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Genome-wide methylation profiling and copy number analysis in atypical fibroxanthomas and pleomorphic dermal sarcomas indicate a similar molecular phenotype

Genome-wide methylation profiling and copy number analysis in atypical fibroxanthomas and... Background: Atypical fibroxanthomas (AFX) and pleomorphic dermal sarcomas (PDS) are lesions of the skin with overlapping histologic features and unspecific molecular traits. PDS behaves aggressive compared to AFX. Thus, a precise delineation, although challenging in some instances, is relevant. Methods: We examined the value of DNA‑methylation profiling and copy number analysis for separating these tumors. DNA‑methylation data were generated from 17 AFX and 15 PDS using the Illumina EPIC array. These were compared with DNA‑methylation data generated from 196 tumors encompassing potential histologic mimics like cutaneous squamous carcinomas (cSCC; n = 19), basal cell carcinomas (n = 10), melanoma metastases originat‑ ing from the skin (n = 11), leiomyosarcomas (n = 11), angiosarcomas of the skin and soft tissue (n = 11), malignant peripheral nerve sheath tumors (n = 19), dermatofibrosarcomas protuberans (n = 13), extraskeletal myxoid chondro‑ sarcomas (n = 9), myxoid liposarcomas (n = 14), schwannomas (n = 10), neurofibromas (n = 21), alveolar (n = 19) and embryonal (n = 17) rhabdomyosarcomas as well as undifferentiated pleomorphic sarcomas (n = 12). Results: DNA‑methylation profiling did not separate AFX from PDS. The DNA‑methylation profiles of the other cases, however, were distinct from AFX/PDS. They reliably assigned to subtype‑specific DNA‑methylation clusters, although overlap occurred between some AFX/PDS and cSCC. Copy number profiling revealed alterations in a similar fre ‑ quency and distribution between AFX and PDS. They involved losses of 9p (22/32) and 13q (25/32). Gains frequently involved 8q (8/32). Notably, a homozygous deletion of CDKN2A was more frequent in PDS (6/15) than in AFX (2/17), whereas amplifications were non‑recurrent and overall rare (5/32). *Correspondence: Christian.Koelsche@med.uni‑heidelberg.de; Andreas. vonDeimling@med.uni‑heidelberg.de Christian Koelsche and Damian Stichel contributed equally to this work Department of General Pathology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Baden‑Württemberg, Germany Department of Neuropathology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Baden‑Württemberg, Germany Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 2 of 8 Conclusions: Our findings support the concept that AFX and PDS belong to a common tumor spectrum. We could demonstrate the diagnostic value of DNA‑methylation profiling to delineating AFX/PDS from potential mimics. How‑ ever, the assessment of certain histologic features remains crucial for separating PDS from AFX. Keywords: Pleomorphic dermal sarcoma, Atypical fibroxanthoma, Sarcomas, Melanomas, Carcinomas, Mimics, DNA methylation, Profiling Background of small blue round cell tumors not otherwise specified Sarcomas of the skin and the adjacent soft tissue com- [21], cancers of unknown primary [22] and nervous sys- prise a heterogeneous tumor group [1]. The classifica - tem tumors [23]. tion of these tumors follows the lineage differentiation AFX and PDS are generally believed to be of mesen- of tumor cells, which is predominantly assessed by their chymal lineage, although a few studies have suggested an expression of lineage specific markers. However, in many epithelial origin [24, 25]. Detailed DNA-methylation pat- cases an unambiguous subtype assignment by histo- terns in AFX and PDS have not been reported yet. We logic and immunohistochemical means is not possible, therefore performed genome-wide methylation profiling and molecular analyses for establishing a final diagnosis and copy number analysis of AFX, PDS and potential his- is required [2]. Unfortunately, certain entities also lack tologic mimics, with a focus on cutaneous squamous car- unequivocal molecular traits, even if more sophisticated cinomas (cSCC) and basal cell carcinomas (BCC) of the molecular approaches such as next generation sequenc- head and neck, alongside of melanomas and 11 soft tissue ing are applied. Atypical fibroxanthomas (AFX) and tumor entities. pleomorphic dermal sarcomas (PDS) belong to the afore- mentioned group of ill-defined tumors and currently Materials and methods remain a diagnosis of exclusion [3]. Sample selection AFX and PDS exhibit overlapping histologic features In total, 228 tumor specimens from different patients, all making a reliable distinction in many cases problematic prototypical examples of their corresponding subtype, [4]. The most important criterion in favor of the diag - were included (Additional file  1: Table  S1). AFX, PDS, nosis PDS is an invading growth pattern into subdermal cSCC and BCC were collected from the Dermatopa- structures, which can be difficult to assess if small biop - thology Bodensee in Friedrichshafen (Germany) and the sies are provided for histopathological diagnostics [5]. Department of Dermatology of the University Hospital Other diagnostic histologic features include necrosis, in Essen (Germany). Melanomas and soft tissue tumors lymphovascular and perineural invasion. However, gen- were collected from the Institute of Pathology of the Uni- eral features of anaplasia such as nuclear pleomorphism versity Hospital in Heidelberg (Germany), in Kiel (Ger- and atypical mitoses are common to both AFX and PDS many), in Jena (Germany), in Nijmegen and in Rotterdam [6, 7]. The distinction of AFX and PDS as different enti - (both the Netherlands), from the Institute of Pathology in ties remains clinically important. AFX has an overall Bamberg (Germany) and from the Department of Pathol- favorable biological behavior compared to the much ogy of the Laboratoire National de Santé (Luxembourg). higher potential for recurrence and metastasis in PDS Diagnoses were based on standard histopathological [3, 6, 7]. Novel diagnostic approaches allowing a clear criteria in conjunction with immunohistochemical and distinction of AFX and PDS would be of great value con- molecular analyses according to the current WHO clas- sidering the steadily increasing incidence of skin cancers sification [1]. The methylation data of melanomas and [8] and promising results of targeted therapies for certain some soft tissue tumors were published previously [12, dermal tumor subtypes [9, 10]. 15, 21]. DNA-methylation profiling has evolved as a powerful method for determining cell differentiation. Array-based epigenotyping technologies nowadays enable large-scale DNA extraction high-throughput studies of DNA methylation patterns. DNA was extracted from formalin-fixed and paraffin- The study of DNA-methylation in different cancers has embedded (FFPE) tumor tissue, thereby only using rep- already revealed molecular subgroups within known his- resentative tumor tissue with highest available tumor tologically defined tumor types [11–18] and led addition - content was chosen for genomic DNA isolation. The ally to the discovery of new tumor types based on unique Maxwell 16FFPE Plus LEV DNA Kit was applied on molecular features [19, 20]. Recently it has been shown to the automated Maxwell device (Promega, Madison, have great diagnostic capabilities determining the lineage WI, USA) according to the manufacturer’s instructions. Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 3 of 8 Tumor DNAs had a total amount of > 100  ng and were Copy-number assessment for segmental/entire chromo- suitable for the array-based DNA-methylation analysis. somal changes was done manually based on array data by a proprietary algorithm based on the R-package conumee Genomewide DNA ‑ ‑methylation data generation after additional baseline correction (https ://githu b.com/ and pre‑processingdstic hel/conum ee). The tumors were subjected to Illumina Infinium 450  k BeadChip or the successor EPIC/850  k BeadChip (Illu- Results mina, San Diego, USA) analysis at the Genomics and Study cohort Proteomics Core Facility of the German Cancer Research Tumor samples from 61 patients with the histopathologi- Center (DKFZ) Heidelberg. DNA-methylation data were cal diagnosis AFX (n = 17), PDS (n = 15), cSCC (n = 19) normalized by performing background correction and and BCC (n = 10) were analyzed together with 11 skin dye bias correction (shifting of negative control probe melanomas and 156 soft tissue tumors. The latter com - mean intensity to zero and scaling of normalization con- prised 11 angiosarcomas, 13 dermatofibrosarcomas pro - trol probe mean intensity to 20,000, respectively). Probes tuberans, 9 extraskeletal myxoid chondrosarcomas, 11 targeting sex chromosomes, probes containing multiple leiomyosarcomas, 14 myxoid liposarcomas, 19 malig- single nucleotide polymorphisms and those that could nant peripheral nerve sheath tumors, 21 neurofibromas, not be uniquely mapped were removed. Probes were 19 alveolar and 17 embryonal rhabdomyosarcomas, 10 excluded if the predecessor Illumina Infinium 450  k schwannomas and 12 undifferentiated pleomorphic BeadChip did not cover them, thereby making data sarcomas of the deep soft tissue. The median age was generated by both 450  k and EPIC comparable for sub- 81 years for AFX, 83 years for PDS, 79 years for cSCC and sequent analyses. In total, 438,370 probes were kept for 77 years for BCC. The AFX, PDS, cSCC and BCC cohort analysis. consisted of 58 primary tumor samples, two recurrent samples and one case with an unknown status. The pre - Unsupervised clustering, t‑SNE analysis, cumulative dominant side of occurrence was the head region (n = 46) copy number plotting and identification of differentially followed by the neck (n = 9). AFX and PDS had a much methylated regions higher incidence in male patients compared to cSCC and For unsupervised hierarchical clustering, we selected BCC. Clinical data are summarized in Table 1. 10,000 probes that showed the highest median absolute deviation (MAD) across the beta values. Samples were Unsupervised genomewide meth ‑ ylation profiling reveals hierarchically clustered using Euclidean distance and distinct signatures in dermal sarcomas and histologic Ward’s linkage method. Methylation probes were reor- mimics dered by hierarchical clustering using Euclidean distance Unsupervised hierarchical clustering and t-SNE analysis and complete linkage. The unscaled methylation levels delineated tumors in methylation classes (Fig.  1), which were shown in a heat map from unmethylated state (blue also kept stable when varying the number of CpGs using color) to methylated state (red color). For unsupervised for this analysis (data not shown). AFX and PDS were 2D representation of pairwise sample correlations dimen- indistinguishable by clustering (Fig. 1a) and t-SNE analy- sionality reduction by t-distributed stochastic neighbor ses (Fig.  1b). cSCC and BCC grouped in close proximity embedding (t-SNE) was performed using the 10,000 most to AFX and PDS. However, both formed homogeneous variable probes, a perplexity of 20 and 2500 iterations. subgroups and therefore were distinct from these in both Table 1 Clinical features of  atypical fibroxanthomas, pleomorphic dermal sarcomas, cutaneous squamous cell carcinomas and basal cell carcinomas Category AFX PDS cSCC BCC Group size (n) 17 15 19 10 Age median (range) [years] 81 (65–93) 83 (60–99) 79 (55–98) 77 (53–87) Male/female 16/1 13/2 12/7 7/3 Tumor location 10 head, skin 14 head, skin 15 head, skin 7 head, skin 7 neck, skin 1 unknown 2 hand, skin 2 trunk, skin 1 neck, skin 1 neck, skin 1 trunk, skin Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 4 of 8 a b ●●● ●● MLS ●●● ●● ●● BCC ●● ● ●● cSCC ●● ●● ● ● ●●●●● ● ●● ●● ● ●● ●● ● AFX/ ●●● ● ● 10 ● ● ● ●● ● ●●● ● PDS/ ● ●● ● ● ●● ●● AS ●● ●● ●● ●● ●●● RMSa #2 ● ●●●● ●●● UPS ● ●●● ● ● ● ●● ●● ●●● DFSP ● ●● ● Mel ●●● ● ●● ●● ●● ●● ●● LMS ● ●● ●● RMSa #1 ●●● ●● ●●● EMCS ● ●●● ●● NF ●● ●● ●●●● −10 ●● ● ● ●●●● ● ●●● ● ●● ● ● Angiosarcoma [AS] (n = 11) Melanoma [Mel] (n = 11) ● ● ● ● ● ● ●●● RMSe ●●● Myxoid liposarcoma [MLS] (n = 14) ●● SWN ● ● Atypical fibroxanthoma [AFX] (n = 17) ● ● ● ●●●● ● ● ●● Basal cell carcinoma [BCC] (n = 10) Neurofibroma [NF] (n = 21) ● ● ● ● Pleomorphic dermal sarcoma [PDS] (n = 15) ● Cutaneous squamous cell carcinoma [cSCC] (n = 19) ● ● ●● ●●● ●● ●● ● Dermatofibrosarcoma protuberans [DFSP] (n = 13) Rhabdomyosarcoma (alveolar) [RMSa] (n = 19) ●● ● ● ● ●●● MPNST ●● Extraskeletal myxoid chondrosarcoma [EMCS] (n = 9) Rhabdomyosarcoma (embryonal) [RMSe] (n = 17) −20 ● ● ● Leiomyosarcoma [LMS] (n = 11) Schwannoma [SWN] (n = 10) ● ● ● ● −20 −10 01020 Malignant peripheral nerve sheath tumor [MPNST] (n = 19) Undifferentiated pleomorphic sarcoma [UPS] (n = 12) ● ● ● ● t-SNE 1 Fig. 1 DNA‑methylation profiling in atypical fibroxanthomas, pleomorphic dermal sarcomas and histologic mimics. Unsupervised hierarchical clustering (a) and t‑Distributed Stochastic Neighbor Embedding (t ‑SNE) analysis (b) of DNA‑methylation data from atypical fibroxanthomas (AFX), pleomorphic dermal sarcomas (PDS) and histologic mimics shows a close epigenetic relation to cutaneous squamous cell carcinomas (cSCC). This AFX/PDS/SCC methylation cluster clearly separated from the methylation clusters of other diagnostic mimics analyses, even though single cases overlapped (Fig.  1). non-recurrent pattern involving 5q21.3 (FER), 8p11.22- Furthermore, we additionally analyzed 167 tumors 23 (FGFR1, TACC1) and 13q34 (LAMP1) in AFX, and encompassing 12 subtypes comprising different sarcoma 11q13.3 (CCND1) and 12q24.31 (KNTC1) in PDS (Addi- entities and melanoma, which may mimic the pheno- tional file 4: Figure S2). type of AFX and PDS. Each of these entities formed a Copy number alterations in cSCC were distributed subtype-specific methylation class. Interestingly, an obvi - similarly to AFX and PDS (Fig.  2c). Chromosomal losses ous outlier case, initially diagnosed as PDS for lack of were frequently encountered on 3p (8/19; 42%), 13q S100 and other melanoma specific staining (Additional (8/19; 42%) and 9p (12/19; 63%). Interestingly, the 19 file  2: Figure S1), repeatedly assigned to the methylation cSCC demonstrated no homozygous deletions of the class of melanomas. Applying a targeted next generation CDKN2A locus (9p). The most frequent gains involved sequencing panel the tumor demonstrated an activat- 3q (4/19; 21%) and 8q (5/19; 26%). Amplifications were ing TERT promoter mutation, a HRAS G12S mutation found in two cSCC involving MYC (8q24.21) and CCND1 as well as a BRAF G466E mutation. Sequencing data are (11q13.3), respectively. given in Additional file 3: Table S2. The copy number profiles of the 10 BCCs showed over - all less frequent chromosomal gains and losses compared to AFX, PDS and SCC (Fig.  2d). Obvious amplifications Cumulative copy‑number profiling revealed and deletions were absent in BCC. overlapping patterns between atypical fibroxanthomas and pleomorphic dermal sarcomas We next generated copy number profiles derived from Discussion the DNA-methylation array data. AFX and PDS (Fig.  2a, Our study demonstrates the predictive power of genome- b) revealed chromosomal imbalances that frequently wide methylation profiling in sarcomas of the skin (AFX/ involved losses of 9p (AFX 11/17; 65% vs. PDS 10/15; PDS) and their histologic mimics. Notably, all examined 66%) and 13q (AFX 11/17; 65% vs. PDS 14/15; 93%). A tumor subtypes exhibit specific epigenetic fingerprints gain of chromosome arm 8q was slightly more frequent with one exception. As expected, unsupervised cluster- in PDS (5/15; 33%) compared to AFX (3/17; 18%). The ing did not sort AFX and PDS into separate methylation homozygous deletion of the CDKN2A locus on 9p was groups. This finding is in line with the hypothesis that more frequent in PDS (6/15; 40%) compared to AFX AFX and PDS are part of a common tumor spectrum (2/17; 12%). Amplifications were rare in both AFX (3/15; with AFX potentially being a precursor lesion of PDS [3]. 20%) and PDS (2/15; 13%). They were distributed in a Distance t-SNE 2 Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 5 of 8 a b Atypical fibroxanthomas (n = 17) Pleomorphic dermal sarcomas (n = 15) 100 100 80 80 60 60 40 40 20 20 0 0 0 0 0 0 20 20 40 40 60 60 80 80 100 100 12 34 56 78 910111213141516171819202 21 2 12 34 56 78 910111213141516171819202 21 2 Chromosomes Chromosomes c d Cutaneous squamous cell carcinomas (n = 19) Basal cell carcinomas (n = 10) 100 100 80 80 60 60 40 40 20 20 0 0 0 0 0 0 20 20 40 40 60 60 80 80 100 100 12 34 56 78 910111213141516171819202 21 2 12 34 56 78 910111213141516171819202122 Chromosomes Chromosomes Fig. 2 Cumulative copy number profiles. Frequency of copy number variations in 17 atypical fibroxanthomas (a), in 15 pleomorphic dermal sarcomas (b), in 19 cutaneous squamous cell carcinomas (c) and 10 basal cell carcinomas (d), assessed by automated aberrations profiling The concept of AFX and PDS comprising a single entity (HNSCC) [34]. It remains to be determined whether this is supported by genetic studies [26, 27]. AFX and PDS finding may be adapted to AFX/PDS and cSCC. If vali - carry similar, but yet unspecific patterns of TP53 and dated in further studies, CDKN2A status might prove as a TERT promoter mutations associated with UV-exposure valuable biomarker in AFX and PDS that might open new such as observed in melanoma, cSCC and BCC [3, 26, 28, therapeutic avenues in a substantial portion of patients 29]. Recently, a next-generation sequencing based study suffering from this disease. on a considerable number of AFX and PDS expanded Our study does not provide a final decision on the the overlapping mutational pattern to NOTCH1/2 and ongoing debate regarding the histogenesis of AFX and FAT1 [27]. However, only a single whole-exome study of PDS. Many experts assume that AFX and PDS derive AFX has been presented so far [30]. Thus, further whole- from a mesenchymal origin [1, 4], whereas others sug- exome/genome studies with larger sample numbers of gest that AFX may derive from an epithelial origin [30, both AFX and PDS will be required to fully understand 35]. This theory was initially introduced by older stud - the genetic underpinnings of these tumors. ies describing clinicopathological similarities between Copy-number aberrations were found in a compa- AFX and cSCC with a sarcomatoid dedifferentiation [36, rable frequency and overlapping distribution in AFX 37]. AFX similar to cSCC and BCC frequently shows an and PDS. This is in concordance with previous stud - association with actinic skin damage and a close prox- ies showing recurrent copy number alterations mostly imity between the epithelium and the neoplastic spindle involving chromosome 8 and 9 [27, 31]. In addition, we cell population, however without an epithelial dysplasia found non-recurrent amplifications in 5/30 cases, which or carcinoma in situ component, which are both features almost equally affected AFX and PDS. In contrast to our and arguments for the diagnosis of a cutaneous spindle findings, a previous study detected amplifications only cell carcinoma with loss of keratin expression [1, 38, 39]. in PDS [31]. Hence, they suggested such markers for a Although we noticed a separation of BCC from cSCC tumor progression towards PDS. However, the study and AFX/PDS by epigenetic profiling and also a remark - cohort was mainly composed of PDS (n = 24) with only able delineation between AFX/PDS and cSCC, DNA- three AFX cases for comparison. methylation profiles of individual AFX, PDS and cSCC Beside amplifications, we also noticed recurrent were overlapping. Thus, the DNA-methylation analysis homozygous CDKN2A deletions in PDS (40%) and less primarily recapitulated the morphology of BCC, cSCC frequently in AFX (12%). CDKN2A deletions have been and AFX/PDS, which is usually quite distinct. recognized as an adverse prognostic marker in a num- Correctly distinguishing AFX/PDS from other tumors ber of tumors, i.e. in melanoma [32, 33]. Furthermore, is critical to allocate affected patients to the correct type a link between the susceptibility to checkpoint inhibi- of treatment and follow-up protocols. The current diag - tors and deletions of CDKN2A was discovered in some nosis of AFX/PDS based primarily on lack of expression cell lines derived from SCC of the head and neck region of certain lineage markers. However, there is a constant Frequency of alterations [%]Frequency of alterations [%] Frequency of alterations [%] Frequency of alterations [%] Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 6 of 8 risk that tumors of other lineages may have lost expres- Additional file 3: Table S2. List of gene mutations revealed by panel sion of diagnostically relevant markers due to dedifferen - sequencing in a pleomorphic dermal sarcoma with discordant DNA‑ methylation profile. tiation and then may be misclassified as AFX/PDS. For Additional file 4: Figure S2. Copy number profiles of the three atypical certain entities, such as the illustrated example where fibroxanthomas and the two pleomorphic dermal sarcomas carrying gene methylation and gene mutation signatures argue for a amplifications. melanoma, misclassification could have significant con - sequences for the patient [40]. Abbreviations Therefore, it would seem prudent to perform molecular MAD: median absolute deviation; t‑SNE: t ‑ distributed stochastic neighbor testing of cutaneous neoplasms when making a definitive embedding; DKFZ: German Cancer Research Center; FFPE: formalin‑fixed and paraffin‑ embedded; ID: internal identifier; Dx: diagnosis; Samp: sample; AS: diagnosis is not possible based on histomorphological angiosarcoma; AFX: atypical fibroxanthoma; BCC: basal cell carcinoma; cSCC: and immunohistochemical assessment alone. cutaneous squamous cell carcinoma; DFSP: dermatofibrosarcoma protu‑ berans; EMCS: extraskeletal myxoid chondrosarcoma; LMS: leiomyosarcoma; MPNST: malignant peripheral nerve sheath tumor; Mel: melanoma; MLS: Conclusion myxoid liposarcoma; NF: neurofibroma; PDS: pleomorphic dermal sarcoma; Our study demonstrates a proof of concept that DNA- RMS: rhabdomyosarcoma; SWN: schwannoma; UPS: undifferentiated pleomor ‑ phic sarcoma; P: primary; R: recurrence; Me: metastasis; U: unknown; f: female; methylation may be a valuable aid in routine diagnostics m: male. of skin tumors posing a diagnostic challenge with con- ventional analytic methods. Our data support the con- Authors’ contributions CK and DSt contributed equally to this manuscript. CK, TM and AvD conceived cept that AFX and PDS are histologically and molecularly the project. CK and AvD wrote the manuscript with input from all co‑authors. closely related and probably belong to a common tumor CK coordinated data generation. MB supervised the DNA‑methylation array spectrum. We observed a CDKN2A deletion in AFX analysis. CK, DSt and DSch analyzed DNA‑methylation array data. CK, KGG, DER, CV, WNMD, IP, MM, ACB, RB, SMP, UF, GM, TM and AvD provided tumor (12%) and PDS (40%), which may represent a potential samples and metadata. MB supervised the methylation data generation. All biomarker, if validated in future studies. Copy number authors analyzed the data and contributed to the final manuscript. All authors analysis and DNA methylation profiling can aid in distin - read and approved the final manuscript. guishing AFX/PDS from other histologic mimics, even Author details though these analyses alone cannot reliably distinguish 1 Department of General Pathology, Institute of Pathology, Heidelberg AFX from PDS. The assessment of histopathological fea - University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Baden‑Würt ‑ temberg, Germany. Department of Neuropathology, Institute of Pathology, tures such as subcutaneous involvement, necrosis, and Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, lymphovascular or perineural invasion still remain criti- 3 Baden‑Württemberg, Germany. Clinical Cooperation Unit Neuropathology, cal in differentiating PDS from AFX. German Cancer Research Center (DKFZ), Heidelberg, Baden‑Württemberg, Germany. German Cancer Consortium (DKTK), Core Center Heidelberg, Hei‑ delberg, Baden‑Württemberg, Germany. Department of Dermatology, Uni‑ versity Hospital Essen, West German Cancer Center, University Duisburg‑Essen and the German Cancer Consortium (DKTK), Essen, North RhineW ‑ estphalia, Additional files Germany. Dermatopathologie bei Mainz, Nieder‑Olm, Rhineland‑Palatinate, Germany. Genomics and Proteomics Core Facility, Microarray Unit, German Cancer Research Center (DKFZ), Heidelberg, Baden‑Württemberg, Germany. Additional file 1: Table S1. Clinical data. ID—internal identifier, Dx— Department of Pediatric Pathology, University Hospital of Schleswig‑Holstein, diagnosis, Samp—sample, AS—angiosarcoma, AFX—atypical fibroxan‑ Kiel, Schleswig‑Holstein, Germany. Department of Pathology, Erasmus Medi‑ thoma, BCC—basal cell carcinoma, cSCC—cutaneous squamous cell cal Center, Rotterdam, The Netherlands. Institute of Pathology, SRH Poliklinik carcinoma, DFSP—dermatofibrosarcoma protuberans, EMCS—extraskel‑ Gera GmbH, Gera, Germany. Luxembourg Centre of Neuropathology etal myxoid chondrosarcoma, LMS—leiomyosarcoma, MPNST—malig‑ (LCNP), Luxembourg City, Luxembourg. Laboratoire National de Santé (LNS), nant peripheral nerve sheath tumor, Mel—melanoma, MLS—myxoid Dudelange, Luxembourg. Luxembourg Centre for Systems Biomedicine liposarcoma, NF—neurofibroma, PDS—pleomorphic dermal sarcoma, (LCSB), University of Luxembourg, Luxembourg City, Luxembourg. NORLUX RMS—rhabdomyosarcoma, SWN—schwannoma, UPS—undifferentiated Neuro‑Oncology Laboratory, Luxembourg Institute of Health (LIH), Luxem‑ pleomorphic sarcoma, P—primary, R—recurrence, Me—metastasis, U— bourg City, Luxembourg. Institute of Pathology, Sozialstiftung Bamberg, unknown, f—female, m—male. Bamberg, Germany. Hopp Childrens Cancer Center at the NCT Heidelberg Additional file 2: Figure S1. Histologic and immunohistochemical fea‑ (KiTZ), Heidelberg, Germany. Division of Pediatric Neurooncology, German tures of a pleomorphic dermal sarcoma with a DNA‑methylation pattern Cancer Research Center (DKFZ), Heidelberg, Baden‑Württemberg, Germany. resembling melanoma. This highly cellular tumor (ID 101138) with brisk Department of Pediatric Oncology, Hematology and Immunology, Univer‑ mitotic activity (green arrows) predominantly presented with a polygonal sity of Heidelberg, Heidelberg, Baden‑Württemberg, Germany. Department to spindle‑shape appearance and a fascicular growth pattern (a). In a of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands. circumscribed area the tumor cells were epithelioid (b). Adjacent subcu‑ Dermatopathology Bodensee, Friedrichshafen, Baden‑Württemberg, taneous fat tissue was infiltrated (c) and vascular invasion was observed Germany. (d). Parts of the tumor were necrotic (e). The tumor cells did not bind S100 specific antibody, whereas peripheral nerve and few histiocytes were posi‑ Acknowledgements tive (f ). The tumor cells were negative for nuclear SOX10 expression with We thank the Microarray Unit of the Genomics and Proteomics Core Facility, peripheral nerve as positive internal control (g), negative for HMB45 (h) German Cancer Research Center (DKFZ), for providing excellent methylation and MelanA (i) protein expression. Scale‑bars equal 100 µm. services. MM would like to thank the Luxembourg National Research Fund Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 7 of 8 (FNR) for the support (FNR PEARL P16/BM/11192868 Grant). We acknowledge 12. Koelsche C, Hovestadt V, Jones DT, Capper D, Sturm D, Sahm F, Schrimpf financial support by Deutsche Forschungsgemeinschaft within the funding D, Adeberg S, Bohmer K, Hagenlocher C, et al. Melanotic tumors of the programme Open Access Publishing, by the Baden‑ Württemberg Ministry of nervous system are characterized by distinct mutational, chromosomal Science, Research and the Arts and by Ruprecht‑Karls‑Universität Heidelberg. and epigenomic profiles. Brain Pathol. 2015;25(2):202–8. 13. Koelsche C, Schrimpf D, Tharun L, Roth E, Sturm D, Jones DTW, Renker Competing interests EK, Sill M, Baude A, Sahm F, et al. Histone 3.3 hotspot mutations in The authors declare that they have no competing interests. conventional osteosarcomas: a comprehensive clinical and molecular characterization of six H3F3A mutated cases. Clin Sarcoma Res. 2017;7:9. Availability of data and materials 14. Pajtler KW, Witt H, Sill M, Jones DT, Hovestadt V, Kratochwil F, Wani K, CpG methylation values are available from the corresponding author upon Tatevossian R, Punchihewa C, Johann P, et al. Molecular classification reasonable request. of ependymal tumors across all CNS compartments, histopathological grades, and age groups. Cancer Cell. 2015;27(5):728–43. Consent for publication 15. Rohrich M, Koelsche C, Schrimpf D, Capper D, Sahm F, Kratz A, Reuss J, Not applicable. Hovestadt V, Jones DT, Bewerunge‑Hudler M, et al. Methylation‑based classification of benign and malignant peripheral nerve sheath tumors. Ethics approval and consent to participate Acta Neuropathol. 2016;131(6):877–87. This study has been performed in accordance with the Declaration of 16. Sahm F, Schrimpf D, Stichel D, Jones DTW, Hielscher T, Schefzyk S, Helsinki. The ethics committee of the medical faculty of Heidelberg University Okonechnikov K, Koelsche C, Reuss DE, Capper D, et al. DNA methylation‑ approved the use of archived tissue specimens (older than 3 years) collected based classification and grading system for meningioma: a multicentre, from the Institute of Pathology of the University Heidelberg for retrospective retrospective analysis. Lancet Oncol. 2017;18(5):682–94. research purposes (reference 206/2005). The patients’ consent was waived due 17. Seki M, Nishimura R, Yoshida K, Shimamura T, Shiraishi Y, Sato Y, Kato M, to the retrospective nature of this study and minimal risk to the subjects. Chiba K, Tanaka H, Hoshino N, et al. Integrated genetic and epigenetic analysis defines novel molecular subgroups in rhabdomyosarcoma. Nat Funding Commun. 2015;6:7557. This work was funded by a Grant (70112499) of the German Cancer Aid (to C.K. 18. Wiestler B, Capper D, Sill M, Jones DT, Hovestadt V, Sturm D, Koelsche C, and A.v.D.). Bertoni A, Schweizer L, Korshunov A, et al. Integrated DNA methylation and copy‑number profiling identify three clinically and biologically rele ‑ vant groups of anaplastic glioma. Acta Neuropathol. 2014;128(4):561–71. Publisher’s Note 19. Sturm D, Orr BA, Toprak UH, Hovestadt V, Jones DTW, Capper D, Sill M, Springer Nature remains neutral with regard to jurisdictional claims in pub‑ Buchhalter I, Northcott PA, Leis I, et al. New brain tumor entities emerge lished maps and institutional affiliations. from molecular classification of CNS‑PNETs. Cell. 2016;164(5):1060–72. 20. Koelsche C, Mynarek M, Schrimpf D, Bertero L, Serrano J, Sahm F, Reuss Received: 27 November 2018 Accepted: 5 February 2019 DE, Hou Y, Baumhoer D, Vokuhl C, et al. Primary intracranial spindle cell sarcoma with rhabdomyosarcoma‑like features share a highly distinct methylation profile and DICER1 mutations. Acta Neuropathol. 2018;136(2):327–37. 21. 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Genome-wide methylation profiling and copy number analysis in atypical fibroxanthomas and pleomorphic dermal sarcomas indicate a similar molecular phenotype

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Biomedicine; Cancer Research; Oncology; Surgical Oncology
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Abstract

Background: Atypical fibroxanthomas (AFX) and pleomorphic dermal sarcomas (PDS) are lesions of the skin with overlapping histologic features and unspecific molecular traits. PDS behaves aggressive compared to AFX. Thus, a precise delineation, although challenging in some instances, is relevant. Methods: We examined the value of DNA‑methylation profiling and copy number analysis for separating these tumors. DNA‑methylation data were generated from 17 AFX and 15 PDS using the Illumina EPIC array. These were compared with DNA‑methylation data generated from 196 tumors encompassing potential histologic mimics like cutaneous squamous carcinomas (cSCC; n = 19), basal cell carcinomas (n = 10), melanoma metastases originat‑ ing from the skin (n = 11), leiomyosarcomas (n = 11), angiosarcomas of the skin and soft tissue (n = 11), malignant peripheral nerve sheath tumors (n = 19), dermatofibrosarcomas protuberans (n = 13), extraskeletal myxoid chondro‑ sarcomas (n = 9), myxoid liposarcomas (n = 14), schwannomas (n = 10), neurofibromas (n = 21), alveolar (n = 19) and embryonal (n = 17) rhabdomyosarcomas as well as undifferentiated pleomorphic sarcomas (n = 12). Results: DNA‑methylation profiling did not separate AFX from PDS. The DNA‑methylation profiles of the other cases, however, were distinct from AFX/PDS. They reliably assigned to subtype‑specific DNA‑methylation clusters, although overlap occurred between some AFX/PDS and cSCC. Copy number profiling revealed alterations in a similar fre ‑ quency and distribution between AFX and PDS. They involved losses of 9p (22/32) and 13q (25/32). Gains frequently involved 8q (8/32). Notably, a homozygous deletion of CDKN2A was more frequent in PDS (6/15) than in AFX (2/17), whereas amplifications were non‑recurrent and overall rare (5/32). *Correspondence: Christian.Koelsche@med.uni‑heidelberg.de; Andreas. vonDeimling@med.uni‑heidelberg.de Christian Koelsche and Damian Stichel contributed equally to this work Department of General Pathology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Baden‑Württemberg, Germany Department of Neuropathology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Baden‑Württemberg, Germany Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 2 of 8 Conclusions: Our findings support the concept that AFX and PDS belong to a common tumor spectrum. We could demonstrate the diagnostic value of DNA‑methylation profiling to delineating AFX/PDS from potential mimics. How‑ ever, the assessment of certain histologic features remains crucial for separating PDS from AFX. Keywords: Pleomorphic dermal sarcoma, Atypical fibroxanthoma, Sarcomas, Melanomas, Carcinomas, Mimics, DNA methylation, Profiling Background of small blue round cell tumors not otherwise specified Sarcomas of the skin and the adjacent soft tissue com- [21], cancers of unknown primary [22] and nervous sys- prise a heterogeneous tumor group [1]. The classifica - tem tumors [23]. tion of these tumors follows the lineage differentiation AFX and PDS are generally believed to be of mesen- of tumor cells, which is predominantly assessed by their chymal lineage, although a few studies have suggested an expression of lineage specific markers. However, in many epithelial origin [24, 25]. Detailed DNA-methylation pat- cases an unambiguous subtype assignment by histo- terns in AFX and PDS have not been reported yet. We logic and immunohistochemical means is not possible, therefore performed genome-wide methylation profiling and molecular analyses for establishing a final diagnosis and copy number analysis of AFX, PDS and potential his- is required [2]. Unfortunately, certain entities also lack tologic mimics, with a focus on cutaneous squamous car- unequivocal molecular traits, even if more sophisticated cinomas (cSCC) and basal cell carcinomas (BCC) of the molecular approaches such as next generation sequenc- head and neck, alongside of melanomas and 11 soft tissue ing are applied. Atypical fibroxanthomas (AFX) and tumor entities. pleomorphic dermal sarcomas (PDS) belong to the afore- mentioned group of ill-defined tumors and currently Materials and methods remain a diagnosis of exclusion [3]. Sample selection AFX and PDS exhibit overlapping histologic features In total, 228 tumor specimens from different patients, all making a reliable distinction in many cases problematic prototypical examples of their corresponding subtype, [4]. The most important criterion in favor of the diag - were included (Additional file  1: Table  S1). AFX, PDS, nosis PDS is an invading growth pattern into subdermal cSCC and BCC were collected from the Dermatopa- structures, which can be difficult to assess if small biop - thology Bodensee in Friedrichshafen (Germany) and the sies are provided for histopathological diagnostics [5]. Department of Dermatology of the University Hospital Other diagnostic histologic features include necrosis, in Essen (Germany). Melanomas and soft tissue tumors lymphovascular and perineural invasion. However, gen- were collected from the Institute of Pathology of the Uni- eral features of anaplasia such as nuclear pleomorphism versity Hospital in Heidelberg (Germany), in Kiel (Ger- and atypical mitoses are common to both AFX and PDS many), in Jena (Germany), in Nijmegen and in Rotterdam [6, 7]. The distinction of AFX and PDS as different enti - (both the Netherlands), from the Institute of Pathology in ties remains clinically important. AFX has an overall Bamberg (Germany) and from the Department of Pathol- favorable biological behavior compared to the much ogy of the Laboratoire National de Santé (Luxembourg). higher potential for recurrence and metastasis in PDS Diagnoses were based on standard histopathological [3, 6, 7]. Novel diagnostic approaches allowing a clear criteria in conjunction with immunohistochemical and distinction of AFX and PDS would be of great value con- molecular analyses according to the current WHO clas- sidering the steadily increasing incidence of skin cancers sification [1]. The methylation data of melanomas and [8] and promising results of targeted therapies for certain some soft tissue tumors were published previously [12, dermal tumor subtypes [9, 10]. 15, 21]. DNA-methylation profiling has evolved as a powerful method for determining cell differentiation. Array-based epigenotyping technologies nowadays enable large-scale DNA extraction high-throughput studies of DNA methylation patterns. DNA was extracted from formalin-fixed and paraffin- The study of DNA-methylation in different cancers has embedded (FFPE) tumor tissue, thereby only using rep- already revealed molecular subgroups within known his- resentative tumor tissue with highest available tumor tologically defined tumor types [11–18] and led addition - content was chosen for genomic DNA isolation. The ally to the discovery of new tumor types based on unique Maxwell 16FFPE Plus LEV DNA Kit was applied on molecular features [19, 20]. Recently it has been shown to the automated Maxwell device (Promega, Madison, have great diagnostic capabilities determining the lineage WI, USA) according to the manufacturer’s instructions. Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 3 of 8 Tumor DNAs had a total amount of > 100  ng and were Copy-number assessment for segmental/entire chromo- suitable for the array-based DNA-methylation analysis. somal changes was done manually based on array data by a proprietary algorithm based on the R-package conumee Genomewide DNA ‑ ‑methylation data generation after additional baseline correction (https ://githu b.com/ and pre‑processingdstic hel/conum ee). The tumors were subjected to Illumina Infinium 450  k BeadChip or the successor EPIC/850  k BeadChip (Illu- Results mina, San Diego, USA) analysis at the Genomics and Study cohort Proteomics Core Facility of the German Cancer Research Tumor samples from 61 patients with the histopathologi- Center (DKFZ) Heidelberg. DNA-methylation data were cal diagnosis AFX (n = 17), PDS (n = 15), cSCC (n = 19) normalized by performing background correction and and BCC (n = 10) were analyzed together with 11 skin dye bias correction (shifting of negative control probe melanomas and 156 soft tissue tumors. The latter com - mean intensity to zero and scaling of normalization con- prised 11 angiosarcomas, 13 dermatofibrosarcomas pro - trol probe mean intensity to 20,000, respectively). Probes tuberans, 9 extraskeletal myxoid chondrosarcomas, 11 targeting sex chromosomes, probes containing multiple leiomyosarcomas, 14 myxoid liposarcomas, 19 malig- single nucleotide polymorphisms and those that could nant peripheral nerve sheath tumors, 21 neurofibromas, not be uniquely mapped were removed. Probes were 19 alveolar and 17 embryonal rhabdomyosarcomas, 10 excluded if the predecessor Illumina Infinium 450  k schwannomas and 12 undifferentiated pleomorphic BeadChip did not cover them, thereby making data sarcomas of the deep soft tissue. The median age was generated by both 450  k and EPIC comparable for sub- 81 years for AFX, 83 years for PDS, 79 years for cSCC and sequent analyses. In total, 438,370 probes were kept for 77 years for BCC. The AFX, PDS, cSCC and BCC cohort analysis. consisted of 58 primary tumor samples, two recurrent samples and one case with an unknown status. The pre - Unsupervised clustering, t‑SNE analysis, cumulative dominant side of occurrence was the head region (n = 46) copy number plotting and identification of differentially followed by the neck (n = 9). AFX and PDS had a much methylated regions higher incidence in male patients compared to cSCC and For unsupervised hierarchical clustering, we selected BCC. Clinical data are summarized in Table 1. 10,000 probes that showed the highest median absolute deviation (MAD) across the beta values. Samples were Unsupervised genomewide meth ‑ ylation profiling reveals hierarchically clustered using Euclidean distance and distinct signatures in dermal sarcomas and histologic Ward’s linkage method. Methylation probes were reor- mimics dered by hierarchical clustering using Euclidean distance Unsupervised hierarchical clustering and t-SNE analysis and complete linkage. The unscaled methylation levels delineated tumors in methylation classes (Fig.  1), which were shown in a heat map from unmethylated state (blue also kept stable when varying the number of CpGs using color) to methylated state (red color). For unsupervised for this analysis (data not shown). AFX and PDS were 2D representation of pairwise sample correlations dimen- indistinguishable by clustering (Fig. 1a) and t-SNE analy- sionality reduction by t-distributed stochastic neighbor ses (Fig.  1b). cSCC and BCC grouped in close proximity embedding (t-SNE) was performed using the 10,000 most to AFX and PDS. However, both formed homogeneous variable probes, a perplexity of 20 and 2500 iterations. subgroups and therefore were distinct from these in both Table 1 Clinical features of  atypical fibroxanthomas, pleomorphic dermal sarcomas, cutaneous squamous cell carcinomas and basal cell carcinomas Category AFX PDS cSCC BCC Group size (n) 17 15 19 10 Age median (range) [years] 81 (65–93) 83 (60–99) 79 (55–98) 77 (53–87) Male/female 16/1 13/2 12/7 7/3 Tumor location 10 head, skin 14 head, skin 15 head, skin 7 head, skin 7 neck, skin 1 unknown 2 hand, skin 2 trunk, skin 1 neck, skin 1 neck, skin 1 trunk, skin Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 4 of 8 a b ●●● ●● MLS ●●● ●● ●● BCC ●● ● ●● cSCC ●● ●● ● ● ●●●●● ● ●● ●● ● ●● ●● ● AFX/ ●●● ● ● 10 ● ● ● ●● ● ●●● ● PDS/ ● ●● ● ● ●● ●● AS ●● ●● ●● ●● ●●● RMSa #2 ● ●●●● ●●● UPS ● ●●● ● ● ● ●● ●● ●●● DFSP ● ●● ● Mel ●●● ● ●● ●● ●● ●● ●● LMS ● ●● ●● RMSa #1 ●●● ●● ●●● EMCS ● ●●● ●● NF ●● ●● ●●●● −10 ●● ● ● ●●●● ● ●●● ● ●● ● ● Angiosarcoma [AS] (n = 11) Melanoma [Mel] (n = 11) ● ● ● ● ● ● ●●● RMSe ●●● Myxoid liposarcoma [MLS] (n = 14) ●● SWN ● ● Atypical fibroxanthoma [AFX] (n = 17) ● ● ● ●●●● ● ● ●● Basal cell carcinoma [BCC] (n = 10) Neurofibroma [NF] (n = 21) ● ● ● ● Pleomorphic dermal sarcoma [PDS] (n = 15) ● Cutaneous squamous cell carcinoma [cSCC] (n = 19) ● ● ●● ●●● ●● ●● ● Dermatofibrosarcoma protuberans [DFSP] (n = 13) Rhabdomyosarcoma (alveolar) [RMSa] (n = 19) ●● ● ● ● ●●● MPNST ●● Extraskeletal myxoid chondrosarcoma [EMCS] (n = 9) Rhabdomyosarcoma (embryonal) [RMSe] (n = 17) −20 ● ● ● Leiomyosarcoma [LMS] (n = 11) Schwannoma [SWN] (n = 10) ● ● ● ● −20 −10 01020 Malignant peripheral nerve sheath tumor [MPNST] (n = 19) Undifferentiated pleomorphic sarcoma [UPS] (n = 12) ● ● ● ● t-SNE 1 Fig. 1 DNA‑methylation profiling in atypical fibroxanthomas, pleomorphic dermal sarcomas and histologic mimics. Unsupervised hierarchical clustering (a) and t‑Distributed Stochastic Neighbor Embedding (t ‑SNE) analysis (b) of DNA‑methylation data from atypical fibroxanthomas (AFX), pleomorphic dermal sarcomas (PDS) and histologic mimics shows a close epigenetic relation to cutaneous squamous cell carcinomas (cSCC). This AFX/PDS/SCC methylation cluster clearly separated from the methylation clusters of other diagnostic mimics analyses, even though single cases overlapped (Fig.  1). non-recurrent pattern involving 5q21.3 (FER), 8p11.22- Furthermore, we additionally analyzed 167 tumors 23 (FGFR1, TACC1) and 13q34 (LAMP1) in AFX, and encompassing 12 subtypes comprising different sarcoma 11q13.3 (CCND1) and 12q24.31 (KNTC1) in PDS (Addi- entities and melanoma, which may mimic the pheno- tional file 4: Figure S2). type of AFX and PDS. Each of these entities formed a Copy number alterations in cSCC were distributed subtype-specific methylation class. Interestingly, an obvi - similarly to AFX and PDS (Fig.  2c). Chromosomal losses ous outlier case, initially diagnosed as PDS for lack of were frequently encountered on 3p (8/19; 42%), 13q S100 and other melanoma specific staining (Additional (8/19; 42%) and 9p (12/19; 63%). Interestingly, the 19 file  2: Figure S1), repeatedly assigned to the methylation cSCC demonstrated no homozygous deletions of the class of melanomas. Applying a targeted next generation CDKN2A locus (9p). The most frequent gains involved sequencing panel the tumor demonstrated an activat- 3q (4/19; 21%) and 8q (5/19; 26%). Amplifications were ing TERT promoter mutation, a HRAS G12S mutation found in two cSCC involving MYC (8q24.21) and CCND1 as well as a BRAF G466E mutation. Sequencing data are (11q13.3), respectively. given in Additional file 3: Table S2. The copy number profiles of the 10 BCCs showed over - all less frequent chromosomal gains and losses compared to AFX, PDS and SCC (Fig.  2d). Obvious amplifications Cumulative copy‑number profiling revealed and deletions were absent in BCC. overlapping patterns between atypical fibroxanthomas and pleomorphic dermal sarcomas We next generated copy number profiles derived from Discussion the DNA-methylation array data. AFX and PDS (Fig.  2a, Our study demonstrates the predictive power of genome- b) revealed chromosomal imbalances that frequently wide methylation profiling in sarcomas of the skin (AFX/ involved losses of 9p (AFX 11/17; 65% vs. PDS 10/15; PDS) and their histologic mimics. Notably, all examined 66%) and 13q (AFX 11/17; 65% vs. PDS 14/15; 93%). A tumor subtypes exhibit specific epigenetic fingerprints gain of chromosome arm 8q was slightly more frequent with one exception. As expected, unsupervised cluster- in PDS (5/15; 33%) compared to AFX (3/17; 18%). The ing did not sort AFX and PDS into separate methylation homozygous deletion of the CDKN2A locus on 9p was groups. This finding is in line with the hypothesis that more frequent in PDS (6/15; 40%) compared to AFX AFX and PDS are part of a common tumor spectrum (2/17; 12%). Amplifications were rare in both AFX (3/15; with AFX potentially being a precursor lesion of PDS [3]. 20%) and PDS (2/15; 13%). They were distributed in a Distance t-SNE 2 Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 5 of 8 a b Atypical fibroxanthomas (n = 17) Pleomorphic dermal sarcomas (n = 15) 100 100 80 80 60 60 40 40 20 20 0 0 0 0 0 0 20 20 40 40 60 60 80 80 100 100 12 34 56 78 910111213141516171819202 21 2 12 34 56 78 910111213141516171819202 21 2 Chromosomes Chromosomes c d Cutaneous squamous cell carcinomas (n = 19) Basal cell carcinomas (n = 10) 100 100 80 80 60 60 40 40 20 20 0 0 0 0 0 0 20 20 40 40 60 60 80 80 100 100 12 34 56 78 910111213141516171819202 21 2 12 34 56 78 910111213141516171819202122 Chromosomes Chromosomes Fig. 2 Cumulative copy number profiles. Frequency of copy number variations in 17 atypical fibroxanthomas (a), in 15 pleomorphic dermal sarcomas (b), in 19 cutaneous squamous cell carcinomas (c) and 10 basal cell carcinomas (d), assessed by automated aberrations profiling The concept of AFX and PDS comprising a single entity (HNSCC) [34]. It remains to be determined whether this is supported by genetic studies [26, 27]. AFX and PDS finding may be adapted to AFX/PDS and cSCC. If vali - carry similar, but yet unspecific patterns of TP53 and dated in further studies, CDKN2A status might prove as a TERT promoter mutations associated with UV-exposure valuable biomarker in AFX and PDS that might open new such as observed in melanoma, cSCC and BCC [3, 26, 28, therapeutic avenues in a substantial portion of patients 29]. Recently, a next-generation sequencing based study suffering from this disease. on a considerable number of AFX and PDS expanded Our study does not provide a final decision on the the overlapping mutational pattern to NOTCH1/2 and ongoing debate regarding the histogenesis of AFX and FAT1 [27]. However, only a single whole-exome study of PDS. Many experts assume that AFX and PDS derive AFX has been presented so far [30]. Thus, further whole- from a mesenchymal origin [1, 4], whereas others sug- exome/genome studies with larger sample numbers of gest that AFX may derive from an epithelial origin [30, both AFX and PDS will be required to fully understand 35]. This theory was initially introduced by older stud - the genetic underpinnings of these tumors. ies describing clinicopathological similarities between Copy-number aberrations were found in a compa- AFX and cSCC with a sarcomatoid dedifferentiation [36, rable frequency and overlapping distribution in AFX 37]. AFX similar to cSCC and BCC frequently shows an and PDS. This is in concordance with previous stud - association with actinic skin damage and a close prox- ies showing recurrent copy number alterations mostly imity between the epithelium and the neoplastic spindle involving chromosome 8 and 9 [27, 31]. In addition, we cell population, however without an epithelial dysplasia found non-recurrent amplifications in 5/30 cases, which or carcinoma in situ component, which are both features almost equally affected AFX and PDS. In contrast to our and arguments for the diagnosis of a cutaneous spindle findings, a previous study detected amplifications only cell carcinoma with loss of keratin expression [1, 38, 39]. in PDS [31]. Hence, they suggested such markers for a Although we noticed a separation of BCC from cSCC tumor progression towards PDS. However, the study and AFX/PDS by epigenetic profiling and also a remark - cohort was mainly composed of PDS (n = 24) with only able delineation between AFX/PDS and cSCC, DNA- three AFX cases for comparison. methylation profiles of individual AFX, PDS and cSCC Beside amplifications, we also noticed recurrent were overlapping. Thus, the DNA-methylation analysis homozygous CDKN2A deletions in PDS (40%) and less primarily recapitulated the morphology of BCC, cSCC frequently in AFX (12%). CDKN2A deletions have been and AFX/PDS, which is usually quite distinct. recognized as an adverse prognostic marker in a num- Correctly distinguishing AFX/PDS from other tumors ber of tumors, i.e. in melanoma [32, 33]. Furthermore, is critical to allocate affected patients to the correct type a link between the susceptibility to checkpoint inhibi- of treatment and follow-up protocols. The current diag - tors and deletions of CDKN2A was discovered in some nosis of AFX/PDS based primarily on lack of expression cell lines derived from SCC of the head and neck region of certain lineage markers. However, there is a constant Frequency of alterations [%]Frequency of alterations [%] Frequency of alterations [%] Frequency of alterations [%] Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 6 of 8 risk that tumors of other lineages may have lost expres- Additional file 3: Table S2. List of gene mutations revealed by panel sion of diagnostically relevant markers due to dedifferen - sequencing in a pleomorphic dermal sarcoma with discordant DNA‑ methylation profile. tiation and then may be misclassified as AFX/PDS. For Additional file 4: Figure S2. Copy number profiles of the three atypical certain entities, such as the illustrated example where fibroxanthomas and the two pleomorphic dermal sarcomas carrying gene methylation and gene mutation signatures argue for a amplifications. melanoma, misclassification could have significant con - sequences for the patient [40]. Abbreviations Therefore, it would seem prudent to perform molecular MAD: median absolute deviation; t‑SNE: t ‑ distributed stochastic neighbor testing of cutaneous neoplasms when making a definitive embedding; DKFZ: German Cancer Research Center; FFPE: formalin‑fixed and paraffin‑ embedded; ID: internal identifier; Dx: diagnosis; Samp: sample; AS: diagnosis is not possible based on histomorphological angiosarcoma; AFX: atypical fibroxanthoma; BCC: basal cell carcinoma; cSCC: and immunohistochemical assessment alone. cutaneous squamous cell carcinoma; DFSP: dermatofibrosarcoma protu‑ berans; EMCS: extraskeletal myxoid chondrosarcoma; LMS: leiomyosarcoma; MPNST: malignant peripheral nerve sheath tumor; Mel: melanoma; MLS: Conclusion myxoid liposarcoma; NF: neurofibroma; PDS: pleomorphic dermal sarcoma; Our study demonstrates a proof of concept that DNA- RMS: rhabdomyosarcoma; SWN: schwannoma; UPS: undifferentiated pleomor ‑ phic sarcoma; P: primary; R: recurrence; Me: metastasis; U: unknown; f: female; methylation may be a valuable aid in routine diagnostics m: male. of skin tumors posing a diagnostic challenge with con- ventional analytic methods. Our data support the con- Authors’ contributions CK and DSt contributed equally to this manuscript. CK, TM and AvD conceived cept that AFX and PDS are histologically and molecularly the project. CK and AvD wrote the manuscript with input from all co‑authors. closely related and probably belong to a common tumor CK coordinated data generation. MB supervised the DNA‑methylation array spectrum. We observed a CDKN2A deletion in AFX analysis. CK, DSt and DSch analyzed DNA‑methylation array data. CK, KGG, DER, CV, WNMD, IP, MM, ACB, RB, SMP, UF, GM, TM and AvD provided tumor (12%) and PDS (40%), which may represent a potential samples and metadata. MB supervised the methylation data generation. All biomarker, if validated in future studies. Copy number authors analyzed the data and contributed to the final manuscript. All authors analysis and DNA methylation profiling can aid in distin - read and approved the final manuscript. guishing AFX/PDS from other histologic mimics, even Author details though these analyses alone cannot reliably distinguish 1 Department of General Pathology, Institute of Pathology, Heidelberg AFX from PDS. The assessment of histopathological fea - University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Baden‑Würt ‑ temberg, Germany. Department of Neuropathology, Institute of Pathology, tures such as subcutaneous involvement, necrosis, and Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, lymphovascular or perineural invasion still remain criti- 3 Baden‑Württemberg, Germany. Clinical Cooperation Unit Neuropathology, cal in differentiating PDS from AFX. German Cancer Research Center (DKFZ), Heidelberg, Baden‑Württemberg, Germany. German Cancer Consortium (DKTK), Core Center Heidelberg, Hei‑ delberg, Baden‑Württemberg, Germany. Department of Dermatology, Uni‑ versity Hospital Essen, West German Cancer Center, University Duisburg‑Essen and the German Cancer Consortium (DKTK), Essen, North RhineW ‑ estphalia, Additional files Germany. Dermatopathologie bei Mainz, Nieder‑Olm, Rhineland‑Palatinate, Germany. Genomics and Proteomics Core Facility, Microarray Unit, German Cancer Research Center (DKFZ), Heidelberg, Baden‑Württemberg, Germany. Additional file 1: Table S1. Clinical data. ID—internal identifier, Dx— Department of Pediatric Pathology, University Hospital of Schleswig‑Holstein, diagnosis, Samp—sample, AS—angiosarcoma, AFX—atypical fibroxan‑ Kiel, Schleswig‑Holstein, Germany. Department of Pathology, Erasmus Medi‑ thoma, BCC—basal cell carcinoma, cSCC—cutaneous squamous cell cal Center, Rotterdam, The Netherlands. Institute of Pathology, SRH Poliklinik carcinoma, DFSP—dermatofibrosarcoma protuberans, EMCS—extraskel‑ Gera GmbH, Gera, Germany. Luxembourg Centre of Neuropathology etal myxoid chondrosarcoma, LMS—leiomyosarcoma, MPNST—malig‑ (LCNP), Luxembourg City, Luxembourg. Laboratoire National de Santé (LNS), nant peripheral nerve sheath tumor, Mel—melanoma, MLS—myxoid Dudelange, Luxembourg. Luxembourg Centre for Systems Biomedicine liposarcoma, NF—neurofibroma, PDS—pleomorphic dermal sarcoma, (LCSB), University of Luxembourg, Luxembourg City, Luxembourg. NORLUX RMS—rhabdomyosarcoma, SWN—schwannoma, UPS—undifferentiated Neuro‑Oncology Laboratory, Luxembourg Institute of Health (LIH), Luxem‑ pleomorphic sarcoma, P—primary, R—recurrence, Me—metastasis, U— bourg City, Luxembourg. Institute of Pathology, Sozialstiftung Bamberg, unknown, f—female, m—male. Bamberg, Germany. Hopp Childrens Cancer Center at the NCT Heidelberg Additional file 2: Figure S1. Histologic and immunohistochemical fea‑ (KiTZ), Heidelberg, Germany. Division of Pediatric Neurooncology, German tures of a pleomorphic dermal sarcoma with a DNA‑methylation pattern Cancer Research Center (DKFZ), Heidelberg, Baden‑Württemberg, Germany. resembling melanoma. This highly cellular tumor (ID 101138) with brisk Department of Pediatric Oncology, Hematology and Immunology, Univer‑ mitotic activity (green arrows) predominantly presented with a polygonal sity of Heidelberg, Heidelberg, Baden‑Württemberg, Germany. Department to spindle‑shape appearance and a fascicular growth pattern (a). In a of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands. circumscribed area the tumor cells were epithelioid (b). Adjacent subcu‑ Dermatopathology Bodensee, Friedrichshafen, Baden‑Württemberg, taneous fat tissue was infiltrated (c) and vascular invasion was observed Germany. (d). Parts of the tumor were necrotic (e). The tumor cells did not bind S100 specific antibody, whereas peripheral nerve and few histiocytes were posi‑ Acknowledgements tive (f ). The tumor cells were negative for nuclear SOX10 expression with We thank the Microarray Unit of the Genomics and Proteomics Core Facility, peripheral nerve as positive internal control (g), negative for HMB45 (h) German Cancer Research Center (DKFZ), for providing excellent methylation and MelanA (i) protein expression. Scale‑bars equal 100 µm. services. MM would like to thank the Luxembourg National Research Fund Koelsche et al. Clin Sarcoma Res (2019) 9:2 Page 7 of 8 (FNR) for the support (FNR PEARL P16/BM/11192868 Grant). We acknowledge 12. Koelsche C, Hovestadt V, Jones DT, Capper D, Sturm D, Sahm F, Schrimpf financial support by Deutsche Forschungsgemeinschaft within the funding D, Adeberg S, Bohmer K, Hagenlocher C, et al. Melanotic tumors of the programme Open Access Publishing, by the Baden‑ Württemberg Ministry of nervous system are characterized by distinct mutational, chromosomal Science, Research and the Arts and by Ruprecht‑Karls‑Universität Heidelberg. and epigenomic profiles. Brain Pathol. 2015;25(2):202–8. 13. Koelsche C, Schrimpf D, Tharun L, Roth E, Sturm D, Jones DTW, Renker Competing interests EK, Sill M, Baude A, Sahm F, et al. Histone 3.3 hotspot mutations in The authors declare that they have no competing interests. conventional osteosarcomas: a comprehensive clinical and molecular characterization of six H3F3A mutated cases. Clin Sarcoma Res. 2017;7:9. Availability of data and materials 14. Pajtler KW, Witt H, Sill M, Jones DT, Hovestadt V, Kratochwil F, Wani K, CpG methylation values are available from the corresponding author upon Tatevossian R, Punchihewa C, Johann P, et al. Molecular classification reasonable request. of ependymal tumors across all CNS compartments, histopathological grades, and age groups. Cancer Cell. 2015;27(5):728–43. Consent for publication 15. Rohrich M, Koelsche C, Schrimpf D, Capper D, Sahm F, Kratz A, Reuss J, Not applicable. Hovestadt V, Jones DT, Bewerunge‑Hudler M, et al. Methylation‑based classification of benign and malignant peripheral nerve sheath tumors. Ethics approval and consent to participate Acta Neuropathol. 2016;131(6):877–87. This study has been performed in accordance with the Declaration of 16. Sahm F, Schrimpf D, Stichel D, Jones DTW, Hielscher T, Schefzyk S, Helsinki. The ethics committee of the medical faculty of Heidelberg University Okonechnikov K, Koelsche C, Reuss DE, Capper D, et al. DNA methylation‑ approved the use of archived tissue specimens (older than 3 years) collected based classification and grading system for meningioma: a multicentre, from the Institute of Pathology of the University Heidelberg for retrospective retrospective analysis. Lancet Oncol. 2017;18(5):682–94. research purposes (reference 206/2005). The patients’ consent was waived due 17. Seki M, Nishimura R, Yoshida K, Shimamura T, Shiraishi Y, Sato Y, Kato M, to the retrospective nature of this study and minimal risk to the subjects. Chiba K, Tanaka H, Hoshino N, et al. Integrated genetic and epigenetic analysis defines novel molecular subgroups in rhabdomyosarcoma. Nat Funding Commun. 2015;6:7557. This work was funded by a Grant (70112499) of the German Cancer Aid (to C.K. 18. Wiestler B, Capper D, Sill M, Jones DT, Hovestadt V, Sturm D, Koelsche C, and A.v.D.). Bertoni A, Schweizer L, Korshunov A, et al. Integrated DNA methylation and copy‑number profiling identify three clinically and biologically rele ‑ vant groups of anaplastic glioma. Acta Neuropathol. 2014;128(4):561–71. Publisher’s Note 19. Sturm D, Orr BA, Toprak UH, Hovestadt V, Jones DTW, Capper D, Sill M, Springer Nature remains neutral with regard to jurisdictional claims in pub‑ Buchhalter I, Northcott PA, Leis I, et al. New brain tumor entities emerge lished maps and institutional affiliations. from molecular classification of CNS‑PNETs. Cell. 2016;164(5):1060–72. 20. Koelsche C, Mynarek M, Schrimpf D, Bertero L, Serrano J, Sahm F, Reuss Received: 27 November 2018 Accepted: 5 February 2019 DE, Hou Y, Baumhoer D, Vokuhl C, et al. Primary intracranial spindle cell sarcoma with rhabdomyosarcoma‑like features share a highly distinct methylation profile and DICER1 mutations. Acta Neuropathol. 2018;136(2):327–37. 21. 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Clinical Sarcoma Research – Springer Journals

Published: Feb 14, 2019

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