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Fan Lin, MD, PhD,etc From the Department of Laboratory Medicine, Geisinger Medical Center Presented by Intern 賴妮均

Journal Reading: Human Kidney Injury Molecule-1 (hKIM-1): 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

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Fan Lin, MD, PhD,etc From the Department of Laboratory Medicine, Geisinger Medical Center Presented by Intern 賴妮均

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  1. Journal Reading:Human Kidney Injury Molecule-1 (hKIM-1): A Useful Immunohistochemical Marker for Diagnosing Renal Cell Carcinoma and Ovarian Clear Cell CarcinomaAm 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

  2. 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

  3. Introduction (2)

  4. Introduction (3)  none of the aforementioned markers is absolutely sensitive and specific for RCC

  5. 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

  6. Material and Method (1) • Case Selection • 264 cases of renal epithelial neoplasm • anonymous and disassociated from any clinical data

  7. Material and Method (2) • TMA sections • 221 cases of nonrenal tumors from various organs to test the specificity of anti-hKIM-1 antibody

  8. 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)

  9. 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%.

  10. 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 blocksLysed tissue homogenatws Electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)Transferred onto polyvinyl diflouride membranes Incubated with AKG7 monoclonal antibody • Evenness of the protein loading: antibody to β-actin

  11. Material and Method (6) • Gene Expression Microarray • tissues were frozen promptly after surgery and stored at -80℃. • hKIM-1 mRNA level

  12. 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

  13. (2/15) in TMA Result (2) • Renal Epithelial Neoplasm

  14. Result (3) • Clear cell RCC, Papillary RCC

  15. Result (4) • Chromophobe RCC

  16. Result (5) • Diffuse immunoreactivity was more frequently observed in high-grade 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)

  17. Result (6) • Metastatic clear cell RCC • The percentages of positive cases were not significantly different among theseanatomic sites.

  18. Result (7) • Non-renal Neoplasms

  19. Result (8) • CCCO

  20. Result (9) • Adenocarcinoma of the colon • Clear cell carcinoma of uterus

  21. Result (10) • Western Blot

  22. 537 285 0 -131 -126 Result (11) • hKIM-1 mRNA in Renal Tumors From Affymetrix Microarrays

  23. Discussion • Do we really need another immunohistochemical marker?

  24. 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

  25. 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

  26. 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 S-transferase-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

  27. Discussion • What are the unique features and utility of this antibody?

  28. 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?

  29. Discussion • clear cell carcinoma of the uterus • only 33% expressed hKIM-1 • not related to chromosome 3 alternation

  30. Adenocarcinoma of the colon • Clear cell carcinoma of uterus

  31. 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 domaincross reaction

  32. Disscusion • Conventional RCC • S-100+/hKIM-1+/MSH2-/CK7- • Chromophobe RCC • MSH2+ (cytoplasmic staining) /CK7+/S-100-/hKIM-1- • Oncocytoma • S-100+/MSH2+ (apical and nuclear staining) /hKIM-1-/CK7-or focal +

  33. 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

  34. Thanks for your attention !

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