Journal Reading:Human Kidney Injury Molecule-1 (hKIM-Human Kidney Injury Molecule-1 (hKIM-1): A Useful Immunohistochemical Mark1): A Useful Immunohistochemical Marker for Diagnosing Renal Cell Carcinoma er for Diagnosing Renal Cell Carcinoma and Ovarian Clear Cell Carcinomaand 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 carcinomasPositive 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

P504S100% of papillary RCCs and some primary and metastatic clear cell RCC

marker for prostatic adenocarcinomaPositive 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) andRon proto-oncogene

Majority of both chromophobe RCC and oncocytoma

frequently absent in clear cell RCC Ksp-cadherin :Positive in normal renal tissue

CD117 (c-kit)chromophobe RCC and some oncocytoma

frequently absent in clear cell RCC

S-100 protein70% of clear cell RCC and 90% of oncocytoma

only observed in 6% of chromophobe RCCPositive 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 mucinl

ike sequence As biomarker for regeneration of renal proximal tubules after tubular injury.3

Material and Method (1)

Case Selection264 cases of renal epithelial neoplasmanonymous 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 constructionH&E stain sections were reviewedremove 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 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

Material and Method (6)

Gene Expression Microarray tissues were frozen promptly after surgery an

d stored at -80 .℃hKIM-1 mRNA level

Results (1) Non-neoplastic renal tissue

Negative: Glomeruli, vessels, inflammatory cells, interstitialcells, lymphoid aggregatesinterstitial 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 high-grad

e 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 c

ases 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 Affy

metrix Microarrays

0

-131

537

285

-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 h

KIM-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 ob

struction and compression (3) atrophic renal tubules; (4) proximal tubules associated with tubular injury,such as hypoxia, ischemia, toxin, inf

ection, 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 S-transfer

ase-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 la

cked 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 uterusonly 33% expressed hKIM-1not 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 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 fo

cal +

Conclusion

HKIM-1 is a relatively sensitive and specific marker for confirming the diagnosis of papillary RCC, cle

ar cell RCC, and metastatic RCCdistinguishing clear cell RCC from chromopho

be RCC and oncocytoma identifying CCCO

Thanks for your attention !