Carcinogenesis in chronic ulcerative colitis : Flow cytotnetry

Flow cytometry is a conceptually straightforward quantitative method that can be used to determine the DNA content (and many ocher parameters) of individual cells. It has been used in chronic ulcerative colitis to evaluate the presence of aneuplo idy as a marker of colon cancer risk. In this paper, the basic principles offlow cytometry are reviewed, the current status of published studies using flow cytometric analysis of DNA content in chronic ulcerative colitis outlined, conclusions from the research are proposed and future research needs it~cntified. Overall, aneuploidy correlates with the current best markers of cancer risk in ulcerative colitis, ie, dysplasia and established cancer. Whether aneuplo1dy will be useful for the prospective identification of individuals at increased risk for colon cancer deserves rigorous evaluation. An important technical issue of potential variability in interpretation of near diplo id peak5 is emphasized. Can J Gastroenterol 1990;4(7):390-396

T Iil: PURPOSE OF THIS ARTICLEIS to review the applic:uion of flow cytometry to the problem of carcino-genes1~ in chronic ulcerative colm,.In this review, methodology will be described including the capacity anJ lim1tatiom of flow cytomctry; the <lau concerning flow cytometric analyMs<i DNA content in chronic ulccrat1vc colitis will be reviewed; reasonabk conclusions wi II he drawn from exm1ng Jara; and finally, some research nce<l 1 in this area will be identified.

FLOW CYTOMETRY METHODOLOGY
The hasic principle of Ov.

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Figure 2) Flow cywmecric analysis of DNA content.In this flc~w cytomecric histogram the intensity of fiuorescence is />lotted on the horiznncal a).is and che number of cells on the ,1erucal axis.An mtemal sumdard (STD) 1s nm wirh each mmple to allow quanmation of each of the fluorescent J,eak.1 The IMJOT population of cells contanl.'itwo copies of each chromosome ( 2N DNA) A small~'T {'c!ak of cells wrth twice as much fluorescence contaim four copre.s of each chromosome ( 4N DNA) and represents the cells in G 2 and M phases of the cell cycle The cells and assays of the cycle are represented /ry the celLs h.ttween the 2N and 4N peak.s of differing fluorescence intens1tie~ ~o that they can be separateJ in an electrical field.This process 1s known as 'fluorescence-activated cell sorting'.In addition to fluorescence intensity, in-formanon about cell volume can he oh-taineJ 1:-,y the Jegree of forwarJ angle I ighr scatter of the excitation lrn;er heam.
The flow cyrometric data can then be displayed in a histogram (Figure 2), plotting the number of cells versus the intensity of fluorescence.Computer methods can be used to calculate the propomon of celb with any given intensity of fluorescence.
Almost all of the studies using flow cytometry in chronic ulcerative colitis have measured DNA content.For the use of flow cyrornetric analysis of DNA content, the isolated cells are inc ubateJ with fluorescent dyes ~uch as propndium iodide, whose binding is proportional co the DNA content.The intensity of fluorescence 1s then proportional co the amount of DNA in each inJividual nucleus as shown in Figure 2. Generally an internal standard i~ run with each sample so that the amount of DNA m each peak can he quantitated.The major peak of cells in normal tissue contains two copies of each chromosome (ZN DNA).These are the cells that are in GO and GI pha:,es of the cell cycle.A small peak of cclb can be seen having twice .ismuch intemrty as the ZN peak.These cells contain four copies of each chromosome (4N DNA) and arc the cells in the G2 and M rhase of the cell cycle.Flow cytomctnc analys is can be used for a large variety of techniques.Flow cytometry has the advantage of heing a 4uant1tat1vc methoJ in that it measures the amount of fluorescence in each in-Jividual cell.It also has the capacity w measure multiple fluore~cenl labels simultaneously using a dual beam flow cyrometer.Thus, two or three quantita-t1ons can be made from the same cell usmg different fluorescent lahels.For example, the amount of DNA and the intensity of growth factor receptor •coul<l be measu red simultaneously co try to correlate growth factor expression with proliferation in individual cells.In addition, by measuring the forward angle light scatter, a measure of cell volume can be determine<l.Some flow cytometers have the capacity to sore populations of cells with varying amounts of fluorescence intensity, thus separating subsets of cells which can be studied further.Although the best resu lts with flow cytometry are obtained when the t1ssue is obtaine<l fresh and prepared immediately, archival blocks of paraffin-embedded nssue can be used for some stu<lies, including DNA analysis.Thus, the presence of aneuploidy can be <letected from such arch ival specimens.
There are also some important limitations to the use of flow cytometry.The technique must be standardized at each institution that develops flow cytometry as a clinical or research tool.This standardization includes not only the equipment but the selection of appropriate software for the interpretation of th flow cytometric results.In addition, for each separate application of flow cytometry to detect a differenl fluorescent label, the method must be standardized so thar normal 'control' levels are determineJ wit hin the population at each individual institution.
For analysis of DNA content, two particular problems (<lebris and aggregation) deserve mention.During preparation of tissue, some nuclei may be fractured and produce debris that registers at the low fluorescence intensity region of the DNA histogram.This debris curve can extend into the regions of the 2N and S phases of the cell cycle and can variably alter the quantitative results of these cell kinetic parameters.Some software ca lculates a debris curve and automatically subtracts it from the 2N or S phase calculations whereas other software docs not; therefore the percentage of cells in the S phase, for example, could vary substantially depend ing upon the type of software used between two institutions.Aggregates of nuclei that are suspended a,

FLOW CYTOMETRIC ANALYSIS OF DNA CONTENT IN CHRONIC ULCERATIVE COLITIS
The initial work that Stimulated interest 111 flow cytometnc analysis of DNA content in chrornc ulcerative col1 t1s came from Hammarherg ct al (1,2).These authors analyzed biopsies from patients with ulcerative colius and founJ a high frequency (90%) of aneuplo1dy in colon cancers in the setting of ulcerative colitis and a low ( less than 5%) frequency of aneuploidy 111 normal tissue.lnrermed,ate levels of aneuploidy were identified in dysplastic mucosa and 111flamed ,1troph1, or hyperplastic mucosa in chronic ulcerative colitis (Figure 4 ).Two subsequent studies from the United K111gdom, however, came to J1vergent conclusions (3,4 ).The results of analysis of individual biopsies in these cwo studies is plotted 111 Figure 5.The study from Leeds showed no significant d ifference in the frequency of aneuploidy from biopsies show111g ulcerative colitis only, dysplasia, or cancer, with a frequency between 18 and 24% (3 ).In contrast, a gradient of 111crcase in frequency of aneuploidy in these three biopsy classifications was seen by the St Mark's group ( 4 ).These observations were par-ticularly disturbing because the studies were done in a very similar manner using archival fixed anJ paraffin-embedded tissues, and using similar flow cytometric techniques.Furthermore, it was initially difficult to envision a systematic difference in preparation or interpretation that might account for the differences because the Leeds group found both a higher frequency of aneuploidy in the biop~ies showing no dysplasia or cancer, and a substantia lly lower frequency of aneuploidy in the dysplastic and cancer biopsies.If there was a systematic difference in the detection or interpretation of aneuploidy, one would expect to show consistent differences in the same direction in all of the groups rather than this disparate result.Subsequently, however, the Leeds group reported that their frequency in cancer had increased to about 46% so that the disparity seemed to be more in the biopsies graded negative for dysplasia or cancer.This is also the case if one analyzes the data with respect to the number of patients rather than the individual biopsies (Figure 6).The percentage of patients with cancer that had aneuploidy was similar between the Leeds and St Marks studies; however the Leeds group found that over 40% of patients with no evidence of cancer had aneuploidy, whereas it was less than 10% in the study from St. Marks.Thus, it seems chat the major difference between the two studies is the finding of a higher frequency of aneuplo idy in the non-neoplastic biopsies in patients by the group at Leeds compared to St Macks.lt is possible that a difference in the c riteria used fo r the classification of aneuploidy between the two studies could account for such differing results.
There have been several other studies that have presented data related to the frequency of aneuploidy in chronic ulcerative colitis.In Figure 7, the results of two studies reporting the correlation between ane uploidy and histology in ind ividual biopsies are shown.In both the study from Bergen, Norway (5) and from Umea, Sweden (6) a substantially higher frequency of aneuploidy was seen in biopsies showing dysplasia than in those negative for couu.lbe interpreted as aneuploidy.On the left, a populauon of cells with a DNA index of/ .2rs plotted.There is a discrete peak of cells with fluorescence intensity approximately I . 2 umes 1he major GO/GI peak.On the right, a large aneuploid />0pulacion is seen with a ONA content a/J/)roximatdJ I .7 times 1he n01711al diploid peak dysplasia.lt is of some interest chat the absolute frequency in the negative group differed by almost two-fold between t he two studies.Two additional groups have reported the results of the relationship between aneuploidy and histology analyzed by patient groups (Figure 8).The study from Stockholm, Sweden ( 7) and the study from Seattle (8) both show a gradient of increasing frequency of aneuplo idy in pauents who only have biopsies chat are negative for dysplasia, those that have one or more biopsies graJeJ as indefinite for dysplasia, and those that have one or more bio psies graded as definite dysplasia.
The bulk of the evi<lence in the literature regarding t he relationship between aneuploid.Y and dysplasia in chronic ulcerative colitis <loes suggest that biopsies that are dysplastic and patients who show dysplasia have a higher frequency of aneuploidy than those with no evidence of d ysplasia.Nonetheless, there is significant discrepancy concerning the frequency of aneuploidy in nondysplastic mucosa.The authors have ha<l the oppo rtunity to perform a large number of flow cytornetric studies in patients with chro nic ulcerative colitb.Patients evaluate<l in these studies include patients entered from the Denver Dysplasia in Ulcerative Colitis study as well as patients enrolled in the C leveland C linic Chronic Ulcerative Colitis registry.
In the authors' studies, colon ic biopsies were obtained from three groups.Controls have come to colonoscopy for a variety of reasons, but were fou nd to have no colonic neoplas ,a and no personal history of chronic ulcerative co11tis, adenoma~ or colon cancer.The other two groups are patienb with chronic ulcerat ive colitis who are either enrolled in a surveillance program o r who h ave come to colectomy.The survei !lance group are patients who have longstanding (greater than eight years), extensive (proxima l to the splenic flexure) ulcerative coli tis.These patients are enrolled in surveillance studies in which yearly colo noscopy is advised and multiple biopsies obtained throughout the colon .The routine procedure in these pa tients is to obtain cwo adjacent biopsies every 10 cm througho ut the colon as well as additional biopsies of any suspic io us lesion.O ne of the biopsies at each site is fixed and prepared for histology and evaluation of dysplasia; the second is prepared for flow cytometric analysis of DNA content.Thus far over 850 biopsies fro m 80 patients in the surveillance group have been evaluated.The colectomy group includes patients with longstanding ulcerative colitis who have come to colectomy for intractable disease, dysplasia or cancer.In this group, two side-by-side four-quadrant biopsies are taken every centimetre through the entire resected spec imen .As in the surveillance group, o ne biopsy at each site is prepared for flow cytometry, the other for histology.
Thus fa r, over 1300 biopsies fro m 12 patients in this group have been evaluated.
The initia l criteria used to define aneuploidy was the presence of a discrete peak of cells distinct from the GO/G l an<l G2/M peaks that conta ined at least 5% of t he total cell population.Using these criteria alone, a high frequency of aneuploidy was found (about 15% of the biopsies).Forty per cent of patients had one or more biopsies that were aneuploid.A large percentage of these putative aneuploid peaks are near d iploid peaks, that is, they are discrete peaks, hut they have DNA contents less than 1.2 times the diplo id peak (Figure 9).
In the surve illan ce group, ove r 80<.)i, of the aneuploid biopsies not sho wing dysplasia fell into the 'near diploid ' group.The frequency of aneuploidy in the nondysplast ic tissue is thus influ enced greatly by whether one interprets the near diploid peaks as aneuploidy.Interobserver variation in the interpre tatio n of near diploid peaks has been no ted in other tissues (9).This difference may well account for much of the variabili ty in the literatu re c ited above.As in the o ther studies in lite ra ture, the present authors achieved a highly significant increase in the freq uency of aneuploidy both in dysplastic biopsies and in pati ents who have d ysplas ia or cancer.
Two other encouraging aspects of these studies sho uld be mentioned.First, patients who come to colectomy beca use of dysplasia or cancer a re ve ry frequen tly found ro have markedly abno rmal flow cytometric analysis with multiple areas of ane uploid populations of d ifferent DNA index.For example, on e of the patients studied came to colectomy because of a cancer fo und at the hepatic flexure.Multi ple areas of dysplas ia were seen in the splenic fle xure as well as in the transverse colon.T he majori ty of these dysp lastic biopsies were a neuplo id in ad<licion, CAN J GASTROENTEROL VOL 4 No 7 NOVEMRl:R 1990 Flow cytometry in chronic ulcerative colitis there were many nondysplast1c biopsies that were a lso aneuplo id.Overa ll, n ine separate aneuploid popul:uions were identified in this colonic mucosa.The presence of multiple aneuploid populations in patients with <lysplasia and cancer h as also been seen by several other groups who have examined this question.These observations suggest that in the late stages of ca rci nogenesis in chronic ulcerative colitis, aneuploidy is a prominent featu re of the colo nic mucosa.Secondl y, the authors have been encouraged by the frequent find ing that not only dysplastic biopsies show aneuploidy, but the same aneuploid population can often be seen in nondysplastic mucosa adjacent to dysplasia.For exa mple, o ne patient had two biopsies at the hepatic flexure which showed dysplasia and were ancuploid with a DNA index of about l.7.In addition, however, four a<ljaccnt nondysplastic biopsies showed the same aneuploid population.This type of observa1 ion suggests the possi bi Ii ty that aneuploidy may appear as a type of field effect and, if th is is so, could decrease the problem with sampling error when looking fo r dysplasia.These observations encourage cont mued evaluatio n of a neuploidy as a promising marker of cancer risk in chronic ulcerati ve colitis.

CONCLUSIONS
The current data suggest that aneuploidy occur~ commonly in ch ronic ulcera tive colitis and that it correlates with the established markers of cancer risk in this setting.Aneuploidy is more common in patients with dysplasia and cancer, but not all patients wi th ncoplasia have detecta ble aneuplo idy.Aneuploidy is also more common in dysplastic and cancerous hiopsie~ than in nond ysp lastic mucosa, but not all neoplastic biopsies a re aneuploid.Aneuploidy abo clearly occurs in non-neoplast ic mucosa.The cl inical meaning of the presence of aneuploidy in nondysplastic mucosa is not yet known and the authors Jo not currently recommend altering clinical decision making on the basis of a single ancuploid population in a patient with ulcerative coli tis.
Nonetheless, ancuplmdy 1s the most promismg of the new markers for the detection of cancer nsk m ulcerative colitis and Jescrves further evaluation.
The data al o suggest that aneuploiJ populations can appear over wide expanses of the colonic mucosa.This kind of obse rvation can be viewed as an example of clonal expansion in this mucosa.It 1s possible th<1t the ulceration and denudation of the mucosa that occurs in ulcera tive colitis a llows the opportunity fur a clone of cells that has growth advantage to repopulate large areas of the colonic mucosa.In the absence of suc h den udatton, perhaps this abnormal clone would be limited to a single crypt anJ the cells woulJ be slougheJ off inro the lumen as they no rma lly a re.In the context of multistage carcmogenesis, one can view the appearance of aneuplo1dy m mult1ple areas of the colo n as an example of such clonal expansion.If this aneuploiJ population were to represent o ne of the genetic events responsible for a seep in carcmogenes1s, its expansion over a wiJer area of the colon would mean that there would be a larger number of cells now a'-'ailable for subsequent carc inogenic steps, making cancer more probable.The most commonly held view is that ane uplo idy does not ibelf represent a spec ific genetic event necessa ry for carc mogenesis, but 1s a mark er of the genomic instability that charac tenzes prema lignant mucosa.
Analysts of the present Jara suggests that variable interpretation of the near d1plo1d peaks may account for some of the diffe rences repo rted m the literature about the relatio nship between aneuploidy, dysplasia and cancer m c hronic ulcerati ve colitis.Inclusion of the near diploid populations in the a uthors' data markedly mcreases the overall frequency of aneuplo idy, particularly m the nondysplnstic group.

RESEARCH NEEDS
From this review of the literature and the present data, 1t appears that there are several impo rtant research needs that sho uld be addressed.From a technical standpoi nt, 1t 1s important tn attempt to define more prec isely the . .' i tgnificance of the near Jiplotd populations.S uch populations coulJ be Jue to near diploiJ aneuploidy o r they could represent somethmg else, such as differential dye bindmg.For example, a population of cells might more avidly bmd the fluorescent dye anJ thus have a marginally higher intensity of fluorescence, appeanng as a near diploid peak.
In adJ1tion, methods of preparation of cells could conceivably produce such near d1plmd peaks.
To compare flow cytometrtc analyses from Jifferent cen tres, 1t will  and carc111omas. &.and J G,1stroentcml 1987;22: 1231-7.6. Ruregard J, Ahsgrcn L, Stcnlmg R, Roos G. DNA concem 111 ulc cnmve be important to standardize both the methodology (sample preparation and stammg) anJ the softwa re used to interpret the flow histograms.This is particu larly important when trying to compare the meaning of quanmat1ve results between different flow cytometry centres.
In the clinical area, the most pressing need is for follow-up stuJies of patients with aneuploidy with or without dysplasia to determme whether aneuploidy is predictive of current anJ/or subseq uent colon can cer nsk.
This can be Jone in the settmg of ongoing prospecttve trials, but in addition, a multicentre case control study using archival speci me ns seems feasible and could po tentia lly a nswer this question more quickly.

SUMMARY
The emerging literature usmg flow cytome try in the setting of ulcerative colitis has thus far prnnarily focused on measurement of DNA rnntent and aneuplo1dy.These studies have sh<lwn a significant relat1onsh1p hetwccn aneuplo 1dy and the current be~t markers of cancer risk tn this population.Aneuplo1dy certainly deserves rigorous test ing to determine 1f its presence will he of clinical value in assessing cancer risk tn patients with c h ronic ulcerative colttis.

Figure 1 )
Figure1) The principal features of a flow cywmeter.Smgle cells are labelled wrth a fluorescent probe and dtrected m single file through a laser beam.Analysis off orward angle light scatter rnn be u.wd to estimate cell volume Fluorescence emission from che excited cell chen provides a dmr1hut1on of fiuarescence intensity.Elecmc charge related co fluorescence mcensity can al10 11<! applied to dro/ilecs contammg single cells.allowing their se/Jaraticm m an electrosratic field Thi.1 /mJCess is known as 'fiuore.scence-acuvateclcell sorcmg' The cells synthcs1Z1ng DNA (those in S phase) nre seen as a plateau of eel b herween the ZN and 4N peab.Aneupl01dy is JefincJ as a population of cells containing an ahnonnal amount of DNA.An aneupl01d cell population can be detected hy flow cycometry as a peak of cells containing an ahnonmd amount of DNA that 1s dbcrete from t he GO/GI pc.ik or tht• GZ/M peak.Such an aneuploid population 1s 111d1cared hy the ,1rrow m Figure3.Th i~ peak 1s diMmcrly separate from the GO/GI peak and has a DNA mdcx which conrnms ahout 1.2 times the normal amount of DNA in d1plo1d cells.

Figure 3 )Figure 4 )Flow
Figure3) This DNA hiswgram shows a distmct peak of cells ( indicated by the arrow) that 1s separait from the major GO/G 1 peak.This populauon of cells con tams approximately I . 2 umes the amount of DNA m the GO/G 1 peak, woula be classified as a near diploul aneuploid population Figure5) The frequency of aneuploidy in chronic ulceratit•e colws biopsies from cwo su«I,cs from che United Kingdom The daca from che Leeds' group (3) is shown as solid bars and chat from the Sr Mark's grnu/1 /4) as cross-hatched han.The />ercencagc of b1ops1e.1 thac arc aneuplmd is plocccd for each of chree groups of b,o/mes 111terprewd as showmg 011/y ulcerative coliu1 without dysplasia (CUC), dysJ>lasia ( DYS) and cmmir (CA) The numbers ac che cop of each column are che numbers of biop11cs analyzed in each group

3 DYSFigure 7 )
Figure 7) The frequency of aneuploidy in b1u/>sies from patients with chronic ulcerative colim from cwoscud1es is presenced.The study from Bergen, Norway is shown as closed bars and the study from Umea, Sweden is shown as cross-hacched bars.The percentage of biopsies chat are aneuploid is ploued far those graded as negative for dysplasia (NEG) and those mcerpreted as dysplasnc (DYS) The number of bwpsies in each group IS shown above the bars