Print version ISSN 0102-6720
ABCD, arq. bras. cir. dig. vol.24 no.4 São Paulo Oct./Dec. 2011
Mário Henrique Osanai; Maria Isabel Albano Edelweiss; Luise Meurer; Maitê de Mello Vilwock; Leandro Totti Cavazzola; Guilherme Ribeiro; Carlos Cauduro Schirmer; Richard Ricachenevsky Gurski
BACKGROUND: The esophageal adenocarcinoma
shows an increasing frequence in the last decades, specially in the developed
countries. The Barrett´s esophagus is accepted as the major premalignant
lesion and the metaplasia-dysplasia-adenocarcinoma sequence presents a lot of
genetic changes since its early events. The alterations in p16INK4a
are frequent in Barrett´s esophagus and esophageal carcinoma.
AIM: To verify the prevalence of the immunohistochemical expression of the p16INK4a protein in patients with esophageal adenocarcinoma.
METHODS: The study population consisted of 37 patients with resected esophageal adenocarcinoma. The p16INK4a protein expression was determined by immunohistochemistry using primary antibody p16INK4aAb-7, clone 16P07 NeoMarkers and assessed according to the Immunoreactive scoring system (IRS).
RESULTS: Of 37 analyzed patients, the most were male (86,5%) and the advanced disease was predominant (stages III and IV = 67,5%). In 12 (32,4%) the immunohistochemistry was positive for p16INK4a.There was no significative relation between the protein expression and the degrees of histological differentiation of the biopsies and surgical especimens (p=0,81) neither with the staging (p=0,485).
CONCLUSION: The lost of the immunohistochemical expression of the p16INK4a protein in this study suggests that p16 is enroled in the carcinogenesis of the adenocarcinoma of esophagus.
Headings: Esophagus. Esophageal neoplasm. Genes, p16.
Several studies have analyzed risk factors and mechanism of progression from metaplasia to dysplasia lesions of the adenocarcinoma14,15,22. The risk of developing adenocarcinoma in patients with Barrett's esophagus (BE) is estimated at one case per 56 to 250 patient-years of follow-up10.
Changes of specific genes related to some types of tumors, show the important role of these genes as potential indicators of prognosis or response to therapy9,10.
Changes in gene p16 INK4a (p16) are the object of study in several tumors such as squamous cell carcinoma of the head, neck and esophagus, adenocarcinoma of the pancreas and gastrointestinal stromal tumors10,24,26,32. Studies in tumors of the pancreas and gastrointestinal stromal tumors reported worse prognosis in the presence of alterations in this gene10,24.
There are many studies about the changes in p16 gene in Barrett's esophagus and esophageal carcinoma, including adenocarcinoma, and allelic loss of p16 gene alteration considered the initial progression of Barrett's metaplasia and hypermethylation, one of the most frequent inactivation of the gene4,16,28. However, there are few publications focusing on the impact of these genetic and epigenetic changes in p16 protein expression in adenocarcinomas of the esophagus4.
Thus, this study aims to assess the prevalence of immunohistochemical expression of p16 INK4a protein in pathological examinations of patients with adenocarcinoma of the esophagus.
This study was submitted and approved by regulatory and ethical guidelines committee of the institution, under protocol number 03-183.
The population studied was obtained by review of medical records of patients from Hospital de Clínicas de Porto Alegre, Brazil with adenocarcinoma of the esophagus.
The inclusion criteria was: patients with pathological diagnosis of adenocarcinoma of the esophagus. The exclusion criteria were: history or presence of other concomitant malignancy, neoadjuvant treatment (chemotherapy or radiation therapy), lack of paraffin blocks for the manufacture of blades for the study.
The variables studied were age, gender, stage, histological grade of differentiation, surgical procedure, the intensity of p16 INK4a protein expression, percentage of nuclei stained for p16 INK4a.
Cuts representative of the tumor to immunohistochemistry were from paraffin blocks of biopsy material and surgical technique using standardize routine of the Pathology Department of Hospital de Clínicas de Porto Alegre. It consisted of deparaffinization and rehydration, antigen retrieval, inactivation of endogenous peroxidase and blocking of nonspecific reactions. The primary antibody, clone 16P07 Neomarkers p16 INK4a was incubated for 12 h at 4° C at a dilution of 1:75, followed by application of streptavidin-biotin complex-peroxidase (LSAB, Dako) and revelation with diaminobenzidine tetraidroclorido Kit (DAB, Dako). The reaction presented as a positive control cervical intraepithelial neoplasia positive for p16, and the negative control performed without the use of primary antibody.
The evaluation of p16 INK4a (p16) was performed according to the scoring system of immunoreactivity (immunoreactive Scoring System - IRS)21 modified and is considered as positive only nuclear staining.
Two staining patterns were: positive - when at least 10% of nuclei were positive in patchy or diffuse through the tissue -, and negative when none or few cells (<10%) showed nuclear staining.
The staining intensity was classified graduating from 0 to 3, where 0 is negative, 1 is weak, 2 moderate and 3 is strong.
The final score of the expression of p16 was obtained by multiplying the two scores.
The tissue was analysed by two pathologists, independently and without knowledge of clinicopathological data. The final result was obtained by review of consensus between the two pathologists.
In Figure 1 can be seen, in several examples of staining, the intensities to better illustrate the method.
In statistical analysis, quantitative data are presented as mean and standard deviation, and categorical by frequency (absolute number) and percentage. The observations, made by two pathologists responsible for assessing the intensity of staining and p16 expression of their agreement, were estimated by Kappa measure of agreement. The distribution of p16 between groups and between histologic stages of disease was compared by the chi-square method. The data were processed and analyzed using SPSS (Statistical Package for Social Sciences) version 13.0.
Were reviewed medical records of 63 patients who met the inclusion criteria. Were included in the study only those who had paraffin blocks with biopsies or surgical specimens compatible for study, forming a population of 37 patients.
The average age was 61.9 years (standard deviation ± 8.6 years). Thirty-two patients were male (86.5%), with a mean age of 61.2 years (SD ± 1.6 years) and five were female (13.5%), mean age of 66 years (SD ± 1.1 years) (Figure 2).
Most patients (67.5%) had disease in stages III and IV. Four cases (10.8%) did not have annotation of the stage.
The surgical procedures with curative intent (total gastrectomy and transhiatal esophagogastrectomy) were performed in 54% of cases. The other underwent palliative operation or did not receive surgical treatment.
The degree of tumor differentiation in five cases was well (13.5%), 21 moderate (56.8%) and in ten undifferentiated (27%).
In immunohistochemical analysis of p16, had good agreement between pathologists regarding the evaluation of the percentage of stained nuclei (Kappa = 0.628).
Regarding the assessment of the intensity of expression of p16 in the nuclei, the agreement was fair (Kappa = 0.392).
When happened disagreement between the observers, the slides were reviewed by two pathologists until a consensus was established. For the final analysis, only the consensual results were considered.
The immunohistochemical expression of p16 was observed in only 32.4% of the cases. In six (16.2%) there was strong expression (score 3), in three (8.1%) it was moderate and in other three (8.1%) poor. In 25 cases (67.6%) loss of expression was found.
Regarding gender, there was protein expression in ten of the 32 men (31.2%) and two in five women (40%) (p = ns). In the staging, the distribution of protein expression was 50% for stage I (expression in two of four patients), 50% for stage II (expression in two of four patients), 23% in stage III (three of expression in 13 patients) and 41.7% in stage IV (expression in five of 12 patients).There was no protein expression in four patients without staging information. Statistical analysis showed no significant relationship between staging and protein expression (p = 0.485)
The distribution of p16 protein expression between the different surgical approaches were as follows: the group submitted to total gastrectomy, there was expression in three of 11 patients (27.3%); in the group undergoing palliative operation, the expression was identified in four of 11 patients (36.4%); in patients submitted to transhiatal resection, four of nine patients (44.4%) expressed the protein and the group of patients who underwent biopsy only, there was an expression of the protein in six patients (16 7%) (p = ns).
There was also no statistic significant relationship between the immunohistochemical expression of p16 and the degree of histological differentiation of tumor, when analyzed the relation to immunoreactivity score (p = 0.81).
The criteria for inclusion and exclusion of cases in this study were rigorously analyzed in order to preserve the representativeness of the sample and the compatibility of the samples with the used technique. This allowed, for example, the inclusion of material obtained from biopsies, in addition to the operative parts of resections performed. Most cases of esophageal adenocarcinoma were diagnosed in advanced stages, with no indication for resection of the organ.
Were excluded patients undergoing neoadjuvant treatment or history or presence of other concomitant malignancy, because these situations could modify the protein expression and the status of the p16 gene16,20.
Several studies show that the analysis of immunohistochemical expression of p16 protein correlates well with other methods of analysis for p16 gene alterations, such as sequencing and amplification of molecular chain reaction (PCR)2,3,4,9,18,19,25,31. This scientific reason justified the use of lower cost method of analysis suitable for the objectives.
Evaluation of the expression of p16 by immunohistochemistry showed a reasonably good agreement between the observers on the criteria for positivity (nuclear staining) and according to the intensity of expression. This agreement may be due to active participation of pathologists in the various studies conducted with patients of the Hospital de Clínicas3.
Was found on literature review only one study published with clear description of the methodology used in immunohistochemical analysis of p16 in adenocarcinoma of the esophagus4. In this study, cases were considered negative when less than 10% of the cells showed nuclear staining. The present study used the same criteria, but added the analysis of the intensity of expression, through the modified score of immunoreactivity.
Changes in the p16 gene were studied in several premalignant lesions7,20,28,29 and numerous tumors16,23, such as colon and rectum25, pancreas10, cervix19, salivary glands18, head and neck9,32, mesotheliomas12, osteosarcomas17, gliomas13 bladder1, gastrointestinal stromal tumors24, ovary5, lung2,3,19, stomach8 and esophagus4,7,13,26,27,29,31.
Numerous changes have been described in the p16 gene, the most frequent deletions and hypermethylation2,3,4,7,8,9,12,13,18,19,25,29,31. These changes are often associated with loss of immunohistochemical expression of p16 protein2,3,9,13,18,19,25.
The molecular processes that accompany the progression of BE to adenocarcinoma led to several studies, establishing it as the main precursor lesion of esophageal adenocarcinoma4,7,28,29. Among the causes of BE, is notable the gastroesophageal reflux disease, with rising prevalence6. Gurski et al.11 in 2003 showed that the antireflux operation provided reversal of metaplastic and dysplastic lesions in BE to form more benign in a significant number of patients.
Changes in the p16 sequence metaplasia-dysplasia-adenocarcinoma sequence shown in some studies4,7,29, correlation between the degree of histologic and immunohistochemical expression of the prevalence of p16 protein. Our study showed no significant association (p = 0.81), but there seemed to be the trend of declining intensity of expression with loss of histological differentiation. It is possible that larger sample to confirm this relationship in esophageal adenocarcinoma.
Another observation that worth to be mentionied is the trend of decreasing proportion of the score of immunoreactivity in stages I, II and III (regional and local progression). In cases with stage IV (distant metastases) there was no such proportionality, suggesting that other genetic processes may play more important role in the process of metastasis, as those involved in cell adhesion and angiogenesis, for example. Further studies with larger numbers of cases and including the analysis of other genes may show the relationship between staging and genetic processes involved.
Papadimitrakopoulos et al.20, in 2001, studied the expression of p16 in premalignant lesions of the upper aerodigestive tract underwent chemoprophylaxis and found no significant correlation between loss of expression with histological progression to cancer. However, no publications were found evaluating this response in BE when treated. Studies including treated cases with EB can clarify how it behaves p16 protein expression, including varying degrees of response. In adenocarcinoma of the esophagus, few studies were done but showed a high prevalence of p16 alterations in this gene (41% of genetic alterations)7 and significant loss of p16 protein expression (loss of expression in 86% of cases)4. In this study, was found loss of immunohistochemical expression of p16 in 67.6% of cases and weak expression in 8.1%. Only 24.3% of cases showed moderate to strong expression.
There was low prevalence (32.4%) of immunohistochemical expression of p16 protein in the cases analyzed, confirming the existing knowledge about the frequent changes of the p16 gene in esophageal adenocarcinoma.
1. Asamoto M, Iwahori Y, Okamura T, Shirai T, Tsuda H. Decreased expression of the p16/MTS1 gene without mutation is frequent in human urinary bladder carcinomas. Jpn J Clin Oncol 1997 Feb;27(1):22-5. [ Links ]
2. Awaya H, Takeshima Y, Amatya VJ, Furonaka O, Tagawa K, Kohno N. Inactivation of the p16 gene by hypermethylation and loss of heterozygosity in adenocarcinoma of the lung. Pathol Int 2004 Jul;54(7):486-9. [ Links ]
3. Belinsky SA, Nikula KJ, Palmisano WA, Michels R, Saccomanno G, Gabrielson E. Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. Proc Natl Acad Sci U S A. 1998; 29;95(20):11891-6. [ Links ]
4. Bian YS, Osterheld MC, Fontolliet C, Bosman FT, Benhattar J. p16 inactivation by methylation of the CDKN2A promoter occurs early during neoplastic progression in Barrett's esophagus. Gastroenterology 2002 Apr;122(4):1113-21. [ Links ]
5. Brown I, Milner BJ, Rooney PH, Haites NE. Inactivation of the p16INK4A gene by methylation is not a frequent event in sporadic ovarian carcinoma. Oncol Rep 2001 Nov;8(6):1359-62. [ Links ]
6. Devesa SS, Blot WJ, Fraumeni JF, Jr. Changing patterns in the incidence of esophageal and gastric carcinoma in the United States. Cancer 1998 Nov 15;83(10):2049-53. [ Links ]
7. Eads CA, Lord RV, Wickramasinghe K, Long TI, Kurumboor SK, Bernstein L. Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res 2001 Apr 15;61(8):3410-8. [ Links ]
8. Ficorella C, Cannita K, Ricevuto E, Toniato E, Fusco C, Sinopoli NT. P16 hypermethylation contributes to the characterization of gene inactivation profiles in primary gastric cancer. Oncol Rep 2003 Jan;10(1):169-73. [ Links ]
9. Geisler SA, Olshan AF, Weissler MC, Cai J, Funkhouser WK, Smith J. p16 and p53 Protein expression as prognostic indicators of survival and disease recurrence from head and neck cancer. Clin Cancer Res 2002 Nov;8(11):3445-53. [ Links ]
10. Gerdes B, Ramaswamy A, Ziegler A, Lang SA, Kersting M, Baumann R. p16INK4a is a prognostic marker in resected ductal pancreatic cancer: an analysis of p16INK4a, p53, MDM2, an Rb. Ann Surg 2002 Jan;235(1):51-9. [ Links ]
11. Gurski RR, Peters JH, Hagen JA, DeMeester SR, Bremner CG, Chandrasoma PT. Barrett's esophagus can and does regress after antireflux surgery: a study of prevalence and predictive features. J Am Coll Surg 2003 May;196(5):706-12. [ Links ]
12. Hirao T, Bueno R, Chen CJ, Gordon GJ, Heilig E, Kelsey KT. Alterations of the p16(INK4) locus in human malignant mesothelial tumors. Carcinogenesis 2002 Jul;23(7):1127-30. [ Links ]
13. Ishii N, Maier D, Merlo A, Tada M, Sawamura Y, Diserens AC. Frequent co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in human glioma cell lines. Brain Pathol 1999 Jul;9(3):469-79. [ Links ]
14. Jacob CE, Bresciani C, Gama-Rodrigues JJ, Yagi OK, Mucerino D, Zilberstein B, Cecconello I. Behavior of gastric cancer in brazilian population. ABCD Arq Bras Cir Dig 2009;22(1):29-3 [ Links ]
15. Jankowski JA, Perry I, Harrison RF. Gastro-oesophageal cancer: death at the junction. BMJ 2000 Aug 19;321(7259):463-4. [ Links ]
16. Kubo A, Nakagawa K, Varma RK, Conrad NK, Cheng JQ, Lee WC. The p16 status of tumor cell lines identifies small molecule inhibitors specific for cyclin-dependent kinase 4. Clin Cancer Res 1999 Dec;5(12):4279-86. [ Links ]
17. Nielsen GP, Burns KL, Rosenberg AE, Louis DN. CDKN2A gene deletions and loss of p16 expression occur in osteosarcomas that lack RB alterations. Am J Pathol 1998 Jul;153(1):159-63. [ Links ]
18. Nishimine M, Nakamura M, Kishi M, Okamoto M, Shimada K, Ishida E. Alterations of p14ARF and p16INK4a genes in salivary gland carcinomas. Oncol Rep 2003 May;10(3):555-60. [ Links ]
19. Nuovo GJ, Plaia TW, Belinsky SA, Baylin SB, Herman JG. In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis. Proc Natl Acad Sci U S A 1999 Oct 26;96(22):12754-9. [ Links ]
20. Papadimitrakopoulou VA, Izzo J, Mao L, Keck J, Hamilton D, Shin DM. Cyclin D1 and p16 alterations in advanced premalignant lesions of the upper aerodigestive tract: role in response to chemoprevention and cancer development. Clin Cancer Res 2001 Oct;7(10):3127-34. [ Links ]
21. Remmele W, Schicketanz KH. Immunohistochemical determination of estrogen and progesterone receptor content in human breast cancer. Computer-assisted image analysis (QIC score) vs. subjective grading (IRS). Pathol Res Pract 1993 Sep;189(8):862-6. [ Links ]
22. Rudolph RE, Vaughan TL, Storer B. Segment Length and Risk for Neoplastic Progression in Patients with Barrett Esophagus. Ann Intern Med 2000 Nov 7;133(9):748. [ Links ]
23. Sasaki S, Kitagawa Y, Sekido Y, Minna JD, Kuwano H, Yokota J. Molecular processes of chromosome 9p21 deletions in human cancers. Oncogene 2003 Jun 12;22(24):3792-8. [ Links ]
24. Schneider-Stock R, Boltze C, Lasota J, Miettinen M, Peters B, Pross M. High prognostic value of p16INK4 alterations in gastrointestinal stromal tumors. J Clin Oncol 2003 May 1;21(9):1688-97. [ Links ]
25. Schneider-Stock R, Boltze C, Peters B, Hopfner T, Meyer F, Lippert H. Differences in loss of p16INK4 protein expression by promoter methylation between left- and right-sided primary colorectal carcinomas. Int J Oncol 2003 Oct;23(4):1009-13. [ Links ]
26. Sturm I, Petrowsky H, Volz R, Lorenz M, Radetzki S, Hillebrand T. Analysis of p53/BAX/p16(ink4a/CDKN2) in esophageal squamous cell carcinoma: high BAX and p16(ink4a/CDKN2) identifies patients with good prognosis. J Clin Oncol 2001 Apr 15;19(8):2272-81. [ Links ]
27. Tokugawa T, Sugihara H, Tani T, Hattori T. Modes of silencing of p16 in development of esophageal squamous cell carcinoma. Cancer Res 2002 Sep 1;62(17):4938-44. [ Links ]
28. Wijnhoven BP, Tilanus HW, Dinjens WN. Molecular biology of Barrett's adenocarcinoma. Ann Surg 2001 Mar;233(3):322-37. [ Links ]
29. Wong DJ, Paulson TG, Prevo LJ, Galipeau PC, Longton G, Blount PL. p16(INK4a) lesions are common, early abnormalities that undergo clonal expansion in Barrett's metaplastic epithelium. Cancer Res 2001 Nov 15;61(22):8284-9. [ Links ]
30. Xing EP, Nie Y, Song Y, Yang GY, Cai YC, Wang LD. Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma. Clin Cancer Res 1999 Oct;5(10):2704-13. [ Links ]
31. Xing EP, Nie Y, Wang LD, Yang GY, Yang CS. Aberrant methylation of p16INK4a and deletion of p15INK4b are frequent events in human esophageal cancer in Linxian, China. Carcinogenesis 1999 Jan;20(1):77-84. [ Links ]
32. Yuen PW, Man M, Lam KY, Kwong YL. Clinicopathological significance of p16 gene expression in the surgical treatment of head and neck squamous cell carcinomas. J Clin Pathol 2002 Jan;55(1):58-60. [ Links ]
Correspondence: Financial source: none Received for publication: 30/03/2011 From Hospital de Clínicas de Porto Alegre
and the Graduate Program in Medicine, Surgery, Faculty of Medicine, Federal
University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
Conflicts of interest: none
Accepted for publication: 16/06/2011
Financial source: none
Received for publication: 30/03/2011
From Hospital de Clínicas de Porto Alegre and the Graduate Program in Medicine, Surgery, Faculty of Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.