Pathology – Research and Practice 210 (2014) 1049–1053

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Clear cell papillary renal cell carcinoma: A chromosomal microarray analysis of two cases using a novel Molecular Inversion Probe (MIP) technology Borislav A. Alexiev ∗ , Ying S. Zou Department of Pathology, University of Maryland Medical Center, Baltimore, Maryland, USA

a r t i c l e

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Article history: Received 9 September 2014 Received in revised form 29 September 2014 Accepted 13 October 2014 Keywords: Renal carcinoma Molecular Inversion Probe

a b s t r a c t Chromosomal microarray analysis using novel Molecular Inversion Probe (MIP) technology demonstrated 2,570 kb copy neutral LOH of 10q11.22 in two clear cell papillary renal cell carcinomas. In addition, one of the tumors had a big 29,784 kb deletion of 13q11-q14.2. There were two variants of unknown significance, a 2,509 kb gain of Xp22.33 and a 257 kb homozygous deletion of 8p11.22. The somatic mutation panel containing 74 mutations in nine genes did not reveal any mutations. Besides identification of submicroscopic duplications or deletions, SNP microarrays can reveal abnormal allelic imbalances including LOH and copy neutral LOH, which cannot be recognized by chromosome, FISH, and non-SNP microarray arrays. To the best of our knowledge, this is the first study demonstrating copy neutral LOH of 10q11.22 in clear cell papillary renal cell carcinomas using the new MIP SNP OncoScan FFPE Assay Kit on formalin-fixed paraffin-embedded tumor samples. © 2014 Elsevier GmbH. All rights reserved.

Introduction Clear cell papillary renal cell carcinoma (CCPRCC) is a novel tumor entity that was recently recognized as a new distinct epithelial tumor within the current classification system [10]. Limited information about the genetic makeup of these neoplasms is available. Molecular genetic studies have revealed that CCPRCCs harbor different genetic alterations from clear cell RCC and papillary RCC [1,2,4–8,10,12,15]. Despite the growing need of cancer researchers and pathologists, obtaining high-quality, whole-genome copy number data from degraded formalin-fixed paraffin-embedded (FFPE)-derived tumor DNA has remained extremely challenging due to the limitations of current methods such as FISH and array CGH technologies. The new OncoScan FFPE Assay Kit delivers an entirely new perspective on the cancer genome from solid tumor samples. Utilizing Affymetrix’ unique Molecular Inversion Probe (MIP) technology, it is capable of analyzing small amounts of highly degraded DNA from FFPE samples quickly and affordably, providing a significant step forward in solid tumor cancer analysis [13,14]. This new product provides whole-genome copy number data with specifically enhanced high resolution in approximately

∗ Corresponding author at: Department of Pathology, NBW85, University of Maryland Medical Center, 22 S Greene Street, Baltimore 21201, MD, USA. E-mail address: [email protected] (B.A. Alexiev). http://dx.doi.org/10.1016/j.prp.2014.10.001 0344-0338/© 2014 Elsevier GmbH. All rights reserved.

900 known cancer genes, loss of heterozygozity (LOH) across the whole genome as well as clinically relevant somatic mutation data – all from a single assay. The aim of this study is to identify potential genetic alterations involved in the biology of CCPRCC and to evaluate the feasibility of this method in archival FFPE samples of renal tumors. Material and methods Two cases with CCPRCC were selected for the study. The cases were retrieved from the files of the Department of Pathology, University of Maryland Medical Center. The criteria used for classification of a tumor as a CCPRCC included the following: (1) diffuse cytoplasmic clarity; (2) papillary, tubular, acinar or cystic architecture; and (3) characteristic linear arrangement of the nuclei away from the basement membrane. Representative tissue sections from the surgical specimens were fixed in 10% buffered formalin and embedded in paraffin. For routine microscopy, 4-␮m-thick sections were stained with hematoxylin-eosin. Immunohistochemical staining was performed using an automated immunostainer (Leica Bond-III, Leica Biosystems, Buffao Grove, IL) and BondRefinePolymerTM biotin-free DAB detection kit. The following antibodies were used: carbonic anhydrase IX (CAIX) (dilution 1:200, mouse monoclonal, Leica), RCC (prediluted, mouse monoclonal, Ventana), vimentin (prediluted, mouse monoclonal, Leica), cytokeratin 7 (CK7) (prediluted, mouse monoclonal,

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Leica), P504s/AMACR (prediluted, rabbit monoclonal, Cell Marque), PAX8 (prediluted, rabbit polyclonal, Cell Marque), cytokeratin 34betaE12 (prediluted, mouse monoclonal, Cell Marque), and TFE3 (prediluted, rabbit monoclonal, Cell Marque). Evaluation of the immunohistochemical staining was performed by light microscopy using a 10× objective lens with the selective use of a 20–40× objective lens for confirmation. The interpretation of immunoreactivity was performed in a semiquantitative manner by analyzing the extent of the staining positivity of the tumor cells. Immunostaining of greater than 10% of tumor cells was required for scoring as a positive case. The interpretation score was as follows: 0 or negative ≤ 10% tumor cell positivity; +1 or weak = 11% to 25% tumor cell positivity; +2 or moderate = 26% to 50% tumor cell positivity; and +3 or strong >50% tumor cell positivity. Fluorescence in situ hybridization (FISH) was performed with centromeric ␣-satellite DNA probes for chromosome 7 (Centromeric Enumeration Probe (CEP) 7, D7Z1, Spectrum Green) and chromosome 17 (CEP 17, D17Z1, Spectrum Orange), as well as TelVysion probes for 3p (locus D3S4559 in Spectrum Green) and 3q (locus D3S4560 in Spectrum Orange) according to the manufacturer’s instructions (Abbott Molecular Inc.). In brief, 4-␮m formalin-fixed paraffin-embedded tissue slides, dried and baked 15 min at 90 ◦ C, were deparaffinized in xylene and washed with 100% ethanol. The slides were pretreated for 55 min in warm (80 ◦ C) citric acid solution (10 mM), 48 min in pepsin (0.2%, at 37 ◦ C), and dehydrated in 70%, 85%, and 100% ethanol washes. Hybridization was performed using the automated Hybrite system (Abbott Molecular Inc.) for 80 ◦ C for 3 min denaturation, followed by 37 ◦ C for 16–18 h hybridization. Then the slides were washed at 45 ◦ C serially in 50% formamide for 10 min, 2× SSC for 5 min twice, and 0.1× SSC for 5 min. After the slides were washed at room temperature in phosphate-buffered detergent for 5 min, the nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Abbott Molecular Inc.). The slides were evaluated under a Zeiss Axioplan 2 microscope (ZEISS Inc.), using a 100× objective. The images were acquired with a CCD camera and analyzed with CytoVision Software (Leica Biosystems Inc.). For each probe, 300 nuclei were scored in areas marked to correspond with hematoxylin-eosin sections by two readers. Cut-off for loss and gain of a signal (chromosome) was determined from assessing variation of probe signals in normal nuclei and set as more than 10% for an interpretation of loss and gain. Chromosomal microarray analysis using MIP SNP assay: Genomic DNA was isolated manually (QIAamp DNA FFPE Tissue Kit; Qiagen) from 5 to 10 sections of 10 ␮m marcodissected sections of the FFPE tissue blocks. DNA was eluted in 30–100 ␮L of Elution Buffer and quantified with PicoGreen (Invitrogen, Carlsbad, CA). A total of 80 ng was subjected to OncoScan FFPE analysis (Affymetrix, Santa Clara, CA), a MIP SNP assay was performed according to the manufacture’s instruction (OncoScan FFPE assay, Affymetrix). Briefly, MIP probes were hybridized to genomic DNA and split into two tubes containing paired nucleotide mixes (AT or GC). In the presence of DNA polymerase and ligase, MIP probes circularized with their complementary nucleotides. Allele discrimination was enzymatically derived and allowed multiplexed assays. Technicalities and the design of probes were described in detail previously [14]. Data that passed quality control criteria of the assay were analyzed using Nexus express for OncoScan 3 software (Biodiscovery, El Segundo, CA) with NCBI build 37 of the human genome. The SNP-FASST2 segmentation algorithm and default settings for significance, number of probes per segment, and gain and loss thresholds were used to identify regions of copy number variation for each sample. Deletion was defined as loss of one copy and homozygous deletion was defined as loss of both copies. Gain was defined with the presence of one/more additional copies. The minimum copy neutral LOH requirement was set to 2,000 kb. Mutation score for the somatic mutation panel containing 74 mutations in nine genes

Table 1 Clear cell papillary renal cell carcinoma. Chromosomal microarray analyses using Molecular Inversion Probe technology. Copy number changes (gains and losses)

Patient 1

Patient 2

Pathogenic changes Loss of 13q11-q14.2 (19,084,823-48,869,013) Variants of unknown significance Loss of 8p11.22 (39,223,656-39,480,366) Gain of Xp22.33 (177,942-2,686,899) Benign polymorphisms Loss of 3q26.1 (162,373,665-162,619,270) Loss of 6q12 (68,234,728-68,505,083) Gain of 13q12.12 (23,561,107-24,884,598) Gain of 16p11.2 (32,624,931-34,201,619) Copy number neutral loss of heterozygosity 1q31.2-q31.3 (192,904,897-197,023,048) 3p22.3 (33,175,854-36,148,330) 3q13.33 (119,016,581-121,528,611) 8q12.3-13.2 (65,021,305-68,408,438) 10q11.22(46,965,151-49,743,008)

(−)

(+)

(−)

(+)

(+) (−)

(−) (+)

(−) (+)

(+) (−)

(−)

(+)

(+)

(−)

(+) (−) (+) (+)

(−) (+) (−) (+)

(BRAF, KRAS, EGFR, IDH1, IDH2, PTEN, PIK3CA, NRAS, TP53) was measured based on differences between the normal wild-type cluster and the test sample. A larger score indicates a higher difference from the wild-type cluster and a higher likelihood of a real mutation. A cutoff at 4 was recommended for BRAF V600E assay to be valid, whilst other mutation assays were recommended at a score of 9. The study met Health Insurance Portability and Accountability Act requirements and has been approved by the Institutional Review Board at the University of Maryland (HP-47559). Results Histologically, the tumors were composed of bland-appearing branched tubules and occasional small papillae lined by clear cells (Fig. 1a and b). Uniform small nuclei were arranged in a linear manner away from the basal aspect of the tubules. The tumors demonstrated strong expression of PAX8, cytokeratin 34betaE12, CK7, vimentin, and CA-IX. RCC, P504S/AMACR and TFE3 stains were negative. The FISH studies revealed a normal two copies for chromosomes 3p, 3q, 7 and 17 in tumor cells (Fig. 1c and d). The summary of SNP microarray findings is listed in Table 1 (Figs. 2 and 3). Both tumors showed 2,570 kb copy neutral LOH of 10q11.22. Patient 2 had a big 29,784 kb deletion of 13q11-q14.2. There were two variants of unknown significance, a 2,509 kb gain of Xp22.33 in patient 1 and a 257 kb homozygous deletion of 8p11.22 in patient 2. Patient 1 also had a 2,972 kb copy neutral LOH of 3p22.3, which is 22,992 kb centromeric from the VHL gene on 3p25.3. The somatic mutation panel containing 74 mutations in nine genes did not reveal any mutations. Discussions There have been several reports of CCPRCC arising in a sporadic setting and in patients with end-stage renal disease [7]. The tumor may also occur in the setting of von Hippel-Lindau (VHL) disease and should be considered in the differential diagnosis of renal tumors with clear cell features arising in this clinical setting [7]. CCPRCC exhibits a unique molecular signature and must be distinguished from clear cell RCC and papillary RCC due to different biological potentials [4,7]. The tumor reportedly lacks trisomies of chromosomes 7 and 17, deletions of 3p25, and VHL gene

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Fig. 1. Clear cell papillary renal cell carcinoma. (a) Case 1 and (b) Case 2. Note branched tubular architecture and clear cytoplasm. Hematoxylin-eosin, 200×. (c) Case 1 and (d) Case 2. FISH with probes for chromosome 3q (in red) and 3p (in green) in the tumor, showing nuclei with two red and two green hybridization signals, consistent with the presence of a normal copy number for 3p and 3q. (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)

Fig. 2. Clear cell papillary renal cell carcinoma. SNP microarray result of case 1 with clear cell papillary renal cell carcinoma showing copy neutral LOH of 10q, and gain of Xp with unknown clinical significance. Red on the left denotes losses, blue on the right denotes gains, and yellow on the cytobands denotes copy neutral LOH. All abnormalities are listed in Table 1. (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)

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Fig. 3. Clear cell papillary renal cell carcinoma. SNP microarray result of case 2 with clear cell papillary renal cell carcinoma showing a big deletion of 13q, copy neutral LOH of 10q, and a homozygous deletion of 8p with unknown clinical significance. Red on the left denotes losses, blue on the right denotes gains, and yellow on the cytobands denotes copy neutral LOH. All abnormalities are listed in Table 1. (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)

mutations [2,8]. A recent study identified multiple somatic mutations in CCPRCC cases, including a recurrent non-synonymous T992I mutation in the MET proto-oncogene, a gene associated with epithelial-to-mesenchymal transition (EMT) [6]. Using a microarray approach, Lawrie et al. [6] found that the expression of mature and pre-miRNAs, as well as snoRNA and scaRNAs, in CCPRCC cases differed from that of clear cell RCC or papillary RCC. The findings suggest that EMT in CCPRCC tumor cells is incomplete or blocked, consistent with the indolent clinical course typical of this malignancy [6]. Previous array-CGH studies detected no chromosomal imbalances in CCPRCC [1]. Using a high-resolution whole genomic SNP microarray analysis, it is worth noting that the frequency of copy number alternations (losses, gains, amplifications) and copy neutral LOH in these 2 samples was remarkably low. However, the copy neutral LOH of 10q11.22 was present in both samples. The copy neutral LOH of 10q11.22 contains 49 RefSeq genes (10 genes are annotated in OMIM entries including SYT15, GPRIN2, NPY4R, ANXA8, RBP3, GDF2, GDF10, PTPN20B, MAPK8, FRMPD2). Among them, 2 are known disease genes (RBP3 associated with retinitis pigmentosa and GDF2 associated with hereditary hemorrhagic telangiectasia). The mitogen-activated protein kinase 8 (MAPK8) gene has been implicated in kidney cancers [13]. The deletion of 13q11-q14.2 found in patient 2 has been described in 14.3% of clear cell RCC patients by SNP array [3]. The big 2,509 kb gain of Xp22.33 found in patient 1 and the small 257 kb homozygous deletion of 8p11.22 in patient 2 are variants with unknown clinical significance. Copy neutral LOH of 3p22.3 in patient 1 does not contain the VHL or the FHIT genes and has only 13 RefSeq genes (known disease gene CRTAP associated with osteogenesis imperfecta). LOHs containing only this 3p22.3 region are not a commonly affected region of LOH on 3p in patients with conventional RCC [3,9,11]. Detailed

sequencing analyses of mutations in these genes especially genes located on copy neutral LOH of 10q11.22 region as well as in more samples might provide insights in the pathogenesis of CCPRCC. Besides identification of submicroscopic duplications or deletions, SNP microarrays can reveal abnormal allelic imbalances including LOH and copy neutral LOH, which cannot be recognized by chromosome, FISH, and non-SNP microarray arrays. Copy neutral LOH is the occurrence of LOH in the absence of allelic loss (copy number ≥2) and has been associated with the duplication of oncogenic mutations with concomitant loss of the normal allele.

Conclusions MIP SNP assay worked very well on FFPE tissue samples. The new OncoScan FFPE Assay Kit is capable of analyzing small amounts of DNA from tumor samples quickly and affordably. This new product provides whole-genome copy number data, LOH and clinically relevant somatic mutation data. To the best of our knowledge, this is the first study demonstrating copy neutral LOH of 10q11.22 in CCPRCC using the new MIP SNP OncoScan FFPE Assay Kit.

Conflict of interest We declare that we have no conflict of interest.

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Clear cell papillary renal cell carcinoma: a chromosomal microarray analysis of two cases using a novel Molecular Inversion Probe (MIP) technology.

Chromosomal microarray analysis using novel Molecular Inversion Probe (MIP) technology demonstrated 2,570 kb copy neutral LOH of 10q11.22 in two clear...
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