Pathological Society of Great Britain and Ireland 163rd Meeting, 3 - 5 July 1991 at

The Queen's University of Belfast

SYNOPSES OF PAPERS Papers and posters presented at this meeting have not been refereed or reviewed by the Journal of Pathology

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Alteration for integrin and stromal protein expression a marker of premalignant change? J. Louise Jones. RosemaryA Walker Oeparfmen!of Pathology.Ciinrcal Sciences Burldmg, Lecester Royal Infirmay P 0 Box 65, Leicesler, LEZ 7 U

The extracailular matrix IS tmportant in modulating the growth and differentiation of epithelia interacting with cells through ~peclfictransmembranereceptors. or integrins This study examinestheexpression of theintegnn subunits u2,as,0, and p4,and the Stromal protein tenascln, ~n53 breast Carcinomas,"on-involved surroundingbreast tissue from 21 of these and 32 narmalibenlgn cases. Frozen tissue and an indirect immunoperaxidasetechnique were used thrwghoui Welldeflned, linearstaining In relationtobasement membranewasseenln28~132normaVbenlgn cases far all integnns. Complete loss of reactivity was seen in 80% of carcinomas Those showing reactivity displayed a predominance of diffuse cytoplasmic stammg not seen on the benlgn cases. Loss of stalnlng was not related to turnour grade or lymph node Status. but cytoplasmlc staining related to loss of structural differentiation. Foro,andB, integrinsthetissuesurraundingcarclnomasbehavedasfornormaVbenlgncases. whilstforasand fiL two thirdsshowedalteredorlackof staining,similartothecorrespondlng tumour.Therewasa band ofstalnlngfor tenawn in carcinomas. with an invene relationshipbetweenintegrin expressionand tenascin reactivity.Thealtered as.f14 integrinexpressionin "an-involved tissue from cancerous breastcould be a useful markerofearlymalignant change.

An in vitro method to examine the caveats involved in cell proliferation analysis using 5-bromo P'deoxyuridine(BrdUrd) in colorectal tumour tissue

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Kate Williamson Isla Hallida ' P W Hamilton Joy Ruddell M Varma P Maxwell 'A Trudgen A Crockard' B J Rowland3

The development of an antibody to BrdUrd has instigated many m vitro and m vivo Studies on tumour cell proliferation Most of these studies have been undertakenwtthom due canslderatlon to problems such as the selective charactensticsof partial denaturation and the uneven access of the analogueto tumour cells A new technique for the infiltration of BrdUrd I" fresh colorectal twmurtissue has been developed Which ellminatesSome of these variables The technique involves enzymatic disaggregation of fresh colorectal tumour tissue into viable single cell suspensionswhich are then infiltrated with BrdUrd This allows all cells equal accessto BrdUrd After a ngorous denaturation procedure a modified ELSA IS used to quantify BrdUrd uptake Ths identified an Optimal infiltrationtimeoftwo hours with an Optimal Concentrationof lo4 M BrdUrd Theseconditions achlevedthegreatest differences m optical density between infiltrated and "on-infiltrated samples and caused minimal cell damage Studieson theconcentrationsoftissue culturemedium constituents revealedthat glutamme concantrattonmaybe a significant factor ~nthe uptake of BrdUrd Nutrient deficiency may also be an important factor m cell proliferation Studies It IS time to clanfy the accuracy of cell Proliferationdata Obtained using monwlonal antibodies to BrdUrd Th89 m ~ t techniow m orovides a witable tool which mav be used to unravel Some of the BrdUrd enlomas

The P53 gene in human thyroid tumours Pat A Wright.' N. R Lemoine? P. E G0retskt.j Fwna S. Wyliie,' Christme Hughes? E. D. Williams.'. D Wynford-Thomas' 'Depanmen!of Patholooqy, Universrhl Of Wales College of Medicine. Heath Park. Card,K CF4 4XN. *MoIecuIarOncoIogyUnr!, HammersmithHospital, Du Cane Road, London. W19 OHS, 3Cepzrfmenr01 Surgeiy Universityof Dusseldorf D ~ 4 w ODusseldorf W Gemany

We have previously reported a mutation of the p53 gene, a putative turnour suppressor gene. in a differentiated human thyroid follicular carcinoma cell Ibne FTC 133, but failed to find any mUtationSby sequencing the main Conservedregions ~nasmalisenesofhumanthyroidcamen Wehavenowextendedthlsanalysls PCRanddnect genomicsequencingofexons5 7 and8ofthep53geneIn20humanthyroldcarclnomas showedonlynomal DNA sequence. lmmunOhiS.whemi~~l~nalysls of 129 human thyroid tUmOUs using a panel p53 antibodies was unequlvmally negative Southernblotbng ~n2OcaSesfalledtodemonstrate any rearrangementordeletlon.However analysisof tissue from the original case from which the FTC 133 cell lhne was derived shows the mutation to be present in thetumourtissueaswellas thederived line, but absent ~nthepatlen~sconstltutlonalDNA Slnca thtscell ilnewasanlyderlved afterl32unSuccessfulanemptswlthother humanfollicularcarcinomas, we proposethat p53 mutation. even If rarely important for human thyroid carclnogeneslsln patlents, may confer on thyrmd folllCUlar tUmOUr cells the ability to grow in Culture We are now transferring exogenous mutant p53 Into thyrold tumOUr cells to test thls hypothesis. If successful.thts Strategywlll pemtt thedevelopment ofthyold turnour Cell lheS whch are more representativeof the human disease.

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Proliferating fractions of choroidal malignant melanomas: comparison with morphological criteria of malignancy Deirdre Devaney. B McDonald.' Hilary McGee. P Kelly Deparfment 01 Patholcgy,Mafer Misencordme Hospifal, Dublm 7, 'Deparfmen!oiNeuropatholcgy, Radcl!fle Infirnay, WwdSlock Road, Oxford

The presenceof the nuclearprotein PCNNCyclin in cells 1s Correlatedwith cell proliferativeactivity. The mOnmlonal antibody PClOrecOgniSesanepltopeotPCNA which ~~re~i~tanttoroutinefomalinfi~ati~nandparaffinemb%dding of tissue Fineenchoraidalmalignantmelanomas were~tainedimmunohistochemicallyforPCNNcyclin. The level of staining was expressedby an index which incorporated semiquantnativeassessmentof bomtheintensltyofstaining and the fraction of positively stained nuclei. The resuns were compared wlm the Conventional Callender Morphological Classification of tumwrs. Within individual tumous of mixed morphology less dmerentiatedareas hada higherpraliforativeIndex.Compansonsbetween tumoun wererenderedmoredifkull byvanation instaining posslbly related to COndltlOnSof flxatlon. Nevertheless. less well dmerentiatedturnours had a h g h w PfOlderatlVe index. Overall. therefore. there IS a correlation between the proliferativeindex of choroidal malignant melanoma measured using PClO and the conventional Callender Morphological Classification.

Recombinant human erythropoietin treatment of tumour-induced anaemia: effects on tumour growth rate and radiosensitivity

Distribution of collagenase and tissue inhibitor of metalloproteinases (TIMP) in colorectal tumours R. E HewB.' I H. Leach.' D. G Powe.' I. M. Clark? T. E Cawston? D R Turner' 'Deparfment oiH!s!opa!hology, Oueen's Medical Centre, Nonmgham. NGIZUH, *RheumalologyResearch Unr!. Addenbrwke's Hosprtal, H!C Road, Cambridge. CBZ 2 0 0

Raised collagenaseactivity calatectal carcinomas has recently been shown to be associated with increased malignant potential.TOdeterminethe tissue distribution of collagenaseand 11s spec~f~c mhlbitor, tissue inhibitor of rnetalloproteinasesFIMP). we canied out an immunohistwhemical Study on colorectal carcinomas (n = 21). adenomas (n = 7) and normal mucosa (n = 6).We found markedly increasedstaining for collagenasein the &nective tissue stroma of carcinomas, as compared with adenomas and normal mucose. Llnle evidence of epithelial cell staining for collagenasewas seen !n any tissue. In carcinomas. both slromal flbrotlasts and collagen fibresstained strongly and stromal staining was stronger close to neoplastic glands. Vascular staining was more prominent in neoplastic than normal tlswes. perhaps reflecting the increased proteolytic activity dunng tumour angiogeness. The pattern of TIMP immunostainlngwas smilar to that of collagenase,although basement membranestaining for TIMP was generally more marked. Another difference was that increased staining for collagenasebut not TIMP. was often seen at the invasive edge of Carcinomas The posstbilttythat collagenolytic activityIsconsequentlyincreasedatmeinvasiveedge.suggeststhatcollagenasemayplayarole in theprOCessOf tummr invasion

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1991 by John Wiley and Sons, Ltd.

347A

J. J. A. McAleer.' D.G. Hmrst."S. R McKwwn,' B. J. Joiner?M P MacManus,'T. R J. Lappin,'

H Bharucha? J. M. Bndges' Departmentsof 'Haemafolwy and zPathology, Queen's Univers~fy.hsbtiite of Cl!nml Scmce, Belfast, B J l Z 6BL. 'CRC Gray Laboiatoy, Norfhwmd. M,ddlesex

Anaemia wwrs frequently in patients with cancer and renders radiation treatment less effecme. Its correction by administration of recombinant human erythropoietin (rHuEpo) may improve treatment outcwne.The KT mammary carcinoma (CaNl), implanted intradermally in male CBA mice, causes anaemia, leucmytosis and splenamegaly, and provides a model to study this problem rHuEpo (0. 5 or 20 Umts) was administered daily by lip Injectbon. Turnours Were irradiated with a 1IMOGy traction of 250kV X-rays and growth delay assessed.Haematmnt levels increased in a dose-dependent manner with rHuEpo dose, resulting ~npolycythaemia at the highest Uose rHuEpo caused no change in white cell or platelet counts: some changes m bone marrow morphology and an increasein spleen stze were observed. Increasing radiation dose caused greater regrowth delay and no significant dmerenca was found between rHuEpo dose groups When growth rate before radiation treatment was examined, volume doubling time forcontrol mice was 2 3 days (SEMO.2)and thasefor5 and 20 Units of rHuEpo were3.5 (0.5) and 4.6 (0.5)days respectively (p < 0.001). This reduction in turnour growth rate with increasing M u E p dose was investigatedby measuring relative turnour blood flow, using the rubidium extraction technique. Tumour blood flow was increased ~nmice which received 20U of rHuEpo as compared to the control group, Whweas livw blood flow was reduced.rHuEpa*caused no change ~nCaNT cell growth when tested m vrfm, (assessed by tntiated thymidine uptake).AlthoughrHuEpoincreased haematwritandreduced tumOurgrowthrateInCaKI, ~ c h a n g e w a s f o u n d r Its. radiation sensitivity.

348A

SYNOPSES OF PAPERS

Tumour marker immunoreactivity of metastatic cancer. An accurate method of identifying primary site of origin?

L 8 H cells of nodular lymphocyticpredominant Hodgkin's disease show

A. R. Gamble, J. A Bell. J E. Ronan, D Pearson, I. 0. Ellis

Christina Schmid, Claire Sargent. P. G. Isaacson

Cify Hospital, HucknallRoad Noflmgham

Department01H~slopaiholoQyUniversity College andMiddlesex School ot Medmne, UnivemtyStreet, London, WC 1 E 6JJ

In order to determine whether Variations m the expression of tUmOUr related antigens can predict the origin of turnours,the,mm""~,eact,",ty~f 100metaslasesand associatedprlmarysitesof ~an0~5typeswasstudied witha panelof antibody markers The panel originally consisted of9 antibodieSinclUdinganew antibody CA 125. CA 19.9, SM 3, Prostatespecific antigen, Thyroglobulin,228 (carcinoembryonicantigen), 198(carcinoembryonicantigenwith nonspecificcrossreactingantigencospecif~city), NCRC 11 (EMAiike)andSM 3iEMAlike) Discrimlnantanalysisof theresultsshowed that 11was pOSSiblelorationaiisethe panel to Ganttbodies These wereCA 125,CA 19 9,SM 3, Prostate specific antigen, Thyroglobulin and 228. Using such a panel the con& primary Site of origin could be predicted in 70% of casesfor men and 54% forwomen In addition a specificityof greaterthan 90% was found for all the tissuetypes (gastrotntestinal,lung, breast, gynaecological,prostateand thyroid) Application Ofthisapproach m patients presenting with metastatic adenocarclnoma of unknown origin can assist ~nthe locabsationof probable pnmarysites and reducethenumberof enenwe and expensiveimagingtechniquesthatarecommonly performed

Gastrointestinalcancer - a new aspect

immunoglobulin light chain restriction

The nodular form of lymphocyte predominant Hodgkin's disease (NLPHD) IS widely accepted to be a B cell neoplasm Despitethis consensus. previousStudieshave failed to demonstratemonotypic lg lightchain expression bytheputativelymalignantL8Hcelis.We havestudied paraffin embedded tissuefrom 18casesaf NLPHDforthe presenceof immunoglobulin(lg) light and heavy chains and J chain In each case L 8 H cells In individual nodules were examined lor the presence of lg light cham restmtion (LCR). Kappa LCR was detected m all L 8 H cells tn all nodules in 5 cases (28%) and in a proporiion (1M 7 % )of nodules in 11 cases (65%). L 8 H cells not showing LCR either failed lostain for light chainsor stained arfefactually with antibodest0 both light chain indicating lg uptake. Df 12 cases stained with antibodies to lg heavy Cham 11 contained IgG and in 1 case no heavy chains were demmstrable Most L 8 H cells lnallcasescontained Jcham. Ourfindingsof monotypic L 8 Hceils mNLPHDin94% of our investigated cases confirm the neoplastic nature Of L 8 H celis and supporf the hypothesis that NLPHD 1s a malignant non-Hodgkin's B cell lymphoma. The presence of kappa light chain restriction in all cases awaits explanation

Geraldine M. Ma&ey, T. C. M Morns. R. C. Curry, Julie A. McCormick. D. Swam, D. H. Alexander Sandra Edgar &parmen1 of HaemafolOQyand SorQeW.Belfast Ctty Hospital, Lisbum Road, Beliasf, BT9 7AD Eleven of 90 patients with gastrointestinal(G.1.T ) carcinoma have been shown to have monocyte esterase deficiency(M E.D.) bycytochemical staining of monocyte preparationsfrom theirpenphera blood This incidenceof 12.2% was signdicantlyhigherthan the p r e ~ 1 0 ~ s l y d e m 0 n 5 tbncidenceol rd i.7% in410patientswithca~ccmoma of OthersystemSandtheincideoceof0.89b in474normal bloOddonors.Theoccurrenceofthedeficiencydoesnot appearto be relatedto thestageof diseaseorits treatment by reSectiOnarchemoradiotherapy We have previously demonstrated an increasedincidence of MED in patients with lymphoproliferatweneoplasiaand autoimmune disease and shown a famd,al incidence of MED m 10 of the 13 families of ME0 patients from these groups. the findingssuggestinganautosomaldominant modeof Inheritance.Wehavesuggestedthatareasonfortheincreased incidenceof MEDin theSegraupsofpatientsmaybethatthedeficiencymaypredisposetothesediseases lnvrfro evidence, namely. that esterase deficient monocytes, in contrast to esterase positive monocytes.Cannot be Stimulatedby lactofenin to kill a turnour cell line (K562).supporis this hypothesis Family Studies of the GlT carcinoma patients with MED have not yet been done but no evidence of an acquired cause for the deficiency has been found. Whether constitutionai or acquired. there IS a definite link bemeen MED and carcinoma arising in the digestive tract.

Demonstration of dominant T-cell receptor gamma gene clonotypes in model systems and normal and diseased skin

The expression of 8-cell antigens by Hodgkin's and Reed Sternberg cells Christina Schrnid. L. Pan. T. DISS.P G. Isaacson Department 01H!stopalhology, Universrly College andM8ddlese.xSchool01 Medmne. UniversrlyStreel, London, WCl E 6JJ TOdetermine the phenotype of Hodgkln's and Reed Sternberg cells (HRS). 20 frozen and 55 paraffin Sections of lymph node Specimensfrom 55 patients With pretreatment Hodgkln's disease(nodularsclerosls Hodgkin's disease, n = 45;mixedcellularityHodgkin'sdisease. n = 10)werestudied byimmunohistochemistryand molecularanalysis usmg Southernblot analysisand the polymerasechar reaction. 48/55cases (87%) Showed positivity of a variable percentage of HRS cells with at least one of the applied 8-cell markers (CDl9. CD20. CD22, CDw75. MB2). 8/55 cases (14 5%) Showed reactivity of a Subpopulationof HRS cells with the applied T-cell ma&ers (CD3, pF1). In the ma~arityafthesecasestheT-cellmahersstainedthecytoplasmofHRSceilsand~nanlyonecasetheT-cellantigens were expressed appropriately on the surface. All cases Which expressed T-cell antigens were also reactive with at least one B-cell marker In 1It8cases rearrangementof immunoglobulinheavy chain geneswas detected All cases were negatlvefor T-cell receptor p chain gene rearrangement. Using the polymerase chain reaction no rearrangement of the bd-2 gene (maforbreakpo1nt)wasdetected in any ofthe 19 casesstudied. Ourresultsprowde strong evidence m suppori of a 8-cell origin of HRS cells

R. B. Goude, Sabeeha Kanm, Janet Gudgeon,Jennifer Garloch

&parjmenls of Pathology and Dermatology,Glasgow Royallninmary, Glasgow, G4 OSF Every cell in a clone of T-lymphocytes has the same clone-Specificrearrangement of T-cell receptor FCR) genes Using a panel of 8 polymerasecham reactions to amplity the more common clonal TCR gamma gene rearrangementsand examiningthe reaction products by electrophoresiswe can detect standard cultured T Cell clones m nanogram quanlitas of DNA even m the presence of 20-100 parts of polyclonal DNA from reactive lymphoidtissue. The method shows that blood and skin from normalsand patientswithvitiligoand PsoriaslSCOntaln multiple dominant TCR gamma gene clonotypes (sets of rearrangementsforming a single electrophoretic band) Those ~nthe skin differ from those in the blood and presumably consist of skin-seekmgmemoryT-cells.

The relationship between wall tension, thickness of the elastic lamellae and intercellular junctions in the media of the thoracic aorta in fetal and adult rats C. L Berry, J. A. Sosa-Melgarejo.S. E Greenwald Departmen1of MorbidAnatomy, The RoyalLmdOn Hospilal, London. E l 788

Expression of the bcl-2 gene in Hodgkin's disease I.W.Thompson. P. J Coates,A. J Norton ol~gy, Hospital, WeslSm,lhfield London, EClA 7BE Departmentso l H i s t o ~ t h o l o g y a n d M ~ d ~ ~ ~ l OSt~ ~Bartholomew's

The importance of the t(14 18) translocation. causing deregulated expression of the bcl-2llg fuslon gene IS establshed m folbde centre cell lymphomas. The tdenttfeatmn of similar bci-2 rearrangements.by an apparently metculous PCR technique, in 17/53 (32%) cases of Hodgkin's Disease (HD) in one series. IS both interestlng and challenging.The significanceof small clones identified asgene reanangementsinvolvingthe immunoglobulin1 0 ~ 1ln. HD tmme. rematns Obscure one problem 1 s.t en the identification of the cells beanng the rearrangement We have thereforeattempted to immun~locali~e the bcl-2 gene product an cryostatsectlons, usmg 2 monOClona1antibodies raisedtoasyntheticpeptide,withastreptawdin biotinperoxidasetechniquB. In21 casesof HD(15NS.4 MC. 1 LD. 1 interioilicular HD). Strong cytoplasmic immunostainingwas ObSeNed In mantlezone lymphocytes butnotgermlnal centre cells (except presumedT cells) in sectmn~of tonsil. and in the cells of 4 cases of fallicular lymphoma In the casesof HD. accompanymgSmallcellswere usuallystrongly bCI-2 posItwe.but ReedSternbergcells were poSitlve inonly321 (14%)cases(2 NS. 1 MC).Th,~techniqueidentifiescellsexpressingthe bcI-2gene. butdoesnotlndicate whetherthis isdue toa t(14.18)translocation in pOSltlVeCellS.These data do not howeversuppofla major direct role for the bCI-2 gene In the prallferatlan of Reed-Stemberg Cells.

TcR V gene usage in malignant and reactive T-cell expansions A. C. Lane, E Hodges, W M. Howell. D B Jones.' J. L Smith RegionalImmunologyUnit and 'University Departmenl01Pathology. SoufhampfonGeneralHosprral, Tremona Road, Swthampton. SO9 4 W A panel of monoclonal antibodies specific for TcR variable M region families has been used to study TcR V region expressionin 33cases 01 malignantand reactiveT-cell expansions,including4 cases of mixed celluiarity Hodgkin's disease (HD) Nine cases demonstrated Significant over-expression of Vg5. Va8. Vu2 and Val 2 families; Vp5 expansionsbeing found in Gcases. Seven of thesecases(3 penpheralT-cell lymphomas (PTCL). 2 HDand2 reactive conditions] wece not associated with clonal TcR gene reanangements,Suggestfngthat the T-cells m these cases were expressing restricted V regions but using drfferent V-D-J combinations. This parallelsthe Situation seen in some autoimmuneconditions and m the response to superantigens.Although the antigenic speciftcity of the expandedT-cellfamilies~nthe7casesIsnotknown, Itwa5foundin4casesthatthesepopulationswereassaciated wlth undedyymg "on-T-cell turnours agamst which the T-cell responsesmay be directed These data suggest that p~lyclonalT-cells expressingrestrictedTcR V genefamilyphenotypes may beexpanded within lymph nodesgivlng morphoiogical appearancesconsistent with PTCL It IS also of interest that restricted TcR V gene usage 1s seen in some cases of HD in addition to PTCL.

Thedistributionof stressesandstralns inthevessel walI1snot uniform.We haveinvestigated theeffectsof thisnonuniformityonthe thicknessof eachelastic lamellaand on the distribution of Interceilularjunctions in the media of the thoracic amta of fetal and adult Sprague-Dawleyrats In fetal aoftas,the internal elastic lamel@IS thicker than the others which in many instances were not properly formed by term. In adult aortas the inner 5 elastic lamellaewere thicker than the rest. IntermediateIUnctlOnSWhlCh are involved in intercellular adheslon and m the transmission d force betweenvascular muscle cells. areconcentrated In areas close to the thickerelastic lamellae.The distribution of n e x ~ ~ j u n c t t (assoctated on~ with inteicellulai communication)follows asimilar panem. The findings are Consistent with theoretical Calc~lationswhich show that the innermost iameilae suppart the highest tension in the wall. In the adult aorta. thestructureismodified toenhancethecapacifytosustainstressIn th8intemaltwo-thirdsafthemedia. This leaves the enema1third of media with a certain ''Weakness'' when exposed to high levels of stress Which may play a role in the location of aonfc dissections occumng m hyperiension

Cytokine and cytokine receptor expression associated with advanced atherosclerosis A. bsa Ramshaw,Julia E Stickland, R J. Balgrie,' Dinah V. Parums Univerjity 01 Oxford, Nufield Department O f Pathology, 'NuKeld Deparlrnentof surge^, John RadclaffeHosprfal,Oxford, OX3 9DU Chronic PeriaOriltlSaccompaniesadvanced human atherosclerosis once the aorilc media is disrupted. This manifests nself as a Spectrum of chronic infiammation consisting of 8 cells, T cells and macrophages (but no polymorphonuclearcells).Theseareoften present as secondaryfallicleswith germinalcentres. Many ofthe B cells andThelpercellSare prollferatmg:HLA DR expressionis abundantand numerousCellSStrOng1yeXpreSS114 receptor (IL4Rec) andlor the 112 receptor (ILZRec),as detected imm~n~hisfochemically. In order to determinewhich cytokines may be controlling this very organized inflammatory reaction, a semi-quantitative assay for screening multiple samples was carriedout Reversetranscription of RNAwaSfollowed by pDlymeraSechainreactionanalysis (PCR) MessageAmplification Phenotyping(MAPPmg). Cytokine and cytokine receptor expression was characterized by screening RNA samples isolated from six cases of abdominal aortic aneurysm Whh associated Chronic penaoriltis. Hut 78 T cell and MCF7 cell lines: 'normal' and patient blood and two 'normall aortas. Oligonucleotideprimers were based on cDNA sequences for cytokines and cytokine ceeceptors. These mduded TNFo. IFNy, ILle. IU, IURac, IL4, IL4Rec.and p-actmasacontrol Detectionof p-actinconfirmed the presenceof ""degraded mRNA for each sample. All of the cytokine and cvtokine recenton studied. exceDt TNFa , were expressed to varying degrees in the inflamed aortic tissue

High frequency of light chain restricted plasma cells in primary B-cell gastric lymphoma demonstrated by double immunoenzyme labelling

Relationshipbetween human fetal lung compliance and gestationalage

S. Nar. H. Navabi. G T. Williams. B. Jasani

S. N Dobbe C E. Golimo. M J. R Dalrymple-Hay,S E Greenwald

Depmmenl olPafhology Universiiy01Wales College O f Medmne, Cardiff, CF4 4XN, Wales

Deparimentot MohrdAnalomy, The RoyalLondon Hospiral, London. E l 1BB

Primary low grade "pleomorphic" 8-cell gastric lymphoma IS characterlsed histologcaily by lymphoid follicles surrounded by large numbers of centrocyte-like (CCL) cells and plasma cells (PCs). Whilst 'monolyplc' light chain restriction IS consistently found in the neoplast$cCCL component, It 1s demonstrable 8n the PC element in only Z U 40% cases. The failure to demonstrate PC monotypia more frequently may be due to interminglingof reactive and theneoplastic Pcs. To investigatethis adouble immunoenzymemethod capableof delineatinglight chain restrictlor amongst PCs in a statistically sensitive manner was applied to 11 cases of primary low grade 8-cell gastric lymphoma diagnosed by conventional criteria. Foci of 25S500 PCs with wrying degrees of light chain restriction weredetected in8cases.ThelndiwdualcaseWLratioswithintheseareaswerecounted to beO.1.2.1.3.0.4.2.8.5, 12.6.24.1 and31 1, respect~vely,allofwhichdifferedsign~f~cantlyfromthemeanratioobseNedforPCs~nidlacent uninvolvedmucasa(mean f 1 SO = 1 07 iO.08) The high(73%)frequencyof light chainrestnctec PCsobSeNed with the more sensitive double immunoenzymemethod indicated that PC differentiation occurs in the neoplastic cells of the majmty of primary low grade B-cell gastnc lymphoma

An,"-whomethodforcalculatingcompllancein humanfetallungsofvariousstagesofdevelopment1sdeecribed.A total of 40 specimens were obtained ranging from 12-38 weeks' gestation. Seventeenof these were sUCCeSSfUlly inflated(aged 14-25weeks)with saline providing setsofrepeatable, static. deflation pressure-volume(P-V)cuNes. The drfferentialof the equations describing each set of curves was used to Calculate total lung compliance. These values were normalisedfor both lung weight and total lung volume and each set of results expressed graphlcaily against gestational age. me total compliance was seen to increase with age, but after normal#sation,was seen in both cases lo fall with age. me tissue was then fixed via the vasculature, mounted and stained using Elastic van Giesen for light microscopical assessment of elastin development with gestation. Elastinwas consistently in the parenchymafrom 32 weeks' and appeared to increase Steadily with age Finally, a small study was conducted into the effectof Artificial lung ExpandingCompound(ALEC)on lung biomechanics. Inthe ALEC inflations considerably less pressurewas requiredtoinflate the lung tothe Samevolume asone filled with air only No such change wasseen in the saline control.

SYNOPSES OF PAPERS Percutaneous biopsy of parenchymallung lesions using a biopty gun Claire M Allen, D M Hansell. Mary N Sheppard

Depanmenls of DiagnosbcRadrology and Lung Palhalogy Royal Bromplon Nahonal Hean and Lung Hosprlal,Sydney Slree:. London SW3 6NP Percutaneousfine needlebiopsy 1s an establisheddiagnostic techniquefor lung lesion^ Afirm diagnOSiSOfbenign versus malignant IS often achieved but hiStologIcal interpretation of small fragments or groups of cells IS difficult Manual cutting (twcut) needlesprovide asuperior histological specimen but are associated with a high complication rate and have been mainly used for pleural lesions This 1s the first prospective study to assess the feasibility of obtaining histological samplesfrom lunglesionswing apoweredcuning needle(Biopty Gun) We have biopsied 33 patients using Biopty Gun There were no major complications Histological diagnoseswere obtained in 30 patients (22 malignant,8 benign) The malignantlesions identified included 7 nan-small cell carcinomas.4 adenocarcmamas. 3 squamous cell carcinomas, 3 bronchiolaalveolarcell carcinomas, 2 8-cell lymphomas, 1 small cell carcinoma. 1 atypical camnoid and 1 metastatic breast Carcmoma. The benign 1es1ons included 1 SarcoidosIs. 1 clyptogenic organising pneumonia. 1 Wegener's, 4 resolving pneumonias with Chronic inflammation The rad~OlogiStlS aSSeSSmentaftheamauntoftissueobtainedconelated withgood histology.01 thethreefalsenegativespecimens obtained the radtdogist noted the inadequacyof the samples ~ntwo cases and the third was a geographic miss Percutaneousbiopsy using the Biopty Gun IS a simple and effective means of obtaining good quality histological material from lung parenchymawith a high degree of diagnostic accuracy for bath benign and malignant lesions

349A

Discriminationof high risk HPVtypes in archival material by nonisotopic in situ hybridisation (NISH) C S Herringtan,A K Graham, K Cooper, J 0 0 McGee Nufield &panmen! of Palhologyand Bacfenology,John RadclrffeHospilal Oxford OX3 9DU It was Shown prev10usIythat the discrimination of human papillamavirus(HPV)types 6 and 11 by NlSH in archival biopsy matenal requires dlfferent Conditions from those predicted by Conventional solufion kinetic analysis The parameterTm' (tissue Tm) was defined in order to describe these differences In this study. these pnnciples were extendedto the discrimination of HPV 16.31 and 33 in 15 casesol ClN The results of NlSHanalysiswerecompared with both immunohistochemistryfor viral capsid protein and PCR typing These data demonstrate that crosshybndisatm of high nsk viral types occurs ~nclinical lesions under Conventionalhybridisation and stnngency WashmgCOndit10nS Thiscross hybridisation isnot due to thepresenceofviralcapsidproteinand1sm0relikelytObe areflectionoftheendpoint uSedInN1SH.I e thepresenceofvisiblesignal.Practcally.multipleNlSHsignalsduet0 closelyrelatedprobesinarchival materialarenot indicativeofmultipleHPVinfectionunlesstheyarepresent either ~nmorphologically discrete areas of the biopsy or their presencehas been confirmed by another molecular technique. Motegenerally,thepresenceof asignal in NlSH usingaparticularprobedoes not implythattheidentity of the target nucleic acid IS that of the probe

Inorganic particulate matter in "normal" lung: a study using light microscopy (LM), scanning electron microscopy (SEM), and energy dispersiveX-ray analysis (EDXA)

Medical students in the autopsy room

I D. Craig, J E. English. 0 Gibson, K Morgan

258 thirdyearmedicalstudents, allof whom Shouldhaveseenatleastoneautospy,weresentasimplequestionnaire about autopsy room COndRiOns. 209 (81.O%) replied. recording their responseson 5-point Liked Scales Analysis Shows that 29 0% thought faces Should be covered during autopsy, and 12 4% thought that genitals Should be covered. about half the group were indifferent to both proposals 35 9 % thought the patient's identrty should be concealed from observers Three Students thought there Should be no Conversation at all. 24 0% thought that Conversation Should be limned to procedures and findings and 44 2 % thought it Should be limited to professional maners;conversely.42 1% thought thereshwldbenolimitson thetopicsdiscwaed NearlySO% thoughtstudents Should beencouragedtoassistbutnotpressuredintadoingsa.3.3%thoughtthatallshould becompelledtoassist, but 12 4% thought that students should onlv obsewe. and not be allowed to aSS1s.t. 9 6% of students thought that they should Oni!iObSeNe autopsies on patients they had clerked. whereas 13 4% thought they Should onlfbe on autopsies on patients they had not clerked. Fmally. 31 6% thought that relations Should give spmfic perm1551on before students observed autopsies. whereas 45.4% thought not

oepairmenls olPalholosV3nd Medcne vlclonaHospilal, Universily Hosprlaland Ihe University Of Wesfem Onlano. London, Onlano. L2partmenI of Palhology 375 Soulh Sireel London Onlano N6A 4G5 Canada

h e possible association between inhaled inorganic matter and Some cases of usual inter5tltial flbrOSIS(UIPI has been of longstanding interest to pathologists and cllnoians. Unfortunately.most analytical techniques are impracticable ~nthe context of a pathology sewice laboratory In an anempt to find a practicable s01Utm to this problem and to establish a baselinefor inorganic particle load in a "normal" population, a study was undertaken using techniqueswhichwauld beavailable ~nmostlargepathologylaboratories Ninecaseswereselectedfram the autopsy SeNICB WhlCh had no known expcsure to lnorganlc dust Sectlons were taken from the nght lung each case(centra1andperipheralparliansaflower. middleand upperlobesjandexamined with theSEMand EDXAand compared with the LM appearance to determine the panicle type and distrlbutm m a "normal" population. A wide range of inorganic matter was ldentlfied canespanding to SIIICB. aluminium and magnesium s4Cate5,rutile and alumina "like" particles varying from < 1p to 15p in size In addition. trace elements including zinc. cadmium and chrOmiumwerealsoobserved.ASllghtincreaSelnabSDlutenumbersofpaltlcles waspresent lntheupperlobe An increase in numberOf particles wasalso noted in areas of fibrosis which were present in two cases (old mflammatory disease)preseumablyrelatedto problems in particle clearance The findings of this pilot study suggest that although the SEM and EDXA will likely prove useful tools in the evaluationof lung biopsy soeclmens. the findinq of lnorqanic material in cases of UIP must be interpreted With Caution

E W Benbow Departmenf of Palhologrcal Sciences, Unrversrtyof Maochesler,Mancheslei M13 9PT

A quantitative study of the effects of fibroblast growth factor on wound strength and cellularity J A Slam,' J A Hunt? J. R G Nash? A. N Kingsnorth.) D F Williams3

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L?epartmenls of Surgery 7Palhologyand %slilule of Medicaland Denla1BCeng!Wnng. Univemty 01 Livemwl

Pulmonaryadenomatosis in humans: an association with peripheral adenocarcinoma F A Carey, W A H Wallace, K M Ken,' R J Fergussan? D Lamb ?partmen1 of Palhology UniversityMedical School. Jevrot Place. Edmburgh EH8 9AG &paitmen: of Palhology, Univeisily olAberdeen Aberdeen.ABSZZD, ZDepanment01 Respralory Medicme City Hospilal Edmburgh EHlO 5% Pulmonaryadenomatosishas been described as adl~tinctivepathologlcalchangeseen in the lungsolexperimental animals often I" aSSoclatlonwlth exposure to Inhaled camnogens Morphologically the lesion IS a clrcumscrlbed area Of replacementof normal alveolar lining cells by a taller more glandulartype of eplthelwm. usually wlthwt significant cytological atypia. We describe 10 cases I" whlch a smtlar change was seen as an m d e n t a l fcndlng~n resectionSpecimensforprimarypulmonary adenocaranoma. The lesions (usually multlpleand each 5 mm orless m diameter)were identified in lung parenchymaat a distance from the tumour and consisted of thickened alveolar walls lined by prominent, distinctly atypical cells morphologicallySlmllar to type I1pneumacytesand cytologically different to the associated turnour Reactivechanges 8" lung involved by obstrmtive pneumonitis were not included !n thts Sews All of the associated tumwra were peripheral adenocarcinamasand all showed a pattern of alveolar wall spread at the tumour periphery Clinically 7 of the patients were female and all were smokers or ex-smokers The slgnlflcance of this lesion in the histogenesis of primary pulmonary ademcarcinoma IS. as yet, unclear

Fibroblast growih factor (FGF) has potent angiogenic and fibrogenlc effects and IS lmpllcated In the formatlon of granulationtissueand healing Few attempts have been made toquantity theseeffectsmnvo. We havestudled arat skinwound modelusing red cellghostsasan FGFvehicle Tensiometv OfthewOUndSShowedamaxlmaleffectof the FGF after seven days when the wound strength was 50% above that m controls (p < 0 01) This effect had disappeared by fourteen days. Computerized image a n a l y s ~using a Joyce Lo& Mini Magiscan measured total nuclear content of areas I" the wounds. permitting a topographic analysis of cellulantyversus distance from the wound centre Cellularltyeffects showed adifferent time coursefrom wound strength. a 34% increaseat fouldays anda42% decreaseat sevendays. relativetocontrols(bothp < 0.05) Attwelvedaysthecellularityeffectwasstill sQnlflCant at a 31 V o decrease but by twenw-one days It had dwappeared The results suggest that FGF causes an early transient increase in cellulardy and more rapid increase in wound strength: most of these cells are macrophagesand fibroblasts Suggesting a connection between thesetwo ObSeNat10ns.

Relapse markers in psoriatic epidermis after systemic cyclosporin therapy Susan M. Andrew,' B D Edwards? J. 0Driscoll.j R J G Chalmers,'F W Ballardie? A J Freemont' 'Depanmenlof PathdogrcalScrences. Unrversity of Miinchester Oxtoid Road Manchesler M 13 9 P i 'Depanmenl of Medmne Mancheslei Royal Infirmary, 'The Skin Hospilal. Salford

Unsuspectedfindings in autopsies performed for sudden unexpected death J P O'Suliivan Departmen1 ofli~slopafhologySf Richard's Hosprlal.Chrcheslei Wesf Sussex. PO19 4SE

A series of 450consecutive personallyConducted autopsiesin patlenfsdying SuddenlyOutside hospnaland where the death was reported to H M Coroner 15 presented Cot deaths were excluded. In 384 cases the death had been reported becausetheattendingdoctarwas unwillingtoissueadeathcertlficate. In theother66cases. deathwasnot duetonaturalcausesandfallowedsuicidearan accident Oftheformetgroup, therewere t36Unsuspected malor findings cn 118 patients (30.7%). that 1s to say findings which were either the cause Of death or would have led to admission to hospital far assessment and possible treatment 1they had been discovered in life The largest group were cardiovascular (56) but there were 22 cases of unsuspected malignant disease There were only five malor unsuspected findings in the group of unnaturaldeaths 7%) and these were in Older sublects (mean age 71.2 years). These results highlight the loss of teaching material in the Coroner's system, at a time when hospnal postmortem rates are in universal decline. This matenal would be of value to medical students and to pathologists In traming. and largely comes from cases of lmle or no medico-legal significance.

The adhesion molecule$ 81 integrin (CD29). 82 integrin (CDl8). and intercellular adhesion molecule-1 (ICAM-1, CD54) are essential to the intimate co-operation of antigen presenting cells (APCs), T cells and keratinocytes Cyclosponn. Which IS an effective treatment for psonas~s,may cause immUnOSUppreSSiOnby altering antigen presentation We have pertormed a quantitative immunohistochemicalassessment of the effect of low dose ~yclosporinon the expression of p1-integnn. p2-integnn and ICAM-f m the epidermis in Chronic plaque psonas1s. Staininglevels werecompared withclinicalresponseasassessed bythePsoriasisAreaSeverihllndex(PAS1 score) pt-lntegnnand CAM expressionon keratioocyteswerenotaltered bytherapy buttherewasasignificantdecrease inthemeanlevelsof P2positivelargedendriticcells(APC5)within theepidermis.BZ-integnn wasnot expressedby keratinacytes There was a strong correlation between p2 expression and PAS1score after three months on cyclosporln and one month off therapy These results lndlcate that P2 stalnlng on large dendrltlc epidermal cells (APCs) IS a marker for the severity of clinical relapse of plaque psonas~sfollowing a course of cyclospann therapy

The number of nucleolar organiser regions (NORs) and their dispersal patterns in borderline cutaneous melanocytic lesions A. T Evans. J M Orrell. A. Grant

Diagnostic use of electron microscopy in autopsy pathology W. G McCluggage, R. Saxena. H M. H. Kamel,C. H S Cameron. P G Toner Deparfmenf of Palhology, Royal Wclona Hospilal Grosvenor Road, Belfasl BT19 6BL A total of 140 postwnortem specimens refened to the Royal Victoria Ho~pdalelectron microSCopy unit dunng the years 1984-1988 havebeen reviewed Thkscompnsed3.4Voof thetotalnumber of sewre-relatedcaaesreferred to theunit.Oulof 14Ocasesreferred.56wereexaminedandrecorded indetail Theremainderdidnotundergoelectron microscopic examination for various reasons, Such as a concIus~vediagnosis being reached by light microscopy alone. and semi-thin Sections Showing Severe tissue mtoIysIs. The most common tssues referred for examination were lung, kidney. liver, brain and head The range Of E M. studies carried out included tranSmiSSion electron microscopy ITEM).scanning electron microscopy (SEMIand X-ray micrOanalySiSon SEM. Theelectron micrographs Wererevlewedwlth respect totlssuepresewatlanand th15Wa5cOrrelatedwlththe timeintervalbetweendeathand autopsy. Electron microscopy was considered, an review. to have been diagnostically useful in 46% of cases an which It was deployed

&paitmen1 of Palhology,N8neneweIIs Hosprtaland Medical Schwl Dundee, DDl 9SY Previousstudies enumeratingSilver stained n ~ c l ~ lorganiser ar regions in problematic cutaneous melanocytic lesions have yielded inconsistent. but generally favourable,resulis. It seems probable that such InconsIstencies. arise largely from differences ~nfixation. staining and counting strategies. Our group, having devised improved methods of AgNOR staining and counting. IS now able to re-examinethe potential role 01 AgNORs in borderline lesions Pilot work demonstrated potentially significant differences in AgNOR dispersal betweenbenign and malignant lesions and I" this study bath AgNOR numbers and dispersal patiems have been evaluated A range of melanocytic lesions including banal naev~.dySplaSticnaevi. typical melanomas, Spiu naevi,atypical spih le~ions and minimal deviation melanomas were collected Virtualty all of the unusual lesions had been diagnosedby specialists in dermatopathology. Using a combined assessment of AgNOR numbers and dispersal, It proved pOSSible to discriminate borderlip lesions from banal naevl and typical melanomas. Benign borderline lesions Such as Spit2 naevi possess numerous NOR$ but display tight clustenng in Contrast to malignant melanomas whereNORdiSperSa1isaprominentfeature DiscriminatInganetypeaf borderlinelesionfromanathercauldnot be achieved however, in practice this distinction IS probably less important than assigning a aoIRary specimen to a benign or malignant group A larger prospective trial of AgNORS in melanocytic lesions 6 currently in progress

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The melanoma histopathologyclub case review: problems in the diagnosis of difficult melanocytic lesions

Expressionof c-myc oncoprotein in disseminated small cell bronchial carcinoma varies with metastatic site

Karen Bless(ng.' N Kfhham? A. Evans? J C. 8nggs.l

s. A. sun. J. R. ~ a s n e y

Departments of Pathology, ' University of A b e i d m . 'Royal Sussex Hospital, Bnghlon. 3Univewty of Dundee. and "Frmchay Hosp!IeI,Bnstol

The Melanoma Histopathology Club IS an association of Bntish histopathologists and dermatologists Who have an interestinmeianoma.Since~sestabiishment 101 cases have beensubminedandcirculatedtoeachmemberofthe 25%werehampatientsundertheageof 15, and over50% fromthose Clubfordiagnosis. Oithecasess~bmined. aged 30years or under. Five 01 the patientswere pregnant.The number of replies per case varied from 2-20. with an average of 14.5. There were problems in ensuring that the circulated section contained all the important diagnostic features that had given rise to concern. Specific recurring areas included terminology lor dysplastic naevus, melanomainchildhood, pregnancyeffects and thedifferential diagnosisof Spitz naevus, pigmented spindle cell naevus and spindle cell melanoma. A lack of consistency in terminology in both benign and malignant melanocyticlesionswasapparent.Inonly20% of thecaseswasthereexcellent agreement asto whetherthelesion was benign armalignant, and in only4cases was identical terminology used by all participants.Twelve patients have developed metastatic disease. of whom 4 had been unanimouslycalled malignant. Mare interestingly there had been universalagreement that 2 of these cases were benign and both were from patients aged only 4 years1The Melanoma Histopathology Club has been educational. It has enabled difficult cases to be Cirwlated nationwide. Childhood lesions. pregnancy effects and the terminology Of Specific entities remain as problems that even Club members have dimculty in resolving.

Depaitmenl ofPathology University of Liverpol, P 0 Box 147, L!vemoOi. L69 3BX

Activation of thec-myconcqene With overexpressionof itsancopratein product o cu rs In bronchlal malignancles ofalltypes, but has beenmost extensivelystudied insmall celicarcmoma,wherensaverexpression~nculturedllnes has beenassociatedwithdevelopmentoffast-growingvanantswhlchlosemuchofthelrendocrlnephenohlpeand aner their morphology. It has been suggested that these vanant lines might be the equlvalent of the large cell bronchial endocrine carcinomas sometimes seen I" YIYO, but this is not proven. We have used th0 Myc 1-9E10 monoclonal antibody and the avidin-biotin technique to study the panem of expression of the p62 oncoprotein product of the c-myc gene in turnour deposits of twelve subjects coming to necropsy with disseminated small cell carcinoma in an attempt to relate 11l o morphological vanablity and metastatic site. Anhough expression bore no relationshipto morphological vanation. it ohen differed markedlyhorn site to slte. Whereas parts of the primary t m w r strongly overexpressedthe protein ~nall but one wbfect, there was considerable vanability between secondarydeposits. OOSSiblVmdicatinaa relatio n ~hi~ betweenc-mycex~resslonand propensltvfor metaStaSlSto certain iocationS.

Tumour cell apoptosis is differentiallyregulated by HPVs and fas M. J.Arends, Y. Donaldson. E. Duvall. A. H. Wyllle, C. C Bird Pathology Depaiimenf. Edmburgh UniversityMed!calSchwl, Tewot Place. Edmburgh EHB 9AG

The epidermal growth factor receptor in human pancreatic cancer N. R. Lemoine.ChnstineM. Hughes. C. M. Barton, G. Kloppel. W. J. GuIhck. P. A. Hall Molmular Palhology Laboralory, ICRF Oncology GIOUP,RPMS, Department 01 Pelholcgy,A u d m K Hospital Jene, Free UmveE!ty ofBwssels, Laarbeklaan 101,B~I090 Brussels; Depaiimm of Htslopathology,RPMS, Hammersmith Hospital, Du Cane Road. London We have previously reported a number of ~ o n s ~ s t e molecular nt abnormalities that occur in pancreatic Cancer. Namely. mutations of Kinten-ras at position 12 occuning early m the disease. abnormalities of p53 and Overexpression of the c-em32 gene product. In addnlon, expression of TGF alpha 1s frequent. In this study we have investigated the expression of the receptor for TGF alpha and EGF receptor (syn. erbB). Formalinfixed. wax embedded examples of normal pancreas(n = to). Chronic pancreatitis (n = 14) and adenacarcinomaof the pancreas (n = 84) were examined using polyclonal antibodies (12E and 1 4 0 raised against synthetic peptides representingresidues 985 to 996 and 11M to 1176 of the EGF receptor. In addition. snap frozen matenal from 23 cases of pancreatic adenocarcinomawere Studied using these antibodies and the monoclonal antibody EGFRl. There was concordance of resultsfromfmren and paraffinsection studies. Ninety-fivepercent OftUmOUrS showed EGF receptor expression. Which was typically cytoplasmic with membrane accentuation. There was coordinate expression of TGF alpha and the EGF receptor. Only islets showed expression of EGFR in normal pancreas, but 10 chronic panmeatitits (even if mtld) expression of the EGF receptor was demonstrable in all duct radicles. These ObSeNatlOnal data further support a role for TGF alDha and the EGF r e c e ~ t oin r aufocrine reaulation of ~ancreatic ductal epithelium. particularly m neoplasla

Evidence that pl integrinsfunction as cell-cell adhesion moleculesin the MCF-7 breast carcinoma cell line C. 0. Bellamy. S. Fleming Depaiiment of Palhology, Edinburgh University Medical Schwl, Ten01 Place, Edmburgh. EH8 9AG

lntegrinsare transmembraneglycaproteins Which function as cell Substratumorcell-cell adhesionmolecules.They are heterodimencthe beta chain being common to Several members of a family While different alpha chains Confer ligand specificity. We have investigated the molecular basis of cell-cell adhesion in the MCF-7 cell line which IS derived from pleural metastasisof a primary breast carcinoma. M ~ n ~ c l o nantibodies al to members ofthe p1 mtegrin family, LCAM, ACAM and vin~ulinwere used in immunofluorescenceexperiments. On cells grown on glass coverslips no reactivity far LCAM. ACAM nor vfnculin was detected but p1 integrin staining was seen over the cell surl=ace.Callswerestainedwithantibodies tomtegrinalphachains 1 4 . Significant reactivitywasonlydetected with theantibody to 02 integnn.Treatmentof CohesivemonolayersOf MCF-7 cells by RGD-containingpeptides resulted in cell separation and eventually loss. 01 adhesion to the wbstratum. Control expenmentsshowed no such effect. These findings are ~ ~ n ~ i s t With e n tfunctional role for p1 integrins m cell-cell adhesion ~nthis breast carcinoma cell iine. m i s contrasts with the findings m normai breast epithelium ~nwhich etther LCAM orACAM reactivity IS found. These changes in the molecular basis of cell adhesion may be important during the development of breast carcinomas.

Expression of ras p21 oncoprotein in small cell bronchial carcinoma is associated with shortened survival S. A. Butt,'J. R. Gosney.' M. A. Gosney,'J. K. Field?M. D. W. Lye' Departments Of 'Pathology.2GenaIncMednne and %iinicaI Dental Soences. UniversilyofLiverPool, L693BX P.O. Box 147,Lve-I,

Mutation ofrasoncogenesleadstooverexpressionafa21 kDmembraneprotefn knownasp21 which playsarolein signal transduction and the control of cell pmllferation. This protein has been extensivelystudied in a number of neoplasms.including bronchial carcinoma, where it is reported to be more ohen over-expressed in non-Small cell than in small cell tUmDUrs. Using the avidin-biotin method and the anti-,as monoclonal antibody yl3-259, we have studied over-expressionof the p21 oncoprotein in tissue biopsy specimensfrom 74 primary bronchla Carcinomas. In keeping with the results of previous studies. over-expressionwas significantly more common ~nnon-small cell than in smali cell carcinoma (P < 0.002). being displayed by 76% of 33 squamous tumours. 72% of 11 adenocarCinomas,400/0aflOiargecellcarcinomasand30% of 20~mallcellles~ons.Although1tbotenorelatlonship to Outcome an the group Of patients with "on-small cell turnours. there was a significant difference in SUNlval according to whether or not it was over-expressed in the sublects with small cell carcinoma (P < 0.05). those with p21-posnive tumours having a much worse prognosis than those in which the protein was not detected.

Expression of p53 protein and cigarette smoking in bronchial carcinoma J. R. Gosney,' S. A. Bun,' M. A. Gosney.?J. K. Field? M . D. W. Lye' Departmentsof %lhology, %enatnc M&mm and %linical Denial Snences, Unrversity 01 L~vepool, P O Box 147, bvepml, L693BX

Tumour growth rate is a key parameter of neoplastic aggression. and is determined by the balance of cell gain and loss.Apoptos~sisama]ormadeoftumourcellloss, but linleisknownof itsregulation. Dieremesin turnourgrowth conferred by HPVtypeS 16and 16 werestudied in aratfibroblast modelsystem. immortaiisedcells weretransfected with HPV 16 and 18 expression vectors. either alone or With activated c-Ha-rasl. Monoclonal cell lines were established. and their vector DNA Content was confirmed by PCR Tumour Cell 111185 ddfered in their growth properties m wvo and m vilro. HPV 18 containing turnours were larger and Showedless apoptosis than those containing HPV 16, although bothshowed moreapoptasls than thenodulestormed bytheparentfibroblastsalone. In all turnours the presenceof ras greatly reduced apoplosis and increasedthe growth rate. Very similar propetlies wereobswvedin culture, and apoptoticratesshowed astranglnveneconelatlonwlthratesofnet Cellgrowth. HPVS appearedIoStim"latetumOurCei1apoptasis, butthiswas suppressedbyras. melowerapaploslsassoclatedwilh HPV 16 compared with HPV 16 may partly explain the more aggressivephenotype of cervical c a n m containing HPV 16.

Abberant expressionof the nuclear membrane protein, statin, in cycling epithelial tumour cells 8. Ansari, P. A. Hall Depaiiment 01 Histopathology,Royal PostgiaduafeMedical School. HammemmllhHospital. Du Cane Road, London. W1Z ONN

It hasbecomeapparent thatanumberof maleculesmaybeexpressed byreStingorqu~escentcellsandarelostwith transition into the cell cycle. In addition to being of biological interest, such molecules may prove useful as operatcanalmahers of quiescent cell populationSin histological material and allow the further characterisationof cellular subpopulations. One such molecule IS statin. a 57kD protein previously reported to be expressed only by cells ~nGo.We have shown bylaserconfocalfluarescence microscopythat thestatin antigen is associatedwith the nuclear envelope with a dstnbution 51m1lar to that of nuclear lamins. Using a monoclonalantibody (5-44) that recognisesslatin we have defined the tissue distribution of statin immunoreactivlty in a range of ContinUOUSly renewing, conditionaily renewing and non-renewingtissues. The distribution Of immunoreactivlty1s essentiallyas would be expected of a maher of quiescent ceils. In contrast. in established pancreatic Carcinomaand other epithelial ceil lines. we have found $latin immunoreactivity ~ncycling cells using biotinylated K 6 7 Slatin double labellingtechniques. In ConcIusion. statin lmmunoreactivitv in normal tissues Correlates with auiescence but this realtionshipIS lost, at least in vifro. 10transformed cells

A tumour promoter induces alterations in vinculin and actin distribution in human renal epithelium Maeve A. Rahilly. S. Fleming Department of Palholcgy. Univeisiry of Edmburgh. Tevmt Place. Edinburgh,EH9 84G

Loss of cell-cell and cell-substratum adhesionare imponantfactors during turnourprogression. Tumour promoten are Compounds Which although not carcinogenic themsews increase the frequency of turnour development in animals previously exposed to carcinogens. We have used the turnour promoter TPA on cultured human renal epithelia ~ells10mimic nwplastlc transformation. Following TPA treatment we haveexamined thedistribution of vlnculin, B1 integrin and actin within thetreatedcells byfluorescencemicroscopy.Treatment of therenal epithelium by TPAcausesarounding up of the cells and a loss of adhesiontoeither laminin Or fibronecttn substrata Fiuorescent microscopic examination reveals a progressive loss of reactivity for vinculin and B1 inlegrin within local Conlacts. These changes are accompanied by a redistribution of the actin microfilaments from Orientated bundles of Stress fibres to a circumferentialarrangement These Changesofareduction in focal contact componentsand disarganised actin cytoskeietan mimic the changes we have previously described in renal Carcinoma.

GlutathioneS-transferase expression in human testicular germ cell neoplasms H. S Klys.' J. Lauder.' D. Whi11#??D. J. Hamson' 'Depaiiment ofPalholcgy University ofEdmbuigh Med~caISchooI,Teviol Place. Edmburgh. EH8 9AG. 'Department of C hca l Oncology, Weslem General Hospital, C r e w Road, Edtnburgh

Glutathiones-transferasesare a diverse group Of enzymes with an important role !n the metabolism 01 faelgn compounds including Some carcinogens and Cancer chemotherapeutic agents. Increasedexpression of GST PI is seen in manyanimal and humancancersandisassociated with resiStanCetocalboplatinandcisplatin in humanlung cancet cell iines. GSTs are involved m steroid hormone transport and metabolism and have a role in the complex metabolic relationshipsbetween Settoliceils and germ cells. We have studied GST isoenzymneexpression by an immunohiStochemicalmethod in 16 Stage I teratomata. 16 stage I1tetatomata, 6 stage I seminoma, 11 cases of intratubulargerm Cell neaplasia(1TGCN)and a groupof Cryptorchidand normaltestes. The Stage I1teratomata had beentreated with cisplatin based therapyand bothprimmaryandpost-therapymetaftatictumourtissuewerestudied. GSTAlphaexpressioncorrelated with morphological evidence of epithelial differentiationin teratomata.Therewas no difference in GST expression between stage I and stage I1 pnmary testicular IumourS nor between primary testicularand post-therapy metastatic tUmOUrS. GSTexpressiondid not correlatewith survwal. GST Pi was strongly expressed in the neoplastic germ cells Of ITGCN but was weak or negative in normal germ cells. This may be Significant in view 01the potential for later contralateral turnour development tn patients treated by cisplatin based to therapy. In Summary, GSTexpression intesticular germ cell turnours reflectedlheirdifferentiationandappsared be unrelatedl o therapy and subsequent survival.

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Electron-microscopicexamination of fine needle aspirates a case in

Arecessivegeneonchromosome17encodesapratein. knownas p53. which narmai~~ctsasanonco-suppressor. point Mutation of the gene may cause synthesis of a mutant p53 protein Which binds to and inactivates11swild-type M . Varma.' C. H. S. Cameron,?L. M. Caughley equivalent.ltis detectable in many malignantneaplasms,including bronchialcarcmama, and has been wocialed with cigarette smoking. M 59 tissue biopsy specimens of primary bronchial malignanciesimmunolabelledfor p53 by Depaitmenls of 'H~slopatholcgy,*Hemonmnroscopy, and3Qiopathdbgy Royal Vclona Hosprlal,Belfast B T l Z 66.4 the asdin-biotin technique using the monoclonaiantibody PAb 1601. 13 (22%) expressedthe protein. of these 13 Northem Ireland Patients. 10 (77%) Smoked more than 20 cigarettes a day and their mean total exposure was 53 pack-years. A case illustrating the usefulness of electron-microscopic examination of fine needle aspirates 1s described Fine Corresponding figures lor those wrth p53-negalive tUmOUrS were 21 (46%) smoking more than 20 cigarenes a day needleaspiration wasperlormedon asubcutanwusnoduleinthechsstwall ofaneldedyman whowassuspmted with a mean total exposure of 36 pack-years. Alihough p53 was expressed more commonly in adenocarcinoma tobesuffering from Bronchogeniccaronomaanclinicalandradiologicalfindings.mesmearswerenotdiagnan~c. (30ROf lo)andsquamouscarc~noma(28~~of29)than~nsmallcelltumours(l0%of2o),thiscouldbeaccountedfor but ~nview of the history af asbestos exposure, the needle washings were submined for eIectrOn-microSCOpic by the smoking history. since patients with non-small cell carcinoma smoked more (a mean af 42 pack-yeas) than examination, Which showed mesathelial differentiation and a diagnosis of metastatic malignantmesotheiioma was those With small cell lesions (mean of 33 pack-years).There was no relationship between p53 expression and suggested. An autopsy perlormed eight months later confirmed the diagnosis of malignant mesotheboma. SUNlYal.

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SYNOPSES OF PAPERS Ploidy of "Pseudosarcomas"by image analysis and flow cytometry

Photodynamic therapy and transitional cell carcinoma

T. Dorman, A ti Ismail, B Cunan, M. Leader

S K Suvama,' R. 0. Plail.?M E. Snell? Marlorie Walker.' M. C Berenbaum'

Pathology Department Hoyal Collegeof Surgeonsm Ireland, Dublln

Nodularfasciitis (NR and dermatofibrosarcamaplOtNbWanS(DFSP)representSpindle Cell tUmOUrSWhlCh Can have many hiStOlogi~allymalignantfeaturessuchas hypercellularltyandhigh mitOtiCCOUntS,but clinically followa benign course. Demoid tUmOUrS and fibromafoses can be densely cellular but usually quite bland histologically and are associated wdh infiltrative marginsand repeated localrecurrences.In addtion irradiatedtissue often Containsmany cellswithcytologicalfeaturesol malignancy ThiSStUdyexaminesthepIoidyofsuchlesionsbyboth olthecunently availabletechniquesusing dlsaggregatedformalin fixed paraffinembeddedtissue Sixteencases 01 NF,6 DFSTs. 7 DTs.29fibromatoses(1ncI~ding palmar, plantar. retrcpentonealand Soh tissue) and 5 miscellaneouscasesincluding 1 leiomyoblastoma, 1 inflammatory pseudosarcomaand plexiform histlocytama. One malignantfibrous histimytoma and t paragangliamaWere included as possible poSLIVe Controls. 10 cases containing numerous irradiation fibroblasts were alm analysed All 'pseudosarcwnas' were euploid by both image analysisand flow cytometry The malignantfibrous histiocytoma was aneuploid by image analysisand flow cytometry and the paragangliomawasaneuplaid by imageanalysis and tetraploid by flow cytometv Themndwonsof thisstudv are that pseudoSarcomas are euploid and aneuploidy would appear to be confined to malignanttUmOUrS of mesenchymalongm.

lmmunohistochemicalanalysis of thirty nasal melanomas J Rode, C M. Milmy.' Toula Georgiau, H. Ayberk, Valerie J.

Lurid'

oepartments of Anatomical Pathology,St Mincent's Hospitaland The Universrfy of Melbourne.AmIraha 'oepartment of forensic Medcme Universrh,of Sheffield, %e lnshtuteof iayngology and Oology, University College and Middlesex School of Medicme London

ThereIslinleinformationonthenatural history Of nasalandparanasal melanOmaandItsrelationshiptomelanomaof other sites We collected 32 cases to study their morphology and antigenic profile after staining for the epithelial markeffiCAM5.2 flow molecularweight keratins)andlp34 (high molecular wefghtkeratins),thegeneralmelanoma and neural markersS1W.neurone specific enoIas8 (NSE). protein gene product 9.5 (PGP) and the melanoma S w i f l C marker HMB 45, the neuroepithelia markers Leu 7 and glial fibnllary acidic protein (GFAP),and the intermediate ftlamentsvimentln VIM) and nsurofilament (NR The following turnour types emerged: I pure Spindle celltype(N = 5),11,purepolyganalcelltumaun(N= 12)comp0sedofpleomorphiccells(N= 3).undwmcellswith an alveolar IN = 3) or sheet arrangement (N = 6).and 111 mixed pleomorphic spindle and polygonal cell tumouffi (N = 13). Two tumouffi proved to be lymphomas An intraepithelialmelanocytic component could be established in 7 cases Within adjacent respiratory or squamous melaplastic epithelium. In 2 cases n e w trunks a~mclatedwith tumOUr contained increasednumbers Of atypical Schwann cells. Consistently expressed were S1W (30130).VIM (29/30)and HMB 45 (29130) Fewer turnours stained for NSE (19130)and PGP (17/30). Pigmentationand staining patternsvariedgreatlyanddidnot show anassociation withcelltumourtype.Threemelanomasshowedmoderate staining for Leu7. Nasal melanomasare phenotypically (and possbly histogenetically)heterogenousSimilar to melanoma of Skin with which they COnsiStentlyappearto share certaindiagnostic antigens Theprognosticvalueof the parametersestablished 1s Cunently under investigation

'Departmentof H~stoplhoiogySt May's Hospital, London, W2,

ioepartment ofurdcgy, s t M a y ' s Hosptal, London, wz

Trans~t~analcellcarc~nomaCTCC)Ofthe bladderremalnsamajorcauseafmolbldltyandmortalityIn theU K.(3Yo all cancer deaths. 10,000 new cases diagnosed per year despite advances In surgery,chemotherapy and radiotherapy). Photodynamictherapy (PDT), whereby high Intensity laser light (at 511 and 578 nm) isdel~veredto Haematoporphynnderivative sensitised tumwr tissue. 1s Underwing evaluaton4" widesptead and m i s t a n t papillarytumOurSand,n casesof rvldespreadseveredysplasra/carnnoma,nufu". Arewewof Spallents (21,7m,ages 4 6 8 0 years. pre- and post therapy) has revealedno change ~nhistological grade and stage ~n7 patients, progressionto invasion in t case and a redunion to mild urothellal atypla alone in 1 case.Local tissue changes following treatment were limnedto oedemaand an acute lntlammatoryreactlon.No metaplastlc.Stromalfibroblast or neNe changes were seen Variations~nbladdw size. both increasedand dlmtnlshed, wwe encountered The histological grading Of TCC following POT Shows little Improvement.but studies are in progress to improve lhght delivery In the bladder, and hence improve treatment outcome.

The expression of cell adhesion molecules in follicular lymphoma A Armour. A. S. Jack Bpartment of Pathology,Umverslty of i

d s .Leeds. LSZ 9JT

Follicular lymphoma IS a disease characterised by widespread lymph node involvementusually at the time of presentation proper tie^ of lymphocyte homing and Circulation appear to be mediated by a variety of cell adhesion molecules. The a m of this study was to compare mrmal nodal lymphocytes with the neoplastic populatlm m folllcularlymphoma. hall caSeSalloftheneaplasticfollicleSexpressed lcaml but thiSYanedfromonlyafewcellsto 70% of cells within a follicle This Dosltlvlty included dendmic r e t i c ~ l ~cells. r n germinal Centrecells and neoplastic lymphocytes. Leu 6 (lymphocytehoming receptor) showed an inverse pattern of expression in all but 3 cases The phenotype (Icaml + L a 87 IS a feature of germinal centres and scanwed paracontcal blasts in reaclive nodes anhough there IS uniformity of expression of lcaml in germinal centres which IS not apparent in any folllcular lyumphoma case. This study showed a loss of lcamt and increasedLeu 8 expression by neoplastic lymphocytes within follicles This may relate to the propensity of this disease to spread widely throughout the lymphatic System

Surface glycan expression of B and T-cells and its alteration on activation P Suttan,' I V Hutchinson.' R . W Stoddart'

Epithelial membrane antigen reactivity in carcinoma, sarcomas, lymphomas and melanomas A. S.Abdulkadir. J. Patel. K. Henry. M. Leader Department of Histopathology,Beaumonl Hospital, Dublh 9. Ireland

Theobjective of thisstudy was toassesstheimmunoreactivityofEMAin arangeol sarcomas andavarietyof other epithelial and non-epithelialturnours.The immunoperoxidase(avidin-biotincomplex)technique wasemployed and a monoclonal EMA antibody was used. TWOhundred and thilty two malignanttumours were examined These included 191 sarcomas,23 Carcinomas.15 lymphomas and 3 malignantmelanomas Twenty-nine of the 191 sarcomaswereImmunoteactive.Theseincluded 13/18~~110~1.Isarcomas. ll7epthelioidsar~0ma~. 3/18malignant schwannomas, lnangiosarcomas and 1/76 malignantfibrous hmaqiomas. Twenty-one of the 23 Carcinomas ~~repo~iti~e.Thenegativecav~includedatransitiona1 cellcarc~nomaofthebladderandarenal cellcarclnoma.All IymphmaSandmalignant melanomaswwenegatwe Thereanivityof EMAinavanetyof tumourssdiscussedand the importance of using this marker as part of a panel of turnour markers IS emphasised

Colon specific antigen and colon ovarian tumour antigen - how specific? T. Dorman. P. Chin B Cuman. M. Leader Pathology &partment Royal College of Surgeons in lmlmd, Dubbn

mi?.study looksat the specificity and Sensitivity Of two recentlyavailableantibodies to colon Specificantigen(CSA) and colon ovanan tumour antigen (COTA).CSAis a heat stablemucin associatedantigenpresentm normalcolonic epithelial cells and IS expressed m greater quantitites tn colon8c adenmarcinomas COTA is a heat Stable antigen present 8n colanicneoplasiaand mumowwananturnours but not ~nnormalcalonicepithelium5psmionsfrom to1 primary adenocarcinomas(30 ovanan, 20 Colo-rectal, 9 gastric. 10 breast. 5 Oesophageal. 10 prostatlc. 13 pancreattc,3endometnal and t gallb1adder)andtwo adenmarcinomas metastatic tothe liverwere incubatedwith anti CSA and anti COTA antibodies using the P.A.P technique with positive and negative controls. While anti CSA positivitywasseenin 19d20~0loni~aden0carcin0ma~(9wsakand lO~tr~"g).~twasalsaseen 1n20af30ovanan (13 weekand 7 strong).6 of 9 gastric (3weakand 3 strong),6 of 10 breaSt(5weak and 1 strong). 5 of 5 Oesophageal (4 weak and 1 strong). 5 of 10 prostatic (all weak). 11 of 13 pancreatic (9 weak and 7 strong). 3 Of 3 endometnal (all weak) and the gallbladder ademcarcinoma. Both liver metastatic adenocarcinomaSwere negative In additim while anti COTA staining was positive m 16 of 20 colonic adenocarcinomaS (6 weak and 10 strong) and 20 of 30 ovarian adenocarcinomas.~twasalsapositive~n5af9gastnc(3weakandZstrong).3allObreast(2weakand 1 StlMg), all 50esophageal(allweak)5of 10pr0~tati~(allweak), 11 0113pa"creatic(lOw8akand t strong),all3endometnal(all weak) and the case of gallbladder endocarcinoma (strons) Both liver metastatic adenocarcinomaswere negative. m e C O ~ C ~ U Sof~ this O ~ study 1s that anti CSA and anti COTA are not adequately specific in the Identification of a ~olonlcor Ovarian origin af an adenocarcinoma and Cannot reliably be applied to the identification Of a metastatic adenocarcinoma of unknown primary Site

Early changes of adenocarcinoma of the nose and paranasal sinuses

'ImmunologyGroup. &partment 01 Cell and Stmctural Bology, 'Departments of Pathoiogcai Saences, Unwrsdy of Mmchester, Manchesfer. M13 9PT

Activation leadsto an alteredfunction and aconcomitant aneration in the chemistry ofthe cell surface,which ~ s site a nchincalbohydrate. Rat lymph nodelymphacytes.eitherunactivatedoraclivatedfot5days1namixedlymphocyte reanlon. were treated with biotinylated IeCtinSfrom a large panel. chosen to probe surfaceglycans Lmlns were revealedWith awdln-phycoerythnn and cell populations were analysed in a fluorescence activated cell-sorter Double-stalnlngwith FITC-monoclonalantlbodles defined the functional lymphocyte Subsets. UEA-1 and LTA boundtOnoce11sof anytype. UnactivatedB-cells boundall theremaininglectins.saveMPA.toagreaterextentthan did UnactlvatedT-cellsandB-cellactlvatlon produced no changeI" glycan sxpresslon.Unactlvated and actlvatedTcells all expressed a2.6-linked Sialyl residues, but o-2.3-linked sialyl expression was hetrogeneous,did not COneSpOndtoanysubsets,and wa~unchangedonanlvatian.Alargegroupoflectinsshowedlow wno binding to UnactlvatedT-cells,but boundonactivatianexactly In parallelto1L-2 receptorexpressionCTacantigen) Thes~ngle Structure GaWGalNAcal,3Galpl,4GlcNAc-) GalIGalNAcat ,3Gal~1,4GlcNAc-)R could account for their binding

Lack of human herpes virus genomes in T-cell rich B-cell lymphomas.An in-situhybridisation study G Khan. P J Coates. A J Norton CepartmentofHislopathology. St Bartholomews Hospital MedicalCollege. West Smdhfield London. EClA 7BE

T-cellnch B-cslllymphamasCTCRBL)areraretumoursIn Wh~chasmallneOplaSticB-cellpopulatlonIsdlSpersedby a polymorphic. reactiveinfiltrate rich I~T-lymphocytesThey may mimic viral bmphadenopathles, penpheralT-cell lymphomasor Hodgkin's dlsease. partlcularfyof the lymphocyte pIedominant type The dstinctton rests on Subtle morphologlcalfeaturesand 8" demonstratlngthe presenceOf monotypic B-Masts. Recentlyclonal EpsteinBamverus genomes have been detecled In three cases of TCRBL *om Hang-Kong In the light of this hndrngand the morphological similarities to Hodgkin's disease we have lnvestlgatedthe role of lymphotropic Herpes Y I N U S ~ SI" 10 cases of TCRBL taken from the file^ of St Bartholomew's Hospflal. Paraffinembedded tissue from elght lymph nodes, two spleens and one small bowel deposil were wallableforstudy. In-srlu hybridisationforcytomegalovirus (CMV).human herpes virus typed (HHV-6) and Epstein-Ban virus (EBV) was performed at a range Of protelnase K digestion times using digoxigenin labelled probes and the Vectw Elde ABC detection s y s t m By contrast with our findings in Hodgkln'sdsease no hybndlsetlonslgnalwas found with the EBV probeorwlth the probesfor CMVand HHV-6 Positivesignalswerefoundwlth appropnatecontrol material. Ourfindingssuggest that these humanherpes viruses do no playarole inthe pathogenessof Westem cases ofTCRBL. Furtherthe lackof EBVgenomesretnforces the similarities betweenTCRBL and cases of bmphocyte predominant Hodgkln's dtsease

Epstein-Barr virus localises to Reed-Sternberg cells in Hodgkin's disease in children

Tanya Levme, L Michaels

P J. Coates. A. J. d'Ardenne, G. Slavm. J E. Kmgslon.' J S. Malpas'

&partmen1 of H,stwathoIogy, University College and Middlesex School of Mndicie. Universty Street. London, WClE

6JJ

Ceparimeni 01 Hrslo~athologyand 'ICRf Department of Paedratnc Oncology,St Bartholomew's Hosprtal, West Srntthfield,London, EClA 7EE

The early Intfa-epithelialchanges of adenocarcinoma of the nose and paranasal Sinuseswere sought In the histological sectionsfrom 30 high grade adenocarcinomas and 4 cyclindric cell Carcinomasfrom this regionnone of which had had previous radiotherapy treatment. 7 of the cases came from wwd-workers. 1 I r m a polishw and 1 from a plasterer Attention was directed to the non-neoplastic epithellm of the Surface and of the sera-muclnous glands. In 14 casesofadenocarcinomaand t of cylindriccell carcinoma surfacechaogeswere detected Thesetook the form of hyperplasmof goblet cells of the rsspiratq epithelwm accompanied by dysplastic changes of other epithelial cdls: m the latter these became enlarged and irregularm Shape with large hyperchromatic nude and prominent nucleoli. lnthsmajorityof IesionsthesechangeswereisolatedIntheepithelium. Changesof thesetypes were never Seen in sefomucinous gland epithelium. In addition similar changes were not noted m the coverlng respiratoryepitheliumin 50casesofnasal polyps evenwhen severely inflamed Thesemsmchangesso described could beof use In screeningbiopsiesorcytologicaI brushlngsforearfy changes In thosewith an OCCUpatiOnal nsk of adenmarcinma

Avanevofstudles haveprov~dedev~denceforaroleof Epsteln-BarrviNs(EBV)InsomecasesofHodgkln'sdlsease In a recent report. the detection of monoclonal EBV and Hodgkln's disease IS more marked ~nchildren and the elderly However,this study did not localise virus to neoplastlc cells, a prerequlslte forassummg any dlrect aetlologlcalrole Weusedlnsrtuhybndlsatlon toldentlfVthesltesof EBV~nfeCtlon~n25caseSot Hodgkin'sdisease Inchildrenundertheageof 15. Waxsactlonswerehybndlsedwlthad,gaxlgenin-labelledEBVprobe,andh~nd~sed Sites identified by a monoclonal antibody to dlgoxm, detected by the ABC-proxidase technique. virus was Identitea ~n five cases (20%). and locallsed to Reed-Stembergcells m each case. H w t W c a l b . the wsdwe samplescomprised 4/20 nodularscleroslng Hodgkin's disease, 114 mlxed cellulamyand 011 lymphocytedeplete& Thisstudysupportstheconcept that EBVls involved lnthepathogeneslsofaproportlonofHodgkin'sdiseaseansmg inchildren OurresUHSdidnot ~0n8rmahlgherlncldenceOfEBV-po~~I~~~Hodgkin'sdlsease~nchildren, which may relatetoditterences~nthemethods used. ormaysimplybeduetothesamplesl2esanalysed Furlherstudleswllbe required to investigatethe role of EBV In chddrens Hodgkm's dlsease.

Helicobacterpy/ori, acquired mucosa associated lymphoid tissue (MALT) and primary gastric B cell lymphoma A C Wotherspoon. C Ortir-Hidalgo. M R Falion. P G Isaacson

Follicular colonization in primary B cell lymphoma of the thyroid P G Isaacson, Athina Papachnstou.T Dlss. L. Pan, D H Wright' Departmen1of H#slopalho!ogyUniversrly Col!egeand MtddlesenScliwf of M&,ane.

Univeisiiy Sfreel London. WCl E

DeparrmenrO! H~sloDalhology.Univeisily College and MiddlPsexSchool of Medicine Univewiy Sireel. London WCtE 6JJ

?&lhwnplon

Pr,marygastr,c low grade cell lymphoma ,s to a,,Se mucOSaaSSOC,ated lympholdt,s5ue Although MALT 1s not present in normal human gastric mucosa, lymphold t,ssue acqulr& ,nresponse lo Colonisationofthemucosa by Helrcob~cleipyioii. We have investigatedthe possibility that thisacquiied lymphoid t1SSue IS 01 MALT type WhlCh may povovlde the background I" which lymphoma can arl~ew e examined gast,,c b~0p5~e5fr0m450casesafH~l~~~bacrerassaciatedgastntis.and biopsyandresectionspecimensfram60casesaf gastrlc 8 cell lymphomaof MALT In 175 cases of HeBCobaCIergaStritiS prominent lymphoidfollicles were idenfilled In 8aftheee B cell clusters were identified within the gastric epithelium.reminiscent Oltheteatures seen 10the dome epithelium Of sm11811inteStinal Peyer'z patch. Thls B cell-eplthelh aSSOclatlOn was not assaclated With the eplthelm cell changes or the glandular destructm seen ~nlymphoeplthellal I ~ S I O ~01SMALT lymphomas. In 54/60 cases of gastric MALTlymphomaHelicobaclercould be identified Oltheremaining6cases.5 weiegastrecfamy specimens 8n Which specimen washing may have contributed to the negativefindings. We suggest that gaStrlc MALT 1s acqulred In responseto local immunological Stimulationas a result of mucosal coloniSatiOnby Hel~cobaclerpylon,and that the development of MALT lymphoma I S a subsequent event

The presenceof neoplastic (light chain restricted)B cell follicles In low grade B c d gaStrolnteStlnal(GI)MmPhomaof mUCOSa associated hmphold tlSSUe (MALTI has been explained On the bas15 of speclftc colonization at reactlve B It has been lowgrade Cellthyro1d foltiC1eS by Ihe neaplastlc cantrocyte-like(CCL) lymPhomaSbeIncludedIn lhecatqoryof MALT lymphomabut the frequent presenceotafoll~culapanemin these t"mours has cOntr'butedto a peKlstence Of Ihe more "Iew arefofl'cle (Fee) We have Ihe and 'nveSflgated the and genotype Of' Of primary low pT*om~nantlyfo~l~Cu~arpanem gradeBcelllymphomasofthethyro'd Wh'Ch were d'St'nguiShed alsodemonstratedfeatures ofMALT1ymphama'ncludtngCCLce115and lymph~'th~'all~'ons.m appearances e and immunoh'Sto'og~ofthefo'l'c'es werethoseofto'l'cularcalon'rat'on described 'nGIMALTMmphomarather thanFCCfoll'cular'~mphoma Thepredomlnant iWg1 ~ane'naffoll~cularcolon~rat~~nconformed tothatdeSlgnated ccLcells show'ng aStnk'ngly h'gh G1lymphomaWlthrep'acementafthefall'cle Cent'e bysl'ghtly using Ihe On DNA prol'ferattonlate No evidence Of the '(14 1' tranSIoCat'an was found In any extracted from fresh (n = 1) or paraffln embedded (n = 9) t15sue mese IlndlngSargue against a FCC lineage lor primary thyroid lymphomas and support their 1ncIus1on8" the MALTcategory

,*

Cytogenetic and molecular investigationsof nodal and extranodal lymphoma H Clark. D 8 Jones, P Jacobs.' D H Wright UnrvenilyDepanmenlof Palhoicgy Leve!E South Block Soulhamplon General Hospilal. TiemonaRoad Soulhamplon SO9 4xY 'RegionalGenerics Laboratory Gen

Univenrly Medcal SChsD!, Tremona Road SOulhamDlOn SO9 4XY

The expression of CD43 in B-cell lymphoma J Treasure,A Lane,' D B Jones. D. H Wright UniveisilyDepanmenlof Pathology Level E, Soulh Black SoulhamplonGeneral Hospital Tremona Road. Soulhampion. SOQ4 x i Regrona! !mmuno!oQyU m f , Souihamplon General Hospital. Tiemona Road Soulhamptoo SO9 4XY

We have prev~ouslypresented data describing the incidence of t(14 18). detected by both cytogenelic and gene So"thampton wasclusterad In the LeucocyteTyp'ng WoI*ShoP as The monoclonaiantlbody~ WR14, rearrangementtechniques. in nodal and extranodal B-cell lymphoma This study has now beenextendedto include a CD43 reagent We used directly biotmylated WR14 in epltope blocking studies on formalin fixed. reactive lymph 101 cases These comprise 71 cases of follicle centre cell lymphoma. 49 with follicular and 77 with diffuse node and haveshawn that antlbody actlvlty IS blocked by the prsence of unlabelledCD43 monoclonalantlbcdms CaSeS was 550,~and 2701~. ,espect,vely h , r t y case5 of f , , t(l 4 18) morphology me,nC,dence of these mesestudleslndlcatethat WR141saCD43reagentwhch blndstoanepltopeidentlcal.or closeto,thatrecognlsed B-cell lymphoma were lnvesllgated, of which 75 were character,sedh~stO~oglCallyMALT kOne by MT1. We haverevlewedfrozen s e c f ~ o n s o fcases ll of hlStologIcally and phenolyplcally deflned 8-cell lymphoma These data SupPo,, the concept cytogenetic showed 111418).detected by Southern blot the centrocyte~likecellcharacter~st~cof MALTdiffers in origin framthatgiving nSetofolliClecentrecell lymphoma The fromthef'lesofSouthampton GenefalHosp'tal These~nc1ude1ymphoc~'c1ymphoma~LL~8~~centrocytic~ymphoma ( c c . 131.cenlroblastic CentrOCVtlC hmphoma. fOlllCular(CB CC. F, 38). dlffuse centroblastlc lymphoma (CB,D. 15) exlranodal blopsles have subsequently been probed wtth a chromOSOme 19 bcl.3 DNA speclflC tD t(14 3 9) and 3 cases 01 centroblastlc lymphomadefined histologically as folllcular and dlnuse All 8 cases of LLand 11 of 13 lq32 913), a t,anslocat,on 5een CLL kOcaSeS of extranodal lymphoma demonstrated a bcl.3 rearrangement additiOntothemolecularstud~es,wehaveanalysed the~nd~v~dualcyogenet~cpanernsoftheenranodatlymphoma O f C C were PoSltIYelOr WR14. 97%0tCB cc, and 80% Of CB, for W R t 4 cases Of biopsies AlthoUghno Singleabnormalily was seen m allcases01MALT, numericalabnormalitiesof chromosomes X, Y . 6 and 18. and Strwtwal abnormalities 01 chromosomes 2 , 8 and 9 were most frequently Obserued Two lymphomasof thyroid demonstrating MALT histology showed a Small duplication of the long arm 01chromasame2 ~ ~~~t~~~ ~ e ~ 5 ~ ~ ~ at the locus of the IL-l gene mmUnOphenOtypiC discrimination of follicle centre from "on-follicle Centre cell lymphoma

~

~~

lmmunohistochemicaldemonstration of granulopoiesis and monopoiesis in bone marrow biopsies

Bone marrow mast cells revisited

B S WIIkins, D B Jones

1 D NOCk. B S Wilkins. D B Jones

Univerrrly Declanmenr of Palho!oQy Level E, South Academic B!ock Soulhamplon General H o s D ! ~Soulhamplor ~! SO9 4XY

We have used a panel of antibode$ to demonstrate Stages of granulocyte maturation by immUnOh~St0ChemiStryI" decalcified.wax-embedded bane marrow trephine biopsies Antibodies reactive with muramidase. u-1 -ant~tryps,n. neutraphil elaslase and CD68 react with early granulocyte precursors. CD15 and calgranulinidentity later granulOCyteS.IhdiYldUaIantibodiesdlfier rn the populations 01cells ldentlfled TheantlbcdteSalso react With cells of mWlOCytellneageandprovide informationconcerning theorganlsatlon afmonopolesls about whlCh liffle IS known In nOrmal marrOw granulopoiesisIS zonal wlth maturation occurring radially around trabeculae and blood vessels This pattern IS exaggerate3in reactive hyperplasia and chronic granulocytic leukaemia (CGL) There IS marked disruption of this zonal organisation in myeloproliferativeand myelodysplastic states, with considerable overlap 01 Patterns between these conditions In chronic mvelomonacvtic leukaemia(CMML)there IS comDlete absence of zonal arrangement of granulopaiesis possibly due to monocytic proliferation obscuring the underlying granulopoletlc Panern We hypotheslsethat monopoles15 normally Occurs 8" a randomly dispersed fashion Within marrow spaces and that by analogy With CGL CMML represents an exaggeration of this normal panern

~

UniveisilyPathology.Soulhamplan Genera! Hosprlal,Southamploo.SO9 4XY Studiesof aspiratedSampleSsuggestthatmastcells(MC)arenormally present in bonemawow but thedisruptionof thetiasue by aspirationdoes not permit aCCurateaSSeSSment Oftheir number or spatial distribution. Increased(MC) numben have been reported in aSSociation with a variety Of lymphopraliterativeand myeloprollferativeconditions 170 COnSeCUtiVe trephine biopsies. representing a range of pathological conditions and morphologically normal marrow, werestainedusing buffered thinnine MCweredetected in47 5% 30 blOpSleSwiththe highest MCcounts were studied further by Computer-ass1stedimage analysis to provide an estimate of MC number per mm2 of nonbony tissue Inselected biopsies. immunohistochemicaldetection of MC using an antitryptase mOnmlonalantiMy (&A11 was compared with metachromatic staining MC number vaned widely in normal marrow The highest numben were found 8n lymphaplasmacytic lymphoma: numben were not consistently raised cn myeloproliferative States In biopsies wlth focal lymphoid infinrates MC accumulated amongst penpheral hmphoid cells We studled the relationshipof MC with focal and diffuse lymphoid infiltrates m further biopsies containing benign lymphoid aggregates. FCC lymphoma, LPL, CLL and myeloma MC were not increasedin CLLor myeloma 8-cells at certain stages of differentiation may produce chemoanractant substances for MC

Germinal centre cell proliferation is T-dependent J R Goodlad'. J C Macaiiney'

The T-cell origin of regressing atypical histiocytosis

Depanmenlol H~slop~lhology SI Thomas H o s ~ I ~iambelh I, Palace Road. London SE I 7EH. 'Depanmmnlol Palhology Thei)lexamrz Hosplal Woodrow Drrve. Reddlch. 898 7UE

D B Jones.'A. D Ramsay,' Ehzabeth Hodges.' M Howell'

Clonal expansion of B lymphocytes within germinal centres produces antigen specific memory B lymphocytes T helper lymphocytesand thelr cyiokmes are thought to COntrOl the germinal centre reaction together with Other factors We have previously demonstrated a biphasic proliferative response in germinal Centres followmg ~mmUn~sat10n with a T dependent antigen To determine the role played by T helper lymphocytes a staihmoklnetlc experiment was performed using the immunosuppressantcyclasporin A to interfere with potentla10 cell - T cell interactionsduring the cour~eof an immune response Eight grOUpSOf6C3HIhe mlcewere rnjectea subcutaneously with 50 m g k g cyclospmn A on 3 Consecutive days prior to immUnisation With Sheep red blood cells and cell blrth rates Of SplenlC germinal centres were determined by Stathmokmet8c experiments on days C-7 aner ~mmun8satton As previously Shown by us in animals untreated With Cyclosparin A. the cell birth rate fell from an initial prelmmunisationvalueof 32 cellsit 000cellsihour to 18 cells11000 celldhouran day 2 followed by a rise1044 cellsit 000 cellslhour On day 4 However, in Cyclosporin A treated animals the cell birth rate (17 5 ce11s/lOOO cells/hour)was signiticantlydepressedbelowthecontrol pre-immunisationlevelandremained suppressedupfaday4fallowed by an abrupt rise These results are consisten1with the hypothesis that T lymphocytes or their pmducts not only drwe r germlnal Centre B lymphocytes the past-lmmunisatlon burst of cell proliferatlan but also the basal I ~ l n O v e Of

4m

Haemopoietic regeneration following chemotherapy for acute leukaemia. Can immunohistochemistryassist in the evaluation of regrowth and residual disease? A C G BoStanci 8 S Wilktns M Ryan D B Jones' Palho!ogyDepanmenl Ankara Un8veiscly T U I W 'UnunlveisilyParhology and 'Haemalology Soulhamplon Genera!Hospilal Souihamplon SO9 4 x i We have studied sequenlial bone mamow trephine biopsies from 20 patients taken before and after treatment lo! acute leukaemla to evaluate the clearance of leukaemic blasts and the re emergence of normal haemopoieseis A panel ofantiboaieswas employed lo assess lineage diStribUfiOnand Stageof dinerentiation othaemopoietic cells neutrophil elastase CD68 Cdt5 and calgianulinidentify granulopolettncells nand 0 slaloglycoprotemery7hioid cells and CDE1 rnegakaryocyietes In agreement wrth previous studxes of leukaernicblasts we found considerable lineageinfcdelctyof antigensexpressed m particular expression of fi-sc%glycoprotein was Seen Cm Several Cases Of oIhe(W1setypical acutemyeloid leukaemia (FA0 MI and M7subtypes) Our antibody panelwasnot a sensmvemeans of detecting low levelsolresidual leukaemia but it establishedthe leukaemicnatureolequivocal cellular n f i t a t e s ~n several cases m e time coune and panem of recoven/ of normal haemowiese. was very variable Permtent dysplasia In the lorm of spatial disturbances of individual haemowlelic lineages and cytolagical abnormalities (especially01 megakaryocytes)was frequently prolonged These features correlated poorly With haemafological parameters assessed lrom peripheral blood

'Unwers#yPalholbgyand 'Regonal ln~~nunology, Souihampton Genera! Hosprl8! Tremona Road, Soulhamofon SO9 Regressingatypical histiocytosis (RAH) 15 a rare Skin Condition with a protracted. indolent clinical course chatacterisedby nodularlesionscomposed of largeatypical cells which wereoriginally thought to be histimytic A 1981paperreportedT-cell receptor gene rearrangment ~na Slngle case of RAH, and postulated that the lesion was ~nfacfacutanw~usT~cellnwplasm ThiSconclusionwasdisputedbyothers We haveexaminedthreecasesofRAH using frozen and paraninsection ImmUnOhiStOChemistn/,and haveperformed gene rearrangement analysis ontwa of the caSeS The atypical cells showed positive staining with a vanety Of T-cell markers includingCD3, CD2. CD5. CD4. BFt, Ft 0101.CD45RO(UCHLl)andCD43(MTtl. andwerenegative with 8-celland macrophagemahen In additi~nthelargecellsexpressedCD30andonecasestainedforepithelialmembraneantigeninamannerslm,larto K i ~anaplastic l large cell lymphoma TWOcases studied by Southern blot hybridization showed T-cell y chain rearrangement. one case also showing T-cell p chain rearrangement wlth the Vy3 and Vy9 lml rearranged The immunoglobulinloci were germ line throughout We conclude that RAH 1s an indolent form of T-cell neoplasm that shares Some morphological and phenotyplcal features with anaplastic large cell lymphoma.

Marginal zone cell lymphoma: a report of three cases

'

Christina Schmid N Kirkham P G Isaacson

Departmen1of Hislopalholcgy univenily Co!legeand MtddlesexSchwl 01 Medicine Unrversiy sheel London WClE hi,

'Depanmeol Of Patho!ogy Roya! Sussex Counly Hospila! Bnghlon EN2 5BF Marginalronelymphocytesofthespleendiffertromthelymphocytesofthemantlezone bytheir specific phenotype (IgM+. IgD-, C D 7 l t . CD73-, K183-.CD5-) Wedescribethreecasesoflowgradenon-Hodgkin'sBcelllymphomas with this phenotype m female patients aged 43, 67 and 77 yr. The patients presented with splenomegaly,anaemia andweight loss.. Bonemarrawwasinvolvedma113casesandmoneaCTscanrevealeOlymphadenopafhy1nchest andabdomen.The histologyof thespleenwascharactenredby broadconc~entncstrandsofmonomo~icmedium sized lyrnphocyles around lymphoid folliclesm 2 ease$and seiectxueiy replacmgfoilxciw m t case. The h k lymph nodesand the bone marrowwereinvolved hysm$rlymphoma 10 paraff~nsecDonstheturnourcellsweieCD70+, K183-, CD21+, and showed light chain restnctmn The proliferation rate using anti FTNA was low In frozen specimens(availableIn2 cases)thecellswere IgM+ ( 1 case) I@+ (1 case. lgD- Kappa+, lambda.CD2O+,CD77+. C O Z l i . CD10-. CD73-. KB61+. UCL4D17+. UCWD3- 2 patlents remain well 5 and 8 months aflw Splenectomy without further treatment. the 43 y old patient died 1 year afiet zpleoectomy having received chemotherapy We believe that the tumour cells of these unusual lymphomas are the neoplasttc counterpart 01 marglnal zone lymphocytes

MALT lymphoma of the bladder

Fatty change of the paediatric myocardium:a minor pathologicalentity?

J Pawade. D H Wnght. P Isaacson'

Naomi Carter, S Variend

Lkpaitmen!ofH!s!opa!hology mdFawl!y oIMedtcne. Level E, Soufh AcademicBlock, SouihamptonGeneral Hospiial TremonaM a d Soulhampton SO9 4W, 'Depanmen! of H~slopa!holcgyUniveisily Collwe and Mcddlesex Schwl 01 Medinne London WC 7E WJ We describe two Cases of bladder lymphoma with CharaCtemt8C histological appearancesof MALT lymphomas In each case the clinicalhistorywasoflang duration and initial biopsies were misinterpretedas Chronic inflammation The morphologicalappearancescompnsedlymphoepithelia lesions(Inonecase). numerouslymphoid follicles and a diffuse infiltrate of monocytoid cells (centrocyte like cells) There was striking plasma cell differentiation and COlonisationof lymphad folllcks by monacytoid cell?. and neoplastic plasma cells. lmmunahistochemistry convincingly demonstrated heavy and light chain restriction ~nall the biopsies from both the cases The bladder 1s developmentallyrelated to the hind gut and this manifests 8" the variety of metaplastic epithelium seen at this Site Hence, ItiSnOtSUrpnSingthatMALTlymphamasseenin thegastrointestinal traCtCana150invOlvethebladder We propose that MALTIymphomaswith c l a ~ ~ i c~linical. al morphologicaland immunohistochemicalfeatures arise in the bladder

Depanmen!01 HiSIOpa!hdOgy Shefield Chrldren s Hospital Westem Bank Shefield S l O Z J h Fatty change of the heart IS a pwrly defined pathological entity which in the adult hean can be caused by severe hypoxia nutritionaldisorders. poisoning by Selected drugs and catecholaminerelease It IS most commonly seen ~n association with coronary artery disease Little data exists with regard to the paediatric hean In 980 pediatric deaths coming to post mortem over a 10 year period. there were 66 cases of myocardialfatty change of varying seventy and distribution detected ~n0 1 1Red0-stained Sections Infectionand congenitaldiSOrdenwereimplicated 1n39deaths Infectionand cangenitaldisordenwereimplicated 1n39deaths.5910fallcasesoffarmchangeand 2 2% Of all deaths Seven ofthe5e~aseshad acombined ~nfecti~u~andcangennalaetiology Othercausesofdeath included turnour, traumaandcomplicationsofbinh 3 6 % of casesofSudden Infant DealhSyndrome(S1DS)hada fatty heart Only one case of the total 66 cases showed deflnlte hlstaloglcal evldence of ischaemic myocardial damage. Insome instances,thedegreeaffattychangemayberelatedtathedurationand seventyoftheundertyng condition Someofthesechildren may havean occunnutritional orenlymedisarderwhich ~sexpressedatacellular level ~nthe form o! fatty change and that contibutes to their early death.

Tissue recruitment of eosinophil leukocytes is due to selective transendothelial migration and not adhesion to the endothelium

Temporal artery biopsy revisited

Clare M Modand, W R Roche

Department olPa!hdugy Level E, Soulh Academrc B!ock. Sou!hamp!on GeneralHospifal,TremonaRoad, Soulhampfon SO9 4W

Departmen! of Pa!ho!cgy Sarlhamplon Univemry GeneralHospiral Soulhamp!on. SO9 4XY

Allergic and ParasItIc diseases are characterlsed by an intense wsinophil leukocyte infiltrate Which 1s disproportionate to the penpheralblood eosinophilCount We have used cunured umbilical vein endothelialcells in leukocyte adhesion and transendothelialmigration assays in order to determinewhich mediators might induce Selectiveeosinophilaccumulationand at what stage they might act. Neutrophilsand eosinophilswerequantified by selective assays of intracellularperoxidases Exposure of endothelial cells to Endotoxin. lnterleukins 1, 3 and 5. Tumaur Necrosis Factor and Granulocyte-MacrophaseColony-Stimulating Factor induced increased adhesion Of both neutrophilsand wsinophils In transendothelialmigrationassays the peptide FMlPheand Leukotriene04 were potent but "on-selective granulocyte attractants. Platelet Activation Factor (PA5 enhanced transendothelial migration of eosinophils selectively. with no effect on neutrophils in a range of Concentrations from t O-" to 1O-'M. PAF C16 and Ct 8 were of equal potency and the effect was completely blocked by a specific PAF antagonist We Conclude that PAF may have a Central role in producing eosinophil-nch inflammatory infinrates by enhancing transendothelial migration.

Saccharide expression on normal and reactive vascular and lymphatic endothelia M C Hawe. R. E Bonshek. R W Stoddan Lkpanmenl olPafholcgcalScrences U " , " ~ ~ , ! y ~ l M ~ Maochesler, " ~ h ~ ~ !M ~ 13 ~ ,9PT Circulatingcells. encauntenngthe endothelialsurface.make contacts 10an environmentrich 8n glycan. A large panel of biotinylated lectins was used to probe far variations in the glycans expressed on endathelia of artenes. veins, arterioles,venules. capillanes,high endothelia vessels and lymphatics m a range of normal and pathological human tissues Formalin-fixed,parafflnembedded specimensfrom the files of ManchesterRoyalInfirmaryand ManChester Royal Eye Hospital were used Lectins were revealed with an avidin-peraxidasesystem No differenceswerefound betweenarterial. arteriolar, venular,veinous or lymphatic endothelia all expressedabundant complex-type N-linked glycans. of several subtypes. Capillaries were highly variable and Showed heterogeneity~ntheir expression O f 1) Outer chain sequencesfn N-linked glycansand 2) mucin-type sequences. both between different. normal organs and Within an organ. implying that the surrounding tissue probably had a regulatory effect Where endothelium was reactive, additional aneratiom wete seen and actively growmg endothelium in granulationtissue expressedhqhshowed a much lower density and narrower range of glycan mannoseSIwCtweS. High endolhelial YBSS~IS expressionthandidad~acenlnormalCapillanes,despite theirknownvery highrateofglycansynthesisandsecretion The IymphocyteILN-homingreceplor(s)would be a component of this restricted porlfolio of glycan

Monocyte marginationin atherosclerosisassociated with immunologicalinjury N. J. C o m b s , P J Gallagher. P. S Bass Departmen! 01 Pathology Sarihampfon University Hospilals Souihamplon SO9 4xY Clinical and experimental evidence indicates that immunological injury IS associated with accelerated atherOSClerOSiS.Allograft recipients may develop acceleratedatheroscleross and anlmalsgwenserum Sicknessand a high fat diet develop more extensive atherOSCleroSisthan controls fed the diet alone We have tested the hypothesis that atherosclerosis~nthese animals IS associatedwith increasedadhesion of monocytes to the aortic endothelium.Chronicswum sicknesswas induced in geneticallyhyperlipidaemicrabbits with nativeanionic bovine serum albumin (nBSA)or highly CatiOniSed protein (CBSA) Rabbits given nBSA showed a Spectrum of glomerular endocapillaryprollferativechange ThasegivencBSA developedearly membranousglomerulopathy In COntrOlSthe numberofmanocytesadherentto theendothelium ranged from 37Y5Isq mm and m animals given SeNm sickness 15-305Isq mm (nBSA)or14~5Isqmm(cBSA)As therewerenosignificant intergroupdifferencestheseiesultsdo not support the hypothesis that immunological injury increases monocyte adhesionto the aortic endothelium

Isolated amyloidosis of the cardiac valves Raid K Al-Talib

Margaret Ashton-Key. P J Gallagher

We havecampared thereparting OftempOralarterybiopsiesbetween 1973-78(135cases)and 198691 (91 cases). The overall incidence Of positive biopsies was 24.5% and 26.5% In each period the number of patients with clear clinical evidence of cranial arteritis was 4 2 % but in 1973-78, 58% and in 198691, 66% of these had positive temporal artery biopsies When the histology was reviewed approximately 6 % of biopsies in each period had been erroneou~lyreported as healed or atypical artentis. In contrast. a true histological diagnosis of artetitis was missed in~nlya~inglepatient Approximately 18% ofall patientswithaclinicaldiagnosisof giant cellarteritisdevelopedan additional symptom or pathological change associated with Steroid treatment Frequent final clinical diagnoses in patients with negativetemporal artery biopsies were transient ischaemic attacks, cerebrovascularaccidents. unexplained headache or migraine. polyarteritis or polymyalgia hwmatica These resuns confirm that one third of patients with deflnite clinical evidence of cranial arteritis will have negative biopsies Pathologists continue to misinterpret normal arterial ageing changes as evidence of healed or atypical arteritis

Expression of adhesion molecules by bronchial epithelium A J d'Ardenne.' J I Devalia, K Jones, N. D Manolitsas,A. E. McAulay,' R J Sapsford, R J Davies

Depanmenf 01 Hslopafhology and Depanmen! 01 Resp8raIoiy Medicne. Sl Banholomew s Hospifal London E C l A /BE Adhesion~fepitheliumto extracellularmatricesis mediatedpartly byafamily of heterodimeric molecules known as megrins We haveexaminedtheexpressionofthealpha-1 toalpha-6integnn Subunits in~unured human branchia epithelial cells. and in bronchial biops8es from normal subjects and atopic asthamtics. We have also studied the cells from expressionof intercellularadhesionmolecule-t (ICAM-1, CD54, rhinovirusreceptor) Br~nchialepithelia surgical Specimens were grown as explant Cultures on glass covenlips. Bronchial biopsies were taken from right upper and middle lobe carinas lmmunastainingwas performed on acetone fixed cells and frozen tissue Sections using alkaline phosphataseand immunoperoxidasetechniques All biopsies showed strong positive staining of epithelium for alphe?, alpha~3and alpha-6 integnns.Stainingforalpha-5 was weak or negativeand epithelium was negativeforalpha-1 and alpha-4 except in twoasthmaticswhere itwas weakhi alpha-4 posIttve.In contrast. cultured bronchial epithelialcellswere posItIvefor all these mtegrinsexcept alpha-1. Epitheliumwas positive for CAM-1 in 91 17 asthmatics but negative 10all Other biopsies. Cultured cells were ~tronglypositive for this molecule It 85 concluded that expression of Some adhesion mOleculeSbv bronchial emhelium may vaw ~nrelation to the cellular environmentand that ths may be imponant tn disease

Low affinity receptors for IgG and IgE on tissue eosinophils in nasal polyps: an immunohistochemicalstudy D C Rawlands.' T T Hansel.'J Cracker' L%panmen! of Pafhology Univeisrtyof Birmingham and Departmenrso! 'Immunology and 3His!opalho!ogy Eas! Birmingham Hosprlal Blood eosinophils are in a relatively inactive State With migration into tissues eosinophils became more activated These activated cells are hypodense compared to most blood eosnoph11s Low affinity receptors for both IgE (FceRII. CD231 and IgG (FcgRIII. CD16) have been documented on activated. hypodense eosinophils This study assessedthe expression of these proteins on ttssue eosmophk derived from nasal wlyps The blood and nasal polyps of seven patients undergoing nasal polypectomy were studied Nornodense and hypodense eosinophils were isolated from venous blood by centrifugation an a discontinuous Percollgradient Percollwassimilarly used to obtainaneosinophilrichpreparationfromcellsearactedoutafthenasalpolyps Cytospin preparationswetem?.de of these samples Frozen sections of each polyp were also prepared lmmunostaining using an alkaline phosphataseianti-alkallnphosphatase detection system demonstrated that neither blood nor nasal polyp ~ lfy p ~ a r e s i m i l a blood rto eosinophilsexpresseddetectableCD23orCDt6 l t i ~ p ~ ~ ~ i b l e t h a t e ~ ~ i n ~npah~ lal lSp O eosinophils and are in a relatively inactive State

Distribution of mast cells and eosinophils in the bronchial mucosa of normal and asthmatic subjects

Departmen! oIPatholcgy,SOuIhampfon Universily Hospdals Soufhamplon. SO9 4XY

P McCullagh, K M Bntten. S J W11son. P H Hawarth, W R Roche

Amyloidcan beidentified 1nupta20%of elderty hearts,especiallyinthemyocardium oftheatrialappendages In a small prapanionofth%lecasesamylaid~salsapresentinthecardiacvalve~butisolatedvalvulardepos,tsarerare. A 73 year old male presented with bilateral leg Weakness and an intradural turnour. He died 3 days after spinal surgeryanddeposltsofaglloblaslomamunlforme wereldentlfledm both thecerebrumand thecord Therewasno hlstory of cardiac disease but all four valves showed translucent verrucous thrkenlngs. These had a uniform eosnophilc hi~lologicalaPpearanCe.stainedwithcongo red and had UltraStNUCtUralfeatUreS of amyloid Amyloid P proteinwas identified immunohistochemically~nthesedeposits but negativemultswere obtained withantibodies t o m . ALand A4 proteins. m w e w a s no evidenceof cerebrovascular ormyocardial amyloidosis The involvementof all four cardiac valves. the stnking absence of amyloid I" other organs and the a ~ ~ o ~ l a twith i o n a widespread glioblastoma are unusuaI but unexplainedfeatures of this case

Palhology and Medrme l Soulhampfoo UoiversiryGeneral Hospifal, Soufhampfan. SO9 4 W

The internal mammary artery (MA) as a coronary bypass graft

Mucosal inflammation1s regardedcurrently as imponant 8n the pathogenesisof bronchial asthma Despite bronchial lavage and biopsy studies. the relationships between mucosal inflammation. bronchospasm and bronchial hyperreactivityare unclear Sincebronchial Smwth muscle has an essentialrolein the pathophydogyof asthma. Bi0p~ie~in~l~ding~moothmu5clefr0m we haveexaminedtheextentto which it ~sin~ol~ed~nallergicinflammation 10 asthmatics (age range 19371who did not use steroids and 4 Controls (age range 2243) were embedded I" Araldite and stainedformast cells ~mOnOCIOnal ant1bodyAAt)and eOSinOphilS(monoclona1antibody EG2). Mast cell numben In the lamina propria and smooth muscle were similar for both asthmatic and ControlSub e m (meanvalues. astham lamina propna 70 81mm2.smooth muscle 72 tImm2, controls 87 5/mm2 and 43 Blmm'respectively) Eosinophilnumbers in the lamina propria were increased 10-fold in the asthmatics (p = 0 008) but there was no Significant increase in the number of eosinaphils rn the bronchial smooth muscle EOSinOphil numbers in the asthmatics correlated positively With FEV, We conclude that the role of muco~alinflammation in the pathophysiologyOf asthma has yet to be determined

S 0 Wharton. N. R. B Caw. G A. Gresham H!s!opa!hdoogyDepartmen!,Addenbrmke S Hosprfal, HMS Road Cambndge. CB2 2W

The IMA 1s used as a bypass of narrowed coronary arteries It IS said to be less prone than vein gratk to develop subsequentocclusivedisease. Thisstudy of pairs of IMAsfrom subjects of vario~sageswas to see if the histological structure ofthevessel might explain immunitytograft disease Sixteen IMAs were fixed in distensionbyformalln at 150 Cm Of water pressure and secllon~taken at the level of each nb (first to StXth). All arteries undelwent Similar changes along thelr length. wlth no Slgniflcant difference between left and rlght lntimal changes were minor conssting of lbro-elastic muscular thickening in all age groups and in those who had died from vascular disease TheinternalelaStiClamellawaswelldefinedat all levels. bUttheexternallamellawasclearlydefinedonlyatthelevel ofthefmhandslxthnbs(I.e. mthedetalartwyJ. Medral thrckne~~decreasedalongthelengthOfthearter~esandInall cases changes from an elastic to a mUSCUlar structure, generally at the level of the fourth or fiflh rib The ratio of medial thickness to numbw of lmdlae in~reasesalong the artery. notably at its point of change Muscle fibre Orientationchangesfrom inter-mixed cir~ularand longitudinalin the elastic part to predominantlycircumferentialin the muscular pan. The pronounced StNCtUral differences of the IMA compared to the Similar sired epicardia coronary arteries. which are muscular. may be Of relevance in explaining their markedly different incidence of atheroma.

Extra-pulmonarypneumocystiscarinii in surgical specimens 0. S. C Rose.' M J. Wilkins. D M Clarke. R Coker? S Lucas,' R. D. Galdin Departmen1olHis!opa!holoQy 2Depanmen!of GenitouonaiyMedicine Sl Maiy 5 Hospifal and School of Medime. Pram Shee! London Depanmen! 01 Hs!sfOpalhology Univeisiry College and MIddIesex School of Medicine RockelellerBudding, Unwesriy Shee! London WClEGJJ Pneumonia due to Pneumocystiscanna IS acommon problem in the Acquired ImmunodeficiencySyndrome (AIDS) We present5 cases ofextra-pulmonary pneumocystosisdiagnosed on routinesurgical specimens(two biopsiesof liver. and one each of gastric mucosa. Small intestine and a pen-anal mass). In each case. the histological features were similar to those seen in the lung. and as in Other material from cases of AIDS, munlple pathology was often found Extra-pulmonalypneumocystasis 1s now being reported from a widerangeof clinical Specialties.One reason forthismcreasemaybe that impraved patient SUIYIY~Iwith"topicaY inhalatiooalpentamidinetherapyallowsvisceral foci of infection to become clinically apparent This hypothesisIS supported by the finding that 4 ofour5cases were taking nebulised pentamidme

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SYNOPSES OF PAPERS

B:T lymphocyte ratios in interstitial lung disease W. W. M. Sawers. W.A. H. Wal1ace.S.A. M. Hawie, 0.Lamb

lmmunoperoxidase markers in routine lung cancer preparations:a study of 44 thoracotomy specimens

Depmmmi of Pathology, Edmburgh Unrvemiy Medical School, Tevior Place, Mnburgh, EHB 9AG

I. D. Craig. R. Inculet, T Mara, 0.Grignon

Sarcad and extrinsic allergicalveolms (EM) are considered to be examplesof hypersensitivityreactionswithin the lung lo known or unknown antigens Broncho-alveolarlavage [BAL) studies on Such patients have shown an increasednumberof T lymphocytes comparedwith other inflammatoryceiis. This has supported theview that these disease processes are predominantly mediated through the cellular arm of the immune system. Using immunocytachemicaltechniques we have examined archival open lung biopsy material lo ascertain the number of B(L26+ve)andT(C03+ve)cells1nsevencaseseach ofsarcoidand EAA WecomparedtheratiootB:Tlymphacytes Inthesehypersensltivitystatestothatobselved mbronchiectasis (a Chronic Suppurative lung condition not thought to In~olvea hypersensitivityaetiology)and in smokers and non-smokerswith no evidence of active pulmonary inflammation Ourresuns havesh0wnthattheB:TlymphocyteratioisnodifferentinEAAandsarcoidfrOmthat seen ~nbronchiectasis and in normal lungs. We believe that thns IS further evidence to suggest that BAL IS an unrepiesentativetechnlquetorlhestudyofinterstitial lung diseasesand that morecansiderationshould begivento the possibility of humoralimmune components in the pathogenesisof EA4 and sarcold

DeparimentsofPaihologyandsu@ery Vlciom Hospmland ihe Universiiyof Western Ontano,London, Oniano, Depanmentof Pathology.375 South Street, London, OntanoN6A 4G5

Pulmonary peptides, noradrenaline and endocrine cells in monocrotaline pulmonary hypertension

A study was undenakento evaluatethe use of immunaperoxidasestains on paraffinembedded tissuelo define the cell type In routine lung cancer preparations. and in particular to identdy a subgroup of turnours showing neuroendocrinedifferentiation. Forty lour consecutivethoracotamycaseswereselected. Followingapilot study of 6 cases. to assess digestion times and potentiaily useful antibodies, the remaining cases were processed using a battery of monOClOnal antibodies: cytokeratr IAElfAE3, 348E12). neuron specitic enolase (NSE), chromogranin. and beta 2 microglobulm. In addition to the 3 carcinoid turnours and 1 oat cell Carcinoma in the study 3 large cell Carcinomasand 5 adenmarcinomas demonstratedpositive neuroendocrinemarkers.Uiirastnrcturally, dense core granules could be demonstrated in Only 2/3 of the large cell Carcinomasand in 1/5 adenocarcmomas.The discrepancy betweenthe lmmunOperOxldaSe staining and electron microscopic features likely reflects the heterogeneityafthese turnours. In thisstudy noneof the turnours co-expressed neuroendocrinemarkersand beta 2 microglabulm. However,the staining panern was inconsistent m the remaining cases High molecular weight cytokeratin (34PE12)was Stronglyposltlvem all casesot squamouscell carcinomaand negativein everyihing else. In summary. monoclonal NSE and chromogranin appeared to provide SuffiCientinformation to identity neuroendocrinedifterentiatianin thecasesexamined m this Study. High molecularweight cytokeratinwasfound to bea usefuldiscriminaforforsquamouscelicarcinoma. Beta2 microglobulin was negativemall the turnoursshowing neuroendocrinedifferentiation.butthe inCOnSistenCyOf staining m non neuroendocrinetumaurs. made it less helpful for routine laboratory use

D ONeill.' R. Fenan. C. Ceconi. A. Rodella. P. Smith,' P. H a r m D. Heath' Oepariment of Caidlology, Universityof Brescra, Italy, Departmentof Paihology,Un8vemPyof Ltverpml

Hypertensivepulmonary Vascular disease was induced m Sprague-Dawleyrals by mlecllons of monocrotalineto determineIts effects upon the number of pulmonary neuroendocrinecells and their peptides. Inone experiment.the Concentrationof noradrenalinein the lung was estimated by chromatography, and that of the peptides bombesm. neurotensinand caknonin gene-related peptide (CGRP) by radiaimmunoassay.There was significantly Ies5 noradrenalineand bornbesin in the lungs of test rats than in controls but the levels of neurotensinand CGRP were unchanged. In a second experiment, pulmonary neuroendocrlnecells In histological sections were labelled with antiserato bombesin,calc,t~tonin,CGRPandproteingenepmduct9 5[PGP 9 S ) a ~ d = ~ " " t ~ d . T h ~ , e nochange w=~ in the number of labelled neuroendocrinecells expressed per unit area of lung or per unit length of airway between test and control rats for calcitonln. CGRP or PGP 9.5. Bornbesin-containingcells could not be identified in either group. An increase~npulmonary neuroendocrinecells could not be identified ~neither group. An increase in pulmonary neuroendacrinecells immunoreactivefor bomtesin and calcitonin OCCU~Sin the early stages of plexOgenicpulmonaryartertopathyin man. Theabsence ofsucha change in mOnOCrOtallneDulmOnaNhYDertenSlOn in the rat suggests that this IS a poor model for the human disease.

Heterogeneity of endocrine differentiation in non-small cell carcinoma of the bronchus J R Gosney.' S. A Bun,' M A Gosney? M.D W Lye'

Departments01 'Path~logyand%enatncMedjnne, University O l L i v e ~ w IP, 0 Box 147, Liverpool, L693BX Although endocrine differentiation IS the essence of Small cell Carcinoma of the bronchus, its occurrence m other morphological ("on-small cell) types of bronchial 1Umour (large cell, squamous and adenmarcdnoma) IS welldescribed However, its prevalencem Such tumours IS uncertam, estimates differing from study to Rudy and accordingto how It IS sought. We have examined, byimmunalabellmg,expression offiveendocrine markerproteins (neuron-specificenolase (NSO, protein gene product [PGP) 9.5, the BB isoenzymeof Creatinekinase [CK-BB), SynaptophysinandS-t 00protein)m60 bronchoscopictissue biopsiesof "on-smallcellcarcinomaandasse~ns varlabiltty Within and between tumour deposits In 16 subjects coming to necropsy with disseminateddisease Exactly half of the tissue biopsy specimens immunolabelledfor one or more marken; one for four, four for three, twenty lor two and five for one. possibly indicating an element of endocrine dtflerentiation inapparent from their morphology. Expressionwas even more prevalent amongst the extensively-sampledturnours at necropsy, but Showed wide variabilityboth Withinthe primarygrowth and between its VamuSdepOsitS.Prevalenceofexpression of theseendocrinemarkersm "on-smallcellcarcinomaoflhebronchusisheavilydependenton theamount Oftissue examinedand the site from which it is taken

Neuroendocrine and mast cells in asbestos-related disease: a quantitative study v

Mudenda, H. M. H. Kamel, P L. Carland. P. Maxwell, H Bharucha

Depaament OIPathology,Royal Wciona Hosptal, Grosuenor Road, Betfast, BTt2 6RL This preliminaryStudy was carried Out to assess the quantitative expression of neuroendocrineand mast cells in adun humanlungsofcasesofasbestos-relateddisease.Tencaseseachofasbestasis,pleuralplaques,carcinoma and mesotheliomawere studied m comparison with ten normalsWnh no history 01 exposure to asbestos The lung sections were stained lor neuroendocrinemarkers neurone Specific enolase (NSO and chromagranln. and, chloroacstate esterase and toluidine blue for mast cells. There was a notable variation in the number of neuroendocrineand mast cells between the control and asbestos-relateddisease group The Variation was also seen between the various asbestos-relateddiseases. Though not statistically sbgnificant.the trend of the Vanatton indicated that the individual diseasesfollow a particular pattern an lhe expresstonOf these two cell POPUIPIOIIS.

Neuroendocrinetumours of lung: combined bronchial carcinoid tumour and small cell (oat cell) carcinoma of lung, a case report C 0 O m N B Goplnathan,R. L. Smith. F. M. B q l TaranakaEase Hosptlai,PnvateBag New Plymoulh, New Zealand

We present the case Of a 41 year old patient who underwent lefl lower lobe lobectomy following a long hlstory of intermlnent leffsidedpleurlticchest painand morerecent historyof acollapsedsegmentofthele~lowerlobeoflung secondary to obstruction from a bronchial turnour A bronchoscopcc blopsy reported as small cell undifferentiated carcinomaof lung wasnot accepted bytheoncologistsbecauseoffhelongcllnicalhlStOry.ThslDbeCtOmySpeclmen revealed a turnour ObStrUcting the len lower lobe bronchus. Hislological examinationof the tumaur revealed combined bronchial carcinoid turnour and small cell (oat cell) carcinoma oflhe lung. Funher immunohistochemical and electron microscopic studies of this combined t ~ m ware i presented. The unusualnature oi this case is emphaslsed Utlllslng a review Of the IlteratUrB ThlS case further highlights the common orlglns Of the bronchial carcinoid turnour and the small cell Carcinoma of lung from the neuroendocrlne(Kullschltsky)cell of the lung

Fibrous tumours of the pleura. An immunohistochemicaland electron microscopic study J. Pawade. C. E. H. DUBouiay.S. Hsrns rA?parimeni of Htstopathdogy,Level E, Souih Academe Block, SouthamptonGeneral Hospiiat, Tiemma Road, southampton, SO9 4 W

We have studied fibrous turnours of the pleura using morphology. immUnOhiStwhemiSlryand eleclronmaroscopy. The findings were compared and contrasted with reactive pleural fibrosis and desmoplastlc mesothelioma.The fibroustumwn hadarangeof hiStologicalappearancesand30%weremalignant ~nnature.Theimmunophemtype was uniform and consistent with positive staining for Vimentin and alpha m w t h muscle actin. This was ~nSharp contrast to findings ~nreactive pleuralfibrosis and desmoplastic mesothelioma.UttraStruCtUral appearancesof the fibrous tumours of the pleura Were Supportiveof a myofibroblastic ongin. We propose that fibrous tumours of the pleura arise from the Submesothelial myofibroblast. The malignantfibrous turnours have a distinct immunohistochemicalprofile and electron microscopic featuresto differentiateL fromthemalignant mesenchymai mesothelioma.

Skin biopsies in general practice - a histopathological appraisal P Oylarah, Lorna McWilliam. Fiona Knax, Naftsa Wilkinson Oepaamentor Palhologicalscien, Univers?, of Manchesierandueparimenf of Histopathology,UnrversityHospital of South Manchestw, Wdhmgton,NellLane, WestDtdsbuw, Manchester,M20 8LR

Since theinlraductionofthenewGeneralPractnianercontractmApril1990,lhere hasbeenasignlicant increasein thenurnbeisof skin biopsiesreceivedmHistopathologydepa~ments In ourdepartment there has beenalhreefold increase in numbers of General Practiioner skin biopsies. The aims of this study were to crnlcally appraise these biopsiesand comparethem tosimilarlysized Skin biapsiesreceivedfrom hospital in-patientsviageneralandplastic surgeons for the SIX months prior to and aner 1st April 1990. Data collected included numbers received, ranae of pathological diagnoses,quality 07 information supplied on the request card, accuracy 01 clinical diagnoses. adequacy of excision, age. sex and sites of lesions The resuns showed a Similar range of pathological diagnoses. The quality of clinical information supplied was comparable in the two groups as was the age and sex of patients. General Practitioner biopsies were less common from the face. Clinical recognition of lesions was somewhat less accurate amongst GeneralPractitionersthan amongst Hospital Surgeons Inadequateexcisionwas more common m General Practnionercases. 5.5% of General Practitioner lesions were found unexpectedly to be premalignant or malignant(eight cases)and all ofthesewere inadequatelyexclsed. Important implicationsemergingfromthisstudy are dlSCUSSed

The histology and immunohistochemistry of small cell mesothelioma F. G. Mayall. A R. Gibbs @parimem ofH!siopalhology, Llandough Hosprtal, Penarih. South Glamorgan, CF6 t m , Depariment Of Pathology, UnlversPy of Wales l%llege of Medmne, Heaih Park. Cardtff, South Glamorgan.

CF4 4XN The objecl of lhis study wasto descnbe the histology and imrnunohistochemistryOf 13 small cell mesothellomas c~ncentratingonreliabledlstinctionsbetweenthemandsmallcellcarc~noma. Wefound that all 13tumoursshowed regiansofmoretypical mesotheliomaif muniple blackswereexamined NOturnoursshowed the Streams,ribbons, rosenes, or haemotaxyphilic blood vessels that are typical of Small cell carcinoma. Miotic figures were relatively scarce and the nuclei had a particularly characteristic open appearanceWtth prominent nucleoli and delicate chromatin. Nuclear moulding was not seen. No turnours produced neutral mucin. lmmunohistochemicalpositivity for neuronspecificenolase(NSOoccurredin 11/13. cytokeratin (CK) in 9f13and Leu-7in4ft3 but nonewas positive fw chromogranin A, carcinoembryonic antigen (CEA)01 leukocyte common antigen (LCA). We concluded that accuratediagnosisof Smallcellmesatheliomaispossibleprovidingtheclinicalpresentation is known. theturnouris adequatelysampled and the microscopycarefully assessed. In Small biopsyspecimens, where thediagnosisis less straightforward. immunohistochemistryfor CEA. and perhaps LCA and chromograninA may be helpful NSE and Leu-7 positwsty IS common m these tumours and might be misleading11 It were intermeted as ieliabie evidence 01 neuroendacrinedifferentiation.

An audit of skin biopsy specimens from general practitioners in Grampian Region: changes in requesting practice and specimen type P. A. J Brown. N. M Kernohan, Louise M. Smart. Susan Robinson, Pratima Savargaonkar,P. Atkinson. K M. Ken Qeparimenl of Pathofogy, University of Aberdeen. Foresterhilt, Abeideen AB9 2ZD In April1990 new contracts lor General Practitionerswere introduced. In particular there is a financial incentive to General Practitionersto pellorm agreaternumberof minorsurgical procedures.AtterPpriI1 Wtherewas a general awarenessamong pathologists, in the Department of Pathology in Aberdeen, that there had been a nse m the numberof Skin specimenssubmined by General Practitioners.Aretrospectivestudyof all skin specimensreceived in four 6 month periods (Apnl lo September)in 1987,1988,1989 and 1990 was made. We found the number of requests hadmorethandoubledin 1990whencomparedwith 1989. whileamuchsmallerrise hadoccurredoverthe pieceedmg years. Most d t h e iesionsexc!sedwere naew and seborrhoiec keraloses although severalmalignancies were removed and we have lwked for changes in the range of lesions being excised and specimenquality.

3554 Model paint as a marker for resection margins in dermatopathology Maureen Y Walsh. J. G. McDermon, C J Carland, Clam M Thornton. E. Ann Blngham' Depanmenr OfPaIhoIogy and 'Dermatologv. Royal Wctona Hospital, Bellas1

Assessmen1ofresactionmarginsofsurgicalspecimens~s becming more important m manyfieldsofpathology. We wwe interested in developing a means of assessingsurgical margins 10dermatopatholqy in both conventionally removed skin ellipses and m Skin ellipses removed during M O ~chemasurgery S technique Many of the conventionally used markers, such as Indian Ink, Alcian Blue and Tipp-ex Correction fluid are difficult to use I" that they are messytoapply. slow todry and show insufficientContrast wiih oneanother both lnthegrass specimenand microscopically Weused"Superman'paint,apaintusedforresinandplastermodels,Wh,chcomeSin awlderange of colours. The paint was easyto apply. did not run and dried qwckiy Becauseofthe variety of colours avaciablewe were able to apply Contrasting c ~ l o u n to the vertical and horizontal axes of B X C ~ S I Oaf~ skm elhpses removed by MOhSchemosurgery.Thepaint prOYidedagaodmarkergrasslyand wasnot affected byfreezinqthetissue Model paint provides another markerlor surgical BXCIJ~II marginsand 1s particularlyusefulfor Moh'schemasurgerywhere horizontal and vertical axes aremarked inorderto assess theadequacyoftheexcision.The model paint may also be usetul in other branches of surgical pathology where resection margins are Important.

Micro-skin grafting using Biobrane@membrane in the pig model P S Reynolds.'J. D Frame? N 2. Wang,'A. Caumbe.2S Cackcrd,' P S Wkhington'

Use of the lower third of human oesophagus as a substrate for the detection of IgA antiendomysialantibodies in patients with dermatitis herpetiformis Patricia Ashe. Ciaire M. Thornton, J C. Sandford, Maureen Y. Walsh Department of Pathology. Royal Victooa Hosp~tai,Belfast IJA antiendomyslalantibody has been reported to occur In a high percentage of untreated patients with dermatitis herpetlformisand ~ o e l i disease. a~ Most reports I" the literature use the lower thlrd of monkey Oesophagus as the

Substrateofchoice for detectionof theantibody Thlssubstrate IS difficult to obtain and we therefore Investigatedthe use of tissues barn the gastrointestrnaitract of rats and rabbits as Substrate. These proved unsuitabla. dmdarly Substrate obtained from the gaslrotntestinai tract at autopsy was not suiiabie. Xsue from a vanery of sRes was obtained from freshly removed Surgtcal Specimensincluding Small and large Intestine,duodenum, stomach and lower third Ofoesophagus Whilesame weak. lnconsstent stammgwasobtained from all othw*BS. thelowerthlrd oftheoe5ophagusgavesuperiorresultsand wasareliablesubstratefor detectingIgAantlendomys$lantibody.We l w k e d at the incidence of I@antmdomysial antlbodles In the Sera of patients wiih clmical, hstological and direct lnmunofiuorerenceproven Omatitis heipetiformaSandcomparedthem with a contml groupwho had a variety of dermatological diseases including pemphlgatd, pemphigus and vasculitis. None of the 30 control patlents had circulating IgAantiendomysialanllbodoes whilst 35 0155 patlents(64%)with dwmams herpetiformlshad ctrculatlng I@ antiendomysial antibodies.

A study of AgNORs and anti-PCNA staining in thick cutaneous melanomas displaying paradoxical behaviour

Department 'SaHenry Sounai Research bboratory and zOepartmentOfMOrbidAnalOmy ihe Royal London Hosprial, 'Department of PiaSt8c Surgeiy Sf Andew's Hospital Billemay EXtenSiYefUII-thBkneSSbumspresenta challengingproblemforthe surgeon 11lnsuffkcient wabieskln lsavallablefar autograning Theexpansion meshautg(ran and mixed avtogran and homqrans allow maximal ml l s a t m of limited donorsites. in addhian. mamltempdiaryskinsubstitutes havebeen proposed including cadaveiaiiografts. pigskin xenogratls and artltlCla1 skln. Blobranee IS a blosynthetlc btlamlnate matenal composed of an outer, ultra-thin layer olsiliconeoverlyingafinenylonmesh. lntegratedpotcinecoliagen peptldesare bonded Withinthismeshtoform the underSUrfaceofthsmembraneSince 1989.Blobraneehas been used at St. Andrew's hospttalas atemporary Skm cover in patients with exlensive burns undergoing immediatefascia1exc~s~ons. A novel mlcro-grafl technbque, involvingtheuseoflinely mincedautograhson the undersulfaceolcadaveric allografts. hasrecentlybeen reported In t hs study we have combined micro-autografts wsth Blobraneeskm 5ubstttute to Obtainraprdhealtngm fullthickness wounds. The results have besn remarkableand we present the grass macroscopic, lkght and electron microscopic appearances~nthe pig model up to 41 days post-graning.

Aquantitative assessment of integrinexpression in normaland psoriatic epidermis N P. Adams,' Susan M Andrew,' B D Edwards,?J ODriscoll? R. J. G Chalmers.' A. J Freemonl'

F W Ballardie,'

'Departmen1o l P a t h o l ~ i c a i S c ~ e nUniversity c, of Maochester, Oxford Road ManChester ,Mi3 9 P i zDepanment Of Medinne, ManChester Royal lnfinnaly m e Skn Hosprlal, Salfom

In psoriasis there IS altered Bpidermaldmwentiattan and increased epidermal turnover both 01 Which involve changesin intercellularadhesion Aquantnative imm~nOh~st0chernicaicompa~~sonof the expressionof the integrin family Of adhesion moIeccuIes rn normal (N = 7) and psanatic M = 121Skins has been made. Mon~cloOalantibOdy Staining of integrin p SUbunltS 1-4 and e subunlts 1 4 were disclosed usmg an andin-blatln peraxidasetechn!que. Keratinocytesexpressed subuniIsfi-1,0-4. a-2.n-3.ando-6m characteristicpanernsInnormalandpsOriaticskins. Large epidermaldendritic cells (antigenpresentingcells)expressedp-2 The psoriatic Skm.showed Increased6-1 a-3.0-6.and p-2expression. buta-2and~-4showednosignificantdifferencefromnormal Thelncreasedlntegrin expression by keratinocytes seems to be a reflection of awered epidermal differentiation rather than increased keratinoc ?turnover Themcreasein ~-2pasitivedendriticceilscauidbeareflectianofalteredanfigen handkngin psonatic

Gin.

Malignant melanoma - a five year histologicalreview Claire M. Thornton. MaureenY. Waish Oepertment of Parhdkgy, Royal V m n a Hosprial Ee~last, m i 2 681 Weexaminedalfprimarycutaneaus malignant melanomasseen rn thmdepartmmtOver a fweyear permd from 1986 to 1990.The total number of tumoum was 354 The number of cases of malignant melanoma both invasive and m M u , increasedfrom 57 cases ~n1966 to 92 cases In 1990. Superfmal spreading melanomawas the most common type of melanoma, accounting for 60% of the total cases This was followed by the nodular melanoma Whch

accountedfor 18%. wlthlentigamallgnamelanomaandacral lent~g~nousmelanomabe~ngtheleast commontypes. Therewasanincreasmg numberoftumourspresentlngwlth aBreslow'sdepthoflessthan t 5mm. thefigurermng from 46% of cases in 1986 lo 64% of cases m 1990 Most cases were stdl of Ciar*e kud ivanhough mcreasing numbersaftumour presented at Clarkelevels I and I1wlth a corresponding reduction ~ncases presenting at Clarke level V More lesions presented with a flat cross SeCtlonal profile ~nthe later years the figures lncreaslngfrom 30% in 1986 1064% in i w o he numbor of I C S ~ showlng S surface ulceratan at predentatlond-reased from 57% In t966to14% In 1990 Themltotlcact~vlty. thedegreeofp~gmentalion.theintensiiyofthe~nnammatorycell infiltrate. the predominant cell type and the mcldence of vascular lnvaslonShowed no change over the study penod

lmmunopathologyof interleukin-2-induceddermatitis Karen Blessing.' Marion I. White? K. G. M Park? S D. Heys? 0 Eremin' Departments 01 %thology

2L%matdogyand 'Surgery Univemty of Aberdeen.Aberdeen, Scotland

Interleukm-2(IL-2). used m the treatment of patients with metastabcdisease fallmg to respond to Conventional treatment, can induce regressionoftumwrbuik in certain patients However,the systemlc administrationofiL-2 is associated wlth a number of toxic effects, including dermatological compl#catlons.These have been poorly documented We have prospectivehl studied the dermatologicalreactions 105 patlents treated wiih IL-2 for metastats Cdorectal ~arconoma.Pre- and past-treatment biopsies were obtwied where possible, arid sect~ons stained with H 8E. Giemsaand PAS:hesh tissues weresubjected to Immunophenotyplng.Therewere3 femaleand 2malepatients.with treatmentrour~sranginginnumbwfrom1-5 Onlyonepatlenthadapastmedlcalhlstoryof any skm complaint (eczema]. Four patlenk suffereda dlffilse eryihematous reaction with mild desquamatm and dryness The other patient developed genwalised erythrodermaand an additlonal photosensitivity type reaction. Histology, anerthe Initialcourse. revealedpatchyspmgioS1S. exocylosis and basal layer epidermaldamagewnh a mild perivascularchronic inflammatorycell mfinrate. with SUhseqUenttreatment there wasthlckenmg ofthe epiderrnls, plgrnent incontinence. dermal oedema and more marked chronic perivascular cell tnfiltrate. immunohistcchemistry revealedmarkedchanges inthe expressionof CDt , HLADr,CAM-1 and CD25 m the derm15. These Changeswere greatly heightsnedwith subsequentliealmsnts. with addnional changestn other T cetl markers. Clearly IL-2 enhancesthe parenchymal expression of antibody-dependent and antigen-mdependentaccessory moleculeswhich are important in focusing the immune response.

Recurrent solitary trichoepithelioma:a report of two cases Naomi Carter' Deirdre M. McKenna' 'Department of H!stopathology Royal HalfamshireH ~ ~ p i t aGlossop l, Road, Shemeld, SlOzJF, 'Oepartmen! OlPathoIogy, Shefield lhivernty MKhcal Schml Fpech HillRoad, Shemeld. S 102RX

Trichoepltheliomaisa hamartomaof the hair germ which can berolitary, or munlpleas part of an knhentedSyndrome Wereport IwOcaSeSofrecurrentSOlitarytrichoepithelioma onewas remavedfrom theanteriot abdominal wall of a 60 year old woman the tumour recurring 8 years after exctslon.the other was removedfrom the left temple 01a 65 y8arold woman. recurring3 yearsanerexcslon. Both turnours ~8reinc~mpletelyexclsed at inltial presentatm The tUmOUrS Consistedof islands of basaloid cells. Some containing centrat areas of kerat!ntsatlon.Surroundedby a loose connective tissue stroma. One lesion contained a central keratinouscyst. Both turnours were posltlve for keratin markers MNF116and PKKt , but negative for HMFGl (so excluding sweat gland differentiation).These turnoursWere Immunohistochemicallyindistinguishablefromtworandomlyssiected basalcell carcinomasshowing squamous differentiation. dstmction betweenthese two lumours 1s often based solely on clm,cal features Recunenceofa solitarytrichoepithelioma~srare,~~~~a~eSShowthatIhelongintervalbefween original incOmOlete excision and recurrence are factors to be borne ~nmind during surgery and subsequent follow-up

356"

SYNOPSES OF PAPERS

Sinusoidal haemangioma: a distinctive pseudosarcomatousvascular tumour

Preservationof tartrate sensitive acid phosphatase(AcP) in paraffin wax blocks

6. D. M. Fletcher,' E. Calonje'

Wlnifred Staley, J. McClure

'Dewrtment of Hrstopathdogy,St. m a s ' s Hospitaland %stitUte of Dermatolcgy (U M.D.S.), LMdon

Dvism of CeliuiarPathdogy, Department of Pathdogcal Sciences, Univwsny of Mmchester, Manchestw, MI3W

Classificationof benignvascular tumours is notoriousiydifliwlt and ciinicopathologicalcorrelationis often imprecise. This almost certainlyreflects the tendency of pathologiststo lump together different lesions under the broadheading 'haemangioma', sometimes with capillarylcavernoussubtyping. Twelvecases of a distinctivesubset of cavernoushaemangiomas,to be knownas sinusoidaihaemangioma,are presented. These presentedinadWs (8 female. 4 male. mean age 49 years. range 2&77). Five arose in the upper limb and five an the trunk (of which two developed in mammarysubcutis). All were solitary and presentedas a bluish cutanmusswellingup to 3.5 cm in diameter of variable duration.One case was associatedwith ipsilateralgynaecomastia. Average foiiow-up of 5.6 years has revealed no tendency far local recurrenceor metastasis. Histologicallythese were subCutaneouS/deep dermal lesionswith a lobular, sieve-likeappearance and focallyill-definedmargins.They were composedof dilated. thin-walledinterwmmunicatingvascular channels with a pseudopapillaryarchitecture.Thrombi were common and two cases showed central infarction.Vascular spaceswere lined by monolayered endothelium Which was often plump and hyperchromaticbut n d mitotic.Distinctionfrom wnventionalcavernous haemangiomaand angiosarcoma (particularlyin breast lesions)is discussed.

Incorporation of functional RNA polymerase promotor sites into PCR products

Histochemicailocalizationof acid phosphatase(AcP) in tissuesis usually demonstratedO n frozen Sections.The standardtestfortartrateresistant acid phosphatase(TRAP)istheadditionof lOmM L-tartaricacidtothe incubating medium. SOmeAcPsurvivesprocessingtoparaffin WaxandthisisthwgMtobetartrateresistant. Frozensections of prostatic carcinoma, mouse liver, kidneyand gut were used. AcP was demonstratedusing an kc-dye cwpling method. The AcP was unaffectedby the addition of 10 mM tartrate. althoughthe AcP was known to be 'tartrate Sensitive'. The addition of 100 mM tartrate or 50 mM sodium fluoride weakened but did not eliminate the reaction. Mouse tissues,prostatic carcinomaand leiomyosarcoma tissues were processedusing variausfixativesand embeddingprocedures. These were tested for AcP and TRAP. The AcP in the muilinucleatedgiant calk of the leiomyosarcoma survived standardformalinfixation and paraffinwax processingand was tartrate resistant.As expectedmouse liver, kidney and gut AcP and prostatic AcP did nd survive most fixativesand embedding procedures. However AcP could be demonstratedin those tissues processedbythe 'AMeX' method. i.e. fixed in acetoneat -2O'C. processedthroughmethyibenzoateandxyianeto paraffinwax. TheAcP.thusimalisedinparaffin blocks was eliminatedby the addition of 10 mM tartrate to the incubatingmedium. If needed the AMeX pracedure PrRSeNesSome tartrate sensitiveAcP in paraffinblocks. AcP that Survives standardfixation and embedding proceduresis iikeiy to be tarfrate resistant.

I.W. Thompson, P. J. Coates, A. J. dArdenne

Demonstration of tartrate resistant acid phosphatase (TRAP) activity in paraffin processed decalcified bone and cartilage

OepartmentsofHistopathologyandMedcal Oncokgy, St. Barthoiomews Hospitai, West Smiihheld,London, €CIA 7BE

Winifred Staiey, J. McClure

The polymerasechainreaction(PCR)isa novel techniquecapableofamplifyingmanifoldspecificDNAsequences. The specificityof the iterativesyntheticreactionis providedbythe hybridisationof a pair d syntheticoligonucleotide primerswith the template molecule. This specificity resides at the 3' ends of the primers: additionaldesired sequences may be appendedto the 5'end. which are then inwrporatedat the ends of the double stranded DNA product. In this fashion imdeukin 1 and lnterleukln5 sequences incwporatingT3 and T7 polymerase promoter seouenceswere amoiifiedtomiawram ouantities.Southern analvsis wnfirmed the soecificitv of the ~roduct.The DNA productWastien used as a i m late forthe transcriptionoismgie strandedRNA lnco 'orating radidabeiled nucleotldes Init~alstudmsindicat~tha~f~ll ienah RNAtranScriotSthusoroducedmav be usegsa orobeformsftu hybridisation.This methodpotentially providesa Simps method for pioducingRNA probes. as an ailernativato subcloningDNAhagmentsinto specialtranscriptionvectors.

Ovrsionof CeNuiar Pathology. Department of Pathological Sciences. UniversityofMmcheste6 Manchester, MI39P7

A comparison of oligonucleotide primer sets for the detection of mycobacteriumtuberculosis by the polymerase chain reaction and their application to paraffin embedded material A. M. T. Clarke,' F. A Lewis.' M. Johnson? P. Hawkey? P. Quirke' Departments of 'PatMcgy and 2Mrcrob~olcgy, Universityof Leeds. Leeds; 3oepartmentof Nwrology. St James's Hosptai. Leads

Foutsetsofknownoligonuclwtideprimen(MPB64ProteinGene:240bp.65 kDProteinGena:383bp, EcoRlpart LambdaPH7311: 158 bpandar~peatedsequencefromcioneM13KE39 123 bp)fwthedetectionof Mycobacteriom tobemulosis by the standardpolymerase chain reaction(PCR) were compared with two Leeds primer sets. The fire set was designedfar 'nestedPCR' fromthe 16s rRNAgene of MywbactenUm fubwculosisand the second set for standardPCR from the 32 kDa protein gene. Under optimisedconditionsthe primer sets were tested against serial 1 : l O dilutions of DNA extracted horn pure cuilure of MycobactenOm tuberculosis.The 16s nestedPCRwaspositivewith0.3fgoftemplateDNA.Thiswasthemostsensitivedetsctionsystem.TheotherPCR detectionlimits were.: MPE 64;3 fg, 65 kD, 30 fg. 32 k 0 350 fg PH7311; 3.5 pg and M13KE393.5 pg. Ail PCR prdocals were appliedto extracts from paraffinprocessedpost mortem lung tissue horntwo cases of pulmonary tuberculosis.AwiderangeofDNAextractionmethodswereempioyedbut no positive resultwasobtainadwith any PCR. All PCRs included positiveand negativec~nfrols.Mycobacteriiai DNA added to the paraffinextractsgave a positive PCR excludingPCR inhibition. PCR for human DNA was positive confirming DNA emaction. Numerous intact mycobacteriawere demonstratedby Ziehl Neelsen stain of smearsfrom all the final extract solutions.The resistanceof paraffinprocessedmycobacteriato DNAextractioncurrentlylimitsthe PCR diagnosis of tuberculosis in routinehistologicalmaterial

Retrospective diagnosis of herpes simplex encephalitis by nested PCR A. M. T. Clarke,' M. Johnson? P. Quirke'

'oepartment OfPathOlogy,Uniwmny Of Leads, W s ; 2DepartmentofNe,eumlogr/.St James's Hospital Trust, Leeds Current methodsforthe identificationof HerpesSimplex Virus (HSVJmay fail to identitythe presence ofthe Virus in biopsy or autopsy material. We have investigated2 autopsycases and 3 neurosurgicalbiopsy cases of clinically suspectedHerpes Simplex Encephalitisby a nestedpolymerase chain reaction(PCR). DNA was extractedfrom routineiyprocessedandparaffinembeddedmaterialbyproteinaseK incubation,phenolchloroformextractionand ethanolprecipitation.NestedPCR was performedusingknownoligonmlaotideprimersdesignedfrom the HSVtype 1 giywprotein D gene from an area with the lowest homology with HSV type 2. Two of 2 autopsyc a w and 2 of 3 neurosurgical biopsy cases ware PCR positivefor HSV.The third neurosurgicalbiopsy case was not confirmed by PCR as beingdue to HSV. Such primerslo HSV allow the rapidretraspeaivediagnosisof Herpes Simplex Encephaliisand Should be of value to neurapathologists.

Tartrate resistant acid phosphataseITRAP)is one of the major enzymessecretedby osteoclasts. The demonstration of this enzyme can be used to identity Osteaclasts and sites of acid phosphatase (Ace activity in bone. Previous experience has Shown that the amount of AcP demonstrablein bone Sections varies depending mainly on prior processingmethods.AcP is usually demonstratedin frozen unfixedtissue sections. Undecaiciliedand fixed decalcifiedSections of bone producedpoor, diffuse results. Double-embeddedundecalcifiedsectionsof bone showed AcP only in osteoclasts.Various elements ot tissue processingprocedureswere examined to find the wnditionsnecessarytoachievemaximumAcP localisation.Tissue blockswerepr-SSBdintowaxusingstandard embeddingprocedures.Threefixativeswereused.10% neutralbufferedfmalin, formalcalciumand ethanol allat +4'Cfor 18 hours.Twodecalcifyingfluidswereemployed,14%ethylenediamine-tetraaceticacid(EDTA)pH7.2at +4%, ZOC ' and37'C.andformicacidIsodium citrateat+4%forW8 hats. FormalinfixedEDTAtreatedtissuaat +4% producedmaximumAcP activity. AcP was Shown in osteociasts,Some ostwcytes, chandrocytes.Cement lines., tide mark, periosteaicellsandlarge macrophage likecellsinbona matraw. Formalinfixedtissuesdecalcified in EDTAatZO'Cand37'C werenegativeforAcPaswerealltissuesfixedinethana1. lnformalcalciumfixedtissueand formic acidlsodium citrate decalcifiedtissuethe AcP reactionwas weaker. with some elements,e.g. chondrocvtic AcP missing.All the AcP preservedthrough paraffinprocessingwas tartrate resistant.

lmmunocytochemistryon resin sections - using LR white resin and DAB enhancement Jocelyn Germain

oepartment of halPathology, The London HosprtalMedrcai College London, El 2AO It is well recognisedthat morphologyand optical reSOiution are vastly improvedwith resin as opposedto paraffin embeddingof tissue. However, difficulties in producingconsistent immunopetoxidasereactionson resin sections have caused many departmentsto abandon thetechnique.Althoughantigenicityis preservedthe lowopticaldensity of Diaminobenzidine(DAE) means that the reactionproduct is barely visible in thin resin Sections. The aim of this study was to develop a methodwhereby antibodieswmmonly used on paraffinsections wuld be successfully applied to 1 pm resin sections.Tissues fixed for 18-24 hrs in 10% formol saline were partially dehydratedand infinratedwithLRWhiteResinat4°Cfollowed bypolymerisationat40Cusingacatalyticmethod. 1 pmSectionswere reacted with polyclonaiand monocionaiantibodms using a standardindirect immunoparoxidase technique. Visualisationwaswith a silveramplificationSystemforDAE(Amenham)appiiedasthetinalstage.Excellent resuns have beenobtainedwith a rangeofantibodiesincludingS100, von WiilibrandFactor. immunoglobulins, UCHLl and L26(DAKOLtd.). Usingthistechniqueitisnowpossibletocombinehighresoiutioniightmicroscopywithaprecise immunocytochemicaireaction.The advantages are Obvious and are particularlyrelevant in the field of lymph node pathology.

lmmunohistochemicaldetection of micrometastasis in cervical lymph nodes in head and neck cancer Deirdre M. McKenna. Hazel Holden, R. W. R. Farreil Department ofPathd@& UniversityofShef6eldMedG31School, SheffieldSlO2Ry: D6partment of Otorhin&ryngolcgy, Roy& Hallamhre Hospital, Shemid, S I O 2JF Routineassessmentof lymph nodes fat metastases uses a single H 8 E stainedequatorialSection. &cult nodal metastases and micrometaslaseshave been demonstratedin axillary lymph node dissectionsusing further H d E serial sectionsand immunohistochemistry. Retrospectiveassessmentof 159negative lymph nodes hom 20 patients With head and neck cancar was carried out using serial sectionsstainedwith H 8 E and a range of immunohiStochemicalepithelialmarkers (EMA, AElIAE3, CAM5.2, PKKI and MNFll6). Five lymph nodes from three patientsrevealed seven micrometastases,three of which were detected on H 8 E Sectionsand four using immunohistochemistry alone. MNFl16 and AEllAE3 provedthe most useful antibodiesin detecting micro- and singlacalltumour metastases.We wncludethat serialsectioningofVmph nOdesfromneckdissectionswili increase turnour pick-up rate, and this pick-up is further enhanced by the use of immunohistochemistry. These resuns may have implicationsfor tumour stagingin head and neck cancer and raise the possibility of using FNA with immunahistochemistry as a pre-operativeinvestigationin these patients.

In situ hybridizationat the ultrastructural level with digoxigenin labelled probes Temporal changes in small intestinalAgNOR numbersfollowing various Adrienne L. Morey.' D. Taatjes? K. Leslie?K. A. Fleming' doses of gamma-irradiation 'Universityof Oxford. Nufik? Department of Pathologv. John Radclfie Hospitai, Oxford OX3 9DU: *UniErsity of Vwmont,CapartmentofPathology. Bufington, Vermont, U.S.A Tissueembeddedat lowtemperatureinLowicryl-K4Mresin hasbeenshowntobesuitableforimmunogoldlabellng of cellularantigens, and to be capableof withstandingthe processingrequiredfor hybridizationof nucleic acid probas.Theaimofthisstudywastoestablish basic wnditionssuitabieforhybridirationofdigoxigeninlabelledDNA probesto Lowicrylembeddedmaterial, bothat the light andelectronmicroscopelevels. CulturedhaemOpoietiicells wereembeddedafler briefaldehyde fixation.DigaxigeninlabelledWhole human DNA or plasmidpER322 (negative control) probeswereappliedtothinandsem-thinsectionsafterproteolytictilgestion and/or denaturationby heat or alkali. Hybridswere detectedin semi-thinsections by standardwlourimetricmethods. and in thin sections by immunogoldtechniques. Elaborate blocking proceduresand prolongedwashes were foundto be unnecessary. Specificnu~l~earsignaiwasseenatbdhthelightandEMisv~iwiththewhaleDNAprobe,revealingdetailsof nuclear DNA distribution not evident in paraffinSections or cytospun preparations.Non-specificbinding and background were minimal. Signal was greatlyreduced if the denaturing step was mined, and was slightly increased by protwlytic digestion, though at the expense of cytoplasmicmorphologicalintegrity.While the sensitivityof this system is limited by the fact that hybridizationoccurs only at the suriace of the section, it is a rapid and specific means of nudaic acid detection,and offers the Dossibilitvof accurate iocalizationof intracellularhuman and viral nucleic acid sequences at a fundamental level.

J. G. McDermon,' H. M. H. Kamei,' Ruth St. C. Giimore? J. S.McCuilaugh?Katherine E. Car?

'ospanmentof Pathology.Oueen's University,Belfast; ?SchwI of Biomednzal Scjance. Owen's University,Betfast Male BSVS mice were treatedwith 5.10.15 or 20 Gy doses of whole body gamma-irradiationand killed at variws timepointsfrom30minutestofivedaysposttreatment. Paraffin sectionshomErunnelifixedjejunalspecimenswere examined histologicallyand the number of AgNORs in various tissue compartmentsassessed. In the cryptal compartment,therewasan early increaseinAgNOR numbenafterthe5 lOand 15Gytreatments.These w~nlsfe1I by fivedaystocontrolvalues or below. Followingthe20Gydose,there wasa drop in cryptalAgNOR numbers. which showed a wntinuing decline, reaching the lowest point 5 days post-irradiation.After 5 and 10 Gythe AgNOR numbers in the muscle wmpartment were not affectedsignificamiythmughoutthe time mrse of the experiment. However, at 15 and 20 Gythe number in muscle dropped Significantly 5.12 and 24 hoursfoilowingtreatment, but hadrecoveradto w n t m levelsbyfivedays.In the lamina propria,AgNOR numbersincreasedinitiailyaflerthe5.10 and 15 Gy treatmentsbut returnedto control values by five days. With 20 Gy the AgNOR numbers showed a significantfall 48 hours after irradiation:this decline continuedup to five days. It is evident that &NOR numbers within the small intestineare affectedfollowing irradiation.The variation in counts is dependent on dose, cell type and time Since irradiation.

SYNOPSES OF PAPERS

357"

Karen M. Britten, W. R. Roche

Menorrhagia associated with retention of spiral arterioles - a possible variant of irregular shedding

oepartment Of Pathology, SouthamptonUnwewty General Hosp~lai,Southampton,SO9 4XY in responseto ever-increasing demands for immunophendyping in inflammatorydisorders, we have developed

oepaments of Pathology and obstetrics and Gymecology, Rotunda and St. J a m ' s Ha?ptfals, Dublh

Comparison of resin embedding techniques for small mucosal biopsies

anernatwefixationand embeddingtechniquesforsmallbiopsy specimens. Bronchialbiopsieswerefixed in buttered formaiin and processedlor embedding in Araidite or were fixed in acetone containing proteaseinhibitors and embedded in the water-soiubk resin glycol methacrylate (GMA). GMA allowed for the investigationof a full phenotypicprofile akin to that which may be wfwmed in frozen sectionwhile yieldingfar superior morphologyand greater numbersof d a n s from Small biopsies. The phenotypic markers included those for T-ceiis (CD3. CD4, CD8,CD25andLFAl),macrophages(CDllC,CD14),mastcelis~B5B6andAAt)andeosinophils(MBP,EGl and EG2). Wehaveaisodemonstratednautrophiieiastase,cytokinesandtheceiiadhesionmolecuiesiCAMl .ELAMand VCAM. Similar high qualty sections were obtainedwith Araidne but the repertoireof antibodieswas restrictedto thoseantibodieswhichcannarmailybeappiiedin paraffin. Wesuggestthatforsmaii biopsieswhichrequiredetailed immunohistochemistry,such as in the areas of transplantationand mucosa immunology, fixation in acetone at -20% with the inclusionof proteaseinhibRorS and processinginto glvcolmethacwiatewith careful temDerature control gives optimum results.

I. A. Robinson, J.

E.Giilan, B. L. Sheppard

Endometrial vascular abnormalitiesin menorrhagia have been pmrly described. This study examined endometrial vesselslrom20womenwho presentedfor hysterectomywith perceivedmenorrhagia(i.e. bioodiossofiessthan 60 mls per menses).20 hysterectomiesperformedfor genuine menonhagia(1.e greaterthan 80 mis b i d loss per menses)and 20 wntroi hysterectomies,performedfor non menStruaVmenormagiaindications. All hysterectomies were devoidof pathologicalfindings which might acwunt for symptomsof excess menstrualioss(p0iyPs. fibroids, endometritis,etc.).Thickwalied spiral arterioles. i.e. wailthicknessequallingiuminal diameter, were quantltiedbythe numberpercm lengthof endometrium and bythenumbsrofiuminai c r o s s s ~ t i o n s o b r v e d perve~~el.Theproduct of these two numbers expresses total vascular complexity. The spiral arterioles in genuine menonhagiashow significant increased t o r t ~ o s t ycompared with wntrois (P < Q.01); wrceived menonhagia cases show an intermediateIncrease (p < 0.05). This increased tortuosity most likely represents retained spiral arterioles from inwmpiete menstrual shedding.

An assessment of one hour fixation methods Darothy E. Hunon, R. I. Davis. J. P. Maxwell, J. G. McDermon HistopathologyLabratoy, Royal Vctona Hosptai, GmsvenorRoad, Belfast, BTIP 6BL ArangeoffreshtissuesfromautopsyandbiOpSysourceswerefixedloraperiodofone hourbyavarietyof methods. These included 10% formaiin at VanOUS temperatures, microwave treatments. Bouin's fluid and a wmmercialiy avaiiabieproduct "Rapid FiX".ThetiSsUeswereroutinelyprocessed. embeddedin paranin wax andstained wilhthe haematoxylinandeosinmethod. An eval"ation~fthefi~alion methodswasaisocarriedoutforimmunocytachemicai Stains. From microscopic examination of the results t was evident that for the haematoxyiin an+eosm stains. 10% formaiinat6O'Cwa~ wnsistentiytheoplimum methodof choice. Farimmunocytochamistryali methodsrasuitedin a poor performanceusing a standardtrypsinisetiontime. However, an acceptable result was achieved from 10% formalinat60°Canda microwave fixative method. byreducingthetrypsinisation time. This. however, requiredstrict WntrOI. Thus it is possible to fix tissue within one hour. by a method which is cost effective and which can be used within limitationsfor immunacytochemistry.

The effect of storage in cytospin collection fluid on nuclear morphology in fine needle aspiration cytology of the breast J. L. Williams. T. J. Stephenson. k J. Shorthouse

HPV genotyping of cervical adenocarcinomas in UK and South Africa using nonisotopic in situ hybridisation K Cooper, C. S. Herrington, Elena SwFong Lo, M F. Evans, J. O D McGee

Numeld Departmentof Pathology and Bactenology,John Radcltfe Hmpdal, Oxford, OX3 9DU USinganonisotopic,nsrlu hybridisation(NiSH)withdigoxigeninlabeiiedprobestoHWtypes6, 11. 18.18.31.33 and35,we haveinvestigated38cervicaiadenocarcinomasonarchival biopsymaterial(U.K.:16andS.A.: 22).Atolai of 17adenocarcinomas(45%)frombothgroupscontainedHPVDNA.IntheU.K.group,5cases(31q~)and3cases (19%) contained HPV 18 and 16 respectively.All 9 cases !n the S.A. gmup contained HPV 18. Hence, HPV 16 was present in 82?6ofthecombined HPV DNApositiveadenocarcinomas.Thisillustratesfurtharthestmngassociation betweenHPV 16 and cervical ademcarcinoma in two geographic locations. The pattern of HPV NlSH signal in ail cancerswasapunctatefdot signal(type2)with anadditionaidiffusesignal (type3)intwocases. This isamdarto a previousdemonstrationof the type 2 NiSH signal in all HPV DNA positive cervical squamous cell cancersand in CaSki and Hela cell lines known to wntain HPVl6 and 18 respectively in an integratedphysical slate. This further demonstrationof the type 2 punctatesignalin all the HPV DNA positiveadenocarcinomasin this series supportsthe hypothesisthatthis signalmorphologymay represent integratedHPV DNA In accordancemth therecentmolecular demonstrationof HPV 18 integrationin cervical adenocarcinomas.

oepartmantof Pathology, Uruvwsdy Of Shefheld, P.O. Box 596 Shefield, SIOZUL Forthe development of quantitativemicroscopy.standardization of specimen samplingand preparationisessentiai. We set out to study the extent and time dependency of storage related artefacts in cytospin fluid (Shandon).We assessedtheeffectof Stwage in cytospin fluidon the nuciearmarphoiqlyof breast FNAC. 10 FNAspecimsnswere coiiected on day 1 and slides were made from each specimen on day 3. day 6 and day 8.These were fixed with a spray fixative. Betweeneach sampk preparation, the cytospin colleaion fluid was kept at 4%. The slides were Fauigen stained and 5 nuclear morphologicalparameters (Area, Perimeter, Form AR. Farm PE. and ConvexityConcavity) weremeasuredusing aSeeScanSOiitairePlus imageanalysis system. There wasasignlticantchange in ail the parametem betwwn day 3 and day 6, and between day 3 and day 8 (p < 0.05). The measurementswere repeatableondifferentoccasionswithoutsignificant difference in theresuns. These resultsindicatethatstorage in cytospin fluid significantlyaners nuclearmorphology. The nuclei becomeprogressively larger and more irregular in shape. ilis therefore important to standardize the Storage conditions of FNA specimens. if accurate objective comparisonsare required.

lnterphase cytogenetics on paraffin embedded material: numerical aberrations of chromosome 11 in squamous carcinoma of the cervix C. S.Hernngt0n.A. K. Graham, J. O D McGse Numeld Departmentof Pathology and Bactenology,John Radclrffe Hospital, Oxford.OX3 QDU

Thedetection of numerical chromosome aberrations m interphasetumourceiisbynonisotopic insitu hybridisation has been previously described but the application d t h s techniqueto paraffin embedded material has been wmpiicatsd bythe requirementfottissuesectioning with the production of partial nuciei. inthisstudy, the analysis 014-20pm thickparaffin sectionsolconventionally processedCaSki ceilsusing both human papliiomavlrus(HPV1 type l e a d achromosome11specificalphoid probe wascompared with resunsoMainedusingintactceils.The use of sectioned materialdid not give signal distributions comparableto those obtained using whale cells. This IS lmmunogold labelling for low temperature scanning electron consistentwnh a mathematical model derivedfor the relationshipbetween Sectionthickness. nuclear Size and microscopy; a salient investigation nuclear retentionin paranln sections. A method was therefore developedfor the extraction and analysis of nuclei fromthick(50pm)paraltinsectionsandappiiedtotheanaiysisof D. M. Lawion, W. B. Oswaid. T J. Lyons, J. McCiure squamouscelicarcinomasofthecervix(n= 11). The number of copies of chromosome11 varied from 2 to 7 and this variation was apparent both between lesions I)lwslon of CallularPathology, Departmentof Pathoicgcal Snences,Unrvemny of Manchesler.Manchester, M I 3 9PT and betweentumour ceiis within the Same ieaon. This IS comparableto resuns obtained w t h cervical carcinoma The identificationof macromolecularcomponentsof hydrated patholqlicai tissues revealed by low temperature derived csll lines. There was. however, no clear relationshipbetweenthe presence of HPV sequences and scanningeiectronmicroscopy(LTSEM) isan emergingfieldof enquiry. In orderthatthetechnicaidetaiisforLTSEM chromosome11 number. These preliminary results suggest that the postulated loss of host suppressionof HPV labeilingmaybeestablishedanexperimentaiprotocoi has beendrawninwhichiabeiparticiesofhighatomicnumber gene function by deletionof genes on chromosome11 does not occur through gross chromosomalabnormalities. are visualized. The experiment comprisesa mcdei System in which bovine serum albumen as a known amigen is dissolvedin phosphatebutteredsalineandadsorbedontonitroceiiuiosemembtane. Theantigenonthemembrane is subsequently reactedmth rabbt anti-cow antibody and then elther proteinA-goid or goat anti-rabbn-gold.Aner treatmentwithadeveiopertoaddaiayerofsiivertothegoidparticies,thepreparationsareanachedtostubs,rapidiy frozen in nitrogenSlush at -21OoC, coated with aluminium and observedon the LTSEM stage at approximately -191°C. BackScaneredeiectronimagingathighacceleratingvoltages(30 kV)isuSBdtodetectsilver-enhancedgoid The effect of cyclosporin A (CyA) on a rabbit model of early stage partic1es.These areciearlyvisuaiized.Thefrozenhydratedpreparationsarestabieunderthe describedwndtions of membranous glomerulonephritis LTSEM operation. in Contrast dry, wnventionai. SEM preparations are beam sensitive; their initiallyobserved Susan H Burroughs. Julie H. Wiiliams. P S.Bass delicate UitrastNctUre quickly dearades an exeosure to even the moderate I15 kW electron beams emDioved in secondary electron imaging. UniversityDepartmntof Paihology, Level E, Soulh Blmk, SoUthamptOn General Hospital, TremonaRoad, Southampton,SO9 4XY

A new approach to systematic storage of pathological specimens for low temperature scanning electron microscopy D. M. Lawtan,W. 0. Oswaid, T. J. Lyons, J. McCiure h w s m of Celluiar Pathology, oepartment of Pathdogmi Snences, Universityof Manchester,Manchester,M I 3 9PT

As greater numbers of pathological specimens are stored for wnvenient future imaging by iow temperature scanning electron microscopy (LTSEM). It IS increasingly important to maximise expensive cryostare capacity. A cassenesystem hasthereforebeendeveiopedwhichincreasesthe hoidingcapacityof cryOStoreSbyafactord2.5 over conventionalbee storage. Each c-he consists of an aluminium disc 44.5 mm in diameter wilh 6 cylindrical wells10 5mmindiametereveniyspacedand1.5mmapart. Specimens. mountedan JEOLstubs,areretainedinthe wells by set Screws. Five cassettesfit into each 60 x 60 mm glass storage jar, gwmg a capacny for a 10 Canister cryastore(w4th4@rs percanister)of 12OOstub-mountedSpecimens An addtionai benefitofthecassettesystem is that each specimen is afforded a mechanically and thermally protected environment. Specimen collection for LTSEM may take place in a distant operatingtheatre or laboratory. lmmobiiiration of Specimens Whilst they are in transit excludes any possibility of their being damaged during normal handling and improves the prospects for survivalof accidents.Afurther benefitisthataparticuiarmountedspecimencannow bemorespeedily identlledand removedfor LTSEM from RS well.

Secondary cytoskeletal abnormalities produced by microinjection and by intracellular debris T. M. Macieod. J. E. Martin, M. Swash, A. Gray oepartment OfMorhdAnafomy, m e Royal London Hospilal, Whitechapei,London, E l 1BB Many pathological condtions are charactensed by the presence of cellular degeneration accompaniedby cytoskeietaiabnormalities. In many such diswders it is not clear whether cytoskeietaiabnormalities are pftmary process or are part of a secondary response to cellular insun by other agents or mechanisms. We have used a fibroblast ceii culture mcdei to study the effects of physicaldistension on the cellular cyioskeielon. inertbeadsof 2 and4pm were introducedintoceilsbyallowingendocytosisor bymicroinjection.Thecytoskeietai responsetothese beads was studied using immunofiuorescence microscopy. Beadsintroduced by endocytosis migratedto the prinuciear regionand over 46 hours becameenmeshed in intermediatefilament and mlcrotubuiaraggregates.Actln microfilamentorganiselionwasnot alfected. Incontrast. microinjectionofbeadsproduced an immediatecollapseof morofilaments,wiUlvimenBnandtubuiin distributionbeing preseNed.The immediateresponsetomicrolnlectlonis similar to the collapse of actin filaments s w n upon thermal stress.This experimentalmodel has shown !hat aggregates of cytoskeietaiproteins may be produced within cells as a secondary responseto intraceiiuiardebris, and that microinjection may induce cytoskeietalabnormalnies similar to those seen In thermal Stress.

Membranous giomeruionephritls(MGN). an immune-mediated disease, IS a frequent caused renal morbidity in man. Cationic bovineSerum albumin (cBSA),given to NZW rabbits in a chronic serum sickness-type protocol. is knowntoinduceglomerularchangessimilartothehumandisease. We~ssedtheenectofashortcourseofthe immunosuppressivedrugCyAon thedevelopmentof early stage.cBSA-inducedMGN. Fourteen male NZWrabbts receivedan iv immunismg dose of 1 mg cBSA and 1 p g E. coliendotoxin. One week later they commenceddaily IY inlectionsaf 25mgcBSAfor21 ~onsec~t~vedays. Three rabbtsweresacftficed atthistime. Sixofthe remaining 11 rabbits were commenced on a short COU~SBof om CyA. whilst continuingto recewe daily doses of cBSA. The 5 remainingrabbitswsregivencBSAonly.After32consecutivedosesofcBSAthesell anlmaisweresacnficed.Ali3 rabbnsgiven 21 doses cBSA Showed early stage MGN and those given 32 doses of the cationic protein showed a more mature, established disease (thickenedglomerular capillary wails w t h diffuse. global. granular depositionof igG/C3 and Subepithelialelectmn dense deposits). Four of the 6 CyA-treated cBSA rabbns showed a marked reduction in giomerular capillary wail C3 deposition. Three of these 4 rabbns had considerably less severe disease ultrastructuraiiy.These results suggest that CyA may aker the course of cBSA-lnduced MGN.

Arterial endothelialitis in renal allograft nephrectomies: a histopathologicaland immunocytochemicalstudy D. R. Gouidesbrough.' R. Axelsen2 'oRoartmentOf Pathology Universityof Edmburgh M&~aiSchooi: zoeparment of Palholcgy, pnncess Alexandra Hospitai. W d b n g a b h a , Austraha Late renal aliogranloss 1s due to arterial intimai proliferationand iumenai narrowing. There are few studies ofthe phenotypesof the intimai cells. We analysed these lesions by light microscopy and immunmyiochemistry using antisera against T-lymphocytes. B-iymphocytes. macmphages, smooth muscle cells, Class ii HLA OR molecules and the proideration antigen PClO 31 vessels were studied from 20 gran nephrectomiesresected between 3 and 171 months post-transplantation.We identified an arterial endolheliaiilis, r-rted in cardiac aliografts, but not emphaszed in renalgraftrejection.4 panernsolarterial pathoiogywwerewgnisd: (1) Endotheiialitisin nnteriobuiar arteries without intimal proliferation,(2)Endothelialiis in larger arteriesaccompanied by intimal prdtferat!on of smooth muscle. (3) '"inactive"lesions with thickened intima (ifoam cells) but no endotheliaiitis,and (4) 'Natural" atherosclerosis of larger arteries. The endothellaidistendedto m U r in s h m w suMving grafts. The pedominant cell was the macrophage. with fewer T-lymphocytes. PClO was expressed in mononuclear ceiis, smwth muscle cells and endothalid ceiis. pmicuiariy. but not exclusively in younger grafls. We ProposBthese ledonsevolve, variably. from an early endotheiiaiitisto late chronic vascular rejmlon or gralt athwmierosis. The predomlnanceof the macrophage at ail stages, suggests it plays a significantroia in the evolution of these lesions.

SYNOPSES OF PAPERS

35an

A comparative study of the effects of stimulation on the distribution of renin-containing cells in human and rat kidneys

Somatic mutation in human colonic epithelium Clare E Fuller, Fiona Campbell, G. T Williams. E. D Williams

D S. Gardmer.' I. Downm.' C Kenyon,?G. 8. M. Lmdop'

PathologyDepartment, Unwemly of Wales Collegeof Mednne. Heath Park, Card6 CF4 4xN

'UnrversityDepartment OfPafhoIcgy, Western hfimary. GlaSgOw,G I 1 6NT 2MRCBkcdPressure Umt, WesternInfirmary, GI~SQOW Gf7 6N7

ThreepanernsofstainingareloundinhumancolonusingatechniquetodemonstrateO-acetylationofsialomucins (mPASJ.8% afindivdualsshowunifwmmPAS-positivity.theremaindwareeitherent~~ymPAS-negativewmPASnegativewithoccasional positivecrypts. We suggestthatthisrepresentspalymorphismof an autosomalgene(0sa) controlling 0-acetylation of sballc acid. isolated crypt-restricted mPAS-posltivkyIn otherwise negativeindivtduais representing somatic mutation of the m a * gene in crypt Stem cells of osa'/osa-individuals. To test this we have Studied colons from 80 patients with rectal carcinoma, half of whom had received4000cGy radiation28 days Preoperataely. RadialiOnd~dnotaffecttheprevalenceofthethreepnenotypesbutincreasedthefrequencyofmPASposltlvecryptsinanegativebackground(t62 ~ 1 0 ~ ~ to*, ~ 6p