Gut 1993; 34: 1566-1571

DNA densitometry of colorectal cancer

G T Deans, K Williamson, P Hamilton, M Heatley, K Arthurs, C C Patterson, B J Rowlands,T G Parks, R A J Spence

AbstractDNA analysis was assessed by densitometryfor 281 cases of colorectal adenocarcinoma.Detection of aneuploidy in a single case rosefrom 65% if one, to 92*5% when three or moresections, were analysed. Although aneuploidtumours had significantly larger nuclear areasthan near diploid tumours (p=0009), densito-metric measurements showed no associationwith clinicopathogical variables. DNA contentdetermined by densitometry was comparedwith that from flow cytometry on 465 tissuesections from 241 cases. Aneuploidy assessedby flow cytometry was significantly associatedwith that determined by densitometry (p<001for all comparisons), ploidy state being similarin 381 sections (82%, x=063, p<0001), and187 cases (77-6%, x=0-57, p<0001). Uni-variate survival analysis showed that DNAdensitometric variables had no significantassociation with survival in (a) all cases, (b)cases without lymph node metastases, or(c) cases without distant metastases. Multi-variate regression analysis of densitometricand clinicopathological variables identifiedDukes's stage, patient age, and tumourdifferentiation as the combination of variablesmost closely related to survival. Densitometricmeasurement of DNA content could notsignificantly improve on the prognostic modelcontaining these three variables. It is con-cluded that, although the assessment ofDNAcontent by densitometry is comparable withthat of flow cytometry, conventional histo-logical variables remain the best predictors ofprognosis in colorectal cancer.(Gut 1993; 34: 1566-1571)

Current prognostic criteria for colorectal cancerare not entirely satisfactory; the TNM classifica-tion' is complex, modifications of Dukes'sclassification2 have led to confusion, while Jass'classification3 is poorly reproducible.4 Combin-ing these with assessments of tumour behaviour,such as DNA content,5 might result in a betterprediction of prognosis. Generally, two tech-niques have been developed for the cytologicalmeasurement of nuclear DNA content. Earlyreports measuring DNA content from sectionpreparations using light microscopy (densito-metry) proved promising,6 but cumbersome.7Emphasis therefore shifted to flow cytometry, inwhich large numbers of cells can be rapidlyanalysed for their nuclear DNA content. Flowcytometry, however, may fail to detect smallnumbers of aneuploid tumour cells, containingabnormal amounts of DNA, if most of thespecimen contains cells with normal (neardiploid) DNA content. Consequently, flowcytometric studies assessing the independent

prognostic value of tumour DNA content incolorectal cancer are conflicting, some reportsimplying that it is of major prognostic impor-tance,8-13 others that it is of little value relative toDukes's stage. 11'9

In contrast with flow cytometry, DNA densi-tometry, determining the DNA content of smallnumbers of tumour cells from stained micro-scope sections, is potentially more specific. DNAcontent assessed by this technique is claimed tobe related to survival in several tumours,2025including colorectal cancer.26 The prognosticvalue of densitometry in colorectal cancer, how-ever, requires verification in a larger series ofcases. A study was therefore performed to estab-lish the role of nuclear DNA content, asdetermined by DNA densitometry, comparedwith currently accepted prognostic criteria incolorectal adenocarcinoma.

Materials and methods

PATIENTS, CLINICOPATHOLOGICAL, AND FLOWCYTOMETRIC DATAAll available paraffin wax embedded histologicalmaterial from 312 patients having surgicalresection of colorectal adenocarcinoma at twoteaching hospitals between January 1974 andDecember 1983 was studied. All material wasreviewed by a single pathologist for Dukes'sstage2 and the histopathological variables com-prising Jass' classification.3 Clinical information,including patient age, sex, tumour site, symp-tom duration, liver function tests, and intestinalobstruction was also determined. Flow cyto-metry, allowing the assessment of the ploidystate and proportion of cells in each phase ofthe cell cycle, was performed on paraffin waxembedded material from each case27 (Table I).

DNA DENSITOMETRIC STUDYFive micron sections were cut from all paraffinwax embedded material and stained using theFeulgen reaction in a method that has beenfound to be reliable.28 3 Integrated opticaldensity (IOD), which reflects Feulgen stainingof a nucleus relative to its size, was used tomeasure nuclear DNA content.The Feulgen stained sections were examined

under oil with an Olympus microscope and theimage transmitted by a high resolution CCDcamera to a VIDAS image analyser (Kontron). Aminimum of 25 lymphocyte nuclei were tracedper section, using a stylus pen and graphic tablet.After correction for background absorption, themean IOD of the lymphocytes was taken asthe value for the near diploid population of thesection and used to set the 2c value on the DNAcontent scale.

Department of Surgery,Belfast City Hospital,The Queen's Universityof BelfastG T DeansT G ParksR A J Spence

Department of Surgery,Royal Victoria Hospital,The Queen's Universityof BelfastK WilliamsonB J Rowlands

Department ofPathology, Royal VictoriaHospital, The Queen'sUniversity of BelfastP HamiltonM HeatleyK Arthurs

Department ofEpidemiology and PublicHealth, The Queen'sUniversity of BelfastC C PattersonCorrespondence to:Mr G T Deans, Department ofSurgery, The Queen'sUniversity of Belfast, BelfastCity Hospital, BelfastBT9 7AB.Accepted for publication24 March 1993

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DNA densitometry ofcolorectal cancer

TABLE I DNA densitometric, flow cytometric, andclinicopathological variables assessed

Densitometric2c deviation index (absolute value)Sc exceeding rate (%)Mean c value (absolute value)Histogram type 1, II, III, IVNuclear area (microns2)Flow cytometricPercentage of cells in the phases of the cell cycle (GO/GI, S, G2/M)

Proliferative index (sum of the S and G2/M phases)Ploidy state (Diploid v non-diploid)

(Diploid v aneuploid v tetraploid)PathologicalDukes's stageTumour typeDifferentiationTubule configurationFibrosisGrowth patternNuclear polarityLymphocytic infiltrationClinicalAgeSexSiteSymptom durationLiver function testsIntestinal obstruction

A, B, C, Dtubular, papillary, mucinouswell, moderate, poorsimple, complex, irregular, nonelittle, moderate, markedexpanding, infiltratingwell, moderately, poorly preservedlittle, moderate, marked

right, left, sigmoid, rectum0-3, 3-6, 6-12, >12 monthsnormal, abnormalpresent, absent

The IOD of a minimum of 50 tumour nucleiwas then determined for each section, correctedaccording to the lymphocyte standards, andplotted as a frequency histogram.

DNA DENSITOMETRY VARIABLESThe histogram of c value distribution for eachsection was classified according to establishedcriteria29: type I: only a small number of cellsdeviate more than a few per cent from the 2c(near diploid) value; type II: two well definedpeaks around the 2c and 4c regions or a distinctmodal value in the near tetraploid (4c) region;type III: peaks around the 2c and 4c regions witha sizeable number of cells with c values similar tothose of lymphocytes in DNA synthesis; typeIV: very irregular DNA aneuploidy, with DNAamounts ranging from 2c to beyond 6c.Near diploid cases were taken as those in

which all sections showed type I histograms.Cases showing type II histograms were classifiedas tetraploid, those with type III or IV histo-grams as aneuploid.By convention, DNA aneuploidy may also be

expressed as the proportion of cells with valuesabove Sc (the Sc exceeding rate). Similarly, themore abnormal the DNA content, the greaterthe number of cells that deviate from the 2cvalue (- that is, the greater the 2c deviationindex). In addition to these assessments ofDNAcontent, mean nuclear area was derived for eachsection (Table I). All analyses were performedfor both the mean and maximum values of eachvariable per case.

In accordance with the clonal concept oftumour development, the ploidy state of theentire tumour was taken as that of the mostaneuploid specimen comprising that tumour.

PILOT STUDIESNuclei sample size - Two sections were randomlychosen from each of 20 randomly selected casesand 50 tumour nuclei measured from eachof three randomly selected fields per section.

Analysis of variance for a nested design deter-mined the components of variance from nucleusto nucleus within a field, from field to field withina section, and from section to section within asubject. The numbers of nuclei, fields, andsections that gave a subject mean with thegreatest precision (- that is, the lowest standarderror) were then calculated to give the number ofnuclei which should be measured per section.32

Well and poorly differentiated tissue - A studywas performed to determine if DNA densi-tometry differs significantly between well andpoorly differentiated colorectal tumour mucosa.Twenty five cases in which three independentobservers identified the tumour as beinguniformly well differentiated were randomlyselected and compared with a similar number ofuniformly poorly differentiated tumours, usingStudent's paired samples t and the X2 tests.

DNA DENSITOMETRIC AND CLINICOPATHOLOGICALVARIABLESThe relation between densitometric and theclinicopathological variables was investigatedusing Pearson correlation coefficients andStudent's independent samples t test. To reducethe risk of chance agreement in the large numberof analyses involved (type I error), significancewas taken at the 1% value.

DNA DENSITOMETRIC AND FLOW CYTOMETRICVARIABLESPloidy state obtained by flow cytometry (neardiploid, tetraploid, aneuploid) was comparedwith that from densitometry (histogram typesI-IV) for each section and for each case using theX2 test and Cohen's x statistic.33 Comparison offlow cytometric ploidy state with the otherdensitometric variables was determined by theStudent's t test. The relation between each of thedensitometric and the other flow cytometricvariables was investigated using Spearman cor-relation coefficients.

SURVIVAL ANALYSISUnivariate - The relation between each of thedensitometric variables and survival wasassessed by the log rank test. Analysis wasinitially performed for all deaths, irrespective ofcause, as the causes ofdeath recorded at certifica-tion may be inaccurate. The analysis was thenrepeated for deaths where the underlying causewas registered as colorectal cancer at deathcertification. Including patients with metastasesmight obscure a significant relation with survivalin better prognosis cases and therefore therelation of densitometric variables to survivalwas repeated for cases (a) without nodalmetastases (Dukes's stages A and B) and (b) forcases without distant metastases (Dukes's stagesA, B, and C combined). Significance was taken atthe 1% value, to reduce the risk of type I errors.Before analysis, continuous variables werecategorised into three equal sized groups usingtertiles and DNA histogram type analysed forindividual types and their combinations.

Multivariate - To determine if DNA densi-

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Deans, Williamson, Hamilton, Heatley, Arthurs, Patterson, Rowlands, Parks, Spence

tometry is of a prognostic value independentfrom known prognostic variables such as

Dukes's stage, the densitometric and clinico-pathological variables were entered into a Cox'sproportional hazards regression analysis. Thisallowed derivation of hazard ratios forcategorical variables and for a 1% increase incontinuous variables. The likelihood ratio teststatistics resulting from the proportional hazardsanalysis34 were referred to the XI distribution, todetermine the combination of variables that bestpredicted prognosis in our dataset. Analysis wasperformed both for all deaths and for colorectalcancer deaths.

Results

PILOT STUDIESNuclei sample size - Densitometric DNA analysisvaried little when more than 10 nuclei per field,or four fields per section were measured. A totalof 50 tumour nuclei, 10 cells from each of fiverandomly selected fields, was therefore chosen as

representative of each section in this study.Well and poorly differentiated tissue - There was

no significant difference in any densitometricvariable between well and poorly differentiatedcolorectal mucosa.

DNA DENSITOMETRIC STUDYClinicopathological data were obtained for all312 patients. Suitable histological material was

available for DNA densitometry on 281 of these(86 8%), while flow cytometry was successful on275. The mean number of sections analysed bydensitometry was 2 02 per case, range one to six.The proportion of DNA aneuploid cases rose

from 65% if one section was analysed to 86-3% iftwo sections, and 92-5% when three or more

sections, were assessed per case.

Forty nine patients (17-4%) showed type Ihistograms in all samples, the numbers of cases

classified as type II, III, and IV histograms being80 (28-5%), 85 (30 3%), and 67 (23-8%), respec-tively. The proportion of cases classified as near

diploid, tetraploid, and aneuploid by flowcytometry were 39 4%, 13-3%, and 47-3%,respectively.

DNA DENSITOMETRIC AND CLINICOPATHOLOGICALVARIABLESThere was no significant association betweenDNA densitometric and any clinicopathologicalvariable.

DNA DENSITOMETRIC AND FLOW CYTOMETRICVARIABLESDirect comparison of densitometry and flowcytometry was possible on 465 sections and 241cases. The coefficient of variation of the midpoint of the G01/GI peak (of all the flowcytometry specimens was 7- 3%. Ploidy stateagreed between the two techniques in 381sections (x2=l77 04, df=l, p<0 001) and 187cases (x2=54-7, df=l, p<0Q001). The corres-

ponding x values were 0-63 and 0 57,

TABLE II Association offiow cytometric ploidy state withdensitometric variables

Mean 95%value Confidence limits p Value

Nuclear area(mean, microns2)

Diploid 35 6 34-4 to 36-8Tetraploid 35 9 32-8 to 39 0 0 009Aneuploid 38-3 37 1 to39 5

2c deviation index(absolute value)

Diploid 1 59 1 39to 1-79Tetraploid 1 66 1-22 to 2-09 0 002Aneuploid 2-13 1-91 to 2-36

Sc exceeding rate(percentage)

Diploid 10-2 7-3 to 13 1Tetraploid 12-1 6-2 to 17 9 0-016Aneuploid 16-6 13-3 to 19-9

Mean c value(absolute value)

Diploid 2-96 2-77 to 3 16Tetraploid 2-99 2 56 to 3 41 0-008Aneuploid 3-41 3-20 to 3-62

representing substantial and moderate levels ofagreement, respectively.35 Progression fromdiploid to aneuploid tumours assessed by flowcytometry was significantly associated withincreasing values of nuclear area, its standarddeviation, 2c deviation index, 5c exceeding rate,and mean c value (Table II). Both 2c deviationindex and 5c exceeding rate were positivelycorrelated to increasing proportions of cells inthe G2/M phase of the cell cycle (p<0005 andp<0001, respectively).

SURVIVAL ANALYSISUnivariate - Analysis for all causes of deathand colorectal cancer deaths produced similarresults. Neither the maximum nor mean valuesof the DNA densitometric variables, nor anycombination of histogram types, was signifi-cantly related to survival (Table III). Similarly,no densitometric variable was significantlyrelated to survival for cases without nodal orwithout distant metastases.

TABLE III Number ofdeaths, hazard ratio, and significancevalues obtained from survival analysis (tertiles) of all causesofdeath (maximum value per case)

Observed Expected Hazarddeaths deaths ratio p Value

2c deviation index(absolute value)

<1-15 57 583 11-15-1 97 60 58 8 1 04 097

>1*98 57 56 7 1*025c exceeding rate(percentage)

<39 58 589 140-12-5 57 58 6 1-01 0-91

>12-5 59 56 5 1-01Mean c value(absolute value)

<2-6 61 58-7 12-6-33 55 579 1.09 0-87

>33 58 574 103Nuclear area(mean, microns2)<33-6% 58 56 9 133-7-38 9 53 59-1 1*14 0 53

>390 63 579 106Ploidy(histogram type)

I 24 25-2 1II 35 37-1 1 02 0-18III 43 42-1 107IV 34 31-8 1 11

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TABLE IV Hazard ratios obtainedfrom multivariate survivalanalysis corrected for age and Dukes's stage. The significantpathological variables are shown for comparison

95%Hazard Confidenceratio limits p Value

DNA Densitometry2c 0.99 0-78 to 1-30 0 75Sc 1 05 0-85 to 1- 12 0-62Mean c 1-02 0-96 to 1-04 0-81Nucleararea 1 00 0-98to 1-02 0 75Ploidy (histogram type)I v II+III+IV 1-16 0-60 to 1-34 0 58I+II v III+IV 1-09 0 65 to 1-29 0-67I+II+III v IV 1-03 0 59 to 1-24 0-75PathologyDifferentiationWell v poor 0 51 0 34 to 0 80Moderate v poor 0 49 0 30 to 0-81 0 003PolarityWell v poorly preserved 0-62 0-44 to 0-89Moderate v poorly preserved 0-78 0 65 to 0-91 0 005ConfigurationComplex v irregular/none 0 50 0 33 to 0 73Simple v irregular/none 0-67 0-46 to 0-98 0-003InfiltrationMarked v little 0-69 0-45 to 1-07Moderate v little 0 70 0-55 to 0-86 0-048

Multivariate - The results derived fromanalysis of all deaths were again similar to thoseof colorectal cancer deaths. Cox's multivariateregression analysis identified increasing patientage and Dukes's stage as the clinicopathologicalvariables most significantly related to poor

survival. Some pathological variables, but no

densitometric variables, were independentlyrelated to survival after age and Dukes's stagewere taken in account (Table IV). 'Whenincluded in continuous (uncategorised) form, no

densitometric variable could add significantly toa model consisting of terms for age, Dukes'sstage, and tumour differentiation, the combi-nation of variables which was found to be best atpredicting survival in colorectal cancer in our

dataset.

DiscussionAlthough early densitometric reports claimedthat aneuploidy may be detected from as few as

three cells,6 this study agrees with most recentreports in showing that assessing 50 nucleipermits a satisfactory representation of the DNAcontent of the section as a whole. Reports on

colorectal tissue using interactive techniquessimilar to this study have measured 50 or 100cells,3638 while reports using automatic machineshave assessed 200 to 1000 cells per case.263942This suggests that, compared with flowcytometry, densitometry is comparatively more

efficient at detecting DNA abnormalities.Studies comparing the two techniques support

this conclusion. The significant association in theassessment of ploidy between densitometry andflow cytometry noted in this study has beenpreviously recognised,2' with correlationcoefficients of 0-83 being reported.' It seems

that DNA densitometry, however, may detectabnormal DNA content more readily than flowcytometry. Bauer et al noted excellent correla-tion between the two techniques in 92 humansolid tumours, although in nine cases, an

aneuploid population was detected by DNA

image, but not by flow cytometric analysis.48 Acomparison of 31 colorectal carcinomas by flowand image cytometry found that, whereas 70% ofthe cases were non-diploid by image analysis,only 56% were similarly classified by flowcytometry.37 Comparable results from this studywere 82-6% and 60-7%, respectively.

Variation of DNA content within the samespecimen (DNA heterogeneity) is well recog-nised in flow cytometry and has been noted indensitometric reports.649 Bocking et al recentlyreported an 81% agreement in the 2c deviationindex between different sections from the sametumour,50 while it is recognised that by densi-tometry, 40% of colorectal cancers are homo-geneously and 60% heterogeneously aneuploid.38In our study aneuploidy increased progressivelywith the number of sections analysed, reaching92-5% when three or more sections were assessedper case. This emphasises the utmost importancein studies of this type that sufficient samples beanalysed to improve the detection of DNAaneuploidy.Flow cytometric reports comparing well and

poorly differentiated tissue have produced con-flicting results, primarily because they relatedthe overall worst grade of the entire specimen tothe worst ploidy state of the entire specimen. Incontrast, this study, although small, directlycompared tumour nuclei in areas of specificallyidentified well or poorly differentiated tissue andshould more accurately reflect the relationbetween ploidy and histological grade. Itsuggests that there is no relation between ploidystate and histological grade in colorectal cancer.As histological grade is considered to be ofprognostic value, this finding may partiallyexplain the failure ofDNA ploidy to be related tosurvival in this study.

It might be anticipated that, because DNAaneuploidy represents increased amounts ofnuclear DNA, aneuploid tumours might havelarger nuclear areas. This study confirms suchan assumption and suggests a strong relationbetween DNA ploidy and nuclear area5; thelarger the nucleus the more likely it is to beaneuploid.There is evidence that diploid cells may pro-

gress to tetraploidy and aneuploidy and that suchaneuploid cells would have a biological advan-tage related to both genome and cell function.52Some densitometric studies on colorectal cancersuggest that ploidy may be of prognostic value.One study suggested that the prognostic differ-ence in rectal cancer between the races iscontributed to by a significant difference inploidy state.53 Other reports, on small numbersof cases and without the use of survival analysis,have found diploid tumours to have a betterprognosis than non-diploid tumours.37 49

Albe et al recently assessed the prognosticvalue of DNA image cytometry on 211 cases ofcolorectal adenocarcinoma.26 Univariate survivalanalysis suggested that DNA ploidy might berelated to patient outcome. Multivariate survivalanalysis, however, showed that no ploidycategory was significantly related to survival.The findings of this study are similar, in thatnone of the DNA densitometric variables, orcategories of ploidy, were significantly related to

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1570 Deans, Williamson, Hamilton, Heatley, Arthurs, Patterson, Rowlands, Parks, Spence

survival, either on univariate or multivariatesurvival analysis. This agrees with most largeflow cytometric studies using multivariatesurvival analysis,'"'9 including our own,54 whichfound that flow cytometry was not of prognosticvalue in colorectal cancer. Therefore, both flowcytometric, and potentially more specific densi-tometric studies, suggest that DNA content is oflittle prognostic benefit in relation to currentlyaccepted criteria in colorectal cancer.

It therefore seems unlikely that DNA analysisusing densitometric or flow cytometric methodswill play a significant prognostic part in themanagement of colorectal cancer patients, prob-ably because they can only detect comparativelylarge changes in tumour DNA content. Densi-tometry may yet prove of benefit in the diagnosisof premalignant conditions,55-58 because itsmorphometric features may be used interactivelyto classify any given cell and therefore replacelarge numbers of flow cytometric parameters.The future assessment of DNA content willprobably lie in gene probes, which can detectchanges in chromosome structure more subtlythan DNA densitometry.9"I In the meantime, itseems that conventional histological variablesremain the best predictors of prognosis incolorectal cancer.

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DNA densitometry of colorectal cancer.

B J Rowlands, T G Parks and R A SpenceG T Deans, K Williamson, P Hamilton, M Heatley, K Arthurs, C C Patterson,

doi: 10.1136/gut.34.11.15661993 34: 1566-1571 Gut

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