Patients and samples
This study was conducted on 28 archival formalin fixed paraffin-embedded (FFPE) specimens from renal metanephric adenomas. FFPE blocks and H&E stained slides were obtained from the departments of pathology at four participating institutions, including Advocate Lutheran General Hospital (Park Ridge, IL, USA), Northwestern Memorial Hospital (Chicago, IL, USA), Children’s Medical Center of Dallas (Dallas, TX, USA), and Charles University Hospital (Plzen, Czech Republic). Fourteen cases were of American origin and 14 of European origin. All samples received for this study and data reported have been de-identified. Because this was a retrospective study, ethics committee ruled that no formal ethics approval was required in this particular study.
The MA specimens and hematoxylin and eosin-stained slides were retrieved and reviewed by expert pathologists at each institution. All pathologic specimens were acquired after partial or complete nephrectomy, and none were diagnosed by needle biopsy. The diagnosis of MA was then re-confirmed by two genitourinary pathologists (Fig. 1) and de-identified representative tissue blocks were further selected for BRAFV600E exon 15 sequencing and FISH analysis. Histologically, the tumors are composed of epithelial cells arranged in tubules and papillary configurations. The relatively small tumor cells have a high nuclear: cytoplasmic ratio, ovoid nuclei, uniformly dispersed chromatin, inconspicuous nucleoli, scant eosinophilic cytoplasm, and ill-defined cell borders with nuclear overlap, Fig. 1, a and b. Mitoses are not conspicuous. Occasional psammomatoid calcifications are seen, Fig. 2. Patient demographics and clinicopathologic characteristics such as: age, gender, tumor size, laterality, and chromosomal analysis results were provided by pathologists at each institution (Table 1).
Fluorescence in situ hybridization (FISH)
Metaphase chromosome spreads and interphase nuclei were prepared on a glass microscope slide in accordance with standard cytogenetic procedure and according to the manufacturer’s instructions. Paraffin embedded tissue slides were cut at 2-μm thickness using a microtome and floated in a protein free waterbath at 40 °C. A concurrent H&E slide was stained and marked by a pathologist to delineate the area of tumor for analysis. Fluorescent in situ hybridization probes were purchased from BlueGnome (Illumina, Cambridge, United Kingdom) and Empire Genomics (Buffalo, NY, USA). Briefly, prepared slides from the tumor were placed in a Coplin jar with 40 mL of 2XSSC pH 7.0 at 37 °C for 15 min. Treatment of slides in 2XSSC was used to artificially age the chromosomes, making them less sensitive to over-denaturation. Next, the slides were dehydrated in 70, 85, and 100% ethanol at room temperature for 2 min each, followed by drying on a 50 °C warmer for 15 min. Paraffin embedded tissue slides were baked in a 60 °C oven for a minimum of 1 h. These slides were then placed on the VP processor (Abbott Molecular, Des Plaines, IL, USA) for de-paraffinization, pretreatment, and protease digestion. A mixture of 3.5 μl of locus specific identifier (LSI) hybridization buffer (Abbott Molecular, Des Plaines, IL, USA), 1 μl of sterile water, and 0.5 μl of probe was prepared for the BlueGnome probes and 4 μl of Empire Genomics buffer was used with 1 μl of probe for the Empire probes for each hybridization area. 5 μl of probe mixture was applied to each hybridization area of patient and control slides. Prepared slides and probes were then co-denatured using the ThermoBrite machine at a denature temperature of 76 °C for 5 min and then hybridized overnight. After hybridization, slides were washed using 40 ml of 0.4X SSC/0.3%NP40 for 2 min, followed by 40 ml of 2X SSC/0.1%NP40 for 1 min to remove any excess or unbound probe. After slides were air-dried, 10 μl of DAPI II counterstain on a 22 × 22 coverslip was applied to the targeted area of the slide.
FISH analysis was performed following standard techniques using a fluorescent microscope with appropriate filters (Olympus, Tokyo, Japan). The number of hybridization signals for each probe was assessed on a minimum of twenty metaphases from fresh tumor slides and from 200 nuclei on the paraffin embedded tissue slides with strong and well-delineated signals, and were further selected for thorough examination using Applied Spectral Imaging (ASI) software (Carlsbad, CA, USA). Karyotypes were described and reported in accordance with the International System Committee for Human Cytogenomic Nomenclature (ISCN) 2016 [22].
Probes used to interrogate the 9q24 region were: RP11-143 M15 (9p24.3-green), RP11-59O6 (9p24.3-orange), RP11-130C19 (9p24.3-green), and RP11-1107A23 (9p24.3-green). Probes used to interrogate the 15q24 region were: RP11-62D2 (15q25.3-orange) and RP11-608H9 (15q25.3-orange).
BRAF mutation analysis
FFPE sections were evaluated for the BRAF mutation on a Roche LightCycler 2.0 instrument (Mannheim, Germany) utilizing the allelic discrimination by real time polymerase chain reaction (PCR), which was performed at the Clinical Laboratory Improvements Amendments (CLIA) certified ACL Laboratories (Rosemont, IL, USA). In short, the appropriate FFPE tissue block was selected by the pathologist. Four-micrometer-thick sections from FFPE tissue blocks were enriched by manual micro-dissection and DNA was isolated by the ZymoPinPiont method (Irvine, CA, USA), according to the manufacturer’s protocol. Exon 15 of the BRAF gene was amplified from 50 ng of genomic DNA by real time PCR using sequence specific primers ordered from Invitrogen-Life Technologies (Carlsbad, CA, USA) (forward primer: 5′-CTCTTCATAATGCTTGCTCTGATAGG-3′, and reverse primer: 5′-TAGTAACTCAGCAGCATCTCAGG-3′). Melting curve analysis was performed by optimized fluorescent probes 5′-FL-TGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATT SpacerC35’-TGGTCTAGCTACAGTGAAATCTC-LC640. The PCR products were amplified in the following conditions: initial denaturation at 95 °C for 10 min; amplification 45 cycles of 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 20 s; melting curves 1 cycle of 95 °C for 2 min, 40 °C for 2 min, and 85 °C at 5 s; and cooling period of 1 cycle at 40 °C for 30 s. The method of BRAF exon 15-mutation analysis interpretation has been previously described [4, 11, 14]. Specifically, our assay detects 28 nucleotide changes involving the following codons: V600E, M, L, R, Q, D, K, A and G, L597V, S, Q, R and L, K601E, del, and N, A598V, A598_T599insV, T599I, T599_V600insT, T599_V600insTT, and V600_K601 > E.
Fisher’s exact analysis was performed for comparison between the differential prevalence of BRAFV600E mutation in patients under the age of 30 and those patients over 30 years of age. A p value <.05 was used to indicate statistical significance.