Identification of Multiple Complex Rearrangements Associated With

ORIGINAL ARTICLE

Identiﬁcation of Multiple Complex Rearrangements Associated with Deletions in the 6q23-27 Region in Se´zary Syndrome Katarzyna Iz˙ykowska1, Mariola Zawada1, Karina Nowicka1, Piotr Grabarczyk2, Floriane C.M. Braun2, Martin Delin2,MarkusMo¨bs3, Marc Beyer3, Wolfram Sterry3, Christian A. Schmidt2 and Grzegorz K. Przybylski1,2

The 6q23-27 region, recurrently deleted in Se´zary syndrome (SS), was characterized at the molecular level in 13 SS patients and SS cell line SeAx. Using fine-tiling comparative genomic hybridization, deletions within the 6q23-27 region were detected in half of the samples (six patients and SeAx). All samples with deletions were further analyzed by ligation-mediated PCR. In addition, in one patient sample and in SeAx, paired-end next-generation sequencing was performed on the HiSeq2000 Illumina platform. Using those techniques, 23 rearrangements associated with the deletions were identified. The majority of rearrangements showed enormous complexity and diversity, including eight inversions, three transpositions, and four translocations (with chromosomes 3, 17, 10, and 12). Fifteen genes were disrupted by those rearrangements, the MYB proto-oncogene three times and the interleukin-22 receptor subunit alpha-2 gene (IL22RA2) twice. All three patients with MYB alterations showed low MYB expression, whereas seven of the remaining patients showed overexpression. Most patients overexpressing MYB also presented increased expression of MYC, HSPA8, and BCL2. Five gene fusions were identified, of which two, CCDC28A–IL22RA2 and AIG1–GOSR1, both in SeAx, were in the same orientation and were expressed at the messenger RNA level. Journal of Investigative Dermatology (2013) 133, 2617–2625; doi:10.1038/jid.2013.188; published online 23 May 2013

INTRODUCTION patients with SS. Recurrent regional gains are usually Se´zary syndrome (SS) is a rare form of cutaneous T-cell observed at 17q, 8q, and 17p, whereas regional losses were lymphoma (CTCL) characterized by erythroderma and the observed at 17p, 10q, 6q, 9q, 10p, 9p, and 19p (Izykowska presence of Se´zary cells (CD3 þ ,CD4þ ,andCD8 ) in skin, and Przybylski, 2011; Steininger et al., 2011). The 6q region lymph nodes, and peripheral blood (Hwang et al., 2008). The caught our attention because it is recurrently deleted in SS etiology and molecular pathogenesis of SS are poorly known. (Espinet et al., 2004; Batista et al., 2006) and correlated to The disease remains fatal, with a median survival between poorer prognosis in CTCL (Fischer et al., 2004). It is also recur- 2 and 4 years, and limited therapeutic options (Prince et al., rently altered in different cancer types, and thus might carry 2009). Chromosomal instability is characteristic of this lym- genes important in oncogenesis (Taborelli et al., 2006). phoma and related to bad prognosis (Caprini et al., 2009), but Using high-resolution fine-tiling comparative genomic only few specific abnormalities that may be directly involved hybridization (FT-CGH) array, we found deletions in the in the development of the disease have been found so far. 6q23-27 region in 6 out of 13 SS patients and in the SS- Over the past few decades, cytogenetic and molecular derived cell line SeAx (Braun et al., 2011). The common cytogenetic findings revealed many genetic alterations in region in those deletions contained the tumor necrosis factor alpha–induced protein 3 (TNFAIP3; A20), which is a putative 1Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; tumor suppressor in CTCL. Besides deletion of the A20- 2Clinic for Internal Medicine C, University Greifswald, Greifswald, Germany encoding gene (TNFAIP3), FT-CGH revealed many different and 3Department of Dermatology and Allergy, Skin Cancer Center, Charite´— breakpoints, which we decided to further analyze at the Universita¨tsmedizin Berlin, Berlin, Germany molecular level. Our previous studies showed that copy Correspondence: Grzegorz K. Przybylski, Institute of Human Genetics, Polish number changes, detected by array comparative genomic Academy of Sciences, Ul. Strzeszynska 32, 60-479 Poznan, Poland. E-mail: [email protected] hybridization, might be associated with small structural Abbreviations: CTCL, cutaneous T-cell lymphoma; FISH, fluorescence in situ aberrations such as inversions or translocations (Przybylski hybridization; FT-CGH, fine-tiling comparative genomic hybridization; et al., 2010). Detailed molecular characterization of DNA LM-PCR, ligation-mediated PCR; mFISH, multicolor FISH; mRNA, messenger changes has the potential to reveal genes being deregulated RNA; NGS, next-generation sequencing; RQ-PCR, real-time quantitative PCR; and possible gene fusions (Przybylski et al., 2005). To SS, Se´zary syndrome sequence each breakpoint and identify possible rearrange- Received 15 October 2012; revised 24 March 2013; accepted 29 March 2013; accepted article preview online 18 April 2013; published online ments, samples with deletions were analyzed by ligation- 23 May 2013 mediated PCR (LM-PCR). In addition, the SeAx cell line was

& 2013 The Society for Investigative Dermatology www.jidonline.org 2617 KIz˙ykowska et al. Complex Rearrangements in Se´zary Syndrome

analyzed by fluorescence in situ hybridization (FISH) with (PPP1R14C). As none of the gene fusions was in right painting probes, and in two samples paired-end next-generation orientation, no fusion transcript was expected. sequencing (NGS) was performed on the HiSeq2000 Illumina In SeAx, LM-PCR was performed first, and subsequently all platform. The number of identified rearrangements and their five identified rearrangements were confirmed by NGS complexity were even higher than expected based on the (Figure 3; Supplementary information 1 online). In addition, results of FT-CGH. NGS revealed a more complex nature of the rearrangement, 22 identified by LM-PCR, including inversion of 6q combined RESULTS AND DISCUSSION with a 270-bp insertion from the third intron of the solute Complex rearrangement pattern revealed by combining FT-CGH carrier family gene SLC27A5 located at 19q13.43. Moreover, with LM-PCR a detailed analysis of the breakpoint at chr6:143,501,752, The main limitation of array comparative genomic hybridiza- associated with translocation t(3;6), led us to identify an tion is that it does not detect structural aberrations, which additional translocation between the region located just might be associated with deletions. Combining FT-CGH with 231 bp proximally from that break and a fragment of chromo- LM-PCR allows to overcome this problem. Using LM-PCR some 17. Most likely, the long arm of the chromosome was with primers specific for 6q23-27, located at the borders of the broken, the proximal part of chr6 was translocated to chr17, deletions visible in FT-CGH, 33 breakpoints (24 in five SS the distal part of chr6 was translocated to chr3, and the short patients and 9 in SeAx) were amplified and sequenced. In one 231-bp fragment was deleted during this rearrangement. The SS patient (249), no rearrangement could be amplified owing t(6;17) fused, in the same orientation, the androgen-induced to the presence of a single large deletion, with its border 1gene(AIG1) and the Golgi SNAP receptor complex member located outside of the region covered by the FT-CGH array. 1 gene GOSR1 (Figure 4). Using this approach, 19 rearrangements were identified, A limitation of LM-PCR is its range of 3–4 kb, which does including 7 deletions, 6 inversions, 3 translocations (with not allow amplification of long tandem repeats with no chromosomes 3, 10, and 12), and 3 transpositions. As a result restriction sites. Therefore, not all breaks visible in FT-CGH of those rearrangements, 11 genes were disrupted on chromo- could be amplified. One such breakpoint, associated with some 6 (MAP7, BCLAF1, PDE7B, SMOC2, IL22RA2, SYNE1, telomere repeats (TTAGGG), was clarified by NGS in SeAx. CCDC28A, AIG1, PERP, REPS1, and MYB). Five rearrange- The process called ‘‘telomere healing’’ is related to the ments were associated with short insertions (27–1,623 bp); stabilization of terminal deletions by the direct addition of in one case, the insertion was originated from a different telomeric repeats onto the end of a broken chromosome chromosome (chr19). All rearrangements associated with (Varley et al., 2000; Ballif et al., 2004). Deficiency in deletions in the 6q23-27 region in SS samples are presented chromosome healing in human cancer cells increases the in Supplementary information 1 online and the nucleotide frequency of chromosome rearrangements leading to tumor sequences in Supplementary information 2 online (GenBank cell progression. accession numbers KC009655–9679). One FT-CGH/LM-PCR To sum up, results of FT-CGH and LM-PCR are concurrent analysis is presented as an example in Figure 1. with paired-end NGS, but the latter method has the advantage of detecting balanced aberrations as well. Both approaches Rearrangements identified by combining FT-CGH and can identify even very small genetic alterations and reveal a paired-end next-generation sequencing direct position of each breakpoint. In our opinion, the Recently, NGS has been used for the identification of genomic techniques we used are too time- and labor-consuming to rearrangements (Chen et al., 2008, 2010; Feldman et al., be currently used in clinical practice. We recommend using 2011; Grossmann et al., 2011; Sobreira et al., 2011; Talkowski those techniques to get a complete picture of the genetic et al., 2011). In this project, one SS patient (312) and SeAx lesions. Once recurrent and relevant rearrangements are cell line were additionally analyzed using NGS; besides described, a simple PCR or array-based approach for clinical confirmation of rearrangements previously identified by use can be developed. LM-PCR, four additional rearrangements were found. In patient 312, breakpoints in the 6q23-27 region were analyzed by Cytogenetic confirmation of rearrangements detected in the PE NGS using the IGV2.1 software instead of LM-PCR. SeAx cell line Two rearrangements of inverted DNA fragments (15 and 16; Most of the rearrangements identified by LM-PCR within Supplementary information 1 online), associated with four 6q23-27 are too small to be detected by cytogenetics. To breaks seen in FT-CGH, were identified. The most likely check whether the chromosomal translocations are present at explanation of those rearrangements is an inversion of the microscopic level, karyotyping and FISH analysis were a 164,252-bp DNA fragment, associated with a large performed in the SeAx cell line. Two whole chromosome 15,024,515-bp proximal and a small 9,496-bp distal deletion painting probes for chromosomes 6 and 3 were used in order (Figure 2). As a result, the iodotyrosine deiodinase gene (IYD) to visualize t(3;6) in SeAx. At least 30 metaphase cells were was split; the major part (exons 1–4) was fused head to head analyzed, and two fusions between chromosomes 6 and 3 with MYB proto-oncogene (exons 1–14), exon 5 and the were detected in all of them: der(3)t(3;6) and der(3)t(3;6;?) majority of exon 6 were deleted, and a small part of the 3’ (Figure 5a). As the latter chromosome contained unstained UTR of exon 6 was fused tail to tail with the last (fourth) exon material from other chromosomes, multicolor FISH (mFISH) of the protein phosphatase 1, regulatory subunit 14C gene analysis was performed in order to explain this complex

2618 Journal of Investigative Dermatology (2013), Volume 133 KIz˙ykowska et al. Complex Rearrangements in Se´zary Syndrome

Dra l Eco RV PvU II Stu l M ssssss ss cccc

G G 2000 bp G G 1000 bp R R

500 bp R

LM-PCR

133 M 140 M 145 M 150 M 155 M 160 M 165 M chr6 Transposition del MYB

FT-CGH inv

135,560,752 MYB

1 2 3 4 5 6 7 8 9 10–12 13 14 15

135,560,050

Figure 1. Complex rearrangements identified by combining fine-tiling comparative genomic hybridization (FT-CGH) and ligation-mediated PCR (LM-PCR). Three different rearrangements, transposition, deletion, and inversion, all involving a duplicated 1,064-kb region, were identified in Se´zary syndrome (SS) patient 247. In the picture, the inversion identified by LM-PCR is presented (rearrangement nr 14, amplified with primers 148,702,669f and 148,702,771f). This inversion and the deletion disrupted the MYB gene, localized at the distal end of the duplicated region. C, control sample; G, germ-line configuration; M, size marker; R, rearrangement.

134 M 140 M 145 M 150 M 155 M 160 M 165 M chr6

MYB

150.5 M 150.560 150.6 M 150.660 150.7 M 150.760 150.8 M

PPP1R14C IYD

MYB 150.757 PPP1R14C 150.767 135,569 150.593 IYD IYD

Figure 2. Combination of fine-tiling comparative genomic hybridization (FT-CGH) and paired-end next-generation sequencing (NGS) for identification of rearrangements in the 6q23-27 region in Se´zary syndrome (SS) patient 312. Region 150.5–150.8 M was enlarged to indicate the inverted 164,252-bp DNA fragment and gene fusions that arose as a result of this rearrangement. IYD, iodotyrosine deiodinase gene. aberration. In addition to the two t(3;6), fusions between the presence of rearrangements between chromosomes 6 and chromosomes 6 and 17 and chromosomes 3 and 17 were 3, and also between chromosomes 6 and 17 in the SeAx cell identified (Figure 5b). In general, mFISH analysis confirmed line. However, many positions were indicated by a question

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136.5 M 140 M 143.5 M 147 M 150.5 M

chr6 t(3;6) (TTAGGG)n del inv inv SYNE1 AIG1 IL22RA2 PERP REPS1 inv CCDC28A

IL22RA2 CCDC28A PERP REPS1 7165423 23 137,532,153 139,143,297 1 1 138,464,027 139,329,571

137.4 M 137.8 M 138.2 M 138.6 M 139 M 139.4 M

inv inv IL22RA2 PERP REPS1 CCDC28A

FT-CGH

Paired-end Biallelic Biallelic sequencing deletion deletion

inv

IL22RA2 PERP CCDC28A REPS1

Figure 3. 6q23-27 rearrangements in Se´zary syndrome cell line (SeAx) cell line identified by fine-tiling comparative genomic hybridization (FT-CGH) and next-generation sequencing. (a) FT-CGH analysis showed deletions in the 6q23-27 region in SeAx; breakpoints were characterized using ligation-mediated PCR (LM-PCR), and five rearrangements were identified; all rearrangements were confirmed using paired-end sequencing, and one breakpoint was identified to be associated with telomere repeats; two gene fusions were identified at the DNA level, yet only IL22RA2/CCDC28A fusion had the orientation to create a possible transcript. (b) Two inversions in SeAx cell line identified by FT-CGH/LM-PCR and paired-end sequencing; biallelic deletions visualized with both techniques.

mark because of the uncertainty in band designation. To align rearrangements were confirmed by PCR on genomic DNA, the mFISH results with the molecular data, NGS results including amplification spanning the 5,798-bp inverted were searched for rearrangements among chromosomes DNA fragment derived from chr3q26.1 (167,027,014- 6, 3, and 17. The sandwich chromosome der(3)t(3;6;17;9) 167,021,217), inserted between the chromosomes 3 and 6. (Figure 5c) was in line with three rearrangements detected Our data indicate that the cytogenetic and molecular by NGS: one (20) in the studied region, 6q24.2 (143,502 k) analyses of the cancer genome, although providing different þ : þ 17q11.2 (25,830 k), and two additional rearrange- information, are compatible. As shown for SeAx, rearrangements, (24) 3q26.1 (167,021 k)-: þ 6q21 (106,549 k) and ments between two chromosomes can be confirmed by (25) 3q13.2 (113,776 k) þ :-3q26.1 (167,027 k), outside of the chromosome painting. In addition, the other way round, for studied region (Supplementary information 1 online). Those the complex ‘‘sandwich chromosome’’ seen in multicolor

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FISH, resulting from a fusion of chromosomes 3, 6, 17, and 9, matching DNA rearrangements could be found.

143,501,500 143,502,000 143,502,500 Expression of genes disrupted in SS Genes with altered expression due to genomic imbalances, as well as gene fusions, might be used in the future as targets for very speciﬁc and effective therapies (Mitelman et al., 2007; t(6;17) Sellers, 2011). t(3;6) In our study, including only seven samples, as a result of identiﬁed rearrangements, 15 genes were directly disrupted. 143,501,521 chr6 chr17 GOSR1 Two of them, the proto-oncogene MYB and interleukin-22 AIG1 receptor subunit alpha-2 gene (IL22RA2), were affected more 1 2 2–6 7 8 9 than once. In three rearrangements, breakpoints were inside chr6 chr3 AIG1 the MYB gene, which encodes a transcription factor, and is often rearranged and overexpressed in cancer, including CTCL 3 456 143,501,752 (Qin et al., 2001; Poenitz et al., 2005; Stenman et al., 2010). To study the consequences of gene disruption at the Figure 4. Translocation t(6;17) in SeAx fusing the AIG1 and the GOSR1 messenger RNA (mRNA) level, expression of three directly genes. Discordant next-generation sequencing (NGS) reads paired with DNA affected genes, MYB, IL22RA2, and PERP, and three genes fragments from chromosomes 3 and 17 are indicated by circles. known to be involved in the C-MYB transcription factor

chr3 chr6 chr17 chr9

3 17 17 3 3 3 6 17 9 6

3q26.1 (167,027–021) 25. 24. 20. 10 20 30 40 50 60 70 80 90 100 110 110 120 130 140 30 ′ 40 ′ 50 ′ 3p25 3p24 3p23 3p22 3p21 3p26 3p14 3p13 3p12 3p11 3q11 3q12 3q13 6q21 6q22 6q23 6q24 17q11 17q12 17q21 17q22 17q23

3pter-3q13.2 3q21-24.2 17q11.2–23 (0–113,776) (106,549–143,502) (25,830–)

Figure 5. Cytogenetic confirmation of the rearrangements identified by next-generation sequencing Se´zary syndrome (SeAx) cell line. (a) Fluorescence in situ hybridization (FISH) analysis of the SeAx cell line with whole chromosome painting probes for chromosome 6 (green) and chromosome 3 (red); chromosome aberrations recognized: der(3)t(3;?), der(?)t(?;3), der(3)t(3;6;?), der(3)t(3;6), der(6)t(6;?), der(?)t(?;6), del(6)(q?). (b) Multicolor FISH (mFISH) analysis of the whole SeAx cell line karyotype; detailed analysis was concentrated on chromosomes 6 and 3; G-banding was performed in order to assess the band designation: der(3)t(3;6;17;9)(3pter-3q13.2::6q22-6q24::17q11-17q23?::9q22.3-9qter); der(3)t(3;6)(3p26-3q13?::6q25?-6q27?); der(17)t(3;17)(3qter- 3q26::17q11-17p12); der(17)t(3;17)(17qter-17p11?::3q21-3qter). (c) Reconstruction of the sandwich chromosome der(3)t(3;6;17;9) detected in mFISH based on molecular results; the presence of the 5,797-bp insert was detected by next-generation sequencing (NGS) but not by mFISH, and it was confirmed on genomic DNA using one primer situated in the 6q21 region (position 106,549,156r) and the second one in the 3q13.2 region (position 113,775,810f).

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4,000 100,000

MYB(Ex1–2) MYC – 42,000 80,000 250,000

131,000 – M M 1,183,000 3,000 T E – 60,000 T – M 2,000 E T 40,000 M E – M 1,000 T – – 20,000 – E – E T T T T 0 0 Hut Hut 249 247 312 246 251 250 245 252 306 311 313 314 315 249 247 312 246 251 250 245 252 306 311 313 314 315 K348 K349 K350 K351 K352 K348 K349 K350 K351 K352 SeAx SeAx

20,000 500,000 MYB(Ex14–15) HSPA8 400,000 148,000 596,000

15,000 3,083,000 2,686,000

300,000 10,000 200,000

5,000 100,000

0 0 Hut Hut 247 249 312 246 251 250 245 252 306 311 313 314 315 249 247 312 246 251 250 245 252 306 311 313 314 315 K348 K349 K350 K351 K352 K348 K349 K350 K351 K352 SeAx SeAx

35,000 3,000 BCL2 PERP 30,000 2,500 25,000 2,000 20,000 1,500 15,000 1,000 10,000

500 5,000

0 0 Hut Hut 249 247 312 246 251 250 245 252 306 311 313 314 315 249 247 312 246 251 250 245 252 306 311 313 314 315 K348 K349 K350 K351 K352 K348 K349 K350 K351 K352 SeAx SeAx

1,400 IL22RA2 1,200 SS patients with del6q 1,000 SS patients without del6q 800 SS cell lines 600 Normal MNC (healthy donors) 400 SS patient with MYB deletion 200 SS patients with MYB rearrangements 0 SS patient with PERP biallelic deletion Hut 249 247 312 246 251 250 245 252 306 311 313 314 315 K348 K349 K350 K351 K352 SeAx

Figure 6. Quantitative expression analysis of genes disrupted by a rearrangement. Expression of three genes (MYB, PERP,andIL22RA2) directly affected by a rearrangement and three genes (MYC, HSPA8, and BCL2) regulated by MYB was analyzed using real-time quantitative PCR with specific TaqMan probes in six Se´zary syndrome (SS) patients with and seven SS patients without 6q23-27 alterations, two SS cell lines (SeAx and HuT-78), and five mononuclear cell samples (MNC) from healthy donors. Each population, as well as the patients with a particular gene rearrangement, is featured by different colors. The gene copy number was normalized to 1 million copies of beta 2 microglobulin (B2MG) reference gene. In the MYC expression diagram, information on MYC amplification (M) and E2A deletions (E) in 8/13 SS patients, published elsewhere by Steininger et al., 2011, positively contributing to MYC expression has been included. No alterations: (–). In addition, TP53 deletions (T) have been indicated, which might suppress the MYC apoptotic pathway. The level of gene expression in cell lines that were out of range was given by numbers.

network,MYC, BCL2, and HSPA8, were analyzed in all SS and for the 30 end of MYB (exons 14–15) was performed. patients by real-time quantitative PCR (RQ-PCR) with speciﬁc Despite predictions, in samples 247 and 312, no difference TaqMan probes (Figure 6). between expression of the 50 and the 30 end was observed, To check whether the loss of MYB 30 UTR, involved in suggesting that deletions of 30 UTR did not result in increased negative regulation of MYB expression by microRNAs (Persson expression of this gene (Figure 6). Those results were con- et al., 2009), resulted in overexpression of the shortened ﬁrmed by comparative PCR with two different primer sets for transcript, RQ-PCR for the 50 end of MYB (exons 1–2) the 50 and 30 end of MYB (data not shown). In concordance

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with published data, in most of the SS samples without MYB The t(6;17) fused the AIG1 and the GOSR1 genes in the alterations (7/10), increased expression of MYB,ascompared same orientation (Figure 4), suggesting a possible expression with normal T cells, was observed (mean 9-fold increase; of a hybrid mRNA. Reverse transcriptase–PCR revealed two P ¼ 0.037). Expression of MYC and HSPA8, positively regu- fusion transcripts being expressed: AIG1(ex1,2)–GOS- lated by MYB, was increased in the majority of samples (8/13) R1(ex2,3,4,5) and AIG1(ex2)–GOSR1(ex2,3,4,5,6,8,9), which (for MYC 7-fold; P ¼ 0.029, for HSPA8 8-fold P ¼ 0.024). have not been reported to date (Supplementary information 2 Increased expression of MYC in samples with low MYB online, Genbank accession numbers KC119451-52). Both expression can be explained by MYC amplification and variants were out-of-frame, leading to premature stop codons deletions of MYC-negative regulator E2A, reported previously in the second exon of GOSR1. Whether any of the five in those patients (Steininger et al., 2011). MYC and HSPA8 transcriptional variants of the two identified fusions is trans- overexpression was even more evident in nine samples with lated into a protein (truncated or translated from alternative MYB overexpression and/or MYC amplification (MYC:9-fold start codons) remains to be determined. increase, P ¼ 0.011; HSPA8 10-fold increase, P ¼ 0.008). As In our previous study (Braun et al., 2011), we reported HSPA8 is also positively regulated by MYC, this might explain deletion of the TNFAIP3 gene (A20) in all samples (six patients their very similar pattern of expression. In 4/7 patients with and SeAx) showing deletions within the 6q23-27 region. To MYB overexpression, 5-fold increased BCL2 expression was our knowledge, interactions between A20 and MYB, IL22RA2, detected, whereas in all six patients with low MYB expression and other disrupted genes have not been studied yet. Whether the mean BCL2 expression was 2-fold lower than that in the A20 loss and the disturbed expression of genes affected by normal T cells, thereby confirming the positive regulations of the complex rearrangements might have a synergistic effect in BCL2 by MYB in SS cells. Low BCL2 expression in the tumor development remains to be determined. majority of cases (9/13) is consistent with previous studies It is predicted that molecularly based individualized cancer showing decreased expression of BCL2 in CTCL, and in some care, defined as the process of selecting treatment for an cases might be due BCL2 deletions reported in SS (Mao et al., individual patient, based on genetic expression, proteomic 2004). Low expression of PERP was observed in all samples profiles, or deregulated cellular pathways of each individual with 6q23-27 alterations showing monoallelic deletions patient, is the future of oncology (Mansour and Schwarz, (Figure 6). In SeAx with biallelic PERP deletion, no expression 2008). Yet, extensive research is still needed to identify the was detected, and in the SS sample 251 with biallelic deletion key genes to be targeted. Our study revealed numerous very low PERP expression, from the accompanying normal complex rearrangements associated with the 6q23-27 cells, was observed. As PERP is a p53 apoptosis effector, lack deletions in SS, disrupting many different genes and thereby of its expression may result in nonfunctional p53 signaling affecting their expression. We did not observe significant (Davies et al., 2011). Indeed, recently impaired apoptosis differences in the survival between the patients with and induction after p53 stimulation was reported in SS cells without 6q alterations. This can be explained by the small (Lamprecht et al., 2012; Manfe et al., 2012), which, at least number of analyzed cases, and the presence of other in some cases, might be due to PERP deletions. chromosomal alterations, not analyzed in this study, which The IL22RA2 gene, encoding a soluble receptor IL-22BP could affect the survival in patients without 6q deletion. This is (Dumoutier et al., 2001), was not expressed in the studied in line with the results of Caprini et al, who did not observe samples, except for the SeAx cell line, where it was rearranged significant differences in the survival for a single alteration with the coiled-coil domain containing 28A gene (CCDC28A) even in a larger cohort of SS (Caprini et al., 2009). Further (Supplementary information 1 online; No 18). Comparative studies are needed to unravel the effect of the identified PCR with two different primer sets for the 50 end (ex1-2) and alterations on the biology of SS cells and their possible role in for the 30 end (ex2-3) of IL22RA2 detected only the 30 end of the malignant transformation. the mRNA, indicating exclusive expression of the rearranged gene. 50 RACE analysis using reverse nested primers located in MATERIALS AND METHODS the second exon of IL22RA2 revealed three fusion trans- Clinical samples cripts with CCDC28A: CCDC28A(ex1,2,3)–IL22RA2(ex2-), Thirteen SS blood samples, diagnosed according to the WHO-EORTC CCDC28A(ex1,3)–IL22RA2(ex2-), and CCDC28A(ex1,2,3)– classification for cutaneous lymphomas, and the SeAx SS cell line IL22RA2(158-bp fragment of in1, ex2-) (Supplementary were included in the study. All patients were included after giving information 2 online; GenBank accession numbers written informed consent. The study was approved by the local ethics KC819274–6). Recently, out-of-frame CCDC28A(ex1)– committee of the Charite´ (Berlin, Germany), and was conducted in NUP98(ex15) and reciprocal in-frame NUP98(ex13)– accordance with the Declaration of Helsinki. All patients were CCDC28(ex2) fusion transcripts have been identified in T-cell pretreated before the sample collection with diverse combinations acute lymphoblastic leukemia, with the latter shown to act as of therapeutic agents. The clinical information of patients and samples an oncogene (Petit et al., 2012). In our study, all three is summarized in Supplementary information 3 online. identified fusions were out-of-frame, leading to premature stop codons in the second exon of IL22RA2. The reciprocal Fine-tiling comparative genomic hybridization IL22RA2-CCDC28A transcript could not be expressed, A custom-designed high-density fine-tiling array of 385,000 oligonu- as the 50 part of IL22RA2 and the 30 part of CCDC28A cleotides, covering the 6q23-27 region, was prepared using the were deleted. Maskless Array Synthesizer technology (NimbleGen Systems,

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Reykjavik, Iceland). Results were analyzed using the SignalMap RACE software (NimbleGen Systems). 50 RACE was performed using the SMARTer RACE cDNA amplification kit (Clontech, Mountain View, CA) and the IL22RA2-specific Ligation-mediated PCR primers, 137,524,542exon2If/6 and 137,524,585 exon2If/6, accord- DNA losses were analyzed by LM-PCR as described in detail ing to the manufacturer’s instructions. elsewhere (Przybylski et al., 2005). Briefly, 1 mg of high-molecular- weight DNA was digested with blunt-end restriction enzymes EcoRV, Reverse transcriptase–PCR and comparative PCR DraI, PvuII, and StuI (New England Biolabs, Ipswich, MA), and 50 mM RNA extraction and reverse transcription were performed as pre- of an adaptor was ligated to both ends of the restriction fragments. viously described (Braun et al., 2011). Reverse transcriptase–PCR was The ligation products were subjected to two rounds of PCR with performed to confirm the AIG1–GOSR1 and CCDC28A–IL22RA2 adaptor-specific and gene-specific primers (Supplementary information fusions at the mRNA level in SeAx, whereas comparative PCR with 4 online), located at the borders of the deleted regions. LM-PCR two sets of primers was used to compare the relative levels of mRNA reaction was performed using long-distance Adv Genomic LA Poly- variants (Supplementary information 4 online). merase (Clontech, Palo Alto, CA) or Amplus DNA Polymerase (EURx, Gdansk, Poland) on a DNA Engine Tetrad 2 thermocycler (Bio-Rad, Real-time quantitative PCR Foster City, CA). Amplification products were separated by gel electro- RQ-PCR was performed using the ICycler thermocycler (Bio-Rad). phoresis, and PCR bands differing in size from the germ-line config- The following genes were analyzed using TaqMan Gene Expression uration were excised from the gel, purified, and directly sequenced. All assays: PERP (Hs00953483_g1), IL22RA2 (Hs00364814_ml), rearrangements were confirmed on genomic DNA by regular PCR. BCL2 (Hs00608023_m1), MYC (Hs00153408_m1), HSPA8 (Hs01683591_g1), and MYB (Hs00920554_m1, exons 1–2; Paired-end NGS Hs00193527_m1, exons 14–15) (Applied Biosystems, Foster City, In two samples (SS patient nr 312 and SeAx cell line), paired-end CA). B2M (Hs00984230_m1) was used as a reference gene. The NGS was performed by Fasteris (Geneva, Switzerland) and LGC analysis was performed for 13 SS patients (six with rearrangements in Genomics (Berlin, Germany) on the HiSeq2000 Illumina platform, the 6q23-27 and seven without rearrangements), and 5 ficoll-purified according to the manufacturer’s instructions. Briefly, high-molecular- mononuclear cells from healthy donors were used as controls. All weight DNA samples were fragmented to about 300 bp in the case of samples were assayed in duplicate. Statistical analysis of the differ- an SS patient and about 900 bp in the case of SeAx. Reads of 100 bp ences in the gene expression was performed using two-tailed were performed from both ends of each fragment. For the patient Student’s t-test for unpaired data. sample 312 67.5 Mio paired reads, 6.5 Gb of mapped sequence were obtained. This corresponds to 2.2 sequence coverage and CONFLICT OF INTEREST 3.4 rearrangement coverage of the genome. For SeAx 77.3 Mio The authors state no conflict of interest. paired reads, 7.73 Gb of mapped sequence was obtained, corre- ACKNOWLEDGMENTS sponding to 2.6 sequence coverage and a high 11.6 rearrange- This work was supported by the National Center of Science, Poland (K.I. and ments coverage (due to longer inserts). At least two nonidentical G.K.P.) and the German Jose´ Carreras Leukemia Foundation (P.G. and discordant inserts with 50 and 30 ends, localized in different regions of F.C.M.B). the genome, were considered to carry a rearrangement. All rearrangements detected by NGS were confirmed by PCR, with primers SUPPLEMENTARY MATERIAL located within the discordant reads and classical Sanger sequencing. Supplementary material is linked to the online version of the paper at http:// www.nature.com/jid SeAx cell line culture and chromosome spreads SeAx cell line was cultured as described before (Braun et al., 2011). 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2624 Journal of Investigative Dermatology (2013), Volume 133 KIz˙ykowska et al. Complex Rearrangements in Se´zary Syndrome

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