Malignant papillary renal tumors with extensive clear cell change: A molecular analysis by microsatellite analysis and fluorescence in situ hybridization

Archives of Pathology & Laboratory Medicine, Sep 2003 by Salama, M E, Worsham, M J, DePeralta-Venturina, M

* Context.-Histologic subtyping of renal cell carcinomas (RCCs) is based not only on cytoarchitectural pattern but also on distinct cytogenetic abnormalities. Some renal tumors demonstrate overlapping morphologic features, rendering histologic subtyping difficult. One such group of tumors is papillary renal neoplasms with extensive clear cell change. Because histologic subtyping has been shown to be of prognostic value, it is important that malignant epithelial renal tumors be accurately subtyped. It is not known if these tumors should be classified as papillary RCC (PRCC) or as conventional/(clear cell) RCC (CRCC).

Objective.-To ascertain if this subgroup of renal neoplasms demonstrates the cytogenetic abnormalities seen typically in PRCC, that is, trisomy 7 and 17 or CRCC, that is, loss of 3p, using microsatellite analysis for loss of heterozygosity (LOH), and fluorescence in situ hybridization (FISH) for trisomies.

Design.-Seven RCCs from 6 patients that showed more than 75% papillary architecture and more than 75% clear cell change were included in the study. Tumor size ranged from 2.5 to 7.0 cm (mean 4.7 cm) and all were confined to the kidney (stage I). DNA was extracted from formalin-fixed paraffin-embedded tissue. FISH was done using In Situ Kits for centromere probes for chromosomes 7 and 17. For LOH, microsatellite analysis using labeled primers for 4 markers in the 3p13 through 3p24.2 region were used. The amplified polymerase chain reaction products were analyzed using an automated DNA sequencer. As compared with normal DNA, LOH in tumor was recognized as a loss of 1 allele, and microsatellite instability as the addition of an extra allele.

Results.-LOH in at least 1 of the markers spanning for 3pl3 through 3p24.2 was detected in 6 of 7 specimens (86%), of which 1 also showed concomitant microsatellite instability. FISH did not demonstrate trisomy for either chromosome 7 or 17. Instead, monosomy 7 was observed in 4 of 6 tumors (67%) and monosomy 17 in all tumors (100%).

Conclusion.-Because malignant papillary renal tumors with extensive clear cell change show molecular changes identical to CRCC, this subgroup of tumors may have to be classified as CRCC. This study underscores the utility of molecular studies in refining light-microscopic criteria in accurate histologic subtyping of RCCs.

The recently refined classification of adult renal epithelial neoplasms is based on morphologic grounds and incorporates current knowledge regarding genetic alterations in these tumors.1,2 Although most cases are easily classifiable into 1 of the 4 recognized histologic subtypes, that is, conventional or clear cell (CRCC), papillary (PRCC), chromophobe (CHRCC), and collecting duct carcinoma (CDC), some malignant renal tumors show overlapping histomorphologic characteristics. We have encountered renal cell carcinomas (RCCs) with predominantly papillary architecture but that were composed extensively of clear cells. Although focal clear cell change had been reported to occur in PRCC3 and focal papillary architecture could be seen in CRCC,4 the cytogenetic abnormalities of these 'hybrid' tumors are not known. Do these tumors show the distinct cytogenetic abnormalities associated with PRCC, that is, trisomy 7 and 17,5 or CRCC, which is characterized by deletion of 3p? Because recent studies have shown that histologic subclassification of RCC has prognostic impact,4 it is important that these hybrid tumors are assigned to their correct histologic classification as supported by their specific cytogenetic alterations.

To our knowledge, there is only 1 study in the literature that addressed this issue. Fuzesi et al from Germany analyzed 3 cases of PRCC with clear cell morphology using classic cytogenetics (standard G-banding techniques) and demonstrated loss of terminal 3p chromosomal segments with no trisomy 17 observed.6 In this study, we used molecular techniques, specifically fluorescence in situ hybridization (FISH) and microsatellite analysis, that is, detection of loss of heterozygosity and microsatellite instability, to determine the genetic alterations in 7 malignant renal tumors with papillary architecture and extensive clear cell change.

MATERIALS AND METHODS

Seven RCCs from 6 patients were selected from the archives of the Department of Pathology at Henry Ford Hospital. These cases were RCC with more than 75% papillary architecture and were composed of more than 75% clear cells (Figure 1). Number of hematoxylin-eosin slides per case ranged from 8 to 40 with a mean of 14. Blocks with tumor and normal renal tissue were selected for molecular analysis.

Fluorescence In Situ Hybridization.-Fluorescence in situ hybridization (FISH) was done using the Chromosome In Situ Kits for centromere probes for chromosomes 7 and 17 (Vysis, Downers Grove, Ill). The hybridization, signal detection, and amplification procedures were performed according to the manufacturer's published protocol with minor modifications. The procedure is outlined in detail in Worsham et al.7 In brief, the procedure is as follows: 4-[mu]m formalin-fixed paraffin-embedded tissue slides, dried and baked overnight at 65[degrees]C, were deparaffinized in xylene and washed with 100% ethanol. The slides were pretreated for 20 minutes in warm (43[degrees]C) sodium bisulfite solution (8 g of sodium bisulfite in 40 mL 2x SSC), washed in 2x SSC pH 7, and dehydrated in 70%, 80%, 90%, and 100% ethanol washes. The sections were digested with proteinase K (25 mg/mL) and dehydrated again in graded ethanol followed by an acetone wash. For hybridization, 3 [mu]L of biotinylated probe (10 ng/[mu]L) in 10 [mu]L of Hybrisol VIII (78% Formamide, 2.4x SSC, and blocking reagents) were pipetted onto the section and sealed under the coverslip with rubber cement, and then the probe and tissue DNA were denatured by heating the slide on a hot plate or in the oven at 90[degrees]C for 12 minutes, and incubated at 37[degrees]C overnight in a humidified chamber. The coverslips were removed, the slides were washed (4 mL 20x SSC, 16 mL distilled water, 20 mL Formamide at 37[degrees]C), and hybridized probes were detected by incubation for 20 minutes with 50-[mu]L fluorescein-labeled avidin and 50 [mu]L rhodamine-labeled anti-digoxigenin. The slides were washed and the avidin signal was further amplified by incubation for 20 minutes in fluorescein-labeled anti-avidin. After washing, the nuclei were stained with 4,6 diamidino-2-phenylindole (DAPI). Coverslips were placed, sealed with nail polish, and the nuclei examined using a fluorescent microscope equipped to detect green, red, and blue fluorescence with filters for rhodamine/DAPI and fluorescein detection. For each probe, 200 to 300 nuclei were scored on 4-[mu]L histologic sections in areas marked to correspond with hematoxylin-eosin sections.