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Chromothripsis and Focal Copy Number AlterationsDetermine Poor Outcome in Malignant Melanoma

Daniela Hirsch1, Ralf Kemmerling2, Sean Davis1, Jordi Camps1, Paul S. Meltzer1,Thomas Ried1, and Timo Gaiser1,3

AbstractGenetic changes during tumorigenesis are usually acquired sequentially. However, a recent study showed that

in 2% to 3% of all cancers a single catastrophic event, termed chromothripsis, can lead to massive genomicrearrangements confined to one or a few chromosomes. To explore whether the degree of genomic instability andchromothripsis influences prognosis in cancer, we retrospectively applied array-comparative genomic hybrid-ization (aCGH) to 20 malignant melanomas that showed, despite comparable conventional clinical andpathologic parameters, a profoundly different clinical course. We compared 10 patients who died of malignantmelanoma 3.7 years (median, range 0.9–7.6 years) after diagnosis with 10 patients who survived malignantmelanoma and had amedian disease-free survival of 14.8 years (range 12.5–16.7 years; P¼ 0.00001).We observed astriking association between the degree of chromosomal instability, both numerical and structural, and outcome.Malignant melanomas associated with good prognosis showed only few chromosomal imbalances (mean 1.6alterations per case), predominantly whole chromosome or chromosome arm gains and losses, whereasmalignant melanomas with poor prognosis harbored significantly more chromosomal aberrations (13.9 percase; P ¼ 0.008). Array-based CGH showed that these aberrations were mostly focal events, culminating in twocases in a pattern consistent with the phenomenon of chromothripsis, which was confirmed by paired-endsequencing. This is the first description of chromothripsis in primary malignant melanomas. Our study thereforelinks focal copy number alterations and chromothripsis with poor outcome in patients with malignantmelanomas (P ¼ 0.0002) and provides a genetic approach to predict outcome in malignant melanomas. CancerRes; 73(5); 1454–9. �2012 AACR.

IntroductionThe clinical course of malignant melanoma, a tumor with

increasing incidence, is difficult to predict. The diagnosis ofmalignant melanomas is based on histology, and disease prog-nosis dependsmainly onmitotic rate, Breslow tumor thickness,and ulceration. Recent studies showed that fluorescencein situ hybridization with a set of specific probes or array-comparative genomic hybridization (aCGH) can help to classifypatients into low- and high-risk groups (1, 2). Despite thisprogress, available data are limited and often problematic tointerpret because of short follow-up observation periods. Initial

findings of our group, however, revealed considerably morechromosomal aberrations inmalignantmelanomaswithmetas-tases than in malignant melanomas without metastases (3).

In most cancers, chromosomal alterations that defineinvasive disease are acquired sequentially during disease pro-gression (4). Recently, however, a phenomenon termed chro-mothripsis was reported (5). Chromothripsis describes a singlecatastrophic cellular event, inwhichoneor a few chromosomes,chromosome arms, or chromosomal subregions are shatteredinto tens to hundreds of pieces and reassembled incorrectlywith the consequence of defined copy number changes. Thispulverization of parts of the genomemight result fromdefectiveand asynchronous DNA replication in micronuclei, which are aconsequence of chromosome segregation errors in mitosis (5,6). Chromothripsis occurs in approximately 2% to 3% of allcancers, yet more frequently in osteosarcoma and chordoma(up to 25%), whereas malignant melanoma is affected in 7.8%,deduced from the analysis of malignant melanoma–derivedcell lines (5, 7). Chromothripsis is associated with a moreaggressive clinical course in multiple myeloma, colorectal can-cer, medulloblastoma, and neuroblastoma (8–11).

However, to date, the significance of chromothripsis forprognosis in patients with malignant melanomas is entirelyunclear. We therefore investigated whether the pattern anddegree of chromosomal instability predict the clinical courseof malignant melanoma.

Authors' Affiliations: 1Genetics Branch, Center for Cancer Research,National Cancer Institute, NIH, Bethesda, Maryland; 2Institute of Pathology,ParacelsusMedicalUniversity, Salzburg, Austria; and 3Institute of Pathology,Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Thomas Ried, Section of Cancer Genomics,Genetics Branch, Center for Cancer Research, National Cancer Institute,NIH, 50SouthDrive, Building50,Room1408,Bethesda,MD20892.Phone:301-594-3118; Fax: 301-402-1204; E-mail: [email protected]; and TimoGaiser, Institute of Pathology, Medical Faculty Mannheim, University ofHeidelberg, Theodor-Kutzer-Ufer 1-3, 68167Mannheim, Germany. Phone:49-621-383-2556; Fax: 49-621-383-2005; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-0928

�2012 American Association for Cancer Research.

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To address this question, we designed a retrospective case–control study using aCGH to map chromosomal gains andlosses in two groups of patients with malignant melanomawith profoundly different survival after long-term follow-up.We confirmed the chromothripsis-like genomic aberrationpatterns that we suspected by aCGH analysis in malignantmelanoma with poor prognosis by paired-end sequencing,which revealed complex inter- and intrachromosomal rear-rangements consistent with chromothripsis.

Materials and MethodsTumor material and clinical dataTwenty formalin-fixed, paraffin-embedded (FFPE) malignant

melanomas, which had been diagnosed between 1992 and 2006with detailed clinical long-term follow-up data, were collectedfrom the archive of the Institute of Pathology, Paracelsus

Medical University Salzburg, Austria. The study was conductedin accordance with the guidelines of the local research ethicscommittee of the Paracelsus Medical University Salzburg andwith preoperative patient's informed consent. The diagnosis ofall 20 tumors was established based on 4-mm-thick hematoxylinand eosin (H&E) stained sections by two board-certified pathol-ogists (R. Kemmerling and T. Gaiser). Tumor staging wasdetermined according to the latest American Joint Committeeon Cancer staging system (7th edition; ref. 12). The 20 selectedmalignant melanomas samples comprised 10 malignant mela-nomas with good prognosis and 10 malignant melanomas withpoor prognosis. Good prognosis was inferred when patientswith malignant melanomas were still alive during the observa-tion interval and had a minimum follow-up of more than 10.0years (median 14.8 years; range 12.5–16.7 years) with cleardocumentation of neither local relapse nor the occurrence of

Table 1. Comparison of clinical and histopathologic characteristics of patients with malignant melanomawith good and poor prognosis

Variable

Malignant melanomaswith good prognosis(n ¼ 10)

Malignant melanomaswith poor prognosis(n ¼ 10) P

Age at diagnosis, yMean � SEM 55 � 3 62 � 5 0.22Median (range) 53 (40–70) 63.5 (35–83) 0.17

SexMale 6 8 0.63Female 4 2

Breslow thickness, mmMean � SEM 2.2 � 0.2 2.9 � 0.5 0.20Median (range) 2.1 (1.4–3.4) 3.0 (0.9–6.0) 0.40

Clark levelII 1 0 0.37III 5 3IV 4 7

UlcerationPresent 4 3 1Absent 6 7

Mitoses, no./mm2

Mean � SEM 1.3 � 0.5 2.5 � 0.6a 0.15Median (range) 0.6 (0.4–4.6) 2.0 (0.9–5.7)a 0.06

AJCC stage at diagnosisIB 5 4 0.53IIA 2 4IIB 3 2

LocusHead/neck 1 0 0.38Trunk 5 3Extremity 3 5N/A 1 2

Follow-up/survival, yMean � SEM 14.5 � 0.5 4.0 � 0.7 0.000000002Median (range) 14.8 (12.5–16.7) 3.7 (0.9–7.6) 0.00001

Abbreviation: AJCC, American Joint Committee on Cancer.an ¼ 9, as in one sample massive pigmentation prevented mitotic count.

Chromothripsis in Malignant Melanoma

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metastases based on regular aftercare examination. In contrast,patients with malignant melanomas were assigned to the poorprognosis group when malignant melanomas–related deathoccurred (median 3.7 years; range 0.9–7.6 years) as proven byeither autopsy or by data from the Salzburg tumor registry. Thetwo different prognostic groups werematched in terms of age atdiagnosis, sex, Breslow thickness, Clark level, ulceration, mitoticrate, disease stage at diagnosis, and location. The groups differedsignificantly in terms of survival (Table 1).

DNA isolationSeven consecutive sections (first and seventh section: 4-

mm-thick, H&E-stained; second to sixth section: 20-mm-thick,unstained) were prepared from each of the FFPE blocks of the20 malignant melanomas cases. On sections 1 and 7, regionswith more than 80% of tumor cells were marked by two board-certified pathologists (R. Kemmerling andT. Gaiser), and tissuewas dissected from sections 2 to 6 for DNA preparation asdescribed previously (13).

Array-comparative genomic hybridizationIsolated FFPEDNAwas labeled using theGenomicDNAULS

Labeling Kit (Agilent) and subsequently hybridized on AgilentSurePrint G3 Human CGH Microarrays 4 � 180 K (Agilent)according to the manufacturer's protocol version 3.1. Briefly,500 ng of tumor DNA and 500 ng of sex-matched humangenomic DNA (Promega) as reference were differentiallylabeledwith ULS-Cy3 andULS-Cy5 (both Agilent), respectively.After hybridizing andwashing according to themanufacturer'sinstructions, slides were scanned with microarray scannerG2565BA (Agilent) and images were analyzed by FeatureExtraction software version 10.7.1.1 (Agilent). The aCGH datawere visualized and analyzed using CGH Analytics software4.0.76 (Agilent) and Nexus Copy Number software version 5(BioDiscovery). The quality of the slides was assessed withcontrol metrics provided by CGH Analytics (Agilent).

Paired-end sequencingIllumina paired-end sequencing libraries were prepared

following the manufacturer's protocol with modifications.

To increase sequence fragment diversity, two libraries wereprepared in parallel and pooled before sequencing. For eachlibrary, 1 mg of DNA was sheared on a Covaris S1 to a meanfragment size of 350 bp. The sheared DNA was end repairedand phosphorylated with T4 DNA polymerase, T4 polynu-cleotide kinase, and Klenow, followed by adenylation usingexo-Klenow Fragment. Illumina paired-end adapters wereligated to the prepared DNA fragments with DNA ligase.Adapter-ligated products were size-selected on a CaliperLabChip XT, retaining fragments of 450 bp � 20%. Theresultant libraries were amplified with Illumina PCR PrimerInPE1.0, PCR Primer InPE2.0, and PCR Primer Indexes. Thetwo indexed, amplified libraries were pooled and sequencedon a single lane of an Illumina HiSeq2000. Reads werealigned to the reference human genome (hg19) usingbwa-0.6.2 and converted to BAM format using samtools(14, 15). Duplicates were removed using the Picard Mark-Duplicates software. The Genomic Analysis of StructuralVariants software was then applied to the 114 millionuniquely mapped read pairs to define potential structuralvariant breakpoints (16). Potential cancer structural variantswere filtered by removing candidates with fewer than fivesupporting read pairs or that had a breakpoint overlappingthose found in a database of normal samples. Data werevisualized using Circos software (17).

Statistical analysisDifferences in clinical and pathologic parameters between

the two different prognostic groups of malignant melano-mas were estimated by the Student t test for the mean,Wilcoxon rank-sum test for the median, and Fisher exact orFreeman–Halton test for categorical variables. Differences interms of survival and the indices of average number of copyalterations (ANCA, calculated as the number of aberrationsdivided by the number of samples, see ref. 4) between bothgroups of patients with malignant melanomas were estimat-ed by the Student t test. The association of the incidence ofgenomic imbalances with outcome as well as the associationof chromothripsis and focal copy number alterations with

Figure 1. Chromosomal aberrations in malignant melanomas with good and poor prognosis. A, frequency plot of copy number gains (green) and losses (red)identifiedby aCGH inmalignantmelanomaswith goodandpoorprognosis. B, number of copyalterations in each sample as a functionof prognosis. Horizontallines show mean number of copy alterations, which differs significantly between malignant melanomas with good and poor prognosis (P ¼ 0.008). MM,malignant melanomas.

Hirsch et al.

Cancer Res; 73(5) March 1, 2013 Cancer Research1456




outcome was determined by Fisher exact test. P < 0.05 wasconsidered significant.

Results and DiscussionWe were able to analyze 18 of the collected 20 malignant

melanoma samples by aCGH (8 malignant melanomas withpoor prognosis and 10 malignant melanomas with good prog-nosis). Two samples were not included because of insufficientFFPE DNA quality.Of the 8 analyzed malignant melanoma cases with poor

prognosis, all 8 (100%) were found to have copy numberchanges by aCGH, a proportion differing significantly fromthe malignant melanoma cases with good prognosis (P ¼0.004), where only 3 of 10 (30%) samples showed aberrations(Fig. 1A, Supplementary Table S1). Independent of prognosis,genomic imbalances characteristic for malignant melanoma,

such as losses of chromosome arms 9p, especially of 9p21.3(CDKN2A/p16 locus), and 10q, were detectable in both groups(18, 19). Yet, we were surprised of the low incidence of genomicimbalances in malignant melanomas with good prognosis, asthese lesions were definitively malignant tumors and notbenign melanocytic lesions as determined by histopathologicexamination. The lesions did not differ from malignant mel-anomas with poor prognosis in terms of age at diagnosis, sex,histopathology, disease stage at diagnosis, and location (Table1). Notably, the few aberrations found inmalignantmelanomaswith good prognosis were all whole chromosome or chromo-some arm gains or losses; this was in strong contrast tomalignant melanomas with poor prognosis, which mostlydisplayed focal copy number gains or losses. This resulted ina far higher number of breakpoints and in an increased ANCAindex (Fig. 1B; ref. 4). The ANCA index, whichwas calculated by

Figure 2. Array-based CGH ideograms of chromosomal gains and losses for malignant melanoma samples with poor prognosis. A and C, genomeviewsof case numbers 14 (A) and20 (C) show thepresenceof genome-widedistributed chromosomal aberrationsand localized regions of chromothripsis-likeaberration patterns. B and D, chromosome views of the chromosomes affected by these chromothripsis-like aberration patterns. B, case number 14;D, case number 20.






dividing the sum of observed copy number imbalances by therespective number of cases, was 1.6 in malignant melanomasassociated with good prognosis and was significantly higher inmalignant melanomas with poor prognosis (13.9; P ¼ 0.008).This indicates that increased genomic instability in malignantmelanomas was associated with poor prognosis, consistentwith previous evidence fromour own laboratory (3). Focal copynumber alterations were present in all (8 of 8, 100%) malignantmelanomas with poor prognosis but were observed onlyonce in the malignant melanomas with good prognosis(gain of 1q21.1-23.3 in case number 11; P ¼ 0.0002). In 2of 8 (25%) malignant melanomas with poor prognosis (casenumbers 14 and 20), these focal copy number alterationsculminated in aberration patterns consistent with therecently discovered phenomenon of chromothripsis (Fig.2; ref. 5). These aberration patterns presented as complexgenomic rearrangements whose positions next to one anoth-er markedly differed from chromosomal aberrations previ-ously described in primary malignant melanomas by us andothers (3, 18, 19). These patterns were confined to segmentalchromosomal regions, rapidly alternating between two orthree distinct copy number states, including high-levelamplifications. To infer the occurrence of chromothripsisfrom copy number profiles, Rausch and colleagues requiredat least 10 changes in copy number involving up to threedistinct copy number states on a single chromosome (11).When applied to our samples, these criteria were fulfilled byboth malignant melanomas with poor prognosis harboringregions of complex aberrations, whereas the vast majority ofchromosomes displayed considerably fewer than 10 copynumber changes per chromosome. Although one fundamen-tal characteristic of chromothripsis is a series of clustered

focal events along a chromosome, we wished to confirm ourinterpretation by identifying the breakpoints of the rearran-gements. To this end, we conducted whole-genome paired-end sequencing on case number 14. This revealed genomicaberration patterns that were similar to the ones shown byStephens and colleagues and included both inter- and intra-chromosomal rearrangements (Fig. 3; ref. 5).

However, chromothripsis-positive malignant melanomasdid not harbor only the characteristic massive local genomicrearrangements but also several chromosomal aberrations,which revealed a recurrent pattern of chromosomal imbal-ances typical for malignant melanomas (Fig. 2A and C; refs, 18,19). This finding is in line with Stephens and colleagues whoalso described the coexistence of chromothripsis with othertypes of chromosomal alterations, likely acquired at distincttime points.

Although the association of both number and structure ofchromosomal aberrations with prognosis is intriguing, thesmall sample size and the low incidence of chromosomalaberrations in malignant melanomas with good prognosismight conceal evidence for focal copy number alterations andchromothripsis in malignant melanomas with good prognosis.Malignantmelanomas is not a rare tumor, but wewould like toemphasize that it is exceedingly difficult to identify a samplecollection for which the clinical long-term follow-up has beenas meticulously documented as in our samples.

The fact that chromothripsis is not only connected withpoor outcome in malignant melanomas but also in multiplemyeloma, colorectal cancer, medulloblastoma, and neuroblas-toma suggests chromothripsis as a possible genetic hallmark ofparticularly aggressive subtypes of various cancers; this couldtranslate to a useful prognostic marker.

Figure 3. Whole-genome Circos plots of case number 14. The outer ring shows ideograms of the chromosomes; the inner ring represents the respectivecopy number changes. The arcs in the center indicate chromosomal rearrangements joining the 2 relevant genomic regions of each rearrangement. A,awhole-genomeCircos plot, showingmassive inter- and intrachromosomal rearrangements, predominantly involving regions of chromosomes 5 and 20. B, azoomed-in version of the Circos plot depicting the most affected regions of chromosomes 5p and 20q.

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Cancer Res; 73(5) March 1, 2013 Cancer Research1458




In conclusion, we could show for the first time that malig-nant melanomas, which were matched according to clinicaland histopathologic features but differed profoundly in termsof prognosis, showed striking disparities in both numerical andstructural chromosomal aberrations. Malignant melanomaswith poor prognosis were associatedwith a significantly higherincidence of genomic imbalances and harbored significantlymore copy number changes than malignant melanomas asso-ciated with good prognosis. In addition, while genomic imbal-ances in malignant melanomas with good prognosis, whenpresent, virtually always affected whole chromosomes or chro-mosome arms, focal copy number alterations and a patternconsistent with chromothripsis were exclusively found inmalignant melanomas with poor prognosis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R. Kemmerling, T. Ried, T. GaiserDevelopment of methodology: D. Hirsch, J. Camps, P.S. Meltzer, T. Gaiser

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Hirsch, R. Kemmerling, P.S. Meltzer, T. GaiserAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Hirsch, R. Kemmerling, S. Davis, J. Camps, P.S.Meltzer, T. GaiserWriting, review, and/or revision of the manuscript: D. Hirsch, R. Kemmer-ling, S. Davis, J. Camps, P.S. Meltzer, T. Ried, T. GaiserAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D. Hirsch, S. Davis, T. GaiserStudy supervision: T. Ried, T. Gaiser

AcknowledgmentsThe authors thank Mr. Buddy Chen for preparing figures and IT-related

support, Dr. Maria R. Gaiser for critical reading of the manuscript, and Prof.Christoph A. Klein, Chair of Experimental Medicine and Therapy Research,Faculty of Medicine, University of Regensburg, Germany, for providing expertadvice.

Grant SupportThe study was supported by the Intramural Research Program of the NIH,

National Cancer Institute. D. Hirsch was supported by the RISE program of theGerman Academic Exchange Service.

ReceivedMarch 21, 2012; revised October 31, 2012; accepted December 4, 2012;published OnlineFirst December 27, 2012.

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2013;73:1454-1460. Published OnlineFirst December 27, 2012.Cancer Res Daniela Hirsch, Ralf Kemmerling, Sean Davis, et al. Poor Outcome in Malignant MelanomaChromothripsis and Focal Copy Number Alterations Determine

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