|Year : 2021 | Volume
| Issue : 5 | Page : 160-165
|Expression patterns and prognostic significances of RRM1 and ERCC1 in pancreatic carcinoma and cholangiocarcinoma
Khushbu Khetan1, Ranjit K Sahoo2, Vandana Baloda1, Shalimar3, Sreenivas Vishnubhatla4, Anoop Saraya3, Nihar Ranjan Dash5, Atul Sharma2, Siddhartha DattaGupta1, Prasenjit Das1
1 Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Medical Oncology, All India Institute of Medical Sciences, New Delhi, India
3 Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India
4 Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India
5 Department of Gastrointestinal Surgery, All India Institute of Medical Sciences, New Delhi, India
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|Date of Submission||09-Jul-2019|
|Date of Decision||22-Dec-2019|
|Date of Acceptance||30-Dec-2019|
|Date of Web Publication||7-Jun-2021|
| Abstract|| |
Background: Aggressive pancreatobiliary tumors often require oxaliplatin-based therapies, instead of standard gemcitabine-based therapy and biomarker studies at diagnosis to decide the appropriate therapeutic regimen. The ribonucleotide Reductase catalytic subunit M1 (RRM1) and excision repair cross-complementing gene-1 (ERCC1) are related to DNA synthesis and repair and essential in this regard. However, apart from the therapeutic benefit, their prognostic implication is controversial. Methods: In this retrospective study, paraffin-embedded tissue from 51 cases of pancreatic cancer and 29 cases of cholangiocarcinoma were evaluated for RRM1 and ERCC1 expression by immunohistochemical technique along with 18 control pancreatic and biliary tissues. The semiquantitatively H score was calculated based on stain distribution and stain intensities. Results: Both RRM1 and ERCC1 expression were high in tumor epithelium than in controls (RRM1: the difference was statistically significant in cholangiocarcinoma (P = 0.008); ERCC1: the difference was statistically significant both in pancreatic and cholangiocarcinoma (P < 0.05)]. However, no correlation was noted between RRM1 and ERCC1-low and high tumors with histological markers of prognosis and overall survival in these patients. Conclusions: The present study adds further evidence against the controversy that if RRM1 and ERCC1 expression in pancreatic and biliary carcinomas have any prognostic significance apart from their proven therapeutic benefits in these tumors.
Keywords: Cholangiocarcinoma, excision repair cross-complementing gene-1, pancreatic adenocarcinoma, prognosis, Ribonucleotide Reductase Catalytic Subunit M1
|How to cite this article:|
Khetan K, Sahoo RK, Baloda V, Shalimar, Vishnubhatla S, Saraya A, Dash NR, Sharma A, DattaGupta S, Das P. Expression patterns and prognostic significances of RRM1 and ERCC1 in pancreatic carcinoma and cholangiocarcinoma. Indian J Pathol Microbiol 2021;64:160-5
|How to cite this URL:|
Khetan K, Sahoo RK, Baloda V, Shalimar, Vishnubhatla S, Saraya A, Dash NR, Sharma A, DattaGupta S, Das P. Expression patterns and prognostic significances of RRM1 and ERCC1 in pancreatic carcinoma and cholangiocarcinoma. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Jun 13];64:160-5. Available from: https://www.ijpmonline.org/text.asp?2021/64/5/160/317914
| Introduction|| |
Pancreatobiliary carcinomas are one of the leading causes of cancer-related deaths worldwide, with one year and five-year survival rates of just 28% and 8%, respectively.,, In India, the prevalence of pancreatic cancer is nearly about 17000 cases per year, out of which approximately 80% patients already have metastatic disease when diagnosed for the first time and only about 10–20% of patients undergo pancreatic resection. Periampullary carcinoma tends to have significantly better prognosis and manifests early due to biliary outflow obstruction as compared to pancreatic carcinoma and bile duct carcinoma. Therefore, it is important to diagnose and separate these entities.,, The overall survival rate in cholangiocarcinoma is only about six months despite aggressive chemotherapy. While the efficacy of gemcitabine-based standard therapy is limited in aggressive tumors, FOLFIRINOX (oxaliplatin), S-1 or oxaliplatin plus 5-fluorouracil plus leucovorin based alternative regimens have shown improved efficacy. FOLFIRINOX is now the preferred regimen for patients who are young and fit and have serum bilirubin level <1.5 mg/dl. Hence, during diagnosis itself, biomarker studies are needed to decide on the appropriate therapeutic regimen keeping in mind the short period of overall survival in such tumors., With regards to gemcitabine and oxaliplatin, the Ribonucleotide Reductase Catalytic Subunit M1 (RRM1) and excision repair cross-complementing gene-1 (ERCC1) biomarkers were particularly found relevant, both of which participate in DNA synthesis and DNA repair.,,, Ribonucleotide reductase (RNR) complex is a multimeric enzyme which converts ribonucleoside diphosphate to deoxyribonucleoside diphosphate and is composed of two subunits, RRM1 and RRM2. During normal cellular replication, RRM1 and RRM2 dimerize and catalyze the production of deoxynucleotide triphosphates (dNTP), which is necessary for DNA synthesis. In vitro studies using lung, breast, colon, biliary tract, and pancreas cancer cell lines have demonstrated gemcitabine resistance in tumor with high expression levels of RRM1 and RRM2, as the deoxycytidine diphosphate, an active intermediate of gemcitabine binds covalently with the RRM1, resulting in less availability of deoxycytidine triphosphate form of gemcitabine which is incorporated into DNA strand and thus prevents further cell replication.,, The ERCC1 is a crucial player in the nucleotide excision repair pathway and excises the bulky intra-strand adducts of DNA during cellular replication. Platinum-based compounds like cisplatin halt the cellular replication by forming similar intra-strand adducts; hence, high ERCC1 expression has been associated with resistance to platinum-based therapeutic regimens. Apart from the therapeutic advantage, the prognostic significance of RRM1 and ERCC1 has been studied by some investigators in tumors such as non-small cell lung carcinoma, bladder cancer, colorectal, and gastric cancers.,,,,,, In studies related to non-small cell lung carcinomas, Zeng et al. found that co-expression, as well as isolated high expression of RRM1 and ERCC1, was associated with better survival. Similar better survival was found by Jian-Wei et al. in non-small cell lung cancer patients with high levels of both ERCC1 and RRM1 expression, and the better response was noted to platinum-based chemotherapy in these tumors. In resected pancreatic carcinomas (n 68), Akita et al. demonstrated differential expression patterns for RRM1 and ERCC1 with good disease-free survival (DFS) and overall survival (OS) rates. However, Valsecchi et al. reported that there is no survival advantage by segregating the pancreatic ductal carcinomas (PDCA, n 94) into RRM1 and ERCC1 high or low tumors. On the other hand, Fisher et al. showed reduced recurrence-free survival (RFS) and decreased OS in ERCC1-high PDCAs (n 95); RRM1 expression in this study was not associated with any survival benefit in PDCA. No survival benefit of ERCC1 and RRM1 expression was noted in biliary carcinomas by this group., Reduced DFS and OS after surgical resection in ERCC1-high PDCA (n 95) were also demonstrated by Mathiel et al. Deng T et al. however could not identify any definite prognostic significance of RRM1 in advanced biliary carcinomas. Hence, it appears that the prognostic implication of RRM1 and ERCC1 expression in pancreatic carcinomas is controversial, and the same was not widely investigated in biliary tract carcinomas. The present study was aimed to see the expression pattern of RRM1 and ERCC1 in pancreatic carcinoma and cholangiocarcinoma and to determine their prognostic significance if any.
| Methods|| |
The Institutional Ethics Committee approved the study. It was a retrospective cross-sectional study. Formalin-fixed, paraffin-embedded (FFPE) archival tissue blocks of patients who underwent surgical resection for PDCA, and periampullary carcinomas were included as pancreatic carcinomas (n = 51). Out of this, 14 cases were of pancreatic carcinoma and 37 cases were of periampullary carcinoma. FFPE blocks of patients with cholangiocarcinoma (n = 29) were also included. Cases with inadequate clinical detail and those having blocks with inadequate tissue were excluded from the study. Besides, FFPE blocks of healthy pancreatic tissue and common bile duct (CBD) resection margin from Whipple's pancreaticoduodenectomy specimens were included as controls (n = 18).
Sections cut from FFPE blocks were dewaxed and rehydrated. Endogenous peroxidase activity was blocked using 4% hydrogen peroxidase followed by antigen retrieval by treating with Tris-EDTA (pH = 9)/citrate (pH = 6). Primary monoclonal antibodies for RRM1 (1:800, pH = 9, Spring Biosciences) and ERCC1 (1:200, pH = 6, Spring Biosciences) were used to incubate the sections at 4°C, in a humid chamber overnight. Universal polymer-based secondary antibody (Skytek Laboratories, USA) was used, and the reaction product was developed with 3, 3“-diaminobenzidine and counterstained with hematoxylin. Appropriate positive and negative controls were used.
Interpretation for RRM1 and ERCC was done on the tumor cells. H scores were generated in these different anatomical regions (stain intensity x stain distribution, i.e., positive percentage cells). Six distribution grades were employed: 0 = no staining (<10% cells positivity); 1+ =10–20% of tumor cell positivity; 2+ =21–40% of tumor cell positivity; 3+ =41-60% of tumor cell positivity; 4+ =61–80% of tumor cell positivity; 5+ >80% of tumor cell positivity. Intensity was graded as: 1+ = mild; 2+ = moderate; 3+ = marked. A final H score of <5 was taken as low expression whereas and >5 was considered as high expression. This categorization was done for the purpose of statistical analysis, and the cutoff was decided arbitrarily. H scores of different patient groups and controls were compared.
Data were analyzed using statistical software STATA-14.0. Qualitative/categorical data were expressed as frequency and percentage. Quantitative data were expressed as mean ± standard deviation and median (minimum-maximum). Fischer exact test was used to test the association between the categorical variable. Mann Whitney test (Ranksum) was used to compare a continuous variable with skewed distribution. Kaplan Meier (survival analysis) was used to find overall survival with associated factors. P < 0.05 was considered statistically significant.
| Results|| |
The median age of patients with pancreatic (PDCA/periampullary) carcinoma was 55.5 ± 10.7 years and 50.8 ± 12.7 years for cholangiocarcinoma. Of the 51 pancreatic carcinoma patients, 43 were male patients, and 8 were female patients, and out of the 29 cholangiocarcinoma patients, 20 were males, and 9 were females.
IHC assessment of expression of RRM1 and ERCC1
Both RRM1 and ERCC1 expression was assessed in the tumor epithelium and in normal epithelium. Fine granular cytoplasmic and nuclear positivity was noted with RRM1 stain, and nuclear positivity was seen with ERCC1 stain [Figure 1]. RRM1 and ERCC1 expressions were high in tumor epithelium as compared to the controls. The result was statistically significant in cholangiocarcinoma where 9 out of 29 (31%) cases showed high RRM1 expression; whereas none of the normal CBD controls showed high expression (0 out of 18) (P = 0.008). In pancreatic carcinomas, 29 out of 51 pancreatic carcinoma cases (56.8%) and 7 out of 18 normal pancreatic controls (38.8%) showed high expression of RRM1 stain [P = 0.2] [Table 1]. ERCC1 immunostaining could be interpreted in 49/51 cases of pancreatic carcinoma, 28/29 cases of cholangiocarcinomas and 16/18 normal CBD controls due to technical reasons. High ERCC1 expression was seen in 34 out of 49 pancreatic carcinoma cases (69.3%) and 6 out of 18 normal pancreatic controls (33.3%) (P = 0.01) and 12 out of 28 cholangiocarcinoma patients (42.8%) and 14 out 16 normal CBD controls (87.5%) (P = 0.005) [Table 1]. The poorly differentiated pancreatic carcinomas and cholangiocarcinomas showed low RRM1 and ERCC1-high expression patterns in comparison to well-differentiated and moderately differentiated tumors; however, this difference was not statistically significant [Table 2]. The benign pancreatic parenchyma adjacent to pancreatic carcinoma focus also showed diffuse and strong nuclear expression of ERCC1 stain [Figure 1].
|Figure 1: Photomicrograph showing RRM1 IHC expression in pancreatic control (A x 100; arrows). Sections from a RRM1-low and RRM1-high tumors noted in figures B (x 200; arrows) & C (x 200; arrows), respectively. Photomicrograph shows ERCC1 IHC nuclear expression in pancreatic controls (D x 100; arrows). Sections from ERCC1-low and ERCC1-high tumors noted in figures E (x 200; arrows) & F (x 200; arrows), respectively|
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|Table 1: Expression patterns of the RRM1 and ERCC1 immunohistochemical stains in pancreatic carcinomas and cholangiocarcinomas|
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|Table 2: Comparison of expression of RRM1 and ERCC1 in pancreatic and cholangiocarcinomas with the histological parameters of tumor aggressiveness|
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Comparison of expression of RRM1 and ERCC1 in pancreatic tumor and cholangiocarcinoma with histological parameters
There was no significant correlation of RRM1 and ERCC1 H scores and various histological parameters such as differentiation, largest tumor dimension, tumor stage, and lymph node metastasis in both pancreatic and cholangiocarcinoma [Table 2].
Correlation of RRM1 and ERCC1 Immunohistochemistry data with the overall survival of the patients: There was no significant correlation of overall survival of the patients with RRM1-high/RRM1-low and ERCC1-high/ERCC1-low pancreatic and cholangiocarcinoma [Figure 2]. On average, the total follow-up period for the patients with pancreatic carcinoma and cholangiocarcinoma was 15.9 months and 15.6 months, respectively. 49.01% of patients with pancreatic carcinoma and 48.2% of the patients with cholangiocarcinoma died till the last available follow-up.
|Figure 2: Overall survival in ERCC1 low versus ERCC1-high pancreatic carcinoma (a), RRM1-low versus RRM2-high pancreatic carcinoma (b), ERCC1-low versus ERCC2-high cholangiocarcinoma (c) and RRM1-low versus RRM1-high cholangiocarcinomas (d)|
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| Discussion|| |
As was aimed, we tried to analyze the expression patterns of the RRM1 and ERCC1 immunohistochemical stains in surgically resected pancreatobiliary carcinomas and divided the tumors included in RRM1-low and RRM1-high, as well as ERCC1-low and ERCC1-high carcinomas based on the defined criteria. We found that the expression of both of these markers was high in pancreatic and biliary carcinoma epithelium than in the control epithelium [Table 1]. However, when the RRM1-high/RRM1-low and ERCC1-high/ERCC1-low marker status were correlated with histological parameters of tumor aggressiveness and overall survival in these patients, no significant correlation was noted between the marker-low and marker-high groups [Table 2].
As has been highlighted earlier, there are certain inconsistencies in the published literature regarding the prognostic implications of RRM1 and ERCC1 in pancreatobiliary carcinoma. Unlike the observations of Akita et al. in pancreatic adenocarcinoma, Zheng et al. and Jian-Wei et al. in non-small cell lung carcinoma, we found that both RRM1 and ERCC1 have no prognostic implications in pancreatic adenocarcinoma and cholangiocarcinoma.,, Though in both the tumor groups, RRM1 and ERCC1 expression was identical; we observed differential expression of RRM1 and ERCC1 stains in pancreatic and cholangiocarcinoma [Table 1]. In literature, low RRM1 expression has been noted with good outcomes in pancreatic carcinoma when treated with gemcitabine, as this enzyme is needed for DNA synthesis. The functional analysis of the ERCC1 gene shows its role in DNA repair and DNA replication by removing the intrastrand adducts. Hence, the co-expression of RRM1 and ERCC1 in these groups of tumors is exciting and different from what has been described in lung non-small cell carcinomas., In addition to protein expression, Valsecchi et al. also analyzed RRM1 and ERCC1 mRNA expression pattern with OS or DFS in patients with resected PDCA, however, they could not identify any prognostic significance of these proteins. On the other hand, Fisher et al. and Mathiel et al. identified reduced RFS and OS after surgical resection in ERCC1-high PDCAs. However, the former group could not identify any survival benefit of RRM1 and ERCC1 expressions in biliary carcinomas, and any implication of RRM1 expression in pancreatic carcinomas like our findings.,, Deng T et al. also did not observe any prognostic significance of RRM1 in advanced biliary carcinomas.
In addition to affirming the results of Valsecchi et al. in pancreatic carcinoma and Fischer et al. in biliary tract malignancy, the current study adds knowledge to the fact that RRM1 and ERCC1 expression possibly do not have any prognostic correlation with histological parameters of tumor aggressiveness [Table 2]. However, there are some limitations of this study; we included a low number of cases with available follow-up data for this analysis. Second, the pancreatic controls included in this study were histologically confirmed healthy tissue collected from the trauma center, where partial pancreatectomy was performed due to abdominal trauma in road traffic accident victims. However, we could not arrange histologically healthy biliary controls. The tissue included as biliary controls were the resected margin of the CBD from Whipple's pancreatoduodenectomy specimens performed for pancreatic head lesions. In all such cases, due to distal obstruction, the CBD had inflammation and periductal fibrosis. Third, we could not validate our results using real-time PCR study, because pancreatic carcinoma tends to have more stromal tissue compared to other cancers, and hence, needs laser capture microdissection of tumor tissue to go for quantitative analysis of gene expression in these tumors.
| Conclusion|| |
In conclusion, the present study adds further evidence against the controversy that if RRM1 and ERCC1 expression in pancreatic and biliary carcinomas have any prognostic significance apart from their proven therapeutic benefits in these tumors.
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Conflicts of interest
There are no conflicts of interest.
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Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]