjacent tissues (in the case of endoscopically excised samples) or from the surgical resection margins of the original specimens. To justify comparisons, lesions from patients with known familial colon cancer syndromes as well as suspected de novo cancers were excluded. The investigation was performed with the consent of the patients given based on adequate information provided to them and approved by the Semmelweis University Regional and Institutional Committee of Science and Research Ethics. This study was performed in accordance with the Helsinki Declaration.
Clinico-pathological variables of lesions and their correlation with PCNA expression difference between pathological alteration and surrounding tissue (PCNA-ED).
Correlation of pathological variables of adenomas with PCNA expression difference between pathological alteration and surrounding tissue (PCNA-ED).
Correlation of recurrence of adenomas with pathological variables and PCNA expression difference between pathological alteration and surrounding tissue (PCNA-ED).
Correlation of clinico-pathological variables of colorectal adenocarcinomas with PCNA expression difference between pathological alteration and surrounding tissue (PCNA-ED).
In the revised Vienna Classification (rVC), histopathologic diagnoses are classified into five categories according to neoplastic severity and depth of invasion. This classification also distinguishes between epithelial neoplastic lesions limited to the mucosa and those invading the submucosa (13). The recommended two-tiered grading system also permits translation of histopathology findings of Western and Japanese pathologists into a uniform system for classification of colorectal neoplastic lesions. Classically, adenomas are divided into tubular, tubulovillous or villous types and demarcation between the three is based on the relative proportions of tubular and villous components, according to the “20% rule” described in the WHO classification of tumors in the digestive tract (14). The stage of colorectal carcinomas was performed according to the seventh edition TNM (15) and the older Dukes classification. The modified Dukes classification system was subsequently complemented by the addition of a fourth stage (D) where the tumor has spread to other organs (16).
Immunoblot analysis of PCNA antigen. Expression of PCNA was determined by immunoblot analysis of tumor tissue and adjacent normal mucosa specimens. Cell nuclear proteins were isolated in the samples using a method described by Dignam et al. (17). Cell nuclear proteins were separated via SDS-PAGE, then blotted to nitrocellulose membranes, and exposed to mouse monoclonal anti-PCNA antibody (clone PC-10, DAKO, Glostrup, Denmark) at 2.4 mg/l. The immunoreaction was detected with the help of horseradish peroxidase-labelled anti-mouse antibody (DAKO) and marked by the development of chemiluminescence (ECL kit, Amersham, Buckinghamshire, UK). Preliminary experiments have shown that nuclear extract samples representing 3 mg of original tissue were sufficient for detecting significant PCNA-ED. Densitometry using OneDScan software of Scanalytics (Fairfax, VA, USA) was used to define the amount of PCNA antigen that could be detected as a result of the immunoblot process. The determined optical density values (OD) were compared with the ones of a positive control, an established American Tissue Culture Collection (ATCC) colon cancer cell line, HT-29. False-positive results, identified through reactivity with the secondary antibody alone, were excluded from the analysis. A positive control was included with each examined sample to ensure validity of the results. As a positive control, nuclear extract protein was aliquoted, kept at −80°C and all aliquots were used only once. If a new positive control nuclear extract was used from another cell nuclear protein isolation, it was calibrated to the previous one. Since an established ATCC cell line was used as standard, and the cell nuclear protein isolation method was reproducible and quick, the variability within the method of the positive controls was less than 10%. The expression level was determined as % of the positive control, both in the tumor sample and the adjacent peritumoral mucosa separately, and was graded with scores as follows: no expression detected at the sensitivity of the method, no band seen in the western blot marked with [−]; less than 25% of the positive control, but detectable band marked with [+] (low expression); 25-50% of positive control marked with [++] (medium expression); more than 50% of the positive control marked with [+++] (high expression).
We studied the difference in PCNA expression in tumors compared to surrounding normal mucosa (PCNA-ED), the latter used as a reference. PCNA-ED was assessed semi-quantitatively by calculating the difference in expression scores between the tumor tissue and the adjacent normal mucosa. Based on this assessment, three categories of PCNA-ED were defined: no PCNA-ED (if there was no PCNA expression either in the tumor or in the surrounding tissue [− and −], or both had low [+ and +], or medium [++ and ++], or high PCNA expression [+++ and +++]), low PCNA-ED (if the difference was only one +), and high PCNA-ED (if the difference was at least ++) (Figure 1).
PCNA expression patterns in the colorectal adenoma–carcinoma sequence. PCNA protein was detected as a 36 kDa band in adenomas and as both 36 kDa and 72 kDa bands (representing dimeric or complex forms) in colorectal adenocarcinomas. Expression levels were analyzed in neoplastic lesions – namely polyps, adenomas, and carcinomas – and compared with adjacent normal mucosa using immunoblotting. Densitometric evaluation categorized expression into four levels: none (−), low (+), moderate (++), and high (+++). The PCNA expression difference (PCNA-ED) was defined as the difference in expression levels between neoplastic tissue and the corresponding adjacent normal mucosa. Cases with equal expression levels in both tissues were categorized as showing no PCNA-ED. A one-category difference (e.g., + vs. − or ++ vs. +) was classified as low PCNA-ED, while a difference of two or more categories (e.g., +++ vs. + or ++ vs. −) was considered high PCNA-ED. S: Surrounding mucosa; A: adenoma; C: carcinoma; CRC: colorectal cancer; PA: pathological alteration (polyp, adenoma, or carcinoma).
Statistical analysis. A two-tailed Student’s t-test was used to compare variable pairs. Patients were grouped based on the presence or absence of PCNA-ED, and the distributions of clinical and pathological variables were compared between these two groups. Chi2-test was used for comparison of distribution between populations showing no PCNA-ED, or showing high or respectively low PCNA-ED. For all tests, p-value <0.05 was considered to be statistically significant. The student’s t-test provided a better picture of the presence or absence of PCNA-ED distribution amongst the patients, whilst Chi2-test proved to be informative regarding the distribution and significance of high or low PCNA-ED modulations. In addition, linear regression was used.
Results
Clinico-pathological variables. All patients were followed up for at least three years after surgery. Patients with colorectal cancer were routinely studied for tumor marker serum levels as well as using diagnostic imaging four times a year.
Sex, age, size, location. Hyperplastic polyps and juvenile polyps were more often diagnosed amongst males, whilst Peutz-Jeghers polyp and those associated with Crohn’s disease were more frequent in females. Inflammatory polyps associated with ulcerative colitis showed an approximately equal sex distribution. Adenomas were more commonly diagnosed in males, whereas colorectal adenocarcinomas were more frequently observed in females (Table I).
In comparison to patients with adenomas, patients with non-neoplastic epithelial polyps were significantly younger, and their tumors were significantly smaller (p<0.05 and p=0.03 with Student’s t-test, respectively). There was no significant difference in the age distribution between patients with adenomas and those with adenocarcinomas (p=0.336 with Student’s t-test); however, adenocarcinomas were significantly larger in size (p=0.009 with Student’s t-test).
No correlation was found between the type of adenoma and the patients’ age; different types of adenomas were characterized by the same mean age (p=0.37 with Student’s t-test). The villous polyps were significantly larger than both tubular (p<0.05 with Student’s t-test) and tubulovillous polyps (p<0.05 with Student’s t-test) (Table I).
The majority of the hyperplastic polyps (86.20%), 57.14% of the hamartomatous polyps, and 74.07% of the inflammatory polyps were located in the distal colon and rectum. In case of adenomas: 5.7% were located in the segment of the large intestine proximal to the ascending colon, 18.48% near the transverse colon, 22.74% near the descending colon, and 53.08% in the rectosigmoid region. Additionally, 51.28% of colorectal adenocarcinomas were located in the rectum (Table IV).
Pathology. The histological diagnosis of 211 adenomas identified 43.12% of the total cases were tubular, 30.33% tubulovillous, and 26.55% villous. 76.91% of the tubular adenomas and 68.75% of the tubulovillous adenomas showed low-grade neoplasia, whilst 39.28% of the villous adenomas showed high-grade neoplasia. Carcinoma in situ was diagnosed in 8.81% of the tubular adenomas, in 12.51% of the tubulovillous adenomas, and in 16.08% of the villous adenomas (Table II). The size of the adenomas correlated with the grade of neoplasia (p<0.05 with Student’s t-test) (Table II). Clear linear relationship was found between the size of the adenoma and the grade of the mucosal neoplasia (R2=0.97 for tubular and tubulovillous and 0.99 for villous adenomas by linear regression) (Figure 2C).
Correlation between grade of mucosal neoplasia, size and PCNA-ED in colorectal adenomas. A) Relationship between grade of neoplasia and PCNA-ED. Degree of dysplasia: 1: mild; 2: moderate; 3: severe; 4: Tis. B) Relationship between size of adenoma and PCNA-ED. C) Relationship between degree of dysplasia and size of adenoma. Degree of dysplasia: 1: mild; 2: moderate; 3: severe; 4: Tis. The relationships in all cases were analyzed by linear regression, and the trend line is visible on the chart for tubular, tubulovillous and villous adenomas.
Among the 156 colorectal adenocarcinoma patients, 41.02% were diagnosed at Dukes B stage, 33.33% at Dukes C, and 25.65% at Dukes D. Most adenocarcinomas (87.17%) were moderately differentiated, while 7.69% were well-differentiated and 5.14% poorly differentiated (Table IV).
Follow-up (recurrence, metastasis, survival). In case of patients with non-neoplastic polyps, no recurrence was found within three years, and overall survival was 100%. In the adenoma group, three patients died from cardiovascular disease, whilst recurrence was observed in 24.17% of the patients. Recurrence showed significant correlation with the size of the adenoma. Adenomas exceeding a threshold of 15 mm in diameter were associated with a significantly increased risk of recurrence (p<0.05 with Student’s t-test). The histological types of adenomas were significantly associated with differences in recurrence rates (p<0.05 with Chi2-test). Recurrence was the most frequent and happened earlier in villous adenomas (p<0.05 with Chi2-test). Both 1-year and 1-3-year recurrence happened statistically more frequently in cases of mucosal high-grade neoplasia as compared to adenomas with low-grade neoplasia (p<0.05 with Chi2-test). Although, Tis had lower frequency of recurrence than adenomas with severe dysplasia, the recurrence happened relatively earlier (Table III).
Recurrence was observed in 30.76% of the adenocarcinoma patients, whilst 10.25% of the patients developed metachronous metastases. More than half (53.84%) of the patients died due to tumor progression (Table IV).
PCNA expression and PCNA-ED in the adenoma-carcinoma sequence. PCNA protein expression was not detectable in all cases of polyps, colorectal adenomas, carcinomas or in the surrounding histologically tumor-free normal mucosa. However, if PCNA expression was detected, the expression was either equal or higher in the pathologically altered tissue than in the surrounding mucosa. Based on this, PCNA-ED was classified according to the description in Patients and Methods. Low expression of PCNA was found in 23% of the normal colon epithelium whereas such expression was moderate in 2% and high in 6% of the cases. In non-neoplastic polyps and adenomas where PCNA expression was detected, only the 36kDa form was present, either in the pathological lesion or the adjacent normal mucosa. In contrast, both the 36kDa and the 72kDa (dimeric or complex) forms were observed in colorectal adenocarcinomas and their surrounding mucosa (Figure 1).
While PCNA-ED was not detected in non-neoplastic polyps, it was observed in 13.73% of adenomas and 33.32% of adenocarcinomas. Among adenomas, the distribution pattern of PCNA-ED was not statistically significant (Student’s t-test: p=0.73; Chi2 test: p=0.7–0.8). Although in villous and tubulovillous adenomas the PCNA-ED was higher than in tubular adenomas, this difference was not statistically significant (p=0.8 with Chi2-test). The PCNA-ED pattern in adenomas and adenocarcinomas was significantly different (p=0.004 with Student’s t-test, p=0.008 with Chi2-test). Both low and high PCNA-ED was more frequent in adenocarcinoma cases than in adenomas (p=0.008 with Chi2-test) (Table I).
Relationship between variables and PCNA-ED pattern in colorectal adenoma. Grade of mucosal neoplasia of adenomas and PCNA-ED. In adenomas, the frequency of PCNA-ED increased with the grade of neoplasia (R2 =0.99 for all types of adenomas by linear regression) (Figure 2A). Both low and high PCNA-ED became more prevalent with higher neoplastic grades, while the proportion of cases without PCNA-ED declined accordingly. Within each neoplasia grade, the distribution of no, low, and high PCNA-ED was consistent across tubular, tubulovillous, and villous adenomas (p=0.7-0.9 with Chi2-test) (Table II).
Size of adenomas and PCNA-ED. An increase in adenoma size was significantly correlated with higher PCNA-ED (p<0.05 with Student’s t-test). This correlation was linear (R2=0.97 for tubular, R2=0.96 for tubulovillous, and R2=0.99 for villous adenomas) (Table II) and (Figure 2B).
Grade of mucosal neoplasia and size of adenomas and PCNA-ED. A clear linear relationship was found between the size of the polyp and the grade of neoplasia (R2=0.97 for tubular, R2=0.96 for tubulovillous and R2=0.99 for villous adenomas by linear regression) (Figure 2C). The grade of neoplasia, the size of the polyp, and the frequency of PCNA-ED detection amongst the patients were correlated (Table II and Figure 2).
Recurrence and PCNA-ED. Recurrence was developed within the first year in all patients with high PCNA-ED. Amongst patients with low PCNA-ED, 95% showed recurrence, 70% of them in 1-3 years. The low PCNA-ED played the prominent role in the group with 1–3-year recurrence, whilst in the group with recurrence within 1 year, the high PCNA-ED was more dominant. PCNA-ED was represented at only 0.57% (only a low PCNA-ED) in cases showing no recurrence within three years. The distribution of PCNA-ED in cases with recurrence was highly statistically significant (with Chi2-test p<0.05). These data suggested a strong correlation between PCNA-ED and the risk of recurrence, whilst high PCNA-ED was highly correlated with early recurrence (Table III).
Relationship between variables and PCNA-ED pattern in colorectal adenocarcinoma. Age and PCNA-ED. Although PCNA-ED is relatively similarly distributed within age groups (p=0.66 with Chi2-test), the high PCNA-ED for the 72 kDa PCNA protein form has shown significantly higher occurrence amongst younger patients (age <50, p<0.05 with Student’s t-test) (Table IV).
Sex and PCNA-ED. PCNA-ED distribution is related with sex. The distribution of PCNA-ED for both the 36 kDa and 72 kDa PCNA protein forms differed significantly between male and female patients (with both Student’s t-test p=0.05 and with Chi2-test p<0.05). PCNA-ED was more frequently found in female patients. Male patients more frequently exhibited high PCNA-ED for the 36 kDa form, while low PCNA-ED was rare. This suggests that when PCNA-ED was present in male adenocarcinoma patients, it typically indicated a more advanced state (Table IV).
Tumor location and PCNA-ED. The distribution pattern of PCNA-ED for both the 36 kDa and 72 kDa PCNA protein forms differed between patients with colon and rectal tumors (p<0.05 with Chi2-test). The colon tumor patients had more frequently the 72 kDa PCNA-ED (p=0.002 with Student’s t-test). For the 36 kDa form, low PCNA-ED was more common in colon tumors, whereas high PCNA-ED was more frequently observed in rectal tumors (p=0.01 with Student’s t-test) (Table IV).
Tumor size and PCNA-ED. The 36 kDa PCNA-ED was more frequent in medium-sized tumors, although high levels of this form were predominantly found in small tumors (p<0.05 with Chi2-test). Differential expression of the 72 kDa PCNA form was significantly more prevalent in tumors larger than 30 mm (p<0.05 with Student’s t-test). Above 30 mm, the PCNA-ED for 72 kDa was more frequent, but it did not further increase with tumor size; tumors of 30-55 mm or above 55 mm showed the same frequency of PCNA-ED (p=0.66 with Student’s t-test) (Table IV).
Necrosis in tumor and PCNA-ED. Extensive necrosis was related to a higher frequency of 36 kDa PCNA-ED (p=2.8×10−4 with Chi2-test if compared with moderate necrosis), which mainly resulted from the increase in cases with low PCNA-ED. Extensive and moderate necrosis correlated with the 72 kDa PCNA-ED in the same way (p=0.99 with Chi2-test, p=0.93 with Student’s t-test). If necrosis was absent in the tumor, PCNA-ED was detected at a low frequency (Table IV).
Histological differentiation and PCNA-ED. Although the distinct differentiation categories have shown distinct PCNA-ED distributions (p<0.05 with Chi2-test and Student’s t-test), no clear relationship was found. This may be due to differences in patient distribution among the grading categories (Table IV).
Modified Dukes stage and PCNA-ED. Dukes C and ‘D’ stages were related with higher PCNA-ED (p<0.05 with Chi2-test and Student’s t-test) than Dukes B stage. The relationship between 36 kDa PCNA-ED and Dukes C or ‘D’ was the same (p=0.45 with Chi2-test), whilst high PCNA-ED of the 72 kDa form was more frequent in Dukes C than Dukes ‘D’ (p<0.05 with Chi2-test) (Table IV).
Depth of tumor invasion and PCNA-ED. The frequency of PCNA-ED for both the 36 kDa and 72 kDa forms showed an increasing trend with greater depth of invasion (T1 to T4) (p<0.05 with Chi2-test). In case of the 36 kDa form, the PCNA-ED appeared to be higher in T4 tumors compared to T3. The 72 kDa PCNA-ED appeared higher in T3 tumors compared to T4. These differences did not reach statistical significance (p=0.07 for the 36 kDa form and p=0.97 for the 72 kDa form, based on comparison of high PCNA-ED with all other categories using Student’s t-test). However, it is important to note that the number of patients differed substantially between the T3 and T4 groups (Table IV).
Lymph node metastasis, lymphatic invasion and PCNA-ED. More than four positive lymph nodes were associated with a higher frequency and greater intensity of PCNA-ED (p<0.05 with Chi2-test). Similarly, a significant increase (p<0.05 with Chi2-test) in PCNA-ED detection and values was observed in cases with lymph vessel invasion (Table IV).
Venous invasion, liver metastasis and PCNA-ED. Venous invasion was significantly associated with both increased frequency and higher values of PCNA-ED (p<0.05, Chi2 test). Liver metastases consistently correlated with a higher frequency of PCNA-ED (p<0.05, Chi2 test). In cases of synchronous metastases, the occurrence of PCNA-ED for the 36 kDa form was more frequent, whereas in metachronous metastases, the intensity of PCNA-ED was greater (p<0.05, Chi2 test; Table IV).
Recurrence and PCNA-ED. PCNA-ED was not related with recurrence within three years (p=0.9 with Student’s t-test). It is noteworthy that the patients who had recurrent disease within three years more frequently exhibited high PCNA-ED (Table IV).
Survival and PCNA-ED. The patients who died within three years had higher PCNA-ED (p<0.05 with Chi2-test), and PCNA-ED was less frequently detected in patients with a survival longer than three years (p<0.05 with Chi2-test) (Table IV).
Discussion
The role of PCNA in colorectal tumorigenesis is not yet clear (18). Several studies have reported a positive correlation between the PCNA labeling index and variables such as adenoma size, grade of mucosal neoplasia, and recurrence risk (6-8) In contrast, other investigators have concluded that PCNA expression alone is not a reliable marker for tumor transformation in the intestinal mucosa (5, 9, 10). For example, Sun et al. (12) found no significant association between PCNA expression and prognosis in colorectal cancer, whereas Paradiso et al. (11) reported a correlation with improved survival in advanced cases.
In our previous study, univariate analysis indicated that PCNA expression in tumor tissue was independent of patient age, sex, tumor location, size, necrosis, histological differentiation, and primary tumor stage. However, high PCNA expression was significantly associated with pN stage, venous invasion, and lymphatic invasion, but not with depth of invasion, recurrence, metastasis, or overall survival (19).
Importantly, in these studies, PCNA expression was evaluated solely within tumor tissue and not in comparison with adjacent normal mucosa. To address this gap, the present study aimed to assess the difference in PCNA expression between pathologically altered and adjacent normal tissue (PCNA-ED) across colorectal polyps, adenomas, and carcinomas. To our knowledge, this is the first study to evaluate PCNA-ED as a potential marker in the colorectal adenoma–carcinoma sequence.
Nishimura et al. (20) reported that approximately 30% of normal colonic mucosa showed PCNA positivity. Similarly, we found PCNA expression in 31% of normal colon epithelium. These findings suggest that elevated PCNA expression in histologically normal mucosa reflects baseline proliferative activity and, by itself, is not indicative of poor prognosis (10).
Whilst in non-neoplastic polyps PCNA-ED was not detected, this difference was already visible in the colorectal adenomas and statistically more often in the colorectal adenocarcinomas. Taking into account the low prevalence of PCNA-ED in adenomas with mucosal low-grade neoplasia, and its high prevalence in adenomas with mucosal high-grade neoplasia, or microinvasive carcinomas, we believe that PCNA-ED reflects cell proliferation changes in the adenoma-carcinoma transformation process. PCNA-ED has not been observed either in hyperplastic polyps, hamartomatous polyps, ulcerative colitis and Crohn’s disease mucosa or in adenomas with mild dysplasia, which suggests that PCNA-ED is not an early feature during colorectal carcinogenesis.
No recurrence was observed within three years among patients with non-neoplastic polyps; however, approximately 25% of patients with adenomas experienced recurrence during the same period. Many data exist to support the fact that size, grade of mucosal neoplasia and histological subtype of adenoma are related to the risk of recurrence and, in particular, subsequent cancer (2, 4). Our observations concerning recurrences essentially corroborate the prevailing concept: size equal to or higher than 15 mm diameter, mucosal high-grade neoplasia, and villous histology strongly increase the likelihood of recurrence. A clear linear relationship was also found between the size of the adenoma and the grade of neoplasia. Our results have shown that as the histological type becomes more villous, high-grade neoplasia becomes more common. This trend is similar to that shown by other authors (21). The increasing grade of the neoplasia, the increasing size of the adenoma and the more frequent detection of PCNA-ED amongst the samples, representing the altered versus the normal tissue, are strongly correlated values. The PCNA-ED was also more common in adenomas with villous elements, although this association did not reach statistical significance.
Generally considered the gold standard for diagnosing colorectal cancer, colonoscopy, has the main advantage of providing diagnosis and definitive treatment of adenomas. Polypectomy followed by surveillance has been shown to significantly reduce the risk of developing colorectal cancer; however, the reduction in risk from surveillance alone after polypectomy remains unclear (22). Although the risk for colorectal cancer may be higher than that suggested by the National Polyp Study, the evidence from observational studies suggests that the high-risk polyps are the major predictors of future advanced adenomas or cancer (22). Our study showed that if the PCNA-ED is high in an adenoma, a more careful follow up is also required, because of the risk of recurrence and/or malignant transformation. Recurrence developed within the first year in all patients with high PCNA-ED between adenoma and adjacent mucosa. We found that low PCNA-ED was more commonly associated recurrence occurring between one and three years, whereas high PCNA-ED was predominantly linked to recurrence within the first year. These data suggest a strong correlation between PCNA-ED and the risk of adenoma recurrence, whilst high PCNA-ED was highly related with early recurrence. Several important risk factors are used to guide the decision for surgical resection after colonoscopic removal of a mucosal high-grade neoplasia (23). Our findings suggest that the detection of high PCNA-ED in these adenomas is an unfavorable prognostic factor that may support the need for surgical referral.
In non-neoplastic polyps as well as in adenomas from mucosal low-grade neoplasia to high-grade neoplasia, where PCNA expression was detected, its’ 36 kDa form was found either in the normal mucosa or in the pathologically altered tissue. Interestingly, in the colorectal adenocarcinomas, where carcinoma cells invaded to the submucosa, the 72 kDa form of PCNA was detected alongside the 36k Da form and was also present in the surrounding histologically tumor-free mucosa. Both low and high PCNA-ED was more frequent in adenocarcinomas compared to adenomas.
Several authors have suggested a worse prognosis for patients below the age of 40 (24, 25). Death within three years was 50% among our patients under 50 years, which is significantly higher than the 27.27% death rate of the older age group. These data suggest that in younger patients the increased occurrence of high PCNA-ED regarding the 72 kDa isoform might be related with their worse prognosis.
It is widely accepted that the prognosis of rectal cancer is worse in male patients than in female patients (26) It is often argued that this may be due to the operative difficulties in performing radical resection in the male pelvis and is not related with the higher invasiveness of the tumor cells (27). In our study, in accordance with the data published in the literature, death within three years was higher in males (35.29%) than in females (27.27%). In the work of Freedman et al. (28), a worse prognosis was found for rectal tumors. The death of our rectal cancer patients within three years was significantly higher (35%), than that of the colon cancer patients (26.31%). The worse prognosis might be in association with the relatively increased occurrence of the high 36 kDa PCNA-ED in rectum tumors.
In the literature, no relationship was found between tumor size and prognosis (24), and no clear relationship could be identified between PCNA-ED and tumor size, either. Necrosis within the tumor was related with increased PCNA expression in tumor compared with peritumoral tissue. No relevant studies addressing this issue were found in the literature. The histological grade has been shown repeatedly to be of independent prognostic significance by multivariate analysis, higher grades being associated with increasing frequency of muscularis propria penetration, more chances of nodal and distant metastasis, and worse prognosis (24).
Thus, almost 90 years after its original publication, Dukes’ classification continues to represent the most accurate indicators in colonic and rectal cancer (24, 25). Dukes C and ‘D’ have a worse prognosis. This is reflected in our findings by the more frequently detected PCNA-ED in Dukes’ C and ‘D’ than in Dukes’ B stage. In contrast, the pTNM system encompasses all major prognostic factors, including the extent of bowel wall invasion and lymph node involvement, which are widely regarded as the most critical indicators of prognosis worldwide (24, 29). Increased frequency and higher PCNA-ED could be found in cases with larger depth of invasion (pT3 and pT4). It is well known that the larger depth of invasion is associated with worse prognosis (30). The increase in both the PCNA-ED value and its frequency along with the increase in pN stage is in accordance with the worse prognosis associated with higher pN value. The univariate and multivariate analyses of published series have shown that the number of lymph node metastases and lymphatic vessel invasion, are the prognostic factors which mainly influence survival (24). Both the presence and higher levels of PCNA-ED are strongly correlated with the number of lymph node metastases as well as lymphatic vessel invasion. The invasion of extramural veins in pericolonic or perirectal fat tissue is associated with a significantly worse prognosis (24). As far as the blood vessel invasion is concerned, a lot of authors have reported an increased incidence of liver metastases and a significant decrease in survival when an extramural component of blood vessel invasion was detected (24). We found that the increased frequency of PCNA-ED as well as its increased levels are related with blood vessel invasion. In our patients, venous invasion at the primary tumor site did not consistently coincide with the presence of synchronous liver metastases. When patients with synchronous liver metastasis (grades H1 and H2) underwent surgery, the 5-year survival rate increased by about 30% (31). Therefore, both early diagnosis and treatment of metastatic tumors are important for improved prognosis. Our findings indicate that the presence of PCNA-ED is associated with synchronous metastasis, whereas higher levels of PCNA-ED are more closely linked to metachronous metastasis. We thus believe that in cases of advanced colorectal cancer where the histological findings show severe venous invasion in the surgical specimen, the PCNA-ED should be analyzed. When high PCNA-ED is observed, closer observation for the presence of liver metastasis should be performed in order to treat the detected liver metastasis accordingly, thus improving prognosis (32, 33).
To assess the risk of recurrence after curative resection, the pathological stage is currently considered as being the most important predictor of disease recurrence. However, it does not consistently predict recurrence in patients within the same stage (34). Based on our findings, if the recurrence appears in the first six months, the PCNA-ED was high (data not shown), whilst if it appeared after the first year, the PCNA-ED was not significant. Approximately two-thirds of our adenocarcinoma patients died due to tumor progression. We found that both detection of any PCNA-ED and its higher levels were related with a shorter survival.
Currently, the benefit of adjuvant chemotherapy is of great controversy in pT3N0M0 colorectal cancer patients (Stage B2 according to Astler-Coller classification) (18, 25, 35). Based on our results, we suggest that high PCNA-ED in these patients might serve as an additional important information that indicates the need of chemotherapy.
This study demonstrates a significant association between increased PCNA expression difference (PCNA-ED) between neoplastic lesions and surrounding tissue, and the risk of recurrence and malignant transformation in adenomas. We demonstrated that moving further in the adenoma-carcinoma sequence, this difference is more frequent. Results of measurements of PCNA-ED may reflect various stages of colorectal tumorigenesis. Our findings suggest that the measurement of PCNA-ED could be used as a marker for colorectal cancer risk; PCNA-ED detection also helps in early diagnosis of colorectal cancer and assessment of the prognosis of the disease. The PCNA expression difference between a neoplastic lesion and its surrounding mucosa could be used to identify patients at high risk for recurrence and to guide decisions on additional therapy after surgery.
Footnotes
A preliminary form of this work has been presented and awarded at the 12th Congress of the European Society of Surgical Oncology, Budapest, Hungary, as well as at the 144th Falk Symposium, Gastroenterology Week, Freiburg, Germany.
Authors’ Contributions
Revekka Harisi MD, PhD, MSc: Conceptualization, Methodology, Investigation, Data curation, Visualization, Writing-Reviewing and Editing. Balazs Jaray MD: Resources. Attila Zsirka MD: Resources. Janina Kulka MD, PhD, DSc: Reviewing. Peter Kupcsulik MD, PhD, DSc: Resources. Laszlo Harsanyi MD, PhD, DSc: Resources. Janos Weltner MD: Resources. Attila Szijarto MD, PhD, DSc: Resources. Jozsef Timar MD, PhD, DSc: Reviewing. Andras Jeney MD, PhD, DSc: Conceptualization, Supervision. Jozsef Dudas MSc, PhD: Methodology, Software, Validation, Formal analysis, Investigation, Writing.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (ChatGPT, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.
- Received June 18, 2025.
- Revision received July 23, 2025.
- Accepted July 28, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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