ROYAL ACADEMY OF MEDICINE IN IRELAND SECTION OF PATHOLOGY Proceedings of Registrar's Prize Meeting held Wednesday 26th January, 1994 employed but none is fully satisfactory. In this study all stillbirths over 1000g birthweight at our unit were analysed over a 20 year period 1973 to 1992 (n=475) using information extracted from clinical case records and necropsy reports where available. All cases with an explained cause were excluded. Our objectives were 1) to obtain the incidence of U.S.B. 2) to correlate this with the incidence of S.I.D.S. given previous studies which show no epidemiological difference between S.I.D.S. and U.S.B. 3) to develop new strategies to examine preserved tissue from U.S.B. using p l a c e n t a l tissue, the polymerase chain reaction and DNA probes. From 1973-82 there were 34 U.S.B. from 27360 births (rate = 1.2/1000 deliveries, 10.6% of total S.B.) and from 1983-92 there were 60 U.S.B. from 25721 births (rate = 2.37/1000 deliveries, 38.9% of total S.B.) representing a significant increase (p < 0.001). From 197382 there were 85 cases of S.I.D.S. and from 1983-92 there were 69. Summating these figures, the total U.S.B. + S.LD.S. from 1973-82 (34+85) = 119 and from 1983-92 (60+69) = 129, almost identical figures and there is a weak but definite negative correlation between U.S.B. and S.I.D.S. (r = -0.22). These figures suggest the fall in S.I.D.S. is related to the rise in U.S.B. and raise a tantalising possibility that U.S.Bs. could be intrauterine cot deaths with a common aetiological background affecting babies at an earlier stage of development. Using the polymerase chain reaction (PCR) and DNA probes which use ribosomal R.N.A. intergenic regions as targets we have investigated archival placental tissue from cases of U.S.B. for listeria monocytogenes as a model for the alpplication of D.N.A. technology in the search for the cause of U.S.B. and potentially S.I.D.S., with early results indicating a possible role for listeria infection in the former.

PROLIFERATIVE HETEROGENEITY IN CARCINOSARCOMAS M. Kennedy, A. Grace, E. C. Sweeney. Histopathology Department, St. James's Hospital, Dublin. Although the epithelial and mesenchymal elements of carcinosarcomas (CS) each exibit varying degrees of anaplasia and mitotic activity, metastases derived from these tumours most frequently consist of the epithelial c~omponent alone. The present study was undertaken to see if this greater metastatic potential was related to a higher growth-rate within the carcinomatous elements. Proliferative activity was assessed on the basis of mitotic count and exibition of proliferation antigens, PCNA and Ki67. To give a more accurate estimation of cell-growth fraction (CGF), the rate of apoptosis was also determined. In four out of five cases of CS (3 lung, 1 breast and 1 gallbladder) the epithelial component demonstrated a significantly higher CGF and, where metastases were documented, these consisted of carcinoma only. CGF in the remaining case was the same in both epithelial and mesenchymal elements and it is of interest that metastases in this case consisted of sarcoma only.

GRANULOMATOUS LYMPHADENITIS IN CROHN'S DISEASE E. E. Mooney, J. Walker. D. O'B. Hourihane. Department of Histopathology, St James's Hospital and Trinity College, Dublin. Crohn's disease (CD) is a multisystem disease of unknown aetiology, characterised by granulomatous inflammation without caseation. Granulomatous vasculitis has been suggested as the pathogenetic mechanism in gastrointestinal CD. On review of sections from resection specmens of CD, granulomas were noted within and adjacent to thin-walled vessels interpreted as lymphatics. This study was designed to explore the relationship of granulomas to lymphatic vessels. Five-micron sections from 10 cases of CD which showed granulomatous inflammatio'n were stained with antibodies to vascular endothelium and vascular wall components to discriminate between lymphatics and capillaries. Results showed that a significant proportion of granulomas were intimately related to lymphatic vessels. This observation lends support to the hypothesis that the pathological features of intestinal CD may be mediated by a chronic obliterative lymphangitis, and that blood vessel involvement may be a secondary phenomenon,

INFECTIVE ENDOCARDITIS 1993 C. O'Sullivan. Department of Microbiology, Mater Misericordiae Hospital, Dublin 7. A series of six episodes (five patients) of culture positive Infective Endocarditis treated in the Mater Misericordiae Hospital during 1993 is presented. Microbiologically, the spectrum of causative organisms ranged from those c o m m o n l y a s s o c i a t e d with infective endocarditis to rare opportunistic organisms. The patients ranged in age from 36 to 87 years. Underlying valve abnormalities were known to be present in two patients, while two others had prosthetic valves. Time from onset of illness to time of diagnosis varied widely. A successful outcome occurred in those treated surgically, medical treatment alone was unsuccessful. These cases were chosen to illustrate the changing clinical presentation of Infective Endocarditis in the 1990s whilst also highlighting important practical management considerations, in particular the need for prompt, close liaison between clinician and microbiologist throughout the course of the illness.

UNEXPLAINED STILLBIRTH AND SUDDEN INFANT DEATH SYNDROME S. Walsh. Department of Histopathology, University College Hospital, Galway. Unexplained stillbirth (U.S.B.) is a poorly documented phenomenen for which various classification systems have been 100

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GENOMIC TYPING OF ACINETOBACTER SPECIES USING DNA AMPLIFICATION FINGERPRINTING (DAF) M. Lynchb, C. Sheehan a, L. BoisseP, B. Cryanb, S. Fanninga. aMedical Sciences Section, Regional Technical College, Cork. bDepartment of Medical Microbiology, Regional Hospital, Cork.

Acinetobacter spp. are important emerging nosocomial pathogens. They are particularly significant in intensive care, special care baby and burns units. Frequently they are resistant to many commonly used antibiotics and are difficult to type for epidemiological purposes by conventional techniques. A new method to characterise these Acinetobacter spp. isolates is described, which utilises a modified polymerase reaction (PCR), capable of generating genomic fingerprints, known as DNA amplification fingerprinting (DAF). In this project thirteen Acinetobacter sppr from intensive care patients were isolated. These were initially characterised using the API-20 NE biotyping system and antibiogram analysis. Purified chromosomal DNA of cultured clinical isolates of Acinetobacter spp. were subjected to DAF using the M13 universal sequencing primers. The polymorphic DNA bands produced, were visualised after agarose gel electrophoresis and ethidium bromide staining. Results demostrated that six of the thirteen clinical isolates represented one group which displayed identical fingerprinting pattern as did a second group of two isolates. The remaining four organisms were all unique. This genotype based method is rapid, simple, reproducible and may have potential as a means of specifically typing Acinetobacter spp. A prospective study of Acinetobacter spp. has been set up in our hospital to determine the distribution of DAF fingerprinting and to help us define their epidemiology.

APOPTOTIC INDICES VARY IN DIFFERENT HUMAN TUMOUR TYPES M. J. Staunton, E. F. Gaffney. St. James's Hospital and Trinity College, Dublin. Apoptosis is the genetically-mediated mechanism controlling deletion of individual cells in normal and diseased tissue. At present there is little data on the degree of apoptosis in different human tumour types. H & E stained paraffin sections of 102 malignant tumours (58 types) were evaluated for apoptotic cells and apoptotic bodies tl~, using the 40X objective with a calibrated eyepiece. An average of 3000 tumour cells per case were counted avoiding necrotic zones. The percentage of apoptotic cells/bodies in the total number of tumour cells examined was designated as the Apoptotic Index (AI) for each case. There was a wide range of AI in the spectrum of tumours examined - from 0 in follicular carcinoma of the thyroid to 27% in small cell carcinoma of lung. 47 tumours had AI less than 1% and 93% had AI less than 7%. Subsequently a further 30 cases were evaluated. 12/12 colonic adenocarcinomas had AI of 0-5.7%, II/12 malignant melanomas had AI of 0-1.4% and 11/12 immunoblastic lymphomas had AI of 0.4-5.7%. Each of the 132 tumours had a consistent overall AI, although there was in several cases, considerable field to field variability. Acceptable inter and intra-observer reproducibility was obtained. These results show that many tumour types have a characteristic AI which reflects innate tumour cell susceptibility to apoptosis and can be e c o n o m i c a l l y determined by light microscopy. Additional information is needed to determine whether variations in AI correlate with tumour cell proliferation, biological behaviour, aberant oncogene/tumour suppres-

Section o f Pathology 101 sor gene expression and standard clinico-pathological variables. Reference

1. Kerr et al. Br. J. Cancer 1972; 26: 239.

NEUROPROLIFERATION IS A FEATURE OF COELIAC DISEASE N. Leonard, D. Hourihane, A. Whelan. Depts. of Histopathology and Immunology, St James's Hospital, Dublin. The mechanisms that lead to villous damage in coeliac disease are unknown. The villus has a delicate neurovascular core which has a role in maintaining villous architecture. In order to study this core in more detail we stained duodenal biopsies from four different disease categories with neuronespecific enolase(NSE). The categories were: untreated coeliac disease (UTCD) (10 biopsies); treated coeliac disease(TCD) (10 biopsies); peptic duodenitis (PD) (8 biopsies) and normal duodenum (N) (10 biopsies). The amount of NSE staining present in an area above the muscularis mucosa was quantified using a Quantimet image analyser. When the groups were compared there was no difference between the untreated and treated coeliac groups but there was a statistically significant difference between the combined coeliac groups versus normal and duodenitis groups. The treated coeliac disease group data split into two very different groups - one with nearly normal NSE values and one with very high NSE values. When the TCD cases were originally selected from file no account was made as to how well the disease had responded to treatment ie was it a fully or poorly responsive coeliac or if the treatment was adequate. The group with high NSE staining corresponded to a group with a poor response to treatment and were therefore added to the UTCD group to form a new group - untreated and partially treated or unresponsive coeliacs. The analysis of this group showed that it had increased NSE staining (p<0.001) when compared to patients responsive to diet, normal controls and patients with duodenitis. These results show that the duodenal mucosa of the untreated and unresponding coeliac patient has increased neuronal tissue when compared with the mucosa from coeliac patients responding to diet, patients with duodenitis and normal mucosa. The data suggests that an increase in neuronal tissue is not simply a result of generalised inflammation as such an increase is not present in the mucosa of peptic duodenitis. These data highlight the possible important role of the neuromusculature and neurovasculature in the maintenance or destruction of the villus in coeliac disease.

INTERPHASE CYTOGENETICS STUDY ON PARTIAL HYDATIDIFORM MOLES P. Faul, P. Kelehan, P. Dervan. Department of Pathology, University College, Dublin, National Maternity Hospital, Dublin. The diagnosis of partial hydatidiform mole is difficult when based on histology alone. Diagnostic accuracy is important particularly in differentiating partial moles from complete moles and from hydropic abortions. We aimed to evaluate the feasibility of identifying chromosome ploidy abnormalities in partial hydatidiform moles using interphase cytogenetic techniques. We performed in situ hybridization (ISH) on 7 cases originally classified as partial moles, 7 cases classified as complete mole and 2 cases classified as

102 Royal Academy of Medicine in Ireland hydropic abortion. All cases had been formalin-fixed and paraffin-embedded. We used a biotinylated chromosome probe specific for chromosome 1 which was visualised with peroxidase stain. In the 7 cases classified as partial mole, triploidy was found in 4 cases and diploidy in 3 cases. After reviewing these 3 cases two cases were reclassified as diploid complete mole and one case as diploid hydropic abortion. In the 7 cases classified as complete mole, diploidy was found in 6 cases confirming diploid complete mole, no ISH signals were obtained in the other case. In the 2 cases of hydropic abortion, triploidy was found confirming triploid hydropic abortion. Maternal decidua served as an internal control in almost all cases. ISH enhances the accuracy of diagnosis of routinely processed partial hydatidiform moles and has the additional b e n e f i t of e n a b l i n g c h r o m o s o m e l~loidy analysis with preservation of morphology.

THE PROGNOSTIC VALUE OF PATHOLOGICAL AND RADIOLOGICAL INDICES OF TUMOUR VASCULARITY IN BREAST CANCER P. Costello~1~,A. McCannt~, E Flanagant2~,J. Stack~2>,E Dervan~ Department of Pathologyt~, University College Dublin and Mater Hospital, and Department of Radiology~2~,Mater Hospital, Dublin. Angiogenesis is a required step in tumourigenesis as it is

I.J.M.S. January, 1995

required for growth of tumours greater than 2-3ram in size. The formation of new vessels also allows the dissemination of tumour cells which precedes the appearance of metastases. In this study we have attempted to correlate the density of neoangiogenesis with the likelihood of metastasis, in patients with lymp node negative breast cancer. Archival formalin-fixed paraffin-embedded tissues from 87 patients were studied. The block most representative of the invasive component was selected, sectioned and stained immunocytochemically for von Willebrand factor (wWF/Factor VIII-related antigen). Sections were scanned at low power to identify "hot spots!' of neo-angiogenesis. Vessels were counted in these areas with a 20X objective and a grid graticnle. Three areas were counted per hot spot with the highest and average values recorded. Vessel counts were correlated with a 2-5 years survival and showed poorer survival in those with higher counts. A further group of 25 patients with breast masses were prospectively studied using gradolinium enhanced MR imaging. This showed characteristic enhancement curves for benign and malignant masses. Immunocytochemistry for vWF of biopsy tissue from these patients demonstrated that greater enhancement corresponded with increased vascularity in malignant tissue. Our findings suggest that increased vascularity in in-vivo imaging may have diagnostic value and in pathological specimens may have prognostic value.