437412

et al.International Journal of Surgical Pathology

IJSXXX10.1177/1066896912437412Luderer

Accuracy of Preoperative Biopsies Compared With Surgical Specimens in the Diagnosis of Colorectal Adenocarcinoma
Loreley A. Luderer, MD1,2,3, Suzana A. S. Lustosa, PhD1,2, Ricardo Artigiani Neto, PhD1, Filipe T. Lopes2,3, and Delcio Matos, PhD1

International Journal of Surgical Pathology 20(4) 355359 The Author(s) 2012 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1066896912437412 http://ijs.sagepub.com

Abstract Objective. Since the first data from a patient with colorectal adenocarcinoma are obtained by biopsy, this study evaluated the accuracy of diagnosis by biopsy as compared with the diagnostic potential of the surgical specimen, considering the histological type, grade of differentiation, and immunohistochemical expression of p53. Methods. The specimens were obtained from 80 patients assisted at Hospital So Paulo. The biopsy and surgical specimen sections were stained by hematoxylineosin and immunohistochemistry and compared by 3 pathologists blinded to the evaluations. Results. The accuracy for the histological types was 88%. The grade of differentiation presented an accuracy of 70% with Kappa = .48. The expression of protein p53 exhibited an accuracy of 68% with Kappa = .22. Conclusion. The preoperative biopsy of colorectal adenocarcinoma presented good accuracy compared with histopathological examination of the surgical specimen, but with weak to moderate effective degree of agreement between the results. Keywords colorectal adenocarcinoma, colorectal biopsy, surgical specimen, accuracy, colorectal carcinoma

Introduction
The issue of the representativeness of the diagnostic sample from preoperative biopsy of a colorectal adenocarcinoma remains controversial, and few studies have addressed this aspect. Therefore, the assumption that fragments obtained by biopsy may effectively represent the tumor in its entirety is yet to be supported. Comparison between the diagnostic potential of biopsy and that of the surgical specimen obtained from complete resection of a colorectal neoplastic lesion might contribute to solving this problem. No studies were found in the literature comparing the biopsy and surgical specimens, in relation to the different histological types. Comparative study between the biopsy and surgical specimens of the same tumor was initially conducted by Kato et al1 in 1989 and later by Burton et al2 in 2003. In both studies, only the grade of cell differentiation of the tumor was taken as a parameter. Other authors also evaluated this relationship, but only in specific histological types, such as villous tumors3 and mucus-secreting tumors.4

The rationale for this study was to examine the potential of preoperative biopsy for colorectal adenocarcinoma features identification and eventually to suggest a prognosis. It was assumed that the material obtained by biopsy, ideally performed, may provide highly valuable diagnostic and prognostic information for treatment of patients. This reasoning was based on whether the biopsy material might be considered as a reliable sample of a population represented by the surgical specimen. Therefore, the main purpose of this study was to determine to what extent
1 Paulista Medical School, Universidade Federal de So Paulo, So Paulo, Brazil 2 Hospital Municipal Dr. Munir Rafful,Volta Redonda, Rio de Janeiro, Brazil 3 UniFOAUniversity Center of Volta Redonda, Rio de Janeiro, Brazil

Corresponding Author: Loreley Aandrade Luderer, MD, Rua Profa, Cllia, 46-Vila Santa Ceclia 27260-500,Volta Redonda, Estado do Rio de Janeiro, Brazil Email: loreleyluderer@uol.com.br

356 the biopsy material, obtained under care conditions and by standardizing the routine attendance, might be considered as representative of the entire tumor, which is analyzed by examination of the postoperative specimen. Since the current approach in case of rectal adenocarcinoma is initial treatment by neoadjuvant therapy and as this procedure may lead to transformation of the tumor tissue into a structure with a significant residual fibrotic component, and even disappearance of the entire neoplastic glandular tissue, the material obtained by biopsy might represent the last opportunity to obtain information concerning the original tumor in these patients. The hypothesis of this study is that the representativeness of the preoperative biopsy sample might be related not only to the morphological and histopathological aspects but also to the molecular analysis related to the protein expression of a tumor marker, by immunohistochemical examination of the material.

International Journal of Surgical Pathology 20(4)

Table 1. Characteristics of the Samples Gender, n (%) Male Female Age in years (M) Location, n (%) Ascending colon Transverse colon Descending and sigmoid colon Rectum 46 (57.5) 34 (42.5) 38-90 (64) 25 (31.3) 3 (3.7) 17 (21.3) 35 (43.7)

Table 2. Interpretation of the Kappa Coefficient Value and Agreement Level According to Altman et al7 Kappa Coefficient Value <0.20 0.21-0.40 0.41-0.60 0.61-0.80 0.81-1.00 Agreement Level None Weak Moderate Good Strong

Materials and Methods

The sample was composed of 160 biopsy and surgical specimens from 80 patients, out of 1703 patients with colorectal adenocarcinoma assisted and operated at Hospital So Paulo from 1997 to 2009. The gender, age range, and location of the neoplasia are presented in Table 1. The study included only specimens for which paraffin blocks of the preoperative biopsy and of the tumor were available and excluded specimens from patients with tumor relapse and those subjected to preoperative radiotherapy and chemotherapy. The sections were stained with hematoxylineosin for histopathology and with streptavidinbiotinperoxidase using anti-p53 antibody (clone DO-7; Dako, Glostrup, Denmark) for immunohistochemistry and were analyzed by 3 experienced pathologists blinded to the diagnosis of the specimens and corresponding biopsies, after which a consensus was reached. Analysis of the histological type was based on the histology of colorectal glandular tumors, and the grade of cell differentiation was scored in 4 types, depending on the presence of glandular structures, according to the World Health Organization.5 For immunohistochemical analysis of protein p53, the sections were examined by light microscopy, counting the positive nuclei and the nuclei stained dark brown, in 10 fields at 200 magnification. The mean of positive and negative values and of the total number of cells was calculated for each case, followed by the percentage of cells with positive nuclei. The immunoreactivity of p53 was expressed by the following scores6: 0 (absent)0% to 10% of stained nuclei; 1 (present)11% to 100% of stained nuclei. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 17.0 for

Windows, and the correlation of interpretation of the Kappa coefficient value with the agreement level according to Altman7 is presented in Table 2.

Results
Concerning the histological type, comparison of the results of the biopsy and the surgical specimen revealed agreement only for adenocarcinoma (90%) and medullary carcinoma (1.25%), without observation of mucinous adenocarcinoma in the biopsy samples, which precluded application of the Kappa test, the Kappa value being high, as well as the accuracy for each type individually (Tables 3 and 4; Figure 1). The degree of agreement between the biopsy sample and the surgical specimen in relation to the grades of differentiation revealed a moderate Kappa coefficient and high accuracy, both collectively and for each grade individually (Tables 5 and 6; Figure 2). Comparison of the biopsy sample and the surgical specimen concerning the immunohistochemical expression of protein p53 evidenced good accuracy and weak Kappa coefficient of agreement (Tables 7 and 8; Figure 3).

Discussion
Analysis of adenocarcinoma evidences that the type exclusively composed of glands prevails in more than 90% of cases. The degree of agreement of the biopsy with these findings was high, with an accuracy of 88.75% in relation to the adenocarcinoma type and an accuracy of

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Table 3. Distribution of the Results of the Agreement Observed Between the Biopsy Sample and the Surgical Specimen in Relation to the Histological Typea Surgical Specimen Biopsy Sample Adenocarcinoma Medullary carcinoma Total
a b

Adenocarcinoma 70 (87.5%) 2 (2.5%) 72 (90.0%)

b

Mucinous Adenocarcinoma 7 (8.8%) 7 (8.8%)

Medullary Carcinoma 1 (1.3%)b 1 (1.3%)

Total 77 (96.3%) 3 (3.8%) 80 (100.0%)

Accuracy = 0.88-0.81, 0.95. Values in italics represent the agreement observed.

Table 4. Accuracy, Sensitivity, Specificity, Positive Predictive Value, Negative Predictive Value, and Kappa Test for the Histological Types Adenocarcinoma and Medullary Carcinoma Adenocarcinoma V Accuracy Se Sp PPV NPV Kappa 0.88 0.97 0.12 0.90 0.33 0.13 CI 0.79-0.94 0.90-0.99 0.00-0.52 0.82-0.96 0.00-0.90 0.00-0.44 V 0.97 1.00 0.97 0.33 1.00 0.49 Medullary Carcinoma CI 0.91-0.99 0.02-1.00 0.91-0.99 0.00-0.90 0.95-1.00 0.00-1.00

Abbreviations:V, value; CI, confidence interval; Se, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value.

Adenocarcinoma (B) e Adenocarcinoma Mucinoso (E); 8,8% Carcinoma Medular (B e E); 1,3%

Carcinoma Medular (B) e Adenocarcinoma (E); 2,5%

Adenocarcinoma (B e E); 87,5%

Figure 1. Graph of the distribution of agreement results observed between the biopsy sample and the surgical specimen in relation to the histological type

97.5% in relation to the medullary type. However, the mucinous type was not diagnosed in the biopsy, because it was observed that greater mucus secretion is found in the deeper regions, thus impairing a percentage above 50 in small fragments of the biopsy, which is different from the findings of Younes et al.4 It is also possible that the low prevalence of adenocarcinoma types may influence the

correlation of the biopsy material with the surgical specimen, especially in the sampling used for this study. In the comparison between the biopsy sample and the surgical specimen in relation to the grades of differentiation, even though the Kappa test presented a moderate coefficient for the different types, it was observed by 2 authors who also analyzed them that this study presented high percentages of accuracy 75%, 75%, 90%, and 100% (for grades from well differentiated up to undifferentiated), even higher than the positive results observed in 19891 of 57.90%, 77.45%, and 85.50% (for grades from well differentiated to poorly differentiated) and the hardly conclusive outcomes observed in 2003,2 which yielded values of 35%, 52%, and 52%, in the same order. In the sections stained by immunohistochemistry, it was observed that p53 exhibits irregular staining within the same tumor and, though positive when there is expression above 11%, it evidences occasionally extensive negative areas. These areas may be punched during biopsy, thus leading to false-positive or false-negative results. Also, this irregular staining is also found in close glands in the same tumor. These observations provide further understanding of the regular degree of Kappa agreement found in the comparison between the biopsy and surgical specimens, despite the accuracy of 68.75%, which is above the average.

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Table 5. Distribution of Degrees of Agreement Observed Between the Biopsy and Surgical Specimens in Relation to the Degree of Differentiationa Surgical Specimen Biopsy Sample Grade 1 Grade 2 Grade 3 Grade 4 Total
a b

Grade 1 13 (16.3%) 8 (10.0%) 21 (26.3%)

b

Grade 2 8 (10.0%) 35 (43.8%)b 1 (1.3%) 44 (55.0%)

Grade 3 4 (5.0%) 3 (3.8%) 7 (8.8%)b 14 (17.5%)

Grade 4 1 (1.3%)b 1 (1.3%)

Total 25 (31.3%) 46 (57.5%) 8 (10.0%) 1 (1.3%) 80 (100.0%)

Accuracy = 0.70-0.60, 0.80; Kappa = 0.48-0.31, 0.65. Values in italics represent the agreement observed.

Table 6. Accuracy, Sensitivity, Specificity, Positive Predictive Value, Negative Predictive Value, and Kappa Test for Differentiation Grades Grade 1 V Accuracy Se Sp PPV NPV Kappa 0.75 0.61 0.79 0.52 0.85 0.39 CI 0.64-0.84 0.38-0.81 0.67-0.89 0.31-0.72 0.73-0.93 0.17-0.61 V 0.75 0.79 0.69 0.76 0.73 0.49 Grade 2 CI 0.64-0.84 0.64-0.90 0.51-0.83 0.61-0.87 0.55-0.87 0.30-0.68 V 0.90 0.50 0.98 0.87 0.90 0.58 Grade 3 CI 0.81-0.95 0.23-0.77 0.91-0.99 0.47-0.99 0.81-0.96 0.33-0.83 V 1.00 1.00 1.00 1.00 1.00 1.00 Grade 4 CI 0.95-1.00 0.02-1.00 0.95-1.00 0.02-1.00 0.95-1.00

Abbreviations:V, value; CI, confidence interval; Se, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value.

grau 2 (B) e grau 3 (E); 3,8% grau 2 (B) e grau 1 (E); 10,0% grau 1 (B) e grau 3 (E); 5,0% grau 1 (B) e grau 2 (E); 10,0% grau 4 (B e E);1,3%

grau 3 (B) e grau 2 (E); 1,3%

grau 1 (B e E); 16,3%

Table 7. Distribution of the Degree of Agreement Between the Biopsy and the Surgical Specimen in the Immunohistochemical Expression of Protein p53 Surgical Specimen Biopsy Sample Absent Present Total
a

Absent 7 (8.8%)a 23 (28.8%) 30 (37.5%)

Present 2 (2.5%) 48 (60.0%)a 50 (62.5%)

Total 9 (11.3%) 71 (88.8%) 80 (100.0%)

Values in italics represent the agreement observed.

grau 3 (B e E); 8,8%

grau 2 (B e E); 43,8%

Figure 2. Graph of the distribution of degrees of agreement observed between the biopsy sample and the surgical specimen in relation to the differentiation grade

Conversely, analysis of the biopsy sample is currently considered to have limited value because its fragments represent very superficial layers of the lesions and are collected from regions that are not very representative and in a nonstandardized manner concerning the technique of endoscopic biopsy.

The literature does not indicate the best region to be biopsied. In 2004, Lavelle et al8 conducted an in vitro study and observed that in ulcerated tumors, biopsies performed at the center of the ulcer or its internal edge presented greater accuracy. The main problem is to be able to identify a necrosis-free area for that purpose during colonoscopy, depending on the size of the lesion. These aspects highlight the need for standardization of the biopsy procedure, aiming to minimize its diagnostic limitations. Considering the diagnostic limitations of these procedures, further studies are needed, mainly involving the aspects observed in the present results.

Luderer et al. Declaration of Conflicting Interests

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Table 8. Accuracy, Sensitivity, Specificity, Positive Predictive Value, Negative Predictive Value, and Kappa Test for Expression of Protein p53 V Accuracy Se Sp PPV NPV Kappa 0.68 0.96 0.23 0.67 0.77 0.22 CI 0.57-0.78 0.86-0.99 0.09-0.42 0.55-0.78 0.39-0.97 0.04-0.40

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.

References
1. Kato T, Kojima H, Hirai T, et al. The correlation between preoperative pathologic diagnosis of a biopsy specimen and postoperative pathologic diagnosis of a tissue specimen involving colorectal cancer patients [article in Japanese]. Gan No Rinsho. 1989;35:1119-1122. 2. Burton S, Eddy B, Li WY, et al. Reliability of preoperative biopsies in the histological grading of colorectal adenocarcinomas. Ann R Coll Surg Engl. 2003;85:23-25. 3. Detry R, Kartheuser A, Hermans BPH, Lagneaux G, Sempoux CH. Colorectal villous tumors. Accuracy of the preoperative biopsies. Acta Gastro Enter Belg. 1999;62:9-12. 4. Younes M, Katikaneni PR, Lechago J. The value of the preoperative mucosal biopsy in the diagnosis of colorectal mucinous adenocarcinoma. Cancer. 1993;72:3588-3592. 5. Hamilton SR, Riboli E, Nakamura S, et al. Carcinoma of the colon and rectum. In: Bosman FT, Carneiro F, Hruban RH, Theise ND, eds. WHO Classification of Tumors of the Digestive System. Lyon, France: International Agency for Research on Cancer; 2010:134-146. 6. Lustosa SAS, Loguillo A, Artigiani R, Saad SS, Goldenberg A, Matos D. Analysis of the correlation between p53 and Bcl-2 expression with staging and prognosis of the colorectal adenocarcinoma. Acta Cir Bras. 2005;20:353-357. 7. Altman DG, Machim D, Bryant TN, Gardner MJ. Statistics With Confidence. 2nd ed. London, England: BMJ Books; 2000. 8. Lavelle MA, Keong NCH, Berresford PA. In vitro biopsy of colorectal carcinomas. Colorectal Dis. 2004;6: 320-322.

Abbreviations:V, value; CI, confidence interval; Se, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value.

p53 ausente (B e E); 8,8% p53 presente (B) e ausente (E); 28,8%

p53 ausente (B) e presente (E); 2,5% p53 presente (B e E); 60,0%

Figure 3. Graph of the distribution of the results of biopsy compared with the surgical specimen concerning the expression of protein p53

Conclusion
The preoperative biopsy of colorectal adenocarcinoma presented good accuracy compared with histopathological examination of the surgical specimen but with weak to moderate effective degree of agreement between the results.

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