comparison of rectal mucosal proliferation measured...

Vol. 4, 7/5-720. (),t,’bi’r/Novt’,nbt’r /995 Cancer Epidemiology. Biomarkers & Prevention 715

3 The abbreviations used are: PCNA. proliferating cell nuclear antigen: W(’M(’.whole-crypt mitotic count: LI. labeling index.

Comparison of Rectal Mucosal Proliferation Measured by Proliferating

Cell Nuclear Antigen (PCNA) Immunohistochemistry and Whole

Crypt Dissection’

Sharon C. Murray, Robert S. Sandier,2 Temitope 0. Keku,Cynthia M. Lyles, Robert C. Millikan, Shrikant I.Bangdiwala, Lawrence L. Kupper, Wuhan Jiang, andMartin H. Ulshen

Departments of Medicine. Epidemiology and Biostatistics. and Center for

(Iastrointestinal Biology and Disease, University of North Carolina at Chapel

Hill. (‘hapel lull. North (‘arolina 27599-7080

Abstract

Rectal mucosal proliferation has been promoted as anintermediate marker for risk of colorectal neoplasia.Proliferating cell nuclear antigen (PCNA)immunohistochemistry has become a standard method tomeasure cell proliferation. Whole-crypt dissection mayprovide a technically simpler method for determiningproliferation within an entire crypt. We conducted astudy to assess the reliability (reproducibility) of whole-crypt dissection in 10 subjects, and we compared whole-crypt dissection to PCNA immunohistochemistry in 91subjects. Reliability of whole-crypt dissection with thesubject as the unit of observation was excellent. Theintraclass correlation coefficient for subjects was 0.93.Biopsy-to-biopsy reliability was lower (r 0.86) andcrypt-to-crypt reliability lower still (r 0.35). There waspoor correlation between measures of proliferation indexusing the two techniques (Kendall’s tau 0.13; P0.08). Compartment analysis based on the percentage of

the total number of labeled cells appearing in each cryptquartile also did not demonstrate a significant correlationbetween the two measures. We conclude that PCNAlabeling index and whole-crypt mitotic count are notcomparable measures of rectal mucosal proliferation.

Introduction

Cellular proliferation in the normal large intestine is generally

confined to the base of colonic crypts. Absorptive and gobletcells migrate upward from putative stem cells at the crypt base(I). An expansion of the proliferative zone has been demon-strated in humans with diseases that predispose to cancer (suchas familial polyposis coli) and in rodents treated with chemicalcarcinogens (2). Expansion of the proliferative zone has alsobeen reported in the mucosa of patients with sporadic adeno-mas, nonfamilial colon cancer, and hereditary nonpolyposis

Received 1 1/21/94: accepted 6/13/95.

� This work was supported in part by Grants ROl CA44684, P3)) DK34987. and

RR (88)46 from the NIFI.: rO whom requests for reprints should he addressed, at Division of Digestive

1)iseases and Nutrition, (‘B # 7(180, 423A Burnett-Womack Building. Universityof North (‘arolina. (‘hapel Ilill. N(’ 27599-7081).

colorectal cancer (3-5). Proliferation measures might, there-fore, be used as biomarkers to identify individuals at increasedrisk for neoplasia or could be used to monitor the effects ofintervention measures to reduce risk (6). Indeed, proposedchemopreventive agents have been shown to decrease the pro-liferative index in both rodents and humans (7, 8, 9).

A number of methods have been used to measure cellularproliferation in the large bowel. The determination of prolifer-ation index by tritiated thymidine autoradiography has beenused for many years but has some disadvantages (7. 10). Thetechnique is somewhat time consuming and requires radioac-

tive material. Bromodeoxyuridine is an analogue of thymidinethat is incorporated into the DNA of replicating cells duringS-phase of the cell cycle (I 1, 12, 13). This technique is fasterthan autoradiography and has been shown to have good corre-lation with tritiated thymidine incorporation (4). PCNA3 is acell cycle-associated protein that is maximally synthesized dur-

ing S-phase (14). Identifying PCNA is a technically simplerprocedure because it does not require an incubation step. Be-

cause of ease of use and comparability to more establishedmethods of measuring cell proliferation, PCNA immunohisto-

chemistry (henceforth PCNA) may largely supplant other tech-niques in large-scale or multicenter studies.

All of these methods involve calculating the proportion oflabeled cells from a cross-section of a colonic crypt. Thewhole-crypt dissection method measures the number and loca-

tion of mitoses in an entire microdissected crypt. The numberof mitoses in an entire crypt has been termed the WCMC. Thereare several theoretical advantages to this technique. Biopsiescan be placed directly into fixative (Carnoy’s solution) andstored in alcohol until analysis: there is no need to maintain

tissue viability or for in si/ru incubation: tissue orientation isunimportant; and hundreds of crypts can be easily counted froma single endoscopic biopsy ( 15). The method is less timeconsuming than PCNA. Preliminary work in fasted and rcfcdrats has suggested that the results of crypt dissection are com-parable to those of the bromodeoxyuridine method (15). Themethod has not been extensively used or validated in average-

risk human subjects.If WCMC were highly reliable and comparable to PCNA

LI, it might offer a much simpler hiomarker for use in inter-vention studies designed to reduce the risk of colorectal ma-lignancy. The 3-fold purpose of this study was: (a) to assess thereproducibility of the whole-crypt dissection method in humantissue obtained with standard endoscopy forceps; (b) to com-pare WCMC to PCNA LI; and, (e) to develop sample size

nomograms that could be used to plan clinical studies.

on May 24, 2018. © 1995 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

http://cebp.aacrjournals.org/

7/6 Comparison of PCNA and Crypt Dissection

Materials and Methods

Biopsies were obtained from consenting patients who were

referred to the University of North Carolina Hospitals for a

clinically indicated colonoscopy. Data are presented on 91

consecutive patients for whom both WCMC and PCNA LI were

calculated: 37 white males, 37 white females, 4 black males,and 13 black females. Six biopsies were taken from the rectum

at the start of the procedure using standard endoscopy forcepsat approximately 8-12 cm from the anal verge. Each biopsywas transferred to bibulous paper (Fisher Scientific Co., Ra-

leigh, NC) with special care not to stretch or tear each speci-men. Four specimens were placed in Steinberg’s Modified

Eagle’s Medium for PCNA, and two specimens were placed inCarnoy’s fixative for later crypt dissection. All specimens forPCNA were processed within 30 mm.

The specimens for PCNA were fixed in 70% ethanol

before processing. Paraffin blocks were prepared according to

routine histological procedures, and care was taken not toexceed 60#{176}C.Five sections were placed on glass slides pre-coated with poly-t.-lysine (Sigma Chemical Co., St. Louis MO).The sections were 5-gm thick and were taken at least 50 p�m

apart so that each section would contain different crypts. Thesections ‘iere dc,.waxeL and rehydrated through graded ethanol

concentrations. Slides were incubated for 12-20 h at 4#{176}CusingPCIO antibody (DAKO Co., Carpinteria, CA) at 1:100 in PBS!BSA. Detection of PCNA was performed using the BioGenex

SterAvigen super sensitive kit for alkaline phosphatase (Bio-

Genex, San Ramon, CA). The kit was used according to themanufacturer’s instructions. The slides were counterstainedwith Mayer’s hematoxylin and mounted with Eukitt (Calibrated

Instruments, Inc., Hawthorne, NY). Positive control slides in

each batch of staining included rat testis and human colon tissuefrom a cancer patient.

Only well-oriented crypts were scored. Crypts were se-Iected for scoring if the base of the crypt touched the muscularis

mucosa. If the muscularis was missing, a crypt was acceptableif its height was uniform with other crypts. For each studysubject, at least S and at most 12 crypts were scored from each

of 2 biopsies. Each of the four biopsies was screened under low

power, and the two slides that had the best orientation wereselected for scoring (i.e., had the highest number of potentially

scorable crypts). Selection was based on orientation and not on

the amount of staining. Beginning with the first scorable crypt

in the first section, successive crypts and successive levels were

counted, if necessary, until at least eight crypts had been scored.If there were fewer than eight scorable crypts in a biopsy, the

next biopsy was selected. If there were fewer than eightscorable crypts in two separate biopsies, blocks were recut andrescored. For each crypt, the total number of cells in the crypt,

the center cell, and the ordinal number of stained nuclei wererecorded. Only deeply stained nuclei were counted.

Tissues for whole-crypt dissection were transferred from

Carnoy’s fixative to 70% ethanol after 2.5-3.0 h. Prior tostaining, the tissue was rehydrated in 5% and 25% ethanol, thenhydrolyzed in I M HCI at 60#{176}Cfor 10 mm in a water bath. The

biopsies were then placed in Schiff’s reagent and incubated in

the dark for 45-60 mm at room temperature. A small piece ofbiopsy was then cut and placed on a microscope slide with 45%

acetic acid. The tissue was gently teased apart under a dissect-

ing microscope, then covered with a coverslip. The coverslipwas gently tapped until the crypts began to separate into dis-

crete crypts. The dissected crypts were examined under a com-pound microscope at X200. To be scored, the top and bottom

of the crypt had to be seen. Broken crypts were not acceptable.

Mitotic cells were identified by nuclear clumping, focusingthrough the entire crypt. Each crypt was also traced on paperusing a drawing attachment (Olympus Corporation, Lake Sue-

cess, NY). All mitoses were drawn within the crypt. For straightcrypts, the height of each crypt was measured from the lowestpoint to the center of a line drawn through the apex. Curved

crypts were measured using a flexible curve. The position of

each mitosis was measured from the crypt base. A total of 10crypts were counted from each of the two biopsies. If there werenot 10 countable crypts in the first piece taken from a givenbiopsy, another piece was taken.

All data were entered and then independently sight-yen-fied. Range checks and checks for impossible values were also

performed.

Reproducibility. The intnareaden reliability of the crypt dis-section method was assessed using data from ten patients whose

slides were read twice by a masked observer. Scatter plots were

generated comparing the values recorded for the first readingand second reading on a pen crypt, per biopsy, and per patientbasis. Intraclass correlation coefficients were calculated in each

of the three cases, using the natural log of the mitotic cell count,as a way of clarifying the degree of agreement etween the twodifferent readings of the same slides. The natural log scale was

chosen because the distribution of the number of cells in mitosistends to be skewed to the right.

Correlation between PCNA and Crypt Dissection Indices.Both PCNA and crypt dissection measurements were obtainedfor 91 patients. WCMC for each individual was calculated as an

average of the number of cells undergoing mitosis in each of 20crypts, 10 from each of 2 biopsies. PCNA LI was calculated for

each individual as the average of the proportion of labeled cellsfrom all scorable crypts in each of two biopsies. A scatter plotcomparing the subject mean values of PCNA LI to WCMC was

generated using SAS/GRAPH (SAS Institute, Cary, NC). Ken-dall’s ‘r, a nonparametnic measure of correlation, and its asso-ciated P were calculated.

Compartment Analyses. Proliferation is not uniformly dis-tnibuted throughout the length of colonic crypts. To evaluate thedistribution of proliferation, we divided crypts into four quar-tiles. For both measures, we calculated the proportion of alllabeled cells appearing in each quartile. For PCNA LI, thisproportion was calculated for each crypt column as the number

of labeled cells in the particular quartile divided by the totalnumber of labeled cells in the crypt column. It was necessary towork with crypt columns rather than crypts because the number

of cells in each column of the crypt was not always the same.The average proportion of labeled cells in each quartile was

computed for each biopsy, and a score for each subject wascomputed as the average of the biopsy scores. The denominatorused in computing this score differs from that usually seen inthe literature for PCNA LI compartment analysis, i.e., the total

number of cells in the compartment. Instead, the number oflabeled cells in the entire crypt column was chosen as the

denominator so that the PCNA compartment scores would becomparable to those obtained using crypt dissection, where wedo not know the total number of cells in each compartment.

For crypt dissection, the height of the crypt was used to

divide the crypt into quartiles. The number of labeled cells ineach quartile (where quartile position is determined by the

distance of the labeled cell from the base of the crypt) dividedby the total number of labeled cells in the entire crypt was

calculated for each quartile in each crypt. These quartile scoreswere then averaged for each of the two biopsies, and the mean

of the biopsy scores was taken as the subject score. Correlation



11 =

I ��ff2\

2�o� + � + �)(z��:� + z�)2

20 25 30

(1)

2nd Rsdm�

25,

�0Lji��j�y:WCMC

�0�s:uc p. �op.y

2,� Rs.a.g

3#{176}IB

25

20

15

0� � � � �-- -�..- �

0 5 10 15 20 25 30

WCMC p� 8.opsy,w R..d.�#{231}

“cue p� S

a�

25

20

,5�

10�

� �

wcMc p� �,� �

Fig. 1. A. WCMC repeatability by crypt. Comparison of P and 2nd cryptreadings with the 45” line through the origin (20 crypts for each of 10 subjects).

B. WCMC repeatability by biopsy. Comparison of � and 2�d biopsy readingswith the 45’ line through the origin (2 biopsies for each of 10 subjects). C.

WCMC repeatability by subject. Comparison of I” and 2� biopsy readings with

the 45” line through the origin (10 subjects).

Cancer Epidemiology, Biomarkers & Prevention 7/7

indices were calculated comparing the average percentage oflabeled cells appearing in each quartile for PCNA and cryptdissection. The overall mean of the subject scores was calcu-lated for each of the quartiles for both PCNA and whole cryptdissection. These overall group mean scores were graphicallycompared using a histogram.

Sample Size Calculations. Minimum sample sizes necessaryto attain 90% power for detecting a 25% difference in the

proliferation measure between two groups were calculated forvarious sample size combinations of subjects, biopsies, andcrypts for whole-crypt dissection. These calculations were

based on a mixed-effects model using the log of the prolifer-

ation measure as the response.The model used included a fixed effect due to group and

random effects due to subject, biopsy, and crypt. Under thismodel, the log of the proliferation measure is assumed to benormally distributed. The hypothesis being tested that there isno difference between the two groups (H0: � 0) and the

alternative hypothesis is two-sided (HA: � � 0). The number of

subjects, ii, required per group is:

where � represents, in log-scale, the minimum absolute detect-able difference between groups, a is the type I error rate, the

power of the test is 1 - �, B is the number of biopsies persubject, and C is the number of crypts per biopsy. Also, �

� and o’�2 are the components of variation due to subjectdifferences, differences between biopsies within subjects, anddifferences between crypts within biopsies, respectively. Thetype I error rate was set to a 0.05, and a group difference of6 = ln(0.75) , which in log-scale corresponds to a 25% differ-

ence in the proliferation measure, was used.Note that if one assumes that oj,2 and r�2 are 0 in the

sample-size formula given above, the result is the familiar

formula for determining the required sample size when corn-paring two groups using a I test. The simpler formula ignores

the variation inherent in measurements taken on the sameindividual, leading to an underestimate of the required samplesize.

Results

Reproducibility. For the crypt dissection data on the 10 sub-jects whose slides were read twice, the mean WCMC for thedata obtained on the first reading was 4.85 (SD 2.83). On the

second reading, the mean WCMC was 4.88 (SD 3.17). In Fig.1, the values obtained upon a second reading of the data areplotted against the original scores on a per-crypt basis, a per-biopsy basis, and a per-subject basis. Note that the number ofcells in mitosis in a crypt ranged from 1 to 24 (Fig. IA). Perbiopsy, the average number of cells in mitosis ranged from 2.1to 12.9 (Fig. 1B). while the per-subject index rangei from 2.6

to 10.1 (Fig. IC). It is clear from the graphs that the reproduc-ibility of this index is better when looked at in terms ofbiopsy-to-biopsy values than in terms of crypt-to-crypt values

and better still when the values are examined per subject. Sincethe biopsy score is an average of crypt scores and the subjectscore is an average of the biopsy scores, this result is as

expected. Calculation of the intraclass correlation coefficient,which measures the degree to which the observations deviate

from the 45#{176}line of equivalency, gives 0.35 for the crypt-to-

crypt comparison, 0.86 for the biopsy-to-biopsy comparison,and 0.93 for the subject-to-subject comparison.

Correlation between PCNA and Crypt Dissection Indices.For the 91 subjects for whom both PCNA and crypt dissection

measures were available, the overall mean PCNA LI was 0.066



PC,””

�x

020

014

a o

002

000

50

40

30

20

10

0

�.. . I

� � �

PCNA WCMC PCNAFig. 2. Scatter plot of the subject mean PCNA LI against the subject mean

WCM(’.

: � .1WCMC PCNA WCMC PCNA WCMC

e0�

Fig. 4. Minimum samples sizes required for 90% power to detect a 25%difference in WCMC between two groups.

7/b’ Comparison of PCNA and Crypt Dissection

AveragePercent

Labeled6O�

let Quartile 2nd Quartile 3rd Quartile 4th Quartile

T�ilol�’ I (‘omparison of the average proportion of “labeled” cells in each

compartment using crypt dissection and PCNA

Proprtion (If labeled cells(‘onlpartment” � ---- .----�- -.-- Kendall’s r P

(‘rypt dissection PCNA

I 48.7 53.4 0.11) (1.1791

2 46.8 27.7 -0.14 0.0577

3 4.4 5.5 0.14 (1.0579

4 0.1 1.7 0.16 0(1924

“ (‘rypts were divided into) four equal compartments by height. starting at the base.

(SD 0.035), and the mean WCMC was 6.58 (SD 3.48). A plotof the subject mean scores for PCNA LI against the subjectmean scores for WCMC for these 91 patients is presented in

Fig. 2. The plot shows little association between the two mdi-ces. Calculation of Kendall’s T confirms this graphical evidenceof little association between the two measures (T = 0.13; P0.0792). Assuming that the subject-to-subject reliability for

WCMC is 0.93 and that the subject-to-subject reliability forPCNA LI is 0.99 (16), the correlation between the two mea-sures (T), corrected for attenuation, is (0.l3/\�”�0.93)*(0.99))0. 14 ( 17). Correcting for attenuation does not greatly affect the

estimate of the correlation between the two measures becausethe subject-to-subject reliability was high in both cases.

Compartment Analyses. The overall mean proportions of Ia-beled cells in each quartile for both PCNA and crypt dissection

are presented in Table I . The correlation between the subjectaverage proportion of labeled cells in each compartment ob-tamed using PCNA and crypt dissection ranged from -0.14(quartile 2) to 0.16 (quartile 4). None of the correlations werestatistically different from zero. A histogram comparing theoverall mean values obtained for each quartile, using bothPCNA and crypt dissection, is displayed in Fig. 3. From the

figure, it is clear that using PCNA, most of the labeled cells arefound near the base of the crypt (first quartile) with much fewerin the second quartile and relatively few in the third and fourthquartiles, at the apex of the crypt. For crypt dissection as well,

the mitoses are concentrated in the first and second quartileswith relatively fewer in the upper quartiles. However, for crypt

dissection, the proportions in the first and second quartiles weresimilar on average.

Fig. 3. Mean percentage of labeling in each quartile, averaged over all subjects,

for PCNA and whole-crypt dissection.

130

20�

100

I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 8 19 20

C-

-- - I eiops0/S,g�en 2 B#{243}op5osIS�ooI 3 Bk,p,,,sIS,,b�&;t 4 B6po�.JS,�en

Sample Size Calculations. Fig. 4 displays the results of the

sample size calculations for crypt dissection when a difference

of 25% between groups is expected. The estimates of the

components of variation (and percentage of total variation) due

to differences between subjects, differences between biopsies

within subjects, and differences between crypts within biopsies

were 0.315 (64.8%), 0.030 (6.1%), and 0.142 (29.1%), respec-

tively. From the graph, it is clear that little is gained by reading

more than six or seven crypts per biopsy. With 90-100 subjects

per group, 95% power could be attained using crypt dissection

with just two biopsies per subject and six crypts per biopsy.

Based on our previous work (16), for a fixed number of sub-

jects, more biopsies are required for PCNA than for crypt

dissection to achieve the same level of power. This is because

the percentage of variation due to differences between biopsies

is lower for crypt dissection than for PCNA.



Cancer Epidemiology, Biomarkers & Prevention 719

Discussion

This study was designed to examine WCMC as a measure of

cell proliferation for use in epidemiological and interventionstudies in the large bowel. As suggested by others, we found

that the crypt dissection technique was simpler to perform thanPCNA immunohistochemistry. Cells in mitosis were easy torecognize and count. Examining an entire crypt, rather than a

cross-section, requires fewer assumptions about proliferative

events within a given crypt.To accurately portray the biology, a diagnostic test such as

crypt dissection must be highly reproducible when repeatedon separate occasions. Although reproducibility is generally

thought to be straightforward, we discovered some interesting

subtleties. Because proliferative measures are generally de-signed to provide information about a given patient, previous

studies have compared the proliferative index, calculated by

averaging individual crypt indices. This average, however, isinherently less likely to vary than the individual crypt indices

that comprise the average. On repeat, some crypts may have a

higher index and some may have a lower index, but the averagemay be about the same. Strictly speaking, one should compare

the index on a crypt-by-crypt basis. Our data suggest that theindividual crypt correlations are not as high. This is understand-

able because the crypt dissection method requires focusingthrough various planes. A given mitotic figure might be over-

looked on repeat. In contrast, previous work with PCNA datahas shown the measure to be reliable both in crypt-to-crypt and

subject-to-subject comparisons (16).We also found a poor correlation between LI determined

using PCNA immunohistochemistry and WCMC. There aretwo possible explanations: (a) the two methods measure

slightly different events. PCNA measures a broader segment ofthe cell cycle than crypt dissection, which might partially ac-

count for the low correlation; and (b) the second possibility isthat the two measures are uncorrelated because WCMC is not

a standardized measure. The PCNA LI is standardized in thesense that it represents the percentage of labeled cells in a

particular crypt. WCMC measures only the total number of

labeled cells. If every crypt contained the same number of cells,

this would not be a problem. However, in our data, the totalnumber of cells in a crypt, using PCNA, ranged from 76 to 206(mean = 124.7; SD 18.9), while the average number of cellsin a crypt for a given subject ranged from 94.7-174.4 (mean

124.2; SD = 14.8). The total number of cells in a crypt is not

the same in every subject or even within a subject. A subjectwith very large crypts could have a high number of mitoses, butthe proportion of cells in mitosis might be low. This inability to

correct WCMC for crypt size is a potential problem.Although it is not possible to adjust the number of mitoses

for crypt size, it is possible to determine the location of mitoseswithin the crypt. Increased proliferation near the crypt apex is

thought to be associated with a greater risk of cancer. By

determining the proportion of labeled cells and mitoses withindiscrete quartiles, it might be possible to compare these twomeasures. However, comparing the subject average proportion

of labeled cells appearing in each quartile for crypt dissection

and PCNA, we found no significant correlation between the

two measures for any of the quartile comparisons. Furthermore,the distribution of the labeled cells within a crypt appeared todiffer for the two methodologies. On average, for PCNA, thegreatest concentration of labeled cells was in the first quartile(crypt base), with smaller proportions of labeled cells in each

successive quartile. For crypt dissection, this trend was not asstrong. Most of the labeled cells appeared in the first two

quartiles, but the overall mean proportions for the first andsecond quartiles were roughly the same (48.7% versus 46.8%).

Looking at a number of the drawings made from dissectedcrypts, we found that in some cases there were no labeled cells

at the bottom of the crypt. This migration of the labeled cells incrypt dissection toward the center of the crypt has been notedby previous authors (18). Crypt dissection measures cells fur-ther through the mitotic cycle than PCNA, allowing more timefor migration. Our analyses confirm that this is often the caseand appear to show that PCNA LI and WCMC are not strictlycomparable measures of proliferation index.

The sample size estimates for crypt dissection are gener-

ally similar to those obtained by others for PCNA (6, 19, 16).If our estimates are slightly higher, this is because we haveaccounted for the added variation due to within-individual

differences in the measures, whereas these differences have notalways been accounted for in sample size calculations in theliterature. Interestingly, we found the percentage of variation incrypt dissection measures due to differences between biopsiestaken on the same subject to be lower than the correspondingpercentage for PCNA. Because of this difference, more biopsieswould be required for PCNA than for crypt dissection, for afixed number of subjects, to achieve the same level of power.

In epidemiological studies, it would not be surprising to

find differences on the order of 25% between groups. Such astudy might require as many as 90 subjects/group. For largerdifferences, fewer subjects would be necessary. Previous stud-ies using cell proliferation as the outcome measure have dem-

onstrated differences with relatively modest numbers of pa-tients (8, 20). In these studies, the variance estimators used in

the test statistics were based on the subject-specific data, ig-noring the within-individual variation. Had their variance esti-mators accounted for the variability between crypts from thesame biopsy and between biopsies from the same subject, theresults might not have been statistically significant. Alterna-tively, differences between laboratories and staining procedureswhich lower the within- and between-subject variation woulddecrease the sample size needed to show a statistically signif-

icant difference between groups.

The results of this study using endoscopic biopsies inhumans are at variance with a previous study in rodents (15).Animal studies generally involve giving animals a carcinogen

or using a fasting/refeeding strategy. Both of these maneuverssignificantly increase proliferation. In relatively healthy, low-

risk human subjects, the indices may be sufficiently low to limitthe ability to show that the two methods are comparable. Thecrypt size in litter mates fed a similar diet may also be lessvariable.

The present study did not assess the utility of PCNA LI

and WCMC as correlates of disease risk. The ultimate valida-tion of any marker is its ability to predict disease outcomes (21).

The real question is whether any of these measures correlateswith risk of developing colorectal cancer or its precursors. The

fact that the two measures did not correlate with each other doesnot imply that they will not be associated with colorectal cancerrisk. Alternatively, even if the two measures were highly cor-related, they might not be good markers.

The concept of using biomarkers to develop new interven-tion strategies is an appealing one. Techniques that work in

animal models cannot necessarily be directly exported to stud-ies in humans (22). Before biomarkers can be adopted, theymust be shown to be reliable. Additional work must be con-ducted to develop statistical methods that are designed to modelthese biological variables. The advantages of the method ofwhole-crypt dissection are considerable, but it remains to be



720 Comparison of PCNA and Crypt Dissection

seen whether this method will be useful to identify groups at

risk for colorectal cancer.In summary, we found poor correlation between the results

of whole-crypt dissection and PCNA immunohistochemistry,two techniques that have become popular to measure epithelial

cell proliferation. The correlation was poor overall and incomparisons of compartment scores. Additional methodologi-cal and statistical research is necessary to determine the reli-ability, validity, and correlation of the various proliferationmeasures.

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1995;4:715-720. Cancer Epidemiol Biomarkers Prev S C Murray, R S Sandler, T O Keku, et al. immunohistochemistry and whole crypt dissection.proliferating cell nuclear antigen (PCNA) Comparison of rectal mucosal proliferation measured by

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