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Comparison of immunohistochemistry analysis methods using cancer stem cell markers
S130
PATHOLOGY 2017 ABSTRACT SUPPLEMENT
statistical difference (p = 0.615). Patients with p53 positive tumours also showed no difference in survival to those without p53 staining (p = 0.741). There was no significant difference between survivals of patients with mismatch repair defect CRCs compared to patients with tumours that showed p53 over-expression. Conclusion: MMR-d colorectal carcinomas and CRCs with p53 over-expression were significantly localised to different sites; and showed significant inverse correlation. They appear distinct groups of tumours with different characteristics. There were no prognostic implications for patients with colorectal cancers by doing the immunohistochemical staining for either mismatch repair or p53. 22. COMPARISON OF IMMUNOHISTOCHEMISTRY ANALYSIS METHODS USING CANCER STEM CELL MARKERS T. Miller1,2, M. McCoy2,3, C. Hemmings1,4, B. Iacopetta1, C. Platell1,2 1 School of Surgery, University of Western Australia, Nedlands, 2 Colorectal Research Unit, St. John of God Subiaco Hospital, Subiaco, 3School of Medicine and Pharmacology, University of Western Australia, Nedlands, and 4St. John of God Pathology, Subiaco, Australia Background: Immunohistochemistry (IHC) studies have produced inconsistent results regarding the prognostic significance of cancer stem cell (CSC) marker expression in colorectal cancer. The method of tissue selection and quantification of expression varies widely between studies. The use of more rigorous methodology for IHC analysis may help to improve the reproducibility of published data. Objective: To compare three different methods for area of tissue selection on results obtained by automated digital analysis using the Aperio software platform. Methods: Formalin-fixed, paraffin-embedded sections of tumour and normal mucosa from 10 specimens of surgically resected stage II colorectal cancers were stained with H&E and with 5 CSC markers using IHC. Regions of interest (ROI) were selected and scored in semi-quantitative fashion. Digital analysis was performed on the selected ROIs, on full-face sections with annotations, and on the whole section. Statistical analysis for concordance was performed using Kendall’s W and tau-b. Findings: The three digital analysis methods showed concordant results for the majority of IHC staining of CSC markers. Discordant results were likely due to the inclusion of stained luminal areas and of non-specific background staining depending on the method of analysis used. Conclusions: The selection of tissue areas for evaluation of biomarker expression can have considerable impact on the results from IHC studies. Proper consideration should therefore be given to these factors during the study design in order to improve the reproducibility of published data. 23. VISUAL QUANTIFICATION OF RENAL INTERSTITIAL FIBROSIS: INTER AND INTRAOBSERVER VARIATIONS J. J. Khoo, W. K. Tey, V. Tan, S. W. Peter, Y. C. Kuang, M. P. L. Ooi Monash University Malaysia, Malaysia
Pathology (2017), 49(S1)
Background and Aims: Assessment of renal interstitial fibrosis is an important evaluation in renal biopsy as it may correlate to renal function and clinical outcome of the patient. However, quantification of renal fibrosis may not be consistent nor reliable. We aim to investigate the inter- and intra-observer variability amongst pathologists in quantification of renal interstitial fibrosis. Methods: Retrospective study using renal biopsies from 24 patients with a range of fibrosis extent. Masson Trichrome stained sections of the biopsies were scanned and 140 images were taken in various areas of the slides. Five histopathologists performed visual scoring of the fibrosis on the images. The exercise was repeated with reshuffled images to study for intra-observer variability. Intraclass correlation coefficient (ICC) and Fleiss kappa test was calculated to measure inter-observer variability and Cohen kappa test for intra-observer variability. Results and Discussion: The patients had a range of renal disease with glomerular filtration rate (eGFR) that varied from 8.7 to 134.7 mL/min. Although eGFR was negatively correlated with renal fibrosis but it was not significant (p = 0.448). The pathologists showed moderate correlation amongst each other in their evaluation of interstitial fibrosis (ICC = 0.6196) but the Fleiss kappa test was low at 0.0349 (p = 0.0025). This suggested that the pathologists while agreeing in different relative scales of quantification of fibrosis, their actual scores for the same images varied significantly amongst each other. The Cohen kappa test showed that all 5 pathologists also did not have a strong agreement between their first and second round of scoring (k = 0.2323 to 0.3129). Conclusions: Pathologists showed poor reliability in their evaluation of renal interstitial fibrosis and had fairly wide variability amongst each other. There appears a need for strict criteria guidelines for quantification of renal interstitial fibrosis to ensure proper and more consistent assessment of renal interstitial fibrosis. 24. RETROPERITONEAL LYMPHANGIOLEIOMYOMATOSIS T. Shein1, A. Field2 1 Sydpath, St Vincent’s Hospital, Sydney, and 2Department of Anatomical Pathology, St Vincent’s Hospital, and Notre Dame University Medical School, Sydney, NSW, Australia Background: Lymphangioleiomyomatosis (LAM) is a rare progressive disease that almost exclusively occurs in women of reproductive age. It is characterized by formation of thin-walled cysts and a hamartomatous proliferation of immature smooth muscle cells and lymphatics. Objectives: Description of a case of a primary retroperitoneal lymphangioleiomyomatosis, and a brief discussion of the diagnosis and management. Methods: Review of histopathology, imaging and literature. Findings: A 31 year old lady presented with a two-day history of abdominal pain and fever. CT scan on admission showed a large, complex, fluid-attenuating lesion in the left retroperitoneal region. A FNAB was performed and drained a large amount of milky, chyle-like fluid from the lesion. There was ongoing chylefluid leakage from this large multicystic lesion and surgical excision was carried out. A large circumscribed spongiform cystic mass covered partially by a glistening grey membranous surface was excised intact. Histologically, the lesion had a thin pseudocapsule in most areas and was composed of ramifying and anastomosing dilated lymphatics lined by D2-40 positive
PATHOLOGY 2017 ABSTRACT SUPPLEMENT
statistical difference (p = 0.615). Patients with p53 positive tumours also showed no difference in survival to those without p53 staining (p = 0.741). There was no significant difference between survivals of patients with mismatch repair defect CRCs compared to patients with tumours that showed p53 over-expression. Conclusion: MMR-d colorectal carcinomas and CRCs with p53 over-expression were significantly localised to different sites; and showed significant inverse correlation. They appear distinct groups of tumours with different characteristics. There were no prognostic implications for patients with colorectal cancers by doing the immunohistochemical staining for either mismatch repair or p53. 22. COMPARISON OF IMMUNOHISTOCHEMISTRY ANALYSIS METHODS USING CANCER STEM CELL MARKERS T. Miller1,2, M. McCoy2,3, C. Hemmings1,4, B. Iacopetta1, C. Platell1,2 1 School of Surgery, University of Western Australia, Nedlands, 2 Colorectal Research Unit, St. John of God Subiaco Hospital, Subiaco, 3School of Medicine and Pharmacology, University of Western Australia, Nedlands, and 4St. John of God Pathology, Subiaco, Australia Background: Immunohistochemistry (IHC) studies have produced inconsistent results regarding the prognostic significance of cancer stem cell (CSC) marker expression in colorectal cancer. The method of tissue selection and quantification of expression varies widely between studies. The use of more rigorous methodology for IHC analysis may help to improve the reproducibility of published data. Objective: To compare three different methods for area of tissue selection on results obtained by automated digital analysis using the Aperio software platform. Methods: Formalin-fixed, paraffin-embedded sections of tumour and normal mucosa from 10 specimens of surgically resected stage II colorectal cancers were stained with H&E and with 5 CSC markers using IHC. Regions of interest (ROI) were selected and scored in semi-quantitative fashion. Digital analysis was performed on the selected ROIs, on full-face sections with annotations, and on the whole section. Statistical analysis for concordance was performed using Kendall’s W and tau-b. Findings: The three digital analysis methods showed concordant results for the majority of IHC staining of CSC markers. Discordant results were likely due to the inclusion of stained luminal areas and of non-specific background staining depending on the method of analysis used. Conclusions: The selection of tissue areas for evaluation of biomarker expression can have considerable impact on the results from IHC studies. Proper consideration should therefore be given to these factors during the study design in order to improve the reproducibility of published data. 23. VISUAL QUANTIFICATION OF RENAL INTERSTITIAL FIBROSIS: INTER AND INTRAOBSERVER VARIATIONS J. J. Khoo, W. K. Tey, V. Tan, S. W. Peter, Y. C. Kuang, M. P. L. Ooi Monash University Malaysia, Malaysia
Pathology (2017), 49(S1)
Background and Aims: Assessment of renal interstitial fibrosis is an important evaluation in renal biopsy as it may correlate to renal function and clinical outcome of the patient. However, quantification of renal fibrosis may not be consistent nor reliable. We aim to investigate the inter- and intra-observer variability amongst pathologists in quantification of renal interstitial fibrosis. Methods: Retrospective study using renal biopsies from 24 patients with a range of fibrosis extent. Masson Trichrome stained sections of the biopsies were scanned and 140 images were taken in various areas of the slides. Five histopathologists performed visual scoring of the fibrosis on the images. The exercise was repeated with reshuffled images to study for intra-observer variability. Intraclass correlation coefficient (ICC) and Fleiss kappa test was calculated to measure inter-observer variability and Cohen kappa test for intra-observer variability. Results and Discussion: The patients had a range of renal disease with glomerular filtration rate (eGFR) that varied from 8.7 to 134.7 mL/min. Although eGFR was negatively correlated with renal fibrosis but it was not significant (p = 0.448). The pathologists showed moderate correlation amongst each other in their evaluation of interstitial fibrosis (ICC = 0.6196) but the Fleiss kappa test was low at 0.0349 (p = 0.0025). This suggested that the pathologists while agreeing in different relative scales of quantification of fibrosis, their actual scores for the same images varied significantly amongst each other. The Cohen kappa test showed that all 5 pathologists also did not have a strong agreement between their first and second round of scoring (k = 0.2323 to 0.3129). Conclusions: Pathologists showed poor reliability in their evaluation of renal interstitial fibrosis and had fairly wide variability amongst each other. There appears a need for strict criteria guidelines for quantification of renal interstitial fibrosis to ensure proper and more consistent assessment of renal interstitial fibrosis. 24. RETROPERITONEAL LYMPHANGIOLEIOMYOMATOSIS T. Shein1, A. Field2 1 Sydpath, St Vincent’s Hospital, Sydney, and 2Department of Anatomical Pathology, St Vincent’s Hospital, and Notre Dame University Medical School, Sydney, NSW, Australia Background: Lymphangioleiomyomatosis (LAM) is a rare progressive disease that almost exclusively occurs in women of reproductive age. It is characterized by formation of thin-walled cysts and a hamartomatous proliferation of immature smooth muscle cells and lymphatics. Objectives: Description of a case of a primary retroperitoneal lymphangioleiomyomatosis, and a brief discussion of the diagnosis and management. Methods: Review of histopathology, imaging and literature. Findings: A 31 year old lady presented with a two-day history of abdominal pain and fever. CT scan on admission showed a large, complex, fluid-attenuating lesion in the left retroperitoneal region. A FNAB was performed and drained a large amount of milky, chyle-like fluid from the lesion. There was ongoing chylefluid leakage from this large multicystic lesion and surgical excision was carried out. A large circumscribed spongiform cystic mass covered partially by a glistening grey membranous surface was excised intact. Histologically, the lesion had a thin pseudocapsule in most areas and was composed of ramifying and anastomosing dilated lymphatics lined by D2-40 positive