Transcript hKIM-1
Journal Reading: Human Kidney Injury Molecule-1 (hKIM1): A Useful Immunohistochemical Marker for Diagnosing Renal Cell Carcinoma and Ovarian Clear Cell Carcinoma Am J Surg Pathol Volume 31, Number 3, March 2007 Fan Lin, MD, PhD,etc From the Department of Laboratory Medicine, Geisinger Medical Center Presented by Intern 賴妮均 Introduction (1) 9th most Common malignant tumors in U.S. Notorious for distant metastasis Lung, lymph node, liver, bone, and brain Subtypes: Conventional; (clear cell) carcinoma Papillary carcinoma Chromophobe carcinoma D/D with oncocytoma (benign) Collecting duct carcinoma Unclassified carcinoma Introduction (2) Immunostaining Markers in RCC CD10 90% of RCCs also detected in many hepatocellular carcinomas, prostatic carcinomas, and urothelial carcinomas Positive in normal renal tissue RCC Ma (RCC marker) 84-92% of primary RCC and 67-84% 0f metastatic RCC Positive in normal renal tissue Coexpression of epithelial membrane antigen and vimentin clear cell RCC MN/CA9 (Carbonic anhydrase IX) 100% of clear cell RCC and papillary RCC Also expressed in cervical dysplasia and carcinoma P504S 100% of papillary RCCs and some primary and metastatic clear cell RCC marker for prostatic adenocarcinoma Positive in normal renal tissue CD9/MPR1), Antimitochondrialantibody (113–1), E-cadherin and vinculin high sensitivity for chromophobe RCC, papillary RCC, and oncocytoma Osteonectin (SPARC) sarcomatoid RCC Introduction (3) B defensin-1 (+), parvalbumin (+), and vimentin (-) 100 % of Chromophobe RCC and oncocytoma glutathione S-transferase-a and carbonic anhydrase II(CAII) Clear cell RCC and chromophobe RCC kidney-specific cadherin (Ksp-cadherin) and Ron proto-oncogene Majority of both chromophobe RCC and oncocytoma CD117 (c-kit) chromophobe RCC and some oncocytoma S-100 protein 70% of clear cell RCC and 90% of oncocytoma frequently absent in clear cell RCC Ksp-cadherin :Positive in normal renal tissue frequently absent in clear cell RCC only observed in 6% of chromophobe RCC Positive in normal renal tissue none of the aforementioned markers is absolutely sensitive and specific for RCC Introduction (4) Human kidney injury molecule-1 (hKIM-1) = TIM-1 (T cell immunoglobin domain andmucin domain protein 1) = HAVCR1 (hepatitis A virus cellular receptor 1) Gene: located in human chromosome 5q Type I transmembranous glycoprotein containing an extracellular immunoglobulinlike domain topping a long mucinlike sequence As biomarker for regeneration of renal proximal tubules after tubular injury.3 Material and Method (1) Case Selection 264 cases of renal epithelial neoplasm anonymous and disassociated from any clinical data Material and Method (2) TMA sections 221 cases of nonrenal tumors from various organs to test the specificity of anti-hKIM-1 antibody Material and Method (3) TMA construction H&E stain sections were reviewed remove 1.0-1.5 mm tissue core from marked area of paraffin block inserted into recipient paraffin block (Beecher Instruments Inc, Sun Prairie, WI) Material and Method (4) Immunohistochemistry Formalin-fixed and paraffin-embedded 4μm sections Antibody to hKIM-1 AKG7 monoclonal antibody Confirmed by ABE3 antibody in CCCO and uterus Positive control: Kidney tissues with acute tubular necrosis Negative control: replacement of the primary antibody with nonimmune mouse serum Interpretation: 2 surgical pathologists indicates negative; 1+, <10%; 2+, 11% to 50%; 3+, >50%. Material and Method (5) Western Blot 4 cases of renal tumor with different subtypes Normal renal tissue from a clear cell RCC case Frozen tissue blocksLysed tissue homogenatws Electrophoresed on 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) Transferred onto polyvinyl diflouride membranes Incubated with AKG7 monoclonal antibody Evenness of the protein loading: antibody to β-actin Material and Method (6) Gene Expression Microarray tissues were frozen promptly after surgery and stored at -80℃. hKIM-1 mRNA level Results (1) Non-neoplastic renal tissue Negative: Glomeruli, vessels, inflammatory cells, interstitialcells, lymphoid aggregates interstitial tissues Positive immunoreactivity was observed in proximal tubules, with a cytoplasmic / membranous staining pattern during acute tubular necrosis 2 of 20 case in TMA showed focal immunostaining occasional proximal tubules Result (2) Renal Epithelial Neoplasm (2/15) in TMA Result (3) Clear cell RCC, Papillary RCC Result (4) Chromophobe RCC Result (5) Diffuse immunoreactivity was more frequently observed in highgrade clear cell RCC (56%) than in low-grade clear cell 2/2 sacromatoid RCC were positive 1 case of Wilms tumor and no immunoreactivity was observed (data not shown) Result (6) Metastatic clear cell RCC The percentages of positive cases were not significantly different among theseanatomic sites. Result (7) Non-renal Neoplasms Result (8) CCCO Result (9) Clear cell carcinoma of uterus Adenocarcinoma of the colon Result (10) Western Blot Result (11) hKIM-1 mRNA in Renal Tumors From Affymetrix Microarrays 537 285 0 -131 -126 Discussion Do we really need another immunohistochemical marker? Discussion Differentiated from normal renal tissue The previously reported biomarkers are expressed in normal renal tissue Murine KIM-1 expressed in actived T cells and linked to air hypersensitivity, but hKIM-1 does not Negative staining in: Non-neoplastic renal tissues Transplanted renal biopsy with acute cellular rejection Positive staining in: (1) proximal tubules adjacent to renal tumors (2) proximal and distal tubules in the tumorous kidney, probably secondary to tumor obstruction and compression (3) atrophic renal tubules; (4) proximal tubules associated with tubular injury,such as hypoxia, ischemia, toxin, infection, etc Discussion More specific to clear cell RCC and papillary RCC Oncocytoma: only 4 of 41 (9.8%) with focally positive stained Chromophobe RCC: all negative CD-10 also expressed in oncocytoma RCC Ma also expressed in chromophobe RCC P504S also expressed in both oncocytoma and chromophobe RCC S-100 also expressed in >90% oncocytoma Discussion More specific to metastatic RCC Han et al: only 1 of 450 nonrenal tumor was positive (HCC) CD10, RCC Ma, S-100, P504S, glutathione Stransferase-a, CD117: well-documented in other nonrenal tumors Why higher positive reaction than previoud Han’s study? CCCO and uterus were not included The previous study only checked TMA Discussion What are the unique features and utility of this antibody? Discussion Expression of hKIM-1 in CCCO (93.8%) Both ovary and uterus serous carcinoma and endometroid carcinoma lacked hKIM-1 expression Dent and coworkers: gene amplification in CCCO was most common at chromosome 3 Von Hippel-Lindau tumor-suppressor genes: located at the chromosome 3p25; and the von Hippel-Lindau tumorsuppressor closely linked to the tumorigenesis of both sporadic and hereditary clear cell RCC hKIM-1 had similar role in both clear cell RCC and CCCO? Anti-hKIM-1Ab may be used in targeted cancer therapy for CCCO? Discussion clear cell carcinoma of the uterus only 33% expressed hKIM-1 not related to chromosome 3 alternation Clear cell carcinoma of uterus Adenocarcinoma of the colon Discussion Focal positive staining in adenocarcinoma of the colon 5 of 40 cases Pattern: apical portions of neoplastic glands Only in well and moderately differentiatied colonic adenocarcinoma the AKG7 anti-hKIM-1 monoclonal antibody:against the extracellular mucin domaincross reaction Disscusion Conventional RCC Chromophobe RCC S-100+/hKIM-1+/MSH2-/CK7MSH2+ (cytoplasmic staining) /CK7+/S-100-/hKIM-1- Oncocytoma S-100+/MSH2+ (apical and nuclear staining) /hKIM-1-/CK7-or focal + Conclusion HKIM-1 is a relatively sensitive and specific marker for confirming the diagnosis of papillary RCC, clear cell RCC, and metastatic RCC distinguishing clear cell RCC from chromophobe RCC and oncocytoma identifying CCCO Thanks for your attention !