Flow cytometric DNA analysis and p53 protein expression show a good correlation with histologic...

Flow Cytometric DNA Analysis and p53 ProteinExpression Show a Good Correlation with HistologicFindings in Patients with Barrett’s Esophagus

Alberto Gimenez, M.D.1

Alfredo Minguela, M.D.2

Pascual Parrilla, M.D., Ph.D.3

Juan Bermejo, M.D.1

Domingo Perez, M.D.4

Joaquın Molina, M.D.5

Ana M. Garcıa, M.D.2

Marıa A. Ortiz, M.D., Ph.D.3

Rocıo Alvarez, M.D., Ph.D.2

Luisa M. de Haro, M.D., Ph.D.3

1 Department of Pathology, Virgen de la ArrixacaUniversity Hospital, Murcia, Spain.

2 Department of Immunology, Virgen de la ArrixacaUniversity Hospital, Murcia, Spain.

3 Department of Surgery, Virgen de la ArrixacaUniversity Hospital, Murcia, Spain.

4 Department of Biostatistics, Facultad de Medi-cina, Murcia, Spain.

5 Department of Gastroenterology, Virgen de laArrixaca University Hospital, Murcia, Spain.

Supported by the Isabel Gomez Aroca Foundation;Royal Academy of Medicine and Surgery of Murcia(Spain).

Address for reprints: Dr. A. Gimenez, Servicio deAnatomıa Patologica, Hospital Universitario Mo-rales Meseguer, Marques de los Velez, s/n, Mur-cia-30008, Spain.

Received November 5, 1997; revision receivedFebruary 13, 1998; accepted February 13, 1998.

BACKGROUND. There is a considerable degree of subjectivity and, therefore, sub-

stantial interobserver and intraobserver disagreement in the diagnosis and grading

of dysplastic lesions in Barrett’s esophagus (BE). The aim of this study was to

evaluate the usefulness of DNA flow cytometry and immunohistochemical staining

for p53 protein as objective methods to complement the conventional histologic

diagnosis of dysplasia in patients with this disease. The most common problems

and the possible advantages of using these procedures are analyzed briefly in this

article.

METHODS. Formalin fixed, paraffin embedded tissue from 55 patients diagnosed

with BE were processed for flow cytometric measurements (ploidy and prolifera-

tion index) and p53 immunostaining.

RESULTS. Both the cytometric data and the positivity of staining for p53 revealed a

statistically significant increase throughout the following sequence: no dysplasia3

indefinite for dysplasia 3 low grade dysplasia 3 high grade dysplasia 3 adeno-

carcinoma. There was also a highly significant correlation between the results of

the cytometric study and the positivity of staining for p53.

CONCLUSIONS. In the future, the use of this procedure could play an important role

in the evaluation of patients with BE. Considering that staining for p53 is techni-

cally simple, economical, and quick, and the materials required are available to

most pathology laboratories, this method appears to be a firm candidate for

application as a biomarker in BE. The authors have shown that it is possible to

obtain adequate results for cytometric analysis with small formalin fixed, paraffin

embedded biopsies if a strict protocol for the acceptance of tissue samples and/or

histograms is observed. Cancer 1998;83:641–51.

© 1998 American Cancer Society.

KEYWORDS: Barrett, Barrett’s esophagus, flow cytometry, proliferation index, pro-liferative activity, cell cycle analysis, p53, oncogene, immunohistochemistry.

Barrett’s esophagus (BE) is a pathologic condition in which thestratified squamous epithelium that normally covers the distal

esophagus is replaced by a metaplastic columnar mucosa, frequentlyassociated with the presence of severe, prolonged chronic gastro-esophageal reflux.1,2 The main interest lies in that it is a premalignantcondition, because it predisposes to the development of esophagealadenocarcinoma.3–5 The incidence of this neoplasia in a patient withBE has been estimated at from 1 in 46 to 1 in 441 patients per year, arisk 30 – 40 times higher than that expected in a similar populationwithout BE.6,7 To detect the neoplasia in these patients at an earlystage, regular endoscopic surveillance is recommended together with

641

© 1998 American Cancer Society

multiple esophageal biopsies in an attempt to detecthistologic dysplasia, a precursor to the development ofadenocarcinoma.8,9

However, there is a considerable degree of subjec-tivity and, therefore, substantial interobserver and in-traobserver disagreement in the diagnosis and gradingof dysplastic lesions,10,11 even among pathologistswho are experts in gastrointestinal pathology. There-fore, dysplasia cannot be considered an ideal biomar-ker of the malignant potential in BE, and there is stilla need for more accurate and objective markers tosignal the risk of a malignant change. DNA flow cy-tometry (FC)12–17 and immunohistochemical stainingfor p53 protein18 –20 have been reported among suchmarkers. The present study aims to evaluate the use-fulness of these two techniques as markers of malig-nancy and to correlate the results obtained with thehistologic evaluation. The degree of correlation be-tween the two techniques when examining identicaltissues is also analyzed.

PATIENTS AND METHODSPatientsThe present study includes histological material from55 patients (45 males and 10 females) with a mean ageof 51.4 years (38 –72 years), who were diagnosed withBE between 1979 and 1996. BE was considered whenthe endoscopic study showed the distal esophagus tobe covered with columnar epithelium, with the pres-ence of “specialized” cylindrical epithelium confirmedhistologically. Eight of the cases had an adenocarci-noma over a BE on the first endoscopy. Another 28cases corresponded to patients with BE who, at somestage in its evolution, presented with dysplastic alter-ations, six of whom developed an adenocarcinomaduring follow-up. The remaining 19 patients werechosen at random from patients with BE who showedno dysplastic alterations at any time in its evolution.

MethodsHistological studyAll the samples used in this study were obtained fromthe histologic archives of the Pathology Department ofthe Virgen de la Arrixaca University Hospital (Murcia,Spain) and corresponded to formalin fixed, paraffinembedded tissue. The tissue samples were processedaccording to the usual histologic techniques. Fromeach endoscopic examination, the tissue fragmentwith the most severe alterations was selected and cat-alogued in one group or another, taking into accountthe highest grade of dysplasia present in the sample.For FC processing, the tissue embedded in a particularblock of paraffin had to have more or less uniformmorphological characteristics (the most severe histo-

logical alteration, according to which the sample wasclassified, was to be represented in at least two-thirdsof the material present in the block). If mixed tissuesamples were observed (e.g., minority dysplastic epi-thelium interspersed with nondysplastic glandular ep-ithelium and squamous-type epithelium), then me-chanical separation was not attempted, and thesample was rejected. A diagnosis of dysplasia was de-fined as an epithelial transformation in a neoplasticsense following the criteria established previously fordiagnosing dysplasia in ulcerative colitis21 and thenmodified for application to BE.10 The slides were his-tologically reviewed and interpreted independently bythree pathologists who had no knowledge of the re-sults of the other techniques used in the study. Incases in which there was no general agreement in thediagnosis and grading of lesions, an attempt was madeto establish a consensual diagnosis with a joint reviewof the preparations. If diagnostic disparity persisted,then the case was rejected from the study. Cases inwhich histologic material was not evaluable (e.g., ne-crotic material from ulcers) or insufficient (paraffinused up) were also rejected. With these criteria, weselected 147 samples for FC processing, of which only95 were regarded as valid, corresponding to 53 pa-tients with the exclusion criteria, as specified in Table1. We also performed 87 immunohistochemical stain-ings for p53, corresponding to 54 patients; in 78 cases,it was possible to perform both techniques on thesame biopsy.

All of the material accepted for the FC study camefrom endoscopic biopsies with the aim of unifying themethod of processing and tissue enzyme digestion aswell as minimizing the risk of contamination by othertissues caused by using large-sized histologic sectionsfrom the resection specimens. We did evaluate somesections of this type in the immunohistochemicalstaining for p53, because they do not present theselimitations.

Flow cytometryWe used a pepsinization technique modified from thatof Hedley et al.22 Two to three 50-mm-thick sections

TABLE 1Exclusion Criteria for Flow Cytometric Analysis

● Scarce celularity obtained (,10,000 nuclei per sample)● Excessive tissue debris (.20%)● Coefficients of variation greater than 8%● Small “shoulders” of the G0/G1 peak● Asymmetric (near-diploid, non-Gaussian) G0/G1 peak; “suspicious” ploidy● Mathematical model innadecuated for your application in the histogram (for

example, nonexponential distribution of debris, S-phase 5 zero, etc.)

642 CANCER August 15, 1998 / Volume 83 / Number 4

were obtained from the paraffin blocks. After depar-affinization in xylene and subsequent rehydration(graded ethanol and distilled water), the tissue wassuspended in distilled water and allowed to hydrateovernight at room temperature. The tissue wasminced mechanically, filtered through a 75-mm nylonmesh, and incubated for 90 minutes at 37°C in a 0.5%pepsin solution (Sigmat Chemical Co., St. Louis, MO),pH 1.5, with intermittent vortexing. After incubation,the suspension was centrifugated for 10 minutes at3900 gravity, the supernatant was removed, and thesamples were resuspended in citrate buffer. They werefiltered again through a 53-mm mesh, and, subse-quently, a simple staining procedure was performedwith propidium iodide, partially using the CellCycle-kit staining equipment (Becton Dickinson Immunocy-tometry Systemst, San Jose, CA), which contained ri-bonuclease A.

Measurements were performed on a FACSort flowcytometer (Becton Dickinson Immunocytometry Sys-temst) equipped with an argon laser set at an emis-sion wavelength of 488 nm and calibrated daily withmarked chicken erythrocytes (QC-particles kit; BectonDickinson). Analysis was performed by using the com-puter program CellFit software (version 2.0.2; BectonDickinson), which counted at least 10,000 nuclei persample.23

If excessive tissue debris was detected (.20%) or ifthe degree of resolution, defined as the coefficient ofvariation of the G0/G1 peak, was greater than 8%,24

then another attempt was made to process the sampleif sufficient material remained; if not, then it wasexcluded from the study. The proportion of cells indifferent cell cycle phases was calculated by using thepolynomial mathematical model, proposed by Deanand Jett,25 with the use of an algorithm that modelsdebris as an exponential.26,27 If necessary, we per-formed an electronic exclusion of doublets and cellaggregates by using the doublet-discrimination mod-ule (DDM) included with the CellFit software. Theproliferative index (PI) was defined as the percentageof cells in the S and G2M phases of the cell cycle (S 1G2M). Diploid samples were regarded as those forwhich the histogram showed a single defined G1 peak.DNA aneuploid samples were defined as those forwhich the histograms presented a second peak in anunexpected location for peaks G1 or G2M.28,29 Thecases with small “shoulders” or asymmetric G0/G1

peaks were also excluded.30 No tetraploid tissues (orwith G2M phases .20%) were found.31 The DNA con-tent for each aneuploid sample was computed by di-viding the mean channel of the aneuploid cell popu-lation by the mean channel of the diploid cellpopulation. When the mathematical model was un-

able to analyze the proportion of cells in different cellcycle regions, the cases were excluded or were repre-sented only by their DNA index.

With these criteria, we analyzed between 10,600and 26,100 nuclei per specimen (mean, 15,800), with amean coefficient of variation of 5.3% (range, 2.7–7.9).All of the cytometric measurements, the acceptance ofa histogram as valid, as well as the electronic exclusionof doublets and cell aggregates were done by stafffrom the Immunology Department of this hospitalindependently and with no knowledge of the his-topathologic diagnosis or of the results of stainingfor p53.

Staining with p53Staining was performed by using the streptavidin-bi-otin-peroxidase method. The primary monoclonal an-tibody against p53 was clone DO-7 (DAKO Diagnos-ticst, Glostup, Denmark), which reacts with both thewild type and mutant forms of this protein. We usedsections of a mammary tumor with known positivityfor p53 as both positive and negative controls (in thelatter case, the tissue fragment was incubated withoutthe primary antibody).

The staining was evaluated independently by asingle pathologist who had no knowledge of the diag-noses previously assigned to each preparation in thehematoxylin and eosin study. Cases were labelled aspositive for p53 if they showed any obvious intensenuclear staining detected in at least 1% of the nuclei inthe tissue area of interest; for this, once the area hadbeen identified under low magnification, we used highmagnification (3400) to examine ten microscopicfields at random, each of which contained at least 50epithelial cells.19 The samples with staining of dubiousinterpretation, with weak intensity, or that were veryfocal were rejected.

Statistical methodComparison of the mean values of the cytometricanalysis between two independent groups (for exam-ple, biopsies without dysplasia vs. biopsies that were“indefinite” for dysplasia) was carried out with thecombined Student’s t test or with the Behrens–Fishertest, according to whether or not there was homoge-neity of variance between the two samples, or with theMann–Whitney nonparametric test when data clearlydeviated from normal despite log transformation.32

Comparison of more than two mean values in inde-pendent samples was made by using a one-way anal-ysis of variance (ANOVA), after which individual com-parisons were made with the Tukey test to find themeans that really differed significantly. In the sameANOVA, we analyzed the linear trend of the means of

Histologic Evaluation for Barrett’s Esophagus/Gimenez et al. 643

the data obtained according to the severity of thehistological diagnosis.32

A limit figure was established as a norm for thecytometric parameters by calculation of the two stan-dard deviations above the mean of biopsies in thegroup without dysplasia, and an ROC curve analysis33

checked that this was the figure that provided the bestrates of diagnostic efficiency (sensitivity and specific-ity) and predictive value, eliminating for these calcu-lations, obviously, the biopsies with a diagnosis of“indefinite” for dysplasia.

To study the relation between qualitative vari-ables (e.g. between positivity or negativity in the stain-ing for p53 protein and histopathological alterations)and comparison of the proportions on independentsamples, and to calculate the sensitivity, specificity,and predictive value of the different parameters, weperformed a contingency table analysis using thePearson chi-squared test and subsequent residualanalysis. We also used the same analysis to study thelinear trend of the proportion of pathological resultsaccording to histological severity. When the frequen-cies were low Fisher’s exact test was used. Comparisonbetween the proportions in paired samples (e.g. com-parison of sensitivity, specificity and predictive valueof the different parameters) was carried out with theMcNemar test.34 In all cases we regarded a differencebetween groups or a relation between variables assignificant when the resulting level of significance (P)was less than 0.05.

RESULTSCorrelation with HistologyBiopsies without dysplasiaIn samples with this histologic diagnosis, there were40 cytometric determinations and 33 stainings for p53,

corresponding to 32 patients. With regard to FC, noaneuploid cell population was detected, and the meanPI (S phase 1 G2M phase) was 4.9 6 0.7% of the cells(median, 4.9%; range, 3.2– 6.7%). If the limit of cyto-metric “normality” (in addition to the absence of anydetectable aneuploid population) is established as themean PI of this group plus 2 standard deviations, thenthe limit is 6.5%. It can be seen in Figure 1 that only 1of the 40 samples without dysplasia (2.5%) exceedsthis limit.

With regard to staining for p53, the results werenegative in the 33 samples studied (see Fig. 2).

Biopsies with indefinite dysplasiaIn samples diagnosed as indefinite dysplasia, therewas a total of 25 valid cytometric determinations and22 stainings for p53, corresponding to 18 patients. Noaneuploid cell population was detected in this groupeither. The mean PI was 6.7 6 2.1% of the cells (me-dian, 6.4%; range, 2.9 –11.6%). Figure 1 shows that thePI is above “normal” in 13 of the 25 samples (52%).Staining for p53 was positive in 6 of the 22 samplesstudied (27%).

Biopsies with low grade dysplasiaWith this diagnosis there were 14 valid cytometricmeasurements and 13 stainings for p53, correspond-ing to 12 patients. No aneuploidies were detected. Themean PI was 10.1 6 3.1% of the cells (median, 9.8%;range, 5.5–19%). The PI was abnormal in 13 of the 14samples (92.9%). Staining for p53 was positive in 8 ofthe 13 samples studied (61.5%).

Biopsies with high grade dysplasiaIn this group, there were five valid cytometric mea-surements and five stainings for p53, corresponding to

FIGURE 1. Correlation between flow

cytometry and histology (solid line indi-

cates the mean proliferative index of the

group without dysplasia 1 2 standard

deviations).


five patients. There were three samples with aneu-ploidy (60%). The respective DNA contents were 1.32,1.30, and 1.62. The mean PI was 22.2 6 8.9% of thecells (median, 20.3%; range, 12.5–35%) in all casesabove normal. Staining for p53 was positive in all fivecases (100%).

Biopsies with adenocarcinomaHistologic material was available from 13 patients, inwhom 11 effective cytometric measurements and 14stainings for p53 were performed. Aneuploidy existedin 9 of the 11 samples processed (81.8%), with DNAcontents of between 1.36 and 1.81. In two of them, themathematical model was unable to calculate the val-ues for the cell cycle phases. The mean PI was 24.1 68.2% (median, 24.3%; range, 14.4 –36.8%) of the cells.The two biopsies without a detectable aneuploid pop-ulation had PIs of 17.7% and 17.2%, respectively.Staining for p53 was positive in 11 of the 14 samples(78.5%).

Table 2 shows that both the cytometric abnormal-

ities (PI, abnormal FC, or aneuploidy) and the positiv-ity of the staining for p53 showed a statistically signif-icant increase throughout the sequence: no dysplasia3 indefinite dysplasia 3 low grade dysplasia 3 highgrade dysplasia3 adenocarcinoma. In Table 3, we seethat the biopsies without dysplasia and with indefinitedysplasia show statistically significant differencesfrom each other and from the other groups both in PIand in positivity for p53. The PI also showed statisti-cally significant differences when biopsies with lowgrade dysplasia were compared with biopsies withhigh grade dysplasia or carcinoma, which did not oc-cur in the staining for p53. Comparison of biopsieswith high grade dysplasia and biopsies with adenocar-cinoma showed no significant differences with any ofthe markers. The sensitivity, specificity, and positiveand negative predictive values of both techniques areexpressed in Table 4.

Regarding the distinction between the most se-vere lesions (high grade dysplasia and/or carcinoma)and the other histological findings—the only distinc-

FIGURE 2. Correlation between stain-

ing for p53 and histology (percentages

of positive biopsies are indicated in pa-

rentheses).

TABLE 2General Results Obtained in the Study

HistologyNo. patients(total 5 55)a

FC analysis(total 5 95)

Stainingsp53 (total 5 87) PI (mean 6 S.D.)

“Abnormal” FCcases (%)

Aneuploidycases (%) p531 cases (%)

Without dysplasia 32 40 33 4.9 (0.7) 1 (2.5) 0 0Indefinite dysplasia 18 25 22 6.7 (2.1) 13 (52) 0 6 (27)Low grade dysplasia 12 14 13 10.1 (3.1) 13 (92.9) 0 8 (61.5)High grade dysplasia 5 5 5 22.2 (8.9) 5 (100) 3 (60) 5 (100)Adenocarcinoma 13 11 14 24.1 (8.2) 11 (100) 9 (81.8) 11 (78.5)Statistical evaluation (linear trend)3 P , 0.0001 P , 0.0001 P , 0.0001 P , 0.0001

FC: flow cytometry; PI: proliferative index.a The total number of patients is less than the sum of the first column, because there are patients in the series with tissue samples in different histological groups, or with tissue samples in the same histological

stage, although samples were taken at different times of their endoscopic follow-up.


tion with immediate clinical significance—very cleardifferences are observed in the results obtained withboth techniques for these two groups of lesions. Thedata referring to these groups are shown in Table 5.

Correlation between Flow Cytometry and Staining for p53Table 6 shows that there is a highly significant corre-lation between the mean PI values or the abnormalityof the cytometric study (PI . 6.5% and/or aneuploidy)and the positivity in the staining for p53. In 51 pa-tients, both techniques were performed at least once.The biopsies from 38 patients (74.5%) coincided fullyfor the two techniques (that is, an abnormal FC wasassociated with positive staining, and vice versa). Inthe remaining 13 patients (25.4%), discordant resultswere obtained at some time (generally an abnormalFC and negative staining for p53). The majority of

these 13 patients (7) had only one sample processed.The other 6 had more biopsies processed, and theresults in 4 of them did indeed coincide in these otherbiopsies.

Patients with Multiple BiopsiesIn Figures 3 and 4, the results are shown in thosepatients who had more than one p53 staining (Fig. 3)or more than one FC (Fig. 4). We can see that, al-though the coincidence is not absolute (i.e., there arecases—Patients 2 and 9 for p53 staining and Patients2, 4 and 5 for FC—in which not all of the samples withthe same range of histologic abnormality obtained thesame result for a particular technique), most resultsare uniform for a particular histologic stage. Further-more, once an abnormal result is observed, it tends topersist in the more advanced stages of tisular abnor-mality (with the exception of Patient 5).

DISCUSSIONIf we consider that most studies confirming a highinterobserver variability in the diagnosis and gradingof dysplastic lesions come from pathologists who areexperts in gastrointestinal pathology, there is an obvi-ous practical interest in having new malignancy mark-ers to confirm the histological diagnosis, especially forpathologists less familiar with the interpretation ofdysplasia in BE. Concerning FC, most studies havefocused their attention on the determination ofploidy.23 Measurement of proliferative activity fromanalyzing the different cell cycle intervals has not re-ceived as much attention until recently, althoughsome studies have shown that this proliferative activ-ity may be a more important prognostic indicator thantissue DNA content.35–37 Our results confirm the im-portance of evaluating tissue proliferative activity us-ing FC. Some authors12,17 have drawn attention to thegood correlation observed between the histologic al-terations in BE and the percentage of cells in differentcell cycle phases, as determined by FC with the pro-cessing of fresh tissue. However, the tendency of DNA

TABLE 3Comparison of the Results of Flow Cytometry (PI) and thePercentages of Positivity in the Immunostaining for p53 betweenEach of the Groups

Groups to compare

P values

Proliferative index p53 Staining

Without dysplasiaIndefinite dysplasia 0.0005 ,0.005Low grade dysplasia ,0.0001 ,0.0001High grade dysplasia ,0.005 ,0.0001Carcinoma ,0.0001 ,0.0001

Indefinite dysplasiaLow grade dysplasia ,0.0005 ,0.05High grade dysplasia ,0.005 0.01Carcinoma ,0.0001 ,0.005

Low grade dysplasiaHigh grade dysplasia 0.01 NSCarcinoma ,0.0001 NS

High grade dysplasiaCarcinoma NS NS

NS: nonsignificant difference.

TABLE 4Values Calculated for the Sensitivity, Specificity, and PredictiveValues of the Different Parameters

Discrimination without dysplasiavs. dysplasia 1 carcinoma

Flow cytometry

Staining forp53 protein (%)

PI(%)

Aneuploidpopulation (%)

Sensitivity 96.7 40 75Specificity 97.5 100 100Positive predictive value 96.7 100 100Negative predictive value 97.5 69 80

PI: proliferative index.

TABLE 5Comparison of the Results for Severe Lesions (High Grade Dysplasiaand Carcinoma) and All Other Groups of Histologic Alterations

ValueHigh grade dysplasiaand/or carcinoma All other

Statisticalevaluation

PI (mean 6 SD) 23.1 (8.5) 7.2 (1.9) P , 0.0001“Abnormal” FC cases (%) 16 (100) 27 (34.1) P , 0.0001Aneuploidy cases (%) 12 (75) 0 (0) P , 0.0001p531 cases (%) 16 (84.2) 14 (20.5) P , 0.0001

PI: proliferative index; FC: flow cytometry.


histograms obtained from material embedded in par-affin to present a larger quantity of tissue aggregatesand debris has been a considerable obstacle to deter-mining the values of proliferative activity reliably.Nevertheless, improvements in and adaptations ofmethods for preparing the material,38,39 together withthe development of software programs that includealgorithms for debris substraction,40,41 have enhancedthis type of analysis. All the same, we tried to beextremely careful in the management of samples andvery demanding when acknowledging a histogram asvalid for inclusion in the study, which meant that thepercentage of samples excluded for these reasons wasmore than one in three of those processed (52 sam-ples; 35.4%), values that are obviously much higherthan those reported in most publications (10 –15%).Less strict criteria for acceptance or the use of morerecent nonexponential algorithms40 would probablyhave allowed us to increase the number of biopsiesprocessed.

It may be worth noting that we found a much

wider range of values for the PIs in severe lesions (highgrade dysplasia and carcinoma); these results are sim-ilar to those obtained by other authors.36,37 However,this variability probably can be attributed to the fre-quent presence of aneuploid cell populations in thesesamples. The aneuploid populations tend to havegreater coefficients of variation (these wide coeffi-cients probably indicate biologic heterogeneity andgenomic instability), and this accounts in most casesfor the variance in the calculated values for the S-phase of the cell cycle.

It has been stated that aneuploidy is probably thereflection of a genomic instability that may culminatein a cancer.42 In fact, 9 of the 11 adenocarcinomasprocessed in our series (81.8%) were shown to haveaneuploid cell populations. Moreover, another threepatients presented with aneuploidies in their esopha-geal mucosa in the absence of carcinoma. The threecases corresponded to patients with high grade dys-plasia in whom a new biopsy taken shortly afterward(between 1 month and 6 months) revealed a neoplas-

TABLE 6Correlation between the Results of Flow Cytometry and Staining for p53

Value p53 Negative (n 5 53) p53 Positive (n 5 25) Statistical evaluation

Mean PI 6 SD (median, range) 6.6 6 4.9 (5.4; 2.9–32) 16.1 6 9.7 (13.5; 3.5–36.8) P , 0.0001“Normal” FC cases (%; n 5 43) 41 (95.3) 2 (4.7) P , 0.0001“Abnormal” FC cases (%; n 5 35) 12 (34.3) 23 (65.7) P , 0.0001Aneuploidy cases (%; n 5 11) 2 (18.1) 9 (81.8) P 5 0.01

PI: proliferative index; SD: standard deviation; FC: flow cytometry.

FIGURE 3. Patients in our series with

more than one stain for p53 protein.

Only in Patient 2 (indefinite for dysplasia)

and Patient 9 (low-grade dysplasia) bi-

opsies with the same histologic findings

had variable results. There was one pa-

tient—Patient 16—who had two pro-

cessed samples of adenocarcinoma.

This patient received surgery for an ear-

ly-stage, well-differentiated adenocarci-

noma. Three years later, he presented

with a poorly differentiated recurrence

histologic samples of which were also

processed. Open squares, p53 negative;

solid squares, p53 positive.


tic degeneration. However, we found no case of aneu-ploidy among the BE patients without dysplasia,which is in line with the results of some authors,17,29

although not with others,12–15 both in this and in othertissues.43 Our results suggest that alteration in thetissue PI could be an indicator of an early genomicinstability, which, with time, will develop into lesionswith a more altered DNA content: aneuploidy. Thediscordance between our results and those obtainedby others who do find aneuploidy in patients withminor histological alterations, and even in patientswithout dysplasia, might be explained in some casesby the variability itself in the diagnosis and grading ofthe dysplastic lesions and, in other cases, by the factthat most of these studies have been conducted infresh material. It has not been explained fully whytissue prepared from material embedded in paraffinusually has a lower proportion of aneuploid sam-ples44 – 46 than when fresh material is used. It has beensuggested that enzyme digestion with pepsin may leadto a selective loss of tumoral cells, because the malig-nant nuclei seem to be larger and have complex nu-clear membranes, which could make them relativelyfragile.28 Other authors have pointed out the presenceof an unusually large proportion of presumably falsehyperdiploid peaks when using fresh tissue.47–50 It isbelieved this may be the result of a deterioration inchromosomic proteins with the subsequent increasein propidium iodide uptake, an apparently consider-able and very common problem in the mucosa of thegastrointestinal tract.47

On the other hand, in the histograms of tissueembedded in paraffin with a large amount of debris or

in cases in which the cell population of interest is inthe minority, the presence of smaller aneuploid pop-ulations may be obscured. Furthermore, although cer-tain guides have been established to the standardiza-tion of the terminology used in FC,30,31 a review byWersto et al.51 of more than 200 articles relating ploidyto the clinical behavior of cancer indicates that thecriteria used for evaluating the histograms have oftenbeen inconsistent and subjective. For example, insome studies, the presence of small shoulders to theright of a G0/G1 population, an asymmetric G0/G1

peak, or simply all doubtful cases are considered to beaneuploid. In this work, these “suspicious” histogramswere rejected.

Concerning immunohistochemical staining forp53 protein, our results show a significant correlationwith the histopathologic alterations, with the percent-age of positive biopsies increasing with the severity ofthe lesions. The p53 gene, which is located in the shortarm of chromosome 17, appears to be the most fre-quent location of genetic alterations in human can-cer.52 Its normal product, or wild type p53 protein,acts by preventing the propagation of genetically dam-aged cells at a cell cycle “checkpoint” between the G1

and S-phases.53,54 Its half-life is very short, which iswhy it cannot be detected with the usual immunohis-tochemical procedures. Alterations in the gene lead tothe expression of mutant forms of the protein that arenonfunctioning and that have a longer half-life,which can be detected immunohistochemically. Thecorrelation that we found between the alterationsdetected by FC and the positivity in the staining forp53 may be explained by the fact that the develop-

FIGURE 4. Patients in our series with

more than one flow cytometry (FC) anal-

ysis. The results are similar to p53 pro-

tein staining. In both techniques, once

an “abnormal” result is observed, it

tends to persist in more advanced

stages of tissue abnormality (the only

exception was Patient 5 for FC). Open

squares, normal FC; solid squares, ab-

normal FC (proliferative index .6.5%

and/or aneuploidy).


ment of cell populations with an increase in the S-and G2M-phases of the cell cycle could be related tothe inactivation of a checkpoint at the G1-phase/S-phase transition.42 Theoretically, therefore, p53 pro-tein overexpression detected on immunohistochemis-try is an indirect method for detecting a p53 genemutation. However, immunohistochemistry mayshow positive staining for the p53 protein in cells thatdo not have p53 mutations. Other factors, such asDNA damage, failure to break down p53 protein, orloss of signals that turn off p53, may increase p53protein expression.55 This, on the other hand, does notmean that the technique cannot be applied to tissueswith wild type protein expression or that conclusionscannot be drawn regarding the significance of thisoverexpression in a tissue or a particular disease. Thechances of the technique becoming clinically applica-ble are greater than for more difficult investigations,such as DNA sequencing, which are suitable currentlyonly for research.

Although certain publications have reportedsmall percentages of samples without dysplasia withpositive immunostaining,20 we, like others,19,56

found no case of this in our series. Although theinfluence of sampling error or differences in sensi-tivity of the antibody used cannot be ruled out com-pletely, it is possible that some of these differencesare related to the criteria used to regard a case aspositive for p53 staining. Whereas some authorsregard a sample with any nuclear staining as posi-tive (without evaluating either the intensity or thenumber of stained nuclei), others require an obvi-ous nuclear staining, which, furthermore, is presentat least in 1% of the nuclei in the tissular area ofinterest.19 In our study, we accepted the latter cri-terion, because it is highly reproducible. When weobserved a biopsy without dysplasia with veryweakly or focal staining, these samples, like thehistograms of dubious interpretation, were rejected.

It may seem surprising that, whereas patients withhigh grade dysplasia reveal positive immunostainingin 100% of cases, only 78.5% of carcinomas are immu-noreactive. However, these data can be comparedwith other reports.19,20 Among the reasons that mightexplain the lower percentage of tumor immunoreac-tivity compared with high grade dysplasia, an increasein the turnover rates of oncoprotein has been sug-gested. This would decrease its half-life, making im-munohistochemical detection difficult or impossible.Other possibilities are the appearance of an additionalmutation or coupling with another protein that causesa structural change in the oncoprotein, making it un-recognizable for the monoclonal antibody used. Fi-nally, p53 may not be produced in sufficient quantity

in the final stages of carcinogenesis for it to be de-tected.20

The data shown in Table 3 indicate that stainingfor p53 would allow differentiation between the sam-ples with or without dysplasia/carcinoma (but notbetween low grade dysplasia, high grade dysplasia,and carcinoma). With the cytometric examination, it isapparently possible to distinguish between all types oflesions, except between high-grade dysplasia and car-cinoma.

With regard to sensitivity, specificity, and pre-dictive value, both techniques show highly reliablevalues. Although the sensitivity and negative predic-tive value of the PI (96.7% and 97.5%, respectively)are statistically higher than those of the stainingfor p53 (75% and 80%; P , 0.01 and P , 0.05respectively; McNemar test), the latter values arevery acceptable, which is why, if we consider thatstaining for p53 is technically simple, economical,and quick and represents a procedure that is avail-able to most pathology laboratories, it seems obvi-ous that the use of this staining could play an im-portant role in the future in the evaluation ofpatients with BE.

Concerning FC, although it is becoming a routineclinical test in many institutions, it does have certaintechnical and interpretative problems. There are sig-nificant interlaboratory differences in the methodsused and in the interpretation of results, not to men-tion different computer analysis programs with vary-ing capacities and possibilities. The most seriousshortcoming currently is the lack of standardizationand quality control.23 There is a need for a set ofwidely accepted evaluation criteria and also for a uni-form methodology to enable us to extrapolate resultsbetween the different institutions and, thus, ensurethe introduction of DNA FC into habitual clinical prac-tice; certainly, a lot of work is being done in thisdirection.57– 61

In any case, the possibility of successfully process-ing small tissue samples fixed in formalin and embed-ded in paraffin enables us to perform cytometry innumerous series of patients with similar characteris-tics and, therefore, to obtain solid data on the behav-iour of BE. This constitutes a unique and very acces-sible in vivo model for studying the pathogenicmechanisms, the cell proliferation and differentiation,and the neoplastic modifications that contribute tothe transformation of an initially normal epidermoidmucosa into a benign metaplastic mucosa and, sub-sequently, into a dysplastic mucosa and, finally, intoan adenocarcinoma.


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