J Clin Pathol 1992;45:979-983

Effects of chemotherapy on ultrastructure ofoesophageal adenocarcinoma

S J Darnton, G N Antonakopoulos, J Newman, H R Matthews

AbstractAims: To compare and contrast the ultra-structural appearance of resected oeso-phageal adenocarcinomas treated withpreoperative chemotherapy with that ofnon-treated resected controls; and todetermine the usefulness ofthis method inthe assessment of the effectiveness of thechemotherapeutic regimen.Methods: Ten resected oesophageal ade-nocarcinomas treated with preoperativechemotherapy--mitomycin-C, ifosfamide,and cisplatin (MIC)-were examined bytransmission electron microscopy andtheir appearance compared with that of 13concurrent untreated resected oesopha-geal adenocarcinomas.Results: The treated adenocarcinomasshowed cytotoxic damage although com-plete tumour eradication was not ach-ieved. In all 10 treated cases a variableproportion of the neoplastic cells showedunusual degenerative and necrotic chan-ges not seen in untreated cases. In themost affected carcinomas the stroma con-tained increased numbers of inflamma-tory cells.Conclusions: This ultrastructural methodis useful for the assessment of the in vivoeffect of MIC.

(7 Clin Pathol 1992;45:979-983)

Adenocarcinoma of the oesophagus or oeso-phagogastric junction is rare but rapidly fatal.'At this hospital neoadjuvant combinationchemotherapy (mitomycin-C, ifosfamide, andcisplatin or MIC) was used before resection ofprimary oesophageal adenocarcinoma in aPhase II study. The clinical details have beenpublished elsewhere.2

Oesophageal ResearchLaboratoryS J DarntonDepartment ofHistopathologyG N AntonakopoulosJ NewmanDepartment ofThoracic SurgeryH R MatthewsEast BirminghamHospital, BirminghamB9 SSTCorrespondence to:Dr S J DamtonAccepted for publication14 May 1992

MethodsBetween December 1989 and July 1991 23consecutive cases of localised adenocarcinomaof the lower oesophagus or oesophagogastricjunction were treated by resection with or

without preoperative chemotherapy.Ten patients (nine men, mean age 58-7

years, range 38-68) received chemotherapy(MIC). Of these, nine patients received twopreoperative pulses with an interval of threeweeks, followed by resection three weeks later.2One additional man aged 70 years receivedonly one pulse of MIC three weeks beforeresection. Thirteen patients (nine men, mean

age 64 2 years, range 50-73) received noneoadjuvant chemotherapy.

Immediately after resection each specimenwas opened longitudinally and pinned out.Photography, mapping, and routine micro-scopic examination were performed. A freshblade was used to take a cube of tumour (ofabout 5 mm size) and also a strip (about5 x 2 mm) of "normal" oesophageal mucosafrom a proximal site on the specimen, as faraway from the tumour area as possible. Thesamples were placed in cold buffered glutar-aldehyde, pH 7-4.

After fixation in glutaraldehyde, the sampleswere cut into 2 mm size cubes. Special carewas taken to select the most viable lookingportions which were then post-fixed in 1%osmium tetroxide in phosphate buffer (pH7 4), dehydrated in ethyl alcohol, and aftertreatment with inhibisol embedded in TAABresin. All stages were prolonged to avoidpossible poor embedding because of the unu-sually large size of the samples. Thick sectionswere stained by toluidine blue and examinedby light microscopy, while thin sections werestained by uranyl acetate and lead citrate andexamined in a JEOL JEM-lOOC transmissionelectron microscope.

In one of the cases treated with MICimmunohistochemistry for PGP 9 5 (ProteinGene Product 9 5, UltraClone Ltd., Rossiter'sFarmhouse, Wellan, Isle ofWight P041 OTE)was performed on the paraffin wax embeddedtissue to confirm the presence of neuroendo-crine cells.

ResultsThe appearance of oesophageal squamousepithelium taken from an area distant from thetumours of treated and untreated cases showedno obvious differences within the three definedlayers-basal, prickle, and functional cells.The appearance of the untreated group of

adenocarcinomas was identical with that whichwe have described in our previous study.

All 10 cases treated with neoadjuvant MICshowed features not seen in the untreatedcases. These features were restricted to theappearance of the neoplastic cells and to thenumber and appearance of inflammatory cellsin the intervening stroma. Smooth muscle cellsand small vessels generally had an unaffected,conventional appearance. In one case thetumour showed heterogeneous differentiationin that it also contained cells of a neuroendo-crine nature. Neurosecretory granules wereconfirmed on a paraffin wax section by the

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Fgure 1 Adjacent conventional cells (c) and unusual cells (u) against a lumen (7).Hypersegmented (n) and pyknotic (p) nuclei. Vacuolated cytoplasm (v) with degeneratemitochondria (m) and a residual body (r).

immunostain PGP 9-5. The neuroendocrinecells had a conventional ultrastructural appear-ance, although the adjacent neoplastic glandu-lar cells were grossly abnormal. In the same

case some desmosomes were seen, a character-istic of squamous rather than glandular differ-entiation, but tonofilaments were not present.The 10 treated cases can be divided into two

groups on the basis of the fine structure of theirtumours. The first group of five cases had a

mixed appearance. Cells with conventional,usual features seen in adenocarcinoma were

admixed with cells displaying features pre-viously not seen in untreated adenocarcino-mas. In the second group of five cases most ofthe neoplastic cells displayed unusual fea-tures.

Group 1: five cases with a proportion ofneoplastic cells showing unusual featuresMany of the tumour cells displayed conven-

~~~~~~:~~~~~~~~~~~~~Figure 2 Adjacent conventional cell (c) and unusual cells. An autophagic vacuole (v)contains nuclear material A nucleolus has an abnormal configuration (n).

tional neoplastic appearances seen in adeno-carcinomas before. Their nuclei were oval orslightly indented with finely dispersed chroma-tin and prominent nucleoli. In a few cells thecondensed chromatin was marginated withreticulate material in the centre of the nucleus.There was abundant cytoplasm rich in orga-nelles, especially rough endoplasmic reticulum(RER).A variable proportion of cells showed

unusual features. This variability was observednot only among cases but also within thetumour of any individual case. These cells werearranged both in clusters as well as beingdispersed between the conventional neoplasticcells (figs 1 and 2).The unusual cells were characterised by

misshapen nucleoli (fig 3). In others, thenuclear chromatin was marginated while themore central areas were occupied by a granularmaterial against a background of low electrondensity. These changes gave the appearance ofnuclear "oedema". Occasional whole nucleiwere hypersegmented (fig 1). Karyopyknosisand karyorrhexis were seen occasionally.

In the cytoplasm of several of these neo-plastic cells were large secondary lysosomes(possibly representing residual bodies (fig 1)and autophagic material (fig 3) or secondarynecrosis of ingested apoptotic bodies). In allcases were cells with large cytoplasmicvacuoles which sometimes impinged on theindented nuclei (fig 1).

In well differentiated areas of the tumoursthe most impressive change, seen in all cases,was the protrusion of finely granular cyto-plasmic blebs into the lumen. Many appearedto be in the process of being discharged (figs4A and B). This feature was consistent with theloss of cellular architecture rather than exo-crine secretion, as microvilli, mucin dropletsand, in one case, a centriole, were seen withinthe granular material of these blebs (fig 4B).

In all cases the stroma and tumour clusterscontained slightly increased numbers ofinflammatory cells including macrophages,lymphocytes, eosinophils and mast cells. Neu-trophils were present in small numbers. Severalmacrophages were seen engulfing amorphousmaterial in the stroma or at the periphery oftumour clusters. These five cases included theone treated with only one pulse of MIC.

Group 2: five cases in which most neoplastic cellsdisplayed unusual featuresMost of the tumour cells showed unusualcellular features, although there was somevariation within tumours.The nuclei and their nucleoli were usually

irregular in shape and the chromatin wascondensed. Karyopyknosis and karyorrhexiswere also seen. Convoluted pyknotic nucleiwere sometimes located against the basalplasma membrane (fig 5). Rupture of thenuclear envelope with discharge of the nuclearcontents into the expanded cisternal networkwas also seen (fig 6).

Cytoplasmic organisation was disrupted inall cases. Many cells contained large vacuolesand plentiful residual bodies filled with elec-

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Effects of chemotherapy on ultrastructure of oesophageal adenocarcinoma

Figure 3 Grosslyabnormal nucleoli from twounusual cells.

tron dense material. All cases showed apicalblebs of granular material. In one case theblebs were so large that they occupied almostthe entire lumen of an acinus. Rupture of thecytoplasmic membrane and discharge of cellcontents into the stroma was common. Cyto-plasmic organelles were disrupted with degen-erate mitochondria containing disintegratedcristae and a matrix of low electron density.The stroma contained abundant macro-

phages loaded with lipid droplets and electron-dense material. These macrophages were seenactively phagocytosing diverse cellular rem-nants (fig 7). Large numbers of lymphocytes,eosinophils, and mast cells, as well as a fewneutrophils, were also present. In all casesmany inflammatory cells, especially macro-phages and lymphocytes, infiltrated thetumour clusters (fig 8). Abundant active fibro-blasts were also present.

Figure 4 Unusual cells.(A) Apical blebsdischarging into thelumen. (B) Apical blebcontaining cytoplasmiccontents, including acentriole (arrowed).

DiscussionAssessment of the response of oesophagealcarcinomas to chemotherapy has been made atthis hospital by barium meal, computedtomography, and light microscopy.34 Electronmicroscopic assessment adds another dimen-sion to observations of changes brought aboutby chemotherapy.An earlier study of the morphology of

oesophageal carcinomas5 reported the hetero-geneous appearance of adenocarcinomas of theoesophagus. In our study samples from areasof the tumours of treated and untreated caseswere selected which included both viable cellsand those showing signs of cell death. Thetreated tumours all showed some signs of thecytotoxic effects of MIC and could be dividedinto two groups depending on the proportionof tumour cells showing unusual features. Theobservation that the case given only one pulse

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xU14',:R -'': - 4-IV 1.s i:-AW 3 w-A S'7 Af - = _ .... \cr-, .o 01-so -s ow 1-.n4 ,-_

Figure 5 Unusual cell with its pyknotic nucleus adjacent to the basal plasma membrane(p). Condensed chromatin (arrows) is released via a ruptured nuclear envelope.

of MIC fell within the group with the lowerlevel of response may possibly be relevantbecause the changes seen in that individualcase were similar to those in four other cases towhom two pulses were administered but num-bers are too small to draw any definite conclu-sion. Variation occurred within all treatedtumours.The only previous investigation of the ultra-

structural effects of chemotherapy on oeso-phageal tumours was on 14 patients withsquamous cell carcinoma.6 Various regimenswere used and ultrastructural changes weredescribed in only one case, and those wererestricted only to nuclear changes-that is,condensation of nucleoli, disintegration of thenucleonema, and reduced electron density ofthe nucleoplasm with dispersion of chromatin.We have seen similar nuclear changes in theadenocarcinomas treated with MIC, but

hypersegmentation of nuclei, abnormally con-figured nucleoli, and breakdown of the nuclearmembrane with resultant spillage of nuclearcontents have also been observed.

Detailed examination has shown other signsof cell disruption, presumably due to thecytotoxic effects of the regimen, because theseabnormalities were not seen in untreatedtumours. There was a complete disruption ofcytoplasmic organisation and of the organelles.The location of pyknotic nuclei against thebasal plasma membrane of cells suggests col-lapse of their cytoskeleton.

Similar signs of cell death have been brieflydescribed in two cases of transitional cellcarcinoma of the human bladder treated withintravesical mitomycin and studied by biopsy7and of resected normal rat bladder.8 Ultra-structural results of in vivo administration ofcytotoxins before resection of human carcino-mas have rarely been reported,6 though similarultrastructural effects of cytotoxins on humancarcinomas transplanted into nude micehave.9The cytotoxic effects of MIC were restricted

specifically to the neoplastic cells of theoesophagus, the normal squamous epitheliumbeing totally unaffected (displaying featuresidentical with those previously reported10).Adjacent smooth muscle and endothelial cellshad a normal appearance. The necrotic chan-ges within the tumour cells cannot, therefore,have been secondary to local tissue ischaemia.The retention of a normal local vasculaturewould be of great therapeutic importance. Anydamage to the vasculature would impededelivery of cytotoxins to the target cells.

Heterogeneity was seen in one case out ofthe 23 in the present series. This case showedneuroendocrine-type cells, unaffected by thechemotherapy, although adjacent glandularcells were grossly abnormal. Such heterogene-ity may further complicate successful treat-ment by chemotherapeutic regimens. Inter-and intratumour variability emphasises the factthat adenocarcinomas do not behave in a

Figure 6 Unusual cellwith its pyknotic nucleusadjacent to the basalplasma membrane (p).Nuclear contents (arrows)spill over into a dilatedcisterna ofRER(arrowhead to ribosomes).

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Effects of chemotherapy on ultrastructure of oesophageal adenocarcinoma

Figure 7 From adisrupted tumour cluster. Alymphocyte (7) adjacent toa macrophage engulfingamorphous material (a).

.

uniform manner.Stromal changes were also seen after treat-

ment. Necrosis of tumour cells presumablyrecruits host inflammatory responses asimmune cells were seen infiltrating thetumours, macrophages engulfed varied mate-rial and active fibroblasts were present. Theseobservations agree with the light microscopicchanges seen in an earlier study.4 It wassuggested that the response of oesophagealsquamous cell carcinoma to chemotherapycould be seen in the shrinkage of tumourclusters and an increase in surrounding inflam-mation and fibrosis.

In no case did viable adenocarcinoma cellscompletely disappear after chemotherapy. It is

impossible to comment on the level of differ-entiation of damaged cells, but individualviable cells were of varying degrees of differ-entiation.

SJD and GNA are supported by the Oesophageal CancerResearch Appeal (OCRA), Birmingham, England. We aregrateful to Miss K H Field for photographic assistance.

1 Matthews HR, Waterhouse JAH, Powell J, McConkey CC,Robertson JE, eds. Clinical cancer monographs. Vol 1.Cancer of the oesophagus. London: The Macmillan PressLtd., 1987:68-71.

2 Allen SM, Duffy JP, Darnton SJ, Cullen MH, MatthewsHR. A phase II study of mitomycin, ifosfamide andcisplatin in operable adenocarcinoma of the oesophagus.Br J Cancer (in press).

3 Walker SJ, Allen SM, Steel A, Cullen MH, Matthews HR.Assessment of the response to chemotherapy in oesopha-geal cancer. Eur J Cardiothorac Surg 1991;5:519-22.

4 Darnton SJ, Allen SM, Edwards CW, Matthews HR.Histopathological findings in oesophageal carcinoma withand without pre-operative chemotherapy. J Pathol 199 1;163: 174A.

5 Newman J, Antonakopoulos GN, Darnton SJ, MatthewsHR. The ultrastructure of oesophageal carcinomas: multi-directional differentiation. A transmission electron micro-scopic study of 43 cases. J Pathol 1992;167:193-8.

6 Iwatsuka M, Yoshida M. A study of the clinicopathologicaleffects of chemotherapy for human esophageal carci-noma. In: Siewert JR, Holscher AH, eds. Diseases of theesophagus. Berlin: Springer-Verlag, 1988:319-22.

7 Stewart RJG, Lawson AH, Weaver JPA. The ultrastructureof transitional cell carcinomas of the human urinarybladder treated with ethoglucid or mitomycin. J Urol1988;139:1355-8.

8 DaskalY, Crooke ST. Morphological effects of mitomycin Con cellular fine structure. In: Carter SK, Crooke ST, eds.Mitomycin C -current status and new developments. NewYork: Academic Press, 1979:48-53.

9 Stenback F, Kangas L, Wasenius V-W. Cell structure andfunction and response to chemotherapy in tumors hetero-transplanted into the subrenal capsule of mice and rats.Eur J Cancer Clin Oncol 1985;21:1523-38.

10 Hopwood D, Logan KR, Bouchier IAD. The electronmicroscopy of normal human oesophageal epithelium.Virchows Arch (Cell Pathol) 1978;26:345-58.

Figure 8 Lymphocytes (1)and a macrophage (m)infiltrating a disruptedcluster of neoplastic cells.

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