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Abstract and Introduction
Abstract
Genetic alteration of the von Hippel-Lindau (VHL) tumor suppressor gene has been linked to hereditary and sporadic clear cell renal cell carcinomas (RCCs). Inconsistent data on immunodetection of the VHL gene product (pVHL) in normal tissues and tumors have been reported. We immunohistochemically reevaluated the usefulness of a specific rabbit polyclonal anti-pVHL antibody in 531 cases of renal and nonrenal neoplasms and normal tissues. Positive immunostaining was observed in nearly 100% of primary renal neoplasms, 95% of metastatic RCCs, and 90% of clear cell carcinomas of the ovary and uterus. In normal tissues, positive immunoreactivity was observed only in renal tubules, exocrine pancreas, islets, and bile ducts. Western blot and reverse transcription–polymerase chain reaction confirmed the immunostaining results. These data indicate that this anti-pVHL antibody is a useful marker in assisting diagnosis of metastatic RCC and may serve as a diagnostic marker for clear cell carcinomas of the ovary and uterus.
Introduction
Renal cell carcinoma (RCC) is the ninth most common malignant tumor in the United States, accounting for approximately 2% of all cancers. In 2006, more than 38,890 new cases of RCC were diagnosed, and an estimated 12,840 people died of this cancer in the United States alone.[1,2] When patients have local disease at initial examination, a favorable prognosis is usually anticipated; however, metastatic RCC has a much worse prognosis.[2] Identification of a metastatic RCC is not always straightforward because RCC is notorious for distant metastases years after the initial diagnosis and treatment. In addition, the morphologic complexity of RCC may create a diagnostic challenge. According to the World Health Organization classification,[3] RCC is subdivided into clear cell, chromophobe, papillary, collecting duct, multilocular cystic renal cell, medullary, mucinous tubular spindle cell, and unclassified RCC. Because of their histologic variants and morphologic complexity, RCCs frequently show histologic features that resemble many other neoplasms. It can be challenging to distinguish them from other metastatic malignancies based on morphologic features alone, especially when a specimen is limited, as in small tissue core biopsies or fine-needle aspiration biopsy specimens.
Many attempts have been made to identify sensitive and specific markers for RCCs. Some successes have been achieved. Coexpression of epithelial membrane antigen and vimentin is frequently present in clear cell RCC.[4] CD10 was detected in approximately 90% of RCCs, but also in many hepatocellular carcinomas, prostatic carcinomas, and urothelial carcinomas.[5,6] The RCC has a diagnostic sensitivity of 84% to 92% in primary RCC and 67% to 84% in metastatic RCC.[5,7,8] Carbonic anhydrase IX (MN/CA9), a tumor-associated antigen, was positive in 100% of clear cell RCCs and papillary RCCs, but it was also expressed in cervical dysplasia and carcinoma.[9] P504S, initially considered to be a specific marker for prostatic adenocarcinoma, has been shown to be positive in 100% of papillary RCCs and some primary and metastatic clear cell RCCs.[10-14] In addition, glutathione S-transferase α and carbonic anhydrase II were demonstrated to be specific markers for clear cell RCC and chromophobe RCC, respectively.[15] Expression of S-100 protein has been shown in 70% of clear cell RCCs but also was positive in many nonrenal tumors.[16]
Recently, we introduced human kidney injury molecule-1 (KIM-1), also known as TIM-1 (T-cell immunoglobulin domain and mucin domain protein 1) or HAVCR1 (hepatitis A virus cellular receptor), for diagnosing RCCs.[17] Our data showed that expression of KIM-1 was present in approximately 75% of primary and metastatic clear cell RCCs and nearly 100% of papillary RCCs. Most clear cell carcinomas of the ovary were also positive for KIM-1.[17] In contrast with previously published findings, KIM-1 is probably one of the most promising biomarkers, with the highest specificity in diagnosing RCCs. With a diagnostic sensitivity of 75%, it is useful but not ideal for detecting metastatic RCCs; therefore, we continue searching for a more sensitive marker.
Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene on chromosome 3p25-26 by mutation, deletion, or hypermethylation is a frequent event in hereditary and sporadic clear cell RCC.[2,18,19] Genetic alteration of the VHL gene seems to be a crucial step in the initiation and progression of clear cell RCC.[2,20] RCC cell lines with biallelic VHL inactivation form RCC when injected into the skin of nude mice; however, the tumors abrogate when VHL function is restored, which indicates that loss of VHL has a role in the initiation and maintenance of the tumor.[21]
The VHL gene encodes 2 biologically active proteins (pVHL) with molecular weights of 30 kd (pVHL30) and 19 kd (pVHL19).[22] The short form of pVHL seems to arise as a result of alternative translation initiation. Only limited data are available on the immunolocalization of pVHL in RCCs, normal tissues, and nonrenal tumors.[23-28] Notably, some of the data are inconsistent with regard to the compartmentalization and distribution of pVHL. Some technical challenges are also encountered.[23] Corless and coworkers[23] reported cytoplasmic staining of VHL in normal epithelial cells of many organs and various types of carcinomas, including clear cell RCC and carcinoma of the lung, prostate, colon, breast, bladder, and thyroid on frozen sections; however, the immunostains did not work on formalin-fixed, paraffin-embedded sections. Schraml et al[27] showed cytoplasmic localization of the VHL protein when using antibody to pVHL30, but nuclear and cytoplasmic staining for the VHL protein was noted when using antibody to pVHL30 and pVHL19.
In the present study, we used a well-characterized rabbit polyclonal antibody against the full-length VHL protein of human origin, attempting to reevaluate the expression pattern, the distribution in normal tissues, and the diagnostic usefulness of pVHL in a large series of specific subtypes of renal cell tumors, metastatic RCCs, and nonrenal tumors, using formalin-fixed, paraffin-embedded tissue microarray (TMA) and routine tissue sections along with Western blots and reverse transcription–polymerase chain reaction (RT-PCR). Our data not only confirmed and expanded the findings in some of the previous reports but also, more important, revealed the additional usefulness of this particular rabbit polyclonal anti-pVHL antibody in assisting diagnosis of metastatic RCC and detecting clear cell carcinoma of the ovary and uterus.Materials and Methods
Case Selection
The study was approved by the institutional review boards at Geisinger Medical Center, Danville, PA, and the coauthors' institutions. We retrieved 526 cases of renal epithelial neoplasms and nonrenal tumors from the archives of the Department of Laboratory Medicine, Geisinger Medical Center, and the departments of pathology from coauthors' institutions. Tissue samples were made anonymous and disassociated from any clinical data. TMA sections and routine large tissue sections were used in the study. The TMA sections included 80 conventional (clear cell) RCCs, 57 papillary RCCs, 15 chromophobe RCCs, 14 renal oncocytomas, 24 cases of nonneoplastic renal cortical-medullary tissue, and 94 nonrenal carcinomas. Cases in TMA sections were different from cases in routine sections. Routine sections from 37 metastatic clear cell RCCs, 11 chromophobe RCCs, 10 oncocytomas, and 213 nonrenal carcinomas from various organs were also included. Nonneoplastic tissues adjacent to the tumors were evaluated as well.
TMA Construction
The H&E-stained sections for the selected cases were reviewed, and the targeted areas were marked on the H&E-stained sections and the corresponding paraffin blocks. One 1.5-mm tissue core for each case was removed using a manual microarray device (Beecher Instruments, Sun Prairie, WI) and inserted into recipient paraffin blocks as described.[15,29]
Immunohistochemical Analysis Using a Rabbit Polyclonal Anti-pVHL Antibody
Immunohistochemical stains were performed on formalin-fixed, paraffin-embedded 4-µm sections of the TMA and routine tissue sections. The tissue sections were deparaffinized, and antigen retrieval was carried out in Proteinase K solution for 10 minutes at room temperature. Six antigen retrieval methods were tested before the Proteinase K retrieval method was chosen (see the "Discussion" section). The sections were incubated in 3% hydrogen peroxidase for 5 minutes to quench endogenous tissue peroxidase. The tissue sections were then incubated with a rabbit polyclonal antibody against pVHL (VHL [FL181]:sc-5575, Santa Cruz Biotechnology, Santa Cruz, CA)[6,21] at a 1:50 dilution for 30 minutes at room temperature. The slides were stained in a DAKO automated immunostainer using a standard EnVision-HRP kit (DAKO, Carpinteria, CA) as previously described.[16] The EnVision-HRP detection kit is designed to significantly reduce or eliminate nonspecific endogenous biotin activity. Immunohistochemical reactions were developed with diamino-benzidine as the chromogenic peroxidase substrate, and slides were counterstained with hematoxylin. Normal kidney tissue served as the positive control sample; negative control samples included replacement of the primary antibody with nonimmune rabbit serum.
Two surgical pathologists (F.L. and H.L.) independently evaluated the immunostained TMA and regular tissue sections. The staining results were recorded in a semiquantitative manner as follows: negative, less than 5% of tumor cells stained; 1+, 6% to 25%; 2+, 26% to 50%; 3+, 51% to 75%; and 4+, more than 75%. The staining intensity was also assessed and recorded as follows: weak, light brown, fine, granular cytoplasmic/membranous staining; or strong, dark brown, granular cytoplasmic/membranous staining. Nuclear staining, if present, was also recorded as a positive result (only when >5% of cells stained).
Immunohistochemical Analysis Using a Mouse Monoclonal Anti-VHL Antibody
To further validate the immunohistochemical staining results from this rabbit polyclonal anti-pVHL antibody, we also performed immunostains on selected TMA sections of renal and nonrenal tumors and VHL-related tumors (hemangioblastoma and pheochromocytoma) using another commercially available mouse monoclonal anti-pVHL antibody. This monoclonal antibody was used in the study by Schraml et al.[27]
Immunohistochemical stains were performed on formalin-fixed, paraffin-embedded 4-µm histologic sections. Six antigen retrieval methods were carried out: (1) target retrieval solution buffer (water bath heating for 20 minutes; cooling for 20 minutes); (2) TUF buffer (water bath heating for 10 minutes; cooling for 10 minutes); (3) EDTA (microwave heating for 20 minutes; cooling for 20 minutes); (4) citrate buffer (microwave heating for 20 minutes; cooling for 20 minutes); (5) Proteinase K, 10 minutes; and (6) no special treatment.
The staining procedure was the same as for the polyclonal antibody. The tissue sections were then incubated with the mouse monoclonal antibody to pVHL (catalog No MS-690-P, clone Ig33, dilution 1:50; LabVision, NeoMarkers, Fremont, CA). The slides were stained in a DAKO automated immunostainer using a standard EnVision-HRP detection kit as previously described.
Western Blot
Two Western blots were performed. Western blot 1 contained 6 renal tumor samples with different histologic subtypes (2 clear cell carcinomas, 2 oncocytomas, and 2 chromophobe RCCs) and the corresponding normal renal tissues from the same cases of clear cell RCCs. Renal tumor tissues were obtained from the central portion of tumors to avoid potential contamination by the adjacent nonneoplastic kidney tissue. Western blot 2 contained 4 nonrenal carcinoma tissues and 1 each of clear cell carcinoma of the ovary, clear cell carcinoma of the uterus, pancreatic adenocarcinoma, and colon adenocarcinoma.
Western blots were performed as previously described.[16] In brief, the frozen tissue blocks containing renal tumors, the corresponding normal tissues, and nonrenal carcinomas were retrieved from the Department of Laboratory Medicine, Geisinger Medical Center, and the departments of pathology at the coauthors' institutions, thawed, cut into tiny pieces, and then lysed in the lysis buffer. Tissue homogenates from each case were electrophoresed on 4% to 20% sodium dodecyl sulfate–polyacrylamide gradient gel electrophoresis. The fractionated proteins were transferred onto polyvinyl difluoride membranes. The membranes were incubated with the same rabbit polyclonal anti-pVHL antibody used for immunohistochemical analysis at a dilution of 1:500. The appropriate secondary antibody conjugated with horseradish peroxidase was used. Immunoreactive proteins were visualized by an enhanced chemiluminescence–Western blotting system (Amersham Pharmacia Biotech, Piscataway, NJ). Incubating with antibody to β-actin and β-tubulin (dilution 1:2,000; Sigma, St Louis, MO) was also performed to evaluate the evenness of the protein loading.
Analysis of VHL Messenger RNA in Renal Tumor Subtypes by RT-PCR
Total RNA was isolated according to the manufacturer's protocol. In brief, renal tumors and normal renal tissues from the same cases as in Western blot 1 were used. The renal tissues were cut into small slices and were homogenized in Trizol solution (Invitrogen, Carlsbad, CA) using a Polytron PT 1200 homogenizer (VWR, Bridgeport, NJ). The tissue lysate was centrifuged, and the supernatant was transferred into a new microcentrifuge tube. Chloroform was added to the lysate. The lysate was spun at 12,000 rpm for 15 minutes at 4°C. The supernatant was transferred to a new Eppendorf tube and incubated with isopropanol for 30 minutes at –20°C. The supernatant was removed and washed with 75% ethanol and spun down. RNA precipitates were dissolved in diethylprocarbonate-treated water. The RNA concentration was measured.
Three sets of VHL primers were synthesized (Integrated DNA Technologies, Coralville, IA) based on a published article,[30] which covered exons 1, 2, and 3. The β-actin primer set was purchased from Stratagene (catalog No. 302010, Stratagene, La Jolla, CA). The 3 sets of VHL primers to cover the 3 VHL exons are listed in Table 1 .
RT-PCR was performed using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit (Invitrogen) according to the protocol from the manufacturer. The complementary DNA was amplified with the aforementioned specific primer sets for 1 cycle. PCR amplification was performed for 40 cycles with the following conditions: denaturing at 94°C for 15 seconds, annealing at 55°C for 50 seconds, and extension at 72°C for 1 minute. PCR products were electrophoresed on 1.2% agarose gel and were detected with the image analysis software, AlphaImager (Alpha Innotech, San Leandro, CA).
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