7 Clin Pathol 1993;46:51-55

Histopathological findings in oesophagealcarcinoma with and without preoperativechemotherapy

S J Darnton, S M Allen, CW Edwards, H R Matthews

AbstractAims: To investigate the pathologicaleffects of preoperative chemotherapy onoesophageal carcinoma.Methods: Qualitative and quantitativechanges in oesophageal carcinoma afterpreoperative chemotherapy were assessedby examination of biopsy specimensbefore treatment and resected speci-mens.Results: Of 13 patients with adenocarci-noma treated with 5-fluorouracil, adria-mycin, and mitomycin (FAM), nineshowed minor histological changes com-pared with 14 control cases. All 12 patientswith squamous carcinoma treated withpreoperative mitomycin, ifosfamide, andcisplatin (MIC) showed noticeable histo-logical changes when compared with the13 control cases. Changes included com-plete ablation (n = 1) and partial regres-sion (n = 5) of the tumour. A quantitativeestimate of the proportion of tumour tostroma showed no difference between con-trol adenocarcinomas and those treatedwith chemotherapy. There was, however, asignificant reduction (p < 0.01) in theproportion of tumour to stroma in thetreated squamous group compared withthe controls. There was no relationbetween the degree of response in squa-mous carcinomas and the degree of differ-entiation of the tumour. Patients in whichsquamous carcinomas responded well, asassessed quantitatively, showed a tend-ency to better survival at one year.Conclusions: Histopathological changesattributable to chemotherapy can beobserved in oesophageal carcinoma. Theresponse of squamous carcinoma to MICis histologically more evident than that ofadenocarcinoma to FAM. A quantitativetechnique may be useful in assessing theeffect of chemotherapy in oesophagealsquamous carcinoma.

(7 Clin Pathol 1993;46:51-55)

The pathological effects of preoperativechemotherapy in oesophageal carcinoma havebeen the subject of review,' but detailedmorphological descriptions of change follow-ing chemotherapy are sparse.2 5 Neoadjuvantchemotherapy trials have used various drugregimens with assessment in terms of reduc-tion in tumour size and survival, though theeffect on long term survival is still uncertain.

At this hospital the results of studies on theeffects of chemotherapy suggest that there isoften a significant histological response whichcannot be detected by barium swallow orcomputed tomography.6 In this study we haveattempted to quantitate the histopathologicalchanges following preoperative chemotherapy.

MethodsBetween January 1988 and July 1990, 55consecutive patients with localised oesopha-geal carcinoma were treated by resection, withor without preoperative chemotherapy, anddivided into four groups as follows.Group 1 comprised 13 patients (12 male, meanage 62X8 years, range 45-71) with adenocarci-noma of the lower oesophagus or oesophago-gastric junction who received two preoperativepulses of 5-fluorouracil (David Bull Labo-ratories, Warwick, England), adriamycin (Dox-orubicin, Farmatalia Carlo Erba Ltd, StAlbans, England), and mitomycin (Mitomy-cin-C Kyowa, Martindale PharmaceuticalsLtd, Romford, England) (FAM) with an inter-val of three weeks, followed by resection threeweeks later.7Group 2 comprised 14 patients (11 male, meanage 63-6 years, range 45-76) also with adeno-carcinomas, but who received no preoperativechemotherapy because of dysphagia resistantto dilatation (n=4), age above 75 years (n=3),refusal (n=3) and specific contraindications tochemotherapy (n=4).Group 3 comprised 12 patients (nine male,mean age 65-3 years, range 55-75) withsquamous carcinoma of the oesophagus whoreceived two preoperative pulses of mitomycin,ifosfamide (Mitoxana, Boehringer IngelheimLtd, Bracknell, England) and cisplatin(Lederle Laboratories, Gosport, England)(MIC) with an interval of three weeks, withresection three weeks later.8Group 4 comprised 13 patients (three male,mean age 67 1 years, range 44-86 years) alsowith squamous carcinoma but who received nopreoperative chemotherapy because of dyspha-gia resistant to dilatation (n = 5), age above 75years (n = 4), refusal (n = 1) and specificcontraindications to chemotherapy (n=3).

Pretreatment endoscopic biopsy and resec-ted specimens were examined in all patientsfollowing fixation in 10% formol saline andstaining with haematoxylin and eosin. Resec-ted specimens were opened longitudinally,pinned out, and mapped prior to processingand three to five full thickness blocks of the

Oesophageal ResearchLaboratoryS J DarntonS M AllenDepartment ofHistopathologyCW EdwardsDepartment ofThoracic SurgeryH R MatthewsEast BirminghamHospital, BirminghamB9 5STCorrespondence to:Dr S J Darnton

Accepted for publication15 July 1992

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Darnton, Allen, Edwards, Matthews

tumour were taken and embedded in paraffinwax. Sections were cut at 3 um thickness.Pathological staging of tumour differentiationand invasion and node status was madeaccording to the UICC classification.9The following comparisons were then

made:(a) Pretreatment biopsy and resected speci-mens in patients treated with chemotherapy(groups 1 and 3).(b) Preoperative biopsy and resected speci-mens in patients not treated with chem-otherapy (groups 2 and 4).(c) Resected specimens in group 1 (adeno-carcinomas with chemotherapy) and group 2(adenocarcinomas without chemotherapy).(d) Resected specimens in group 3 (squamouscarcinomas with chemotherapy) and group 4(squamous carcinomas without chemother-apy).

In addition to a qualitative assessment, aquantitative morphometric measurement wasmade on the resected specimens to determinethe proportion of tumour to stroma. A sectionwas selected from the central area of eachtumour, or in the case of grossly ablatedtumours, from the centre of the healed area.The densest region of tumour compared withbackground stroma was located empiricallyand a submucosal field observed at a magnifi-cation of xlO0. A camera lucida permittedsimultaneous observation of a movable styluson a bit pad. The proportion of tumour area tostroma was measured using the digitisingtablet system and stylus (Summagraphics BitPad Two, Summagraphics Corp., Fairfield,Connecticut, USA). Areas within the fieldwere outlined by the stylus whose position andmovement were recorded with the aid of amicroprocessor (Acorn Archimedes 440/1Computer, Acorn, Cambridge, England) andthe appropriate software ("Digit AnalysisPackage", BP Hayes Software, Berkhamsted,England). The proportion of total tumour areato the area of background stroma (the area ofthe microscopic field) was then expressed as apercentage.The mean and standard errors of these

percentage values were taken for each patientgroup. Student's t test on unpaired data wasused to test values for groups 1 and 2, withpopulations of a normal distribution, at the95% confidence level. Wilcoxon's rank sumtest for non-parametric unpaired data was usedto test the values for groups 3 and 4, becausethe distribution in group 3 was not normal atthe 95% confidence level.

ResultsQualitative changes seen after chemotherapyincluded complete ablation or partial regres-sion of the tumour, regeneration of surfaceepithelium over previously ulcerated tumourareas, pronounced fibrosis and a chronicinflammatory reaction (figs 1 and 2).(a) Pretreatment biopsy and resected speci-mens in nine adenocarcinomas treated withFAM 9 (out of 13 in group 1) showed onlyminor histological differences. In all 12 squa-

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Figure IA Case A: well differentiated squamouscarcinoma. Pretreatment biopsy specimen.

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Figure IB Case A: resected specimen followingchemotherapy (MIC). There is complete ablation oftumour with re-epithelialisation, local chronicinflammation, and submucosal fibrosis.

mous carcinomas, however, there were pro-nounced differences between pretreatmentbiopsy and resected specimens consisting ofablation or regression of tumour, disappear-ance of surface ulceration with healing of theepithelium and fibrosis, and areas of chronicinflammatory exudate localised aroundtumour islands when present.(b) Pretreatment biopsy and resected speci-mens in patients not treated with chem-otherapy (groups 2 and 4) showed nohistological differences.(c) Comparison of resected specimens ingroups 1 (adenocarcinomas treated withFAM) with resected specimens in group 2(untreated adenocarcinomas) showed that nineout of 13 tumours in group 1 showed minorhistological differences while the other fourwere identical in appearance.(d) Comparison of resected specimens ingroup 3 (squamous carcinomas treated with

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Histopathological findings in oesophageal carcinoma with and without preoperative chemotherapy

Figure 2A Case B: welldifferentiated squamouscarcinoma. Pretreatmentbiopsy specimen.

Figure 2B Case B:resected specimen followingchemotherapy (MIC).There is pronouncedregression of tumour tosmall foci of tumour cells(arrowed) surrounded by a

chronic inflammatoryexudate.

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4Squamouscarcinomacontrols(n = 13)

Figure 3 Staging of the resected specimens (by tumour invasion depth and local nodes)in the four patient groups.

MIC) with resected specimens in group 4(untreated squamous carcinomas) showed thatall 12 tumours in group 3 showed considerabledifferences from the untreated tumours. In onepatient there was complete ablation of thetumour (fig 1B); in five there was pronouncedtumour regression, with only minute foci ofmalignant cells scattered in the submucosa ormuscularis propria, frequently surrounded bya mixed chronic inflammatory cell infiltrate(fig 2B). All 12 specimens showed a localisedfibrotic response and a localised chronicinflammatory cell infiltrate, and 10 showedregeneration of the surface epithelium by a thinstratified squamous layer.The staging of resected specimens by

tumour and node status is shown in fig 3.Groups 1 and 2 showed a similar range ofstaging, but group 3 patients showed a lowerstaging when compared with group 4 con-trols.By quantitative analysis the mean proportion

of tumour to stroma in the adenocarcinomastreated with FAM (group 1) was 73-25% (CI10-94). This did not differ significantly fromthat of the untreated adenocarcinomas (group2) which was 71-56% (CI 12-58). The meanvalue for the proportion oftumour to stroma inthe group of squamous carcinomas treatedwith MIC (group 3) was 32-54% (CI 16.46)compared with 64-44% (CI 9d13) in theuntreated cases. This difference was significant(p < 0 01) (fig 4). In this group there appearedto be two clearly distinguishable subgroups;one group of six patients (group 3a) wheremore than 50% of the field consisted oftumour and another group of six patients(group 3b) where less than 19% of the fieldwas occupied. The differentiation of the pre-treatment biopsy specimens in these two sub-groups was as follows: group 3a contained twopoorly, one moderately, and three well differ-entiated tumours; group 3b contained twopoorly, one moderately, and three well differ-entiated tumours. Numbers are too small forformal statistics but group 3b showed a tend-ency to better survival at one year-five out ofsix surviving, with the sixth a postoperativedeath, compared with three survivors out of sixin group 3a, one death being unrelated and twodue to recurrence of tumour.

DiscussionThe study has shown that qualitative andquantitative responses to chemotherapy can beobserved histopathologically in oesophagealcancer. Qualitative and morphometric tech-niques confirmed that the treatment of adeno-carcinomas with FAM (group 1) was much lesseffective than treatment of squamous carcino-mas with MIC (group 3).

Qualitative differences were seen followingboth chemotherapeutic regimens but wereminor in group 1 compared with those ingroup 3, in both numbers of responders andextent. Previous light microscopic studies havedescribed chemotherapeutic response in termsof the presence or absence of microscopictumour, necrotic tumour,2 or of necrosis only.3

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4Squamouscarcinomacontrols(n = 13)

Figure 4 Scatter diagram showing the proportion of tumour area to area of backgroundstroma as a percentage for individuals in the four patient groups. Mean values are shown.Group 1 versus group 2, not significant. Group 3 versus group 4, significant (p < 0-01).

There has been one electron microscopic studyreporting chemotherapeutic effect in terms ofnecrotic features of nuclei.4 Xian et al observedtumour necrosis and a stromal lymphoidresponse in oesophageal squamous carcinomatreated with traditional Chinese herbs. Likethem, we also noted the frequent presence of a

chronic mixed inflammatory cell infiltrate sur-rounding tumour areas after chemotherapy,but necrotic tumour areas were seen in onlytwo of 13 cases in group 1 and one of 12 casesin group 3. Persisting tumour cells appearedviable even with tumour regression to smallaggregates of cells (in group 3). It may be thatduring the chemotherapy treatment period ofsix weeks there was tumour cell death followedby in inflammatory response. Islands of inflam-matory exudate were seen as haloes surround-ing tumour islands in resected specimens fromthe treated groups. Healing of areas previouslyoccupied by tumour appeared to be by fibrosiswith overlying surface re-epithelialisation.

Routine histological examination of a resec-

ted specimen can determine completeresponse to chemotherapy, although samplingerrors are possible unless serial sections are

taken through the entire specimen. Assessmentof partial response in a resected specimen byqualitative examination alone is more difficult.The Japanese system of reporting the histopa-thological response to chemotherapy in oeso-phageal tumours'° relies on the estimation ofthe proportion of necrotic to viable tumourcells. Their response grade Efl has one third ofthe tumour occupied by degenerate tumourcells; grade Ef2 is a degenerating tumouroccupied by one third viable cells and gradeEf3 is a completely necrotic tumour with no

viable cells. We consider this methodology to

be inappropriate as in our experience reduc-tion of tumour bulk in a healing oesophagusleaves small foci of apparently viable tumourwith no necrotic areas. The presence of degen-erate and necrotic tumour seen in other stud-ies, and occasionally in this study, could besecondary to local ischaemia rather than repre-senting a direct effect of chemotherapy. Thisdiscrepancy in results is possibly due totumours being studied at different intervalsfollowing chemotherapy or because more oftheir data relate to tumours treated by radio-therapy. It is not possible to determine this asthe necessary details are not stated in thepublication.'0The morphometric technique used in this

study quantitates a partial response to chem-otherapy by measuring the actual ratio oftumour to background stroma in the treatedgroups when compared with the untreatedgroups. Quantitatively, the proportion oftumour to stroma was not significantly reducedby FAM chemotherapy in adenocarcinoma,despite minor qualitative changes seen in nineout of the 13 patients. The MIC regimen,however, significantly reduced the proportionof tumour to stroma in squamous tumours. Inthese patients there appeared to be two clearlydivided subgroups in terms of degree ofresponse. The numbers are small but it couldbe that these two subgroups represent a differ-ent susceptibility to MIC. The degree ofdifferentiation of the tumour found at pretreat-ment biopsy bore no relation to susceptibility.Good quantitative responders, however,showed a tendency to improved survival at oneyear.

The similar staging in the treated anduntreated adenocarcinomas confirms the sta-tus of group 2 cases as representative controlsfor group 1, bearing in mind the low level ofhistopathological response. When comparingthe treated squamous carcinomas with theircontrols, the former showed a lower staging.This is of interest since staging of IIA andbetter (nine out of 12) represents local nodenegative patients with invasion into the adven-titia or less. This could indicate a reduction instaging as a result of the chemotherapy butcannot be proved in the individual patient asresection is only performed once. The assump-tion of a chemotherapeutic effect of MIC onsquamous carcinoma staging is reasonable asthe observed reduction in the ratio of tumourto stroma provides indirect confirmatory evi-dence. The fact that there was no such loweredstaging or reduction in tumour to stroma ratioin the adenocarcinomas treated with FAM alsotends to corroborate this.

In conclusion, histopathological changesattributable to chemotherapy can be observedin oesophageal carcinoma. The response ofsquamous carcinoma to MIC chemotherapy ishistologically more evident than that of adeno-carcinoma to FAM chemotherapy. Qualitativeobservation of these changes may provideuseful information about the response tochemotherapy, but it is of necessity impreciseand subject to observer bias. Quantitativeanalysis, particularly in relation to the propor-

u I

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Histopathological findings in oesophageal carcinoma with and without preoperative chemotherapy

tion oftumour to stroma, is a simple techniquewhich is more accurate and extends our abilityto evaluate objectively the effect of chem-otherapy on squamous carcinoma of theoesophagus.

This study was supported by the Oesophageal Cancer ResearchAppeal (OCRA), Birmingham, England.

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2 Hilgenberg AD, Carey RW, Wilkins EW, Choi NC, Mathi-sen DJ, Grillo HC. Preoperative chemotherapy, surgicalresection, and selective postoperative therapy for squa-mous cell carcinoma of the esophagus. Ann Thorac Surg1988;45:357-63.

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