4. Identification of distinctive protein expression patterns in colorectal adenoma

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PATHOLOGY 2011 ABSTRACT SUPPLEMENT

1. INTRAGENIC POLYMORPHISM USING PCR-RFLP IN CARRIER DETECTION OF HEMOPHILIA A IN INDIAN POPULATION Suresh Babu, Nuzhat Husain, S.T. Raza, Nuzhat J. Faridi, Praveen Kumar, Ashutosh Kumar Department of Pathology, CSM Medical University Lucknow, India Background: Hemophilia A is a common X linked recessive disorder. Carrier detection has been done using RFLP markers but heterozygosity rates vary in different ethnic groups. We examined usefulness of Intragenic markers including BclI, HindIII, Xbal for carrier detection in North Indian families with Hemophilia A. Design: Prospective case series including 143 families, 150 Cases with Hemophilia and 238 relatives. On the basis of the FVIII bioassay study groups was divided into 80, 50 and 13 cases each of severe, moderate and mild hemophilia respectively. DNA was extracted using Qiagen DNA extraction Kit. Gene polymorphism was identified by the specific PCR/RFLP followed by gel electrophoresis. Expected frequency of Heterozygosity was calculated by using (1a2 þb2) where a ¼ positive allele frequency and b ¼ negative allele frequency. Results: Using PCR/RFLP polymorphism detection at Intron 18 (Bcl1), Intron 19(HindIII) and Intron 22 (Xba1) 101 of 132 families were informative. Conclusion: Intragenic polymorphisms at BclI, Hind III, XbaI locus are informative in 78.5% Indian families with Hemophilia A. Although direct mutation detection is idea, linkage analysis is of value when mutation in families has not been identified. It is also useful for tracking of the origin of de novo mutations. The procedure is simple, cost effective, with low infrastructure and expertise requirement and can be implemented in developing countries for carrier screening and prevention of Hemophilia A. 2. PINEOBLASTOMA – A CASE REPORT INCLUDING GRADING CRITERIA AND CYTOLOGICAL FEATURES Andrew Bettington, Thomas Robertson Department of Anatomical Pathology, Princess Alexandra Hospital, Brisbane, Queensland, Australia Background: Neoplasms of the pineal region are rare accounting for 0.5-1% of all intracranial neoplasms and of these pineal parenchymal tumours comprise 15–30%. The World Health Organisation (WHO) classification scheme recognises three entities within the group of pineal parenchymal tumours – pineocytoma (WHO grade 1), pineal parenchymal tumour of intermediate differentiation (WHO grade 2 or 3) and pineoblastoma (WHO grade 4). Due to their rarity, there is no consensus with respect to the grading of these tumours and no definite criteria are outlined by the WHO. Case presentation: We present a case of a pineal tumour in a 47 year old man. The case illustrates the difficulty in separating pineal parenchymal tumour of intermediate differentiation from pineoblastoma. A proposed grading system is discussed, with tumour morphology, mitotic rate and NFP staining being the criteria used in this system. In our case, the presence of a diffuse growth pattern, up to 7 mitoses per ten high power fields and negative staining for neurofilament protein led to a diagnosis of pineoblastoma. Cytology of cerebrospinal fluid is also included. Conclusion: Pineal parenchymal tumours are rare entities and grading criteria are poorly defined. Further research is necessary to validate the proposed grading system or develop new criteria to

Pathology (2011), 43(S1)

differentiate lower grade tumours from pineoblastoma in borderline cases. 3. IS IT POSSIBLE TO PERFORM 2 MM SLICING OF SENTINEL LYMPH NODES? D. Botros, R. G. Wright, G. Baxter, S. Sharifi Pathology Queensland – Gold Coast Hospital, Southport, Queensland, Australia Background: The National Breast and Ovarian Cancer Centre has advised 2 mm slicing of sentinel lymph nodes prior to a sectioning protocol. This method seeks to identify all macro-metastases and the theoretical basis of this is well established. The detailed methodology of the lymph node slicing has not been extensively investigated. Aims/Methods: This study examines slicing of inert material and the consequences of a 2 mm slicing strategy. Cubes and cuboidal structures of play-dough with a thickness of 10 mm were prepared and participants were asked to slice these into 2 mm thick sections. The difficulty in measuring 2 mm slices using callipers is well known, so a surrogate measure of slice weight was used. Each cube or cuboidal structure was weighed with an ideal slice being 20% of the original mass. Instruments available in all laboratories (scalpels and feather blades) were used. Results were recorded and tabulated using the surrogate measure of slice weight reflecting thickness. Three groups of participants were involved in this study (pathology consultants, pathology registrars and laboratory scientists/technicians). Results: This study found a significant difference between the three groups of participants but not the instrument used. Also, there was a significantly different fifth slice. Conclusions: We propose that the method does not allow even slicing of inert material and that similar results would be obtained in sentinel lymph nodes. We consider that the current methodology is flawed because of the extreme variability in the final slice and that an alternative method or a multi-slicer could be used. A controlled trial could be devised to consider these methods further. 4. IDENTIFICATION OF DISTINCTIVE PROTEIN EXPRESSION PATTERNS IN COLORECTAL ADENOMA C. Clarke1, F. F. Lam2, L. Jankova3, O. F. Dent2, M. P. Molloy4, S. Y. Kwun1, P. H. Chapuis2, G. Robertson3, P. Beale5, S. Clarke5, E. L. Bokey2, B. P. C. Lin1, C. Chan1 1 Department of Anatomical Pathology, Concord Hospital and Discipline of Pathology, University of Sydney, 2Department of Colorectal Surgery, Concord Hospital and Discipline of Surgery, University of Sydney, 3Cancer Pharmacology Unit, ANZAC Research Institute, 4Australian Proteome Analysis Facility and Department of Chemistry and Biomolecular Sciences, Macquarie University, and 5Department of Medicine, Concord Hospital; Discipline of Medicine, University of Sydney, Sydney, NSW, Australia Background: As a precursor lesion, adenoma is ideally suited for proteome profiling to investigate early colorectal cancer (CRC) development. The ability to recognise premalignant lesions may have important applications in cancer prevention. Aim: To identify protein expression patterns that can distinguish colorectal adenoma from normal tissue. Methods: Twenty paired samples of adenoma and normal mucosa were analysed by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MALDI-TOF/TOF MS) to detect proteins with

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ABSTRACTS

2-fold differential expression. Protein expression was validated and localised by immunohistochemistry (IHC). Results: 2-DE identified four up-regulated (Annexin A3, S100A11, S100P and eIF5A-1) and three down-regulated (Galectin-1, S100A9 and FABPL) proteins in adenomas. These proteins have important functions in cell differentiation, proliferation and metabolism.The finding of decreased S100A9 and galectin-1 expression in adenoma was unexpected given the previous reports of their up-regulation in CRC. Our observations however, were validated by IHC and explained by the cellular localisation of these proteins, which are linked to tumour-associated inflammatory and stromal responses respectively, features seen in CRC but not in adenoma. Conclusions: Proteomic study of adenomas provides unique biomolecular insights into early colorectal carcinogenesis, however findings need to be validated and interpreted in the light of IHC tissue localisation. This study identifies a distinctive pattern of protein expression that differentiates colorectal adenoma from both CRC and normal mucosa. 5. MOLECULAR PROFILING OF BACTERIAL DNA ISOLATED FROM FORMALIN-FIXED PARAFFIN-EMBEDDED (FFPE) TISSUE: A COMPARATIVE STUDY C. L. O’Brien1, G. E. Allison1,2, P. Pavli1,3, J. E. Dahlstrom1,4 1 Medical School and 2Research School of Biology, ANU; Departments of 3Gastroenterology and 4Anatomical Pathology, Canberra Hospital, ACT, Australia There is increasing use of FFPE tissues for molecular profiling. We sought to determine the effects of fixation on our ability to detect bacterial DNA in tissues. Aim: To investigate the effect of FFPE, and two de-paraffinisation methods on DNA yields, bacterial DNA amplification and molecular fingerprinting. Methods: Intestinal tissue and corresponding gut microflora were collected from mice and chickens. Triplicate samples from 14 animals were either snap frozen at –80oC (FS); or fixed in 10% formalin prior to processing. The FFPE samples were de-paraffinised using either the traditional method (TM) or a microwave method (MW). DNA was extracted from all tissues using a modified commercial kit protocol. DNA yields were determined, and amplification of bacterial 16S rDNA fragments compared. Denaturing gradient gel electrophoresis (DGGE) was used to generate ‘fingerprints’ of PCR amplicons to obtain an estimate of number of species of gut microbiota present. Results: DNA yields from FFPE-TM and -MW tissues were 11% and 35% respectively of those from FS. 16S PCR products were obtained from 93% of FS, 7% of FFPE-TM and 57% of FFPE-MW samples. DGGE profiles showed FS yielded PCR fingerprints with the most bands (avg 22 bands) followed by FFPE-MW (avg 3 bands). Conclusion: Compared to FS, FFPE tissue yields less bacterial DNA, and is less likely to enable the generation of PCR amplicons or DGGE bands for molecular profiling. The MW method yielded twice the DNA of TM. The amplification success of the 260 bp 16S rDNA fragment is poor when DNA has been extracted from FFPE, regardless of de-paraffinisation method. 6. MYELOID SARCOMA OF THE TESTIS WITHOUT INVOLVEMENT OF BONE-MARROW BASED DISEASE Laurence Galea, Vanaja Muthurajah, Jack Metz Dorevitch Pathology, Ballarat Base Hospital, Victoria, Australia

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Myeloid sarcoma is a neoplasm of immature myeloid cells occuring in an extramedullary site. It rarely occurs in the testis and if so, is almost always associated with recurrent, co-existing or is the initial presentation of bone-marrow based disease. This is usually acute myeloid leukaemia and less commonly, myelodysplastic or myeloproliferative disorders. We describe a case of myeloid sarcoma as an isolated mass in the testis of a seventy-six year old; without past or current history of acute myeloid leukaemia. We discuss the histologic, cytochemical and immunohistochemical findings that brought us to the diagnosis. The differential diagnoses considered in this case include plasmacytoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, precursor lymphoblastic lymphoma/leukaemia and metastatic amelanocytic melanoma. The literature was reviewed in order to calculate the range as well as mean interval time between first presentation of myeloid sarcoma and acute myeloid leukaemia and to calculate how long cases defined as myeloid sarcoma isolated to testis had been followed-up. Our aim is to determine how long such a patient should be followed-up for in order to accurately diagnose the entity of myeloid sarcoma confined to testis without bone-based disease. 7. TRENDS IN APPENDICECTOMIES AT ROYAL NORTH SHORE HOSPITAL Rosanne Robosa1, Anthony Gill1,2 1 Royal North Shore Hospital and 2University of Sydney, Sydney, NSW, Australia Appendectomies are one of the most common surgical procedures in adults and children. This study analysed records of specimens from appendectomies at Royal North Shore Hospital (RNSH) in 2009 and compared this data to published figures. In 2009 there were 588 appendicectomies recorded in the RNSH database (mean age 32.5, female 55%) Acute appendicitis was found in 80.61% of appendectomies (mean age 33.09, female 52.74%), non-inflammed appendices were found in 19.39% of appendectomies (mean age 30.02, female 64%). Co-existing pathologies such as adenoma, carcinoid, granulomatous inflammation and enterobius vermicularis comprised of 4.25% of cases. On a whole the trends in appendicectomies at RNSH are reflective of global trends. 8. THE HOSPITAL AUTOPSY RATE HAS FALLEN DRAMATICALLY Amy Doldissen1, Aldo Severino2, David Bourne2, Anthony Gill1,2 Faculty of Medicine, University of Sydney and 2Department of Anatomical Pathology, PaLMS, Royal North Shore Hospital, Sydney, NSW, Australia 1

Hospital autopsy rates have been declining worldwide. There is limited data on the rate of decline of autopsies in Australian hospitals. We studied the autopsy rates in Royal North Shore Hospital, a major Australian teaching hospital, over the last 47 years (1963–2009). Autopsy rates (defined as the percentage of in-hospital deaths which culminated in non-coronial autopsy) have steadily declined from 45% in the 1970 s to 21% in the 1980 s, 9% in the 1990 s and 3% in the decade 2000 to 2009. The absolute autopsy numbers reached a peak in 1979 at 389 autopsies and decreased to 30 in 2009. Although the number of adult autopsies has declined, the number of perinatal autopsies performed has remained stable over

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