j coloproctol (rio j). 2 0 1 4;3 4(3):136–140

Journal ofColoproctology

www.jco l .org .br

Original Article

Clinical-pathological and p53 protein expressionstudy in dysplasia associated with ulcerativecolitis�,��

Antônio Baldin Júniora,b,∗, José Ederaldo Queiroz Telles c,d,e, Renato de Araújo Bonardia,f,Heda Maria Barska dos Santos Amarantea,g, Rosimeri Kuhl Svoboda Baldina,d,e

a Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazilb Coloproctology Unit, Hospital de Clínicas, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazilc Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazild Hospital de Clínicas, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazile Department of Clinical Pathology, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazilf Department of Surgery, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazilg Department of Internal Medicine, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazil

a r t i c l e i n f o

Article history:

Received 19 November 2013

Accepted 4 February 2014

Available online 16 June 2014

Keywords:

Ulcerative rectocolitis

Dysplasia

Protein p53

a b s t r a c t

Background: The association between ulcerative colitis and adenocarcinoma determined

strategies for patient follow-up and early detection of dysplastic and neoplastic lesions.

Aims: To analyze the incidence of dysplasia in patients with ulcerative colitis, compar-

ing clinical data of patients with and without dysplasia and check immunohistochemical

expression of p53 protein in dysplasias.

Materials and methods: We analyzed biopsy samples and clinical data of 124 patients with

ulcerative colitis at Hospital de Clínicas, Federal University of Paraná, Curitiba, Brazil.

Results: Dysplasia incidence was low (9.67%) and all cases with low-grade dysplasia. Patients

clinical data comparison with and without dysplasia did not show significant statistical dif-

ferences with regard to the race, age at the start of the disease, age at last biopsy, duration and

anatomic extent of ulcerative colitis. Significant difference was found between males and

females with predominance of males (58.34%) for dysplasia. Seventeenth biopsy samples of

12 patients with dysplasia, 5 (29.4%) were p53 positive.

Conclusions: From these results it is concluded that the incidence of dysplasia was low, higher

in males and there was positivity of p53 protein in dysplasia.

© 2014 Sociedade Brasileira de Coloproctologia. Published by Elsevier Editora Ltda. All

rights reserved.

� Study conducted at the Department of Pathology, Hospital de Clínicas, Universidade Federal do Paraná, Curitiba, PR, Brazil.�� Presented as a poster at the XXV Brazilian Congress of Pathology, Natal, RN, 2005.

∗ Corresponding author.E-mail: antoniobaldinjr@ufpr.br, ksbaldin@ig.com.br (A. Baldin Júnior).

http://dx.doi.org/10.1016/j.jcol.2014.02.0092237-9363/© 2014 Sociedade Brasileira de Coloproctologia. Published by Elsevier Editora Ltda. All rights reserved.

j coloproctol (rio j). 2 0 1 4;3 4(3):136–140 137

Estudo clínico-patológico e da expressão da proteína p53 nas displasiasassociadas à retocolite ulcerativa

Palavras-chave:

Retocolite ulcerative

Displasia

Proteína p53

r e s u m o

Racional: A associacão entre retocolite ulcerativa e adenocarcinoma determinou estratégias

para seguimento dos pacientes e deteccão precoce das lesões displásicas e neoplásicas.

Objetivos: Analisar a incidência de displasia nos pacientes com retocolite ulcerativa,

comparar dados clínicos dos pacientes com e sem displasia e verificar a expressão imunois-

toquímica da proteína p53 nas displasias.

Material e Métodos: Foram estudados os exames anatomopatológicos e dados clínicos de 124

pacientes com e sem displasia, portadores de retocolite ulcerativa no Hospital de Clínicas

da Universidade Federal do Paraná.

Resultados: A incidência de displasia foi de 9,67% e todos os casos foram de displasia de

baixo grau. Na comparacão dos dados clínicos dos pacientes com e sem displasia não houve

diferenca estatisticamente significativa com relacão à cor, idade no início da doenca, idade

na última biópsia, extensão da doenca e tempo de evolucão da doenca. Houve diferenca

estatística com predomínio de pacientes do sexo masculino (58,34%) em relacão ao feminino

para displasia. Dos 17 exames avaliados de 12 pacientes com displasia, em 5 exames (29,4%)

a expressão da proteína p53 foi positiva.

Conclusões: Desses resultados conclui-se que a incidência de displasia foi baixa, maior no

sexo masculino e houve positividade da proteína p53 nas displasias.

© 2014 Sociedade Brasileira de Coloproctologia. Publicado por Elsevier Editora Ltda.

Todos os direitos reservados.

I

Umon8taiisTttasGtadaU

cdbisc

onset of the disease (ulcerative colitis) and the time of identi-

ntroduction

lcerative colitis (UC) falls within the group of chronic inflam-atory bowel diseases of unknown cause. With outbreaks

f remission and exacerbation, UC has an incidence of 3–20ew cases per year per 100,000 inhabitants.1 UC with over

years of evolution represents an important risk factor forhe development of dysplasia and subsequent development ofdenocarcinoma.2,3 The increased replacement (turnover) ofntestinal epithelial cells damaged by chronic inflammations considered a risk factor for the development of dyspla-ia and adenocarcinoma in patients with long progression.4

he risk of developing colorectal cancer varies from 5.5%o 13.5%, and risk factors include the extent of disease andhe progression time.5 Monitoring and follow-up of patientsre performed by means of colonoscopies and serial biop-ies, but these cover less than 0.05% of the colic surface.6

enetic alterations have been studied to characterize dysplas-ic lesions and for an early detection of carcinogenesis.5 Ofll the markers studied, the expression of p53 protein hasemonstrated significant correlation with duration of disease,s a risk factor of developing colorectal cancer associated withC.7

Although the expression of p53 is a late event in colorectalarcinogenesis, it is considered an early event in the onset ofysplasia associated with UC.8 The onset of expression of p53,y an abnormal protein, in the nucleus of epithelial cells of the

ntestinal mucosa occurs even without the presence of dyspla-ia and precedes the development of dysplasia and colorectalancer.9

Material

A retrospective study was performed since 2004 in 124patients with clinical and endoscopic diagnosis of UC fol-lowed at the inflammatory bowel diseases outpatient service,Hospital de Clinicas, Universidade Federal do Paraná, andwho underwent pathological examination of the colonicmucosa. This study was approved by the Research EthicsCommittee, Hospital de Clinicas, with CEP/HC 732.151/2003-10 protocol. In the database of the Department of Pathologyof the same hospital, all patients and their respective examswere reviewed. A clinical data survey was conducted for age,gender, race, progression time of the disease until the time ofexamination, and extent of disease. Pathological lab workupincluded endoscopic biopsies and surgical specimens. Thepathological reports in which were mentioned “indefinite fordysplasia (IND)”, “presence of dysplasia” or “adenocarcinoma”were selected.

In cases with dysplasia and in two cases of colic adeno-carcinoma, immunohistochemical reactions for nuclear p53protein were performed.

Method

This study included 124 patients with ulcerative colitis,followed-up by biopsies, and the time elapsed between the

fication of dysplasia was registered. Patients without dysplasiahad recorded their follow-up to the last biopsy. The study vari-ables were: race, gender, age at onset, age at last biopsy and

j). 2 0 1 4;3 4(3):136–140

Fig. 1 – Area of dysplasia. Note: Dysplastic nuclei dark

dysplasia-free time (p = 0.0242). The progression of disease inpatients with and without dysplasia showed no difference(p = 0.8055).

138 j coloproctol (rio

site of disease. The explanatory variables were dichotomizedby testing the null hypothesis of distributions of dysplasia-free time (time elapsed from disease progression to diagnosisof dysplasia, or to the last biopsy) equal in both classificationsversus the alternative hypothesis of different distributions ofdysplasia-free time. The statistical test used was consideredthe Cox-Mantel test. The evaluation of the progression time ofthe groups with and without dysplasia was performed usingthe non-parametric Mann–Whitney test. p values <0.05 wereconsidered statistically significant.

The indefinite cases for dysplasia and those with dysplasiawere reviewed with the use of a discriminant classification tocharacterize dysplasia as atypia or repair.10 After reviewingthe pathological examination reports, the cases were clas-sified as “with dysplasia”, “without dysplasia” or “indefinitefor dysplasia”. The paraffin-impregnated material was cut in4-mm thick-sections. Preparation of immunohistochemistryslides and their deparaffinization and hydration with xyleneand decreasing concentrations of ethanol were performed, aswell as antigen retrieval with citrate buffer. Mouse monoclonalantibody anti-p53 protein in previously paraffined materialwas used. This a monoclonal antibody of immunoglobulin G2class, that binds to both the wild-type and to the mutatedprotein. The antibody was used at a dilution of 1:100. Areaction using the streptavidin–biotin-peroxidase complex,using colon adenocarcinoma p53-positive as positive con-trol, was performed. A counting of brown-stained nuclei bymeans of an image analyzer (Image-Pro Plus® – The provensolutionTM Version 4.5.1.23 for Windows 98/NT/Me/2000/XPCopyright©1993–2002 Media Cybernetics Inc.) was performed.Positive cells with nuclei strongly brown-stained were countedby the program by color difference versus nuclei in blue (neg-ative cells). Lesions with less than 10% positive cells werenegative for p53; between 1% and 25%, positive +, between26% and 50%, positive ++, greater than 50%, positive +++.11

Results

Thirty-eight (30.65%) male and 86 (69.35%) female patientswere recruited. Nine (7.26%) were black or mestizo patientsand 115 (92.74%) were Caucasians. Clinical data (age, age atonset of disease, disease duration, and number of biopsies)are shown in Table 1.

Regarding the extent of disease, 78 (62.90%) patients hadonly involvement of left colon and 46 (37.09%) exhibited pan-colitis.

Of the 124 patients, in 12 (9.67%) we found 20 patholog-ical examinations diagnosed with low-grade dysplasia (LGD)or IND, 17 with LGD (Fig. 1) and 3 with IND. No examinationwith high-grade dysplasia was found.

Two female patients with adenocarcinoma had concurrentdysplasia. One patient developed adenocarcinoma in hepaticcolonic flexure, after 8 years of disease progression, with stageT2 N0 M0; Astler-Coller B1; Dukes A. Another patient pre-sented with rectal adenocarcinoma with signet ring cells areas

after 12 years of disease progression, with stage T2 N1 M0;Coller Astler-C1-2; Dukes C.

Of the 17 tests evaluated (from 12 patients with dysplasia),in 5 (29.4%) the result was p53-positive (Fig. 2) and in 12 (70.6%)

blue-stained. HE 400×.Source: Department of Pathology, UFPR.

was p53-negative. An examination with positivity + (24% ofcells), 1 with positivity ++ (39% of cells) and 3 with positivity+++ (> 50% of cells). Two cases of dysplasia were associatedwith a mass (DALM), with a positivity +++. The female patientwith a rectal tumor was p53-positive +++ and the other patienthad her tumor in right colon flexure was p53-negative.

In the statistical analysis with dichotomized variables,dysplasia-free time did not correlate with race (p = 0.9467) norwith the extent of the disease (p = 0.1551). The age before orafter 15 years at disease onset and patient age <40 years or ≥40years at the last biopsy did not correlate with dysplasia-freetime (p = 0.8882 and p = 0.7920 respectively). In Fig. 3, it appearsthat males had higher incidence of dysplasia in relation to

Fig. 2 – Dysplasia p53-positive. Note: Dysplastic nucleibrown-stained brown before marking for cell count.Immunohistochemistry for p53, 400×.Source: Department of Pathology, UFPR.

j coloproctol (rio j). 2 0 1 4;3 4(3):136–140 139

Table 1 – Clinical data.

Variable n Minimum Maximum Median Mean Standarddeviation

Age at last biopsy 124 10.00 81.00 39.00 39.71 13.48Age at the beginning of the disease 124 3.00 79.00 32.50 33.90 13.56Disease duration at last biopsy 124 1.00 36.00 5.00 6.81 5.97Nr. of biopsies 124 1.00 10.00 3.00 3.60 2.17

D

TwtOpvo

dLora

cchidlr

cwar

Table 2 – Frequency distribution of disease duration atlast biopsy.

Follow-up (years) Without dysplasia With dysplasia

Frequency Percentage Frequency Percentage

<5 50 44.64 4 33.335–9.9 35 31.25 5 41.6710–14.9 16 14.29 3 25.0015–19.9 7 6.25 – –≥20 4 3.57 – –

Source: The Author.

iscussion

he medical attention is focused on the monitoring of patientsith UC, and the dysplasia found in pathological examina-

ions became the most significant predictive factor of cancer.12

ne study found 13.1% of dysplasia in 590 patients undergoingroctocolectomy and determined that the positive predictivealue for colonic cancer of a preoperative finding of dysplasiaf any grade is 50%.13

Low-grade dysplasia can be detected in 17% of patientsuring follow-up with endoscopic biopsies.14 In this study,GD was detected in 12 patients (9.67%). Higher incidencef dysplasia in patients with concomitant adenocarcinoma iselated; in our sample, two patients who had adenocarcinomalso had LGD.13

Some authors found a five -fold higher incidence of can-er in patients with pancolitis than in patients with left sideolitis.15 Another study found 18 patients with LGD; 17 of themad pancolitis (94%).16 In this study, disagreeing with the find-

ngs of the authors above mentioned, of the 12 patients withysplasia, seven (58.30%) had pancolitis and five (41.70%) had

eft colitis. The extent of disease did not influence the occur-ence of dysplasia with statistical significance (p = 0.1551).

The time of disease progression is cited as a risk factor forolorectal cancer.15,17 In our sample there was no agreement

ith this observation (Table 2). The comparison of groups with

nd without dysplasia showed no significant difference withegard to time of disease progression (p = 0.8055).

Male Female

Dysplasia-free timeMale x female

Complete Censored

Ulcerative rectocolitis duration (years)

Cum

ulat

ive

popu

latio

n of

cas

es w

ithou

t dys

plas

ia

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95

1.00

4035302520151050

P=0.242 (cox-mantel)

Fig. 3 – Dysplasia-free time and gender.Source: The author.

Total 112 100.00 12 100.00

Source: The Author.

In this study there was a predominance of white patients,which is also observed in the United States.18 In this series,the group with dysplasia had 11 white patients and one blackpatient. However, when analyzing the influence of race andfree dysplasia-free time, there was no significant difference(p = 0.9467).

The expression of the nuclear protein p53 has been studiedas a marker for displasia.7 Although the mutation of the p53gene is considered to be a late event in the carcinogenesis ofsporadic colonic cancers, in the cancer associated with colitis,the mutation appears as an earlier event.7,9 In this sample,five exams with LGD were p53-positive (29.4%). This finding isconsistent with the literature, where LGD positivity indexes of25–50% are found.9,19

In this study, the tests with DALM showed dense stain-ing (+++) for p53. The presence of a DALM injury correlates in43% of cases with a concurrent finding of adenocarcinoma.2

A strong expression of p53 is found in DALM and not inadenoma.20

In the United States, women are more affected by UCthan men.18 Accordingly, we found a female predominancein patients with ulcerative colitis (69.35%). The European epi-demiological study was discordant from the USA study andfound higher incidence in men in the age groups above 35years.21 In our sample, we found more men in the group withdysplasia, and the statistical analysis showed a significantdifference (p = 0.0242). Some studies have found a higher pro-portion of cases with dysplasia and colorectal cancer in males,but there was no statistical assessment of the significance ofthis difference in relation to gender.22–24

Conclusions

The incidence of dysplasia was lower than that in the liter-ature. Patients with colorectal carcinoma had concomitant

j). 2 0

r

1

1

1

1

1

1

1

1

1

1

2

2

2

2

24. Riegler G, Bossa F, Caserta L, Pera A, Tonelli F, Stumiolo GC,

140 j coloproctol (rio

dysplasia. The incidence of dysplasia was significantly higherin males. The expression of p53 protein in dysplasias was con-sistent with the literature.

Conflicts of interest

The authors declare no conflicts of interest.

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