CDKN2A but not TP53 mutations nor HPV presencepredict poor outcome in metastatic squamous cellcarcinoma of the skin

Heidi V.N. Kusters-Vandevelde1, Arjanne Van Leeuwen2, Marian A.J. Verdijk2, Maurits N.C. de Koning3,

Wim G.V. Quint3, Willem J.G. Melchers4, Marjolijn J.L. Ligtenberg2,5 and Willeke A.M. Blokx2

1 Department of Pathology, Canisius Wilhelmina Hospital, 6500 GS Nijmegen, The Netherlands2 Department of Pathology, Radboud University Nijmegen Medical Centre, The Netherlands3 DDL Diagnostic Laboratory, Voorburg, The Netherlands4 Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, The Netherlands5 Department of Human Genetics, Radboud University Nijmegen Medical Centre, The Netherlands

Genetic alterations in metastatic cutaneous squamous cell carcinoma (CSCC) which might serve as prognostic biomarkers are

not well investigated. We investigated the mutation status and protein expression of the CDKN2A (INK4a-ARF) and TP53 genes

in metastatic CSCCs and correlated this with clinicopathological variables, HPV presence, and survival data. Sequence analysis

was performed on formalin-fixed and paraffin-embedded tissue of 35 metastases and their primary tumors, and was

correlated with immunohistochemical stainings for p53, p16 and p14. Beta-PV and alpha-PV DNA was detected using PCR-

based assays. Kaplan–Meier and Cox regression methods were used for survival assessment. CDKN2A was mutated in 31% of

the metastases and their primary tumors, while the TP53 gene was mutated in 51% of the metastases. P53 protein

expression was significantly associated with missense type of mutations (p 5 0.002). No persistent HPV types were detected.

CDKN2A mutations were significantly associated with disease-specific death (p 5 0.001). A significant difference was

observed in disease-specific survival between patients with or without a CDKN2A mutation (p 5 0.010), while this was not the

case for TP53. At univariate Cox’s regression analysis tumor size (p 5 0.010), invasion depth (p 5 0.030) and CDKN2A

mutations (p 5 0.040) were significantly related to shorter disease-specific survival. At multivariate Cox’s regression only

tumor size had an adverse effect on survival (p 5 0.002). In conclusion, our study indicates that the CDKN2A mutation status

might be of prognostic value in metastatic CSCCs. In most cases, CDKN2A and TP53 mutations are early genetic events in

CSCC tumorigenesis. The possible role of HPV in metastatic CSCC needs further exploration.

Cutaneous squamous cell carcinoma (CSCC) is the secondmost common malignancy in Caucasians, after basal cell car-cinoma, and its incidence is still rising.1 Generally the risk ofmetastasis is reported up to 5%, while immunocompromisedpatients have a larger tendency to metastasize (5–10%).1,2

The outcome of these patients once metastasized is poor,

with a 5-year survival rate between 25 and 50%.3,4 Geneticalterations in CSCCs which might serve as prognostic bio-markers are not well investigated.

TP53 mutations are the most frequent genetic alterationsin CSCC with incidences of up to 50%.5,6 TP53 mutationsare early events in skin carcinogenesis, with a similar muta-tion frequency of 50% in actinic keratosis, a precancerousskin lesion that potentially can progress to CSCC.7

Mutations in the CDKN2A (INK4a-ARF) locus arereported in CSCC with frequencies ranging up to 24% insporadic CSCC.8–10 This gene maps to chromosome 9p21,which by alternative transcripts encodes for 2 cell cycle regu-latory proteins, p16INK4a (Exons 1a, 2 and 3) and p14ARF

(Exons 1b, 2 and 3).10,11 The exact role of the CDKN2A genein skin carcinogenesis is not well understood. Especially inmetastatic CSCCs alterations in the CDKN2A gene have notbeen investigated. In a small previous study,12 we found ahigh frequency of CDKN2A mutations in metastatic CSCCs(75%) when compared to frequency spectra reported in theliterature in primary sporadic CSCCs (24%). CDKN2A andTP53 mutations have been shown to have a prognostic effectin different tumor types.13–16 In CSCC, the influence of

Key words: cutaneous squamous cell carcinoma, metastasis, TP53,

CDKN2A, HPV

Abbreviations: alpha-PV, alpha papilloma-virus; beta-PV, beta

papilloma-virus; CSCC, cutaneous squamous cell carcinoma; HPV,

human papilloma-virus; ICI, immunocompetent individual; ISI,

immunesuppressed individual; LiPA25, line probe assay; PM-PCR

RHA, PM-PCR reverse hybridization assay; SPF10, short PCR

fragment.

DOI: 10.1002/ijc.24871

History: Received 22 Apr 2009; Accepted 3 Aug 2009; Online 8 Sep

2009

Correspondence to: Heidi V.N. Kusters-Vandevelde, Department of

Pathology C66, Canisius Wilhelmina Hospital, P.O. Box 9015, 6500

GS Nijmegen, The Netherlands, Fax: þ31-24-3658844,

E-mail: h.kusters@cwz.nl

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Int. J. Cancer: 126, 2123–2132 (2010) VC 2009 UICC

International Journal of Cancer

IJC

CDKN2A and TP53 mutations on survival has not beeninvestigated yet. One of the purposes of this study is to inves-tigate the role of these genes on survival in a group of meta-static CSCCs.

The exact role of HPV in CSCC remains unclear.17–19 Upto now, only few studies included HPV testing in skintumors and their metastasis.20,21 Comparison of the preva-lence of HPV in both metastasis and primary tumor mightlead to the detection of HPV types that are persistently pres-ent, possibly indicating a more oncogenic role for these HPVtypes.

In this study we assessed the frequency of CDKN2A andTP53 mutations in a series of 35 metastatic CSCCs and stud-ied possible relations between mutation status and proteinexpression of both tumor suppressors. In addition, it wastested whether metastatic CSCCs demonstrate persistentHPV infections. The results were correlated with clinicopa-thological and survival data.

Material and MethodsPatients and histopathology

We retrieved formalin-fixed and paraffin-embedded tissue ofprimary CSCCs, their lymph node metastasis and normal tis-sue from 35 patients out of the archives of 14 departments ofpathology in the Netherlands, by a search question in theDutch nationwide histopathology and cytopathology datanetwork and archive (PALGA).22 Available patient character-istics were sex, age and location of primary tumor and me-tastasis. Data on immune status were only in part available.Histology of all samples was reviewed by a single pathologist(HK). The SCCs were classified in ‘‘well,’’ ‘‘moderately’’ and‘‘poorly’’ differentiated.23 Tumor size and depth of invasionwas assessed. Survival data from time of diagnosis of the pri-mary tumor were obtained.

Immunohistochemistry

Immunohistochemistry for p14, p16 and p53 was performedon all metastases and primary tumors, as previouslydescribed in detail.24

Quantification of immunohistochemical results

Immunoreactivity was scored semi-quantitatively: 0 (nega-tive), 1þ (up to 10% of tumor cells positive), 2þ (10-50%positive tumor cells) or 3þ (>50% positive tumor cells). Ascore of 1þ or higher and only nuclear/nucleolar stainingwas considered positive. Scoring was performed withoutknowledge of clinical outcome and molecular data.

DNA isolation and PCR reactions for mutation analyses

Mutation analyses were performed on the metastases and, incase a mutation was detected, the corresponding primary tumorwas examined. The analysis of the primary tumors was re-stricted to the fragments containing the mutations identified inthe metastases. DNA isolation and PCR reactions were per-

formed as previously described.12 For the DNA isolation about3 sections of 10 lm per cm2 formalin-fixed paraffin-embeddedtissue were manually dissected from unstained sections to obtaina tumor cell percentage of at least 60%, using HE colored sec-tions as a reference. All primers used in the analysis of TP53and CDKN2A contained either an M13 forward or an M13reverse consensus sequence. Sequence analysis of the PCR prod-ucts was performed using M13 consensus primers. All primersand sizes of the PCR products are previously described.12 Theentire open reading frames of CDKN2A and Exon 5 up to andincluding Exon 8 of TP53 were analyzed in DNA isolated fromthe metastases. Mutations were confirmed on an independentPCR product. Only somatic alterations that were not present innormal tissue of the patient were included in the study.

Alpha-PV detection and genotyping by SPF10-LiPA25All metastases and primary tumors were tested. The com-bined SPF10-LiPA25 system for detection and genotyping ofHPV has previously been described in detail.24,25 The SPF10-PCR system amplifies a 65-bp fragment of the L1 open read-ing frame, allowing for detection of at least 43 HPV types. Incase of positive PCR result, subsequent HPV genotyping wasperformed via a reverse hybridization line probe assay(LiPA), allowing for simultaneous typing of HPV 6, 11, 16,18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56,58, 59, 66, 68, 70 and 74.25

Beta-PV detection and genotyping by the PM-PCR RHA

method

All metastases and primary tumors were tested. The PM-PCR RHA method has previously been described indetail.24,26 The method was designed for the identification of25 established beta-PV types, namely genotypes 5, 8, 9, 12,14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75,76, 80, 92, 93 and 96.

Statistical analyses

Fisher’s exact test (SPSS 16.0 for Windows) was used to eval-uate the associations between mutation status and clinicopa-thological variables (sex, differentiation grade, sun-exposedskin site, immune status and HPV status), between HPV sta-tus and clinicopathological variables (sex, differentiationgrade, sun-exposed skin site and immune status) andbetween mutation status and immunohistochemical results.Student’s t test was used to compare mean age, tumor sizeand invasion depth with mutation status and protein expres-sion. Survival data included 4 groups of patients: patientswho died of metastatic CSCC (dead of disease), patients whodied of other (non-CSCCs related) causes, patients alive attime of follow-up and patients who were lost to follow-up.The minimal follow-up time was 2 years. In the analysis ofdisease-specific survival, patients who died of metastaticCSCCs were classified as dead of disease. With Fisher’s exacttest we tested possible associations between mutation statusand disease-specific death (CSCC-related death), using 2 � 2

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2124 CDKN2A in metastatic CSCC

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contingency tables (v2-test). The effect of mutation statusand clinicopathological variables on disease-specific survivalwas assessed by uni-and multivariate analyses by means ofCox’s regression, using a forward procedure. Survival timewas calculated from time of diagnosis of the primary tumorto time of death. In case survival data were not available tous, these patients were excluded from the statistical analyses.The influence of CDKN2A and TP53 mutations on disease-specific survival was assessed by the Kaplan–Meier methodand the survival curves were compared using the log ranktest. Patients dead from non-related disease or patients aliveat time of follow-up were censored. The mean survival timein relation to mutation status was compared in ANOVAtables. p values of less than 0.05 were considered as statisti-cally significant.

ResultsPatient and clinicopathological characteristics

Our patient group included 33 men and 2 women. Mean agewas 74.8 years (range 58–89). Four patients were immuno-suppressed individuals (ISI). The clinicopathological featuresof these patients are listed in Table 1.

Mutation analysis of the CDKN2A and TP53 genes

We performed mutation analyses of the CDKN2A and TP53genes in a series of 35 metastatic CSCCs. For practical rea-sons, we performed the sequence analyses on the metastasesand in case a mutation was present, the corresponding pri-mary tumor was tested. In 11 metastases the CDKN2A genewas mutated (31%) (Table 2). Of these 11 metastases 2 me-

tastases contained a double mutation (Patient 4 and 25),resulting in a total of 13 somatic CDKN2A mutations in 11metastases. All 13 mutations detected in the metastases werealso present in the primary tumors. Ten mutations occurredin Exon 2 (p14/p16) (Figs. 1c and 1d). One of the doublemutations (Patient 4) contained, besides an Exon 2 mutationan additional mutation in Intron 1A, which is unlikely tohave an effect on splicing and thus is not likely to affect theprotein. In Patient 3 and 15, an identical mutation in Intron1A/B was present, affecting the splice site of Exon 2. Fivemutations consisted of a single C to T nucleotide change and1 double substitution CC to TT was detected, consistent witha UVB fingerprint type of mutation (6/13, 46%). In 18 me-tastases the TP53 gene was mutated (51%) (Table 2). Of these18 metastases 4 metastases contained a double mutation(patients 4, 18, 27, 28) and 1 metastasis had 3 missensemutations (Patient 8), resulting in a total number of 24 TP53mutations in 18 metastases (Figs. 1g and 1h). Two of thedouble mutations consisted of 2 missense mutations (Patients18 and 28) and the other 2 double mutations of a nonsensemutation combined with a silent mutation (Patient 27) andan Intron 6 mutation affecting splice donor site (Patient 4).Five mutations consisted of a single C to T nucleotide changeand 2 mutations showed a double substitution CC to TT,consistent with UVB fingerprint type of mutation. TheseUV-related mutations (7/24, 29%) occurred in tumors arisingon sun-exposed areas. 18 of the 24 mutations detected in themetastases were also present in the primary tumors. In 5 pri-mary tumors definite confirmation of the detected TP53mutation in the metastasis was not possible due to lowerquality of DNA and/or due to lower percentage of tumorcells. In Patient 32, the frame shift mutation found in Exon 6was not present in the primary tumor. In 5 metastases amutation in both the CDKN2A gene and the TP53 gene waspresent (14.3%). In 24 of 35 metastases either a TP53 muta-tion and/or a CDKN2A mutation was present (69%).

Immunohistochemistry versus mutation status

Immunohistochemistry was performed on all metastases andprimary tumors, and compared with mutation status, and ifappropriate with type of mutation. For p16, internal positivecontrol cells included inflammatory cells and melanocytes(Figs. 1a and 1b). For p53, normal epidermis showed slightnuclear staining in a few scattered basal keratinocytes. P14was very low or not expressed in normal tissue. Positive im-munostaining in tumor cells for p16 was present in both thenucleus and cytoplasm, whereas the localization of p14 andp53 was restricted to the nucleus. P14 staining showed nucle-olar staining (Figs. 1i and 1j). The percentage of positiveimmunostainings in the metastases are presented in Table 3.The percentage of positive tumor cells for the 3 immunos-tainings was similar in primary tumor and its metastasis(data not further shown). P16 staining was positive in 15 of35 metastases (43%), while p14 staining was positive in 10 of35 metastases (29%). No significant association was found

Table 1. Clinicopathological characteristics

No. of patients (%)

Sex

Male 33 (94)

Female 2 (6)

Immune status

ICI1 24 (69)

ISI2 4 (11)

n.a.3 7 (20)

Sun-exposed skin sites 32 (91)

Differentation grade

Moderately 20 (57)

Poorly 15 (43)

Well 0

Mean

Age (yrs) 74.8 (range 58–89)

Tumor size (mm) 19 (range 5–35 mm)

Invasion depth (mm) 11 (range 4–18 mm)

1ICI, immunocompetent individual. 2ISI, immunesuppressed individual.3n.a, data not available.

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Int. J. Cancer: 126, 2123–2132 (2010) VC 2009 UICC

Table

2.CDKN2AandTP

53mutationanalysiswithcorresp

ondingim

munohistoch

emistry

Mutationanalysis

Immunostaining1

CDKN2A

TP53

p16

p14

p53

Pat

MetastasisExo

n/intron,

codon,base,AAch

ange2

Primary

tumor

MetastasisExo

n/intron,

codon,base,AAch

ange2

Primary

tumor

Metastases

3Intron1A/B

(p16)c.151-1G>A(p14)c.317-1G>A

Confirm

ed

––

––

–

4Intron1A(p16)c.150þ8

G>C

Confirm

ed

Exon6c.637C>T,

3–

––

Exon2(p16)c.238C>T,

p.Arg80X(p14)c.404C>T,

p.Pro135Leu

Confirm

ed

p.Arg213X

Intron6c.672þ1

G>A

3

7Exon2(p16)c.151-1_151delinsA

A(p14)

c.317-1_317delinsA

AConfirm

ed

––

––

>50%

8–

–Exon8c.833C>T,

p.Pro278Leu

Confirm

ed

––

>50%

Exon8c.845G>A,p.Arg282Gln

Confirm

ed

Exon6c.568_569delinsTT,

p.Pro190Phe

Confirm

ed

9–

Exon7c.734G>T,

p.Gly245Val

Confirm

ed

––

>50%

14

––

Exon5c.466del,p.Arg156fs

Confirm

ed

––

–

15

Intron1A/B

(p16)c.151-1G>A(p14)c.317-1G>A

Confirm

ed

Exon5c.535C>T,

p.His179Tyr

Confirm

ed

>50%

>50%

>50%

17

––

Exon6c.599del,p.Asn

200fs

Confirm

ed

––

–

18

––

Exon6c.590T>

A,p.Val197Glu

Confirm

ed

––

10–50%

Exon7c.772G>A,p.Glu258Lys

Confirm

ed

19

––

Exon5c.513del,p.Glu171fs

4–

––

21

Exon2(p16)c.330G>A,p.Trp110X(p14)c.496G>A,

p.Gly166Arg

Confirm

ed

––

––

>50%

22

Exon2(p16)c.172C>T,

p.Arg58X(p14)c.338C>T,

p.Pro113Leu

Confirm

ed

Exon7c.741_742delinsTT,

p.Arg248Trp

3–

–>50%

23

––

Exon5c.396G>T,

p.Lys132Asn

Confirm

ed

>50%

–>50%

24

––

Exon7c.700T>

C,p.Tyr234His

Confirm

ed

––

>50%

25

Exon2(p16)c.238C>T,

p.Arg80X(p14)c.404C>T,

p.Pro135Leu

Confirm

ed

––

5–10%

5–10%

>50%

Exon2(p16)c.290T>

G,p.Leu97Arg

(p14)c.456T>

G,p.¼

26

Exon2(p16)c.341C>T,

p.Pro114Leu(p14)c.507C>T,

p.¼

Confirm

ed

Exon5c.417G>C,p.Lys139Asn

35–10%

––

27

––

Exon6c.617T>

A,p.Leu206X

Confirm

ed

>50%

>50%

–

Exon7c.756C>T,

p.¼

Confirm

ed

28

––

Exon5c.455C>G,p.Pro152Arg

Confirm

ed

10–50%

–>50%

Exon7c.742C>T,

p.Arg248Trp

Confirm

ed

29

––

Exon8c.827_829delinsTC,p.Ala276fs

Confirm

ed

>50%

5–10%

–

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2126 CDKN2A in metastatic CSCC

Int. J. Cancer: 126, 2123–2132 (2010) VC 2009 UICC

Table

2.CDKN2AandTP

53mutationanalysiswithcorresp

ondingim

munohistoch

emistry(Continued).

Mutationanalysis

Immunostaining1

CDKN2A

TP53

p16

p14

p53

Pat

MetastasisExo

n/intron,

codon,base,AAch

ange2

Primary

tumor

MetastasisExo

n/intron,

codon,base,AAch

ange2

Primary

tumor

Metastases

30

Exon2(p16)c.341_342delinsTT,

p.Pro114Leu(p14)

c.507_508delinsTT,

p.Arg170Cys

Confirm

ed

––

10–50%

–>50%

32

––

Exon6c.597_603dup,p.Arg202fs

Notpresent

–5–10%

5–10%

33

Exon2(p16)c.172C>T,

p.Arg58X(p14)c.338C>T,

p.Pro113Leu

Confirm

ed

Exon5c.469G>T,

p.Val157Phe

Confirm

ed

––

>50%

34

Exon2(p16)c.290T>

G,p.Leu97Arg

(p14)c.456T>

G,p.¼

Confirm

ed

––

––

5–10%

35

––

Exon7c.700T>

C,p.Tyr234His

Confirm

ed

5–10%

–>50%

Total

13CDKN2Amutationsin

11metastases

AllCDKN2A

mutations

Confirm

ed

24TP

53mutationsin

18metastases

18of24TP

53

mutations

Confirm

ed

9pos

5pos

16pos

1%

nuclearstainingforthemetastases,

p.¼

:nomenclature

forasilentmutationaccordingto

therulesoftheHumanGenomeVariationSociety

(HGVS).Fordetailssee:http://w

ww.genomic.

unim

elb.edu.au/m

di/mutnomen/.

2Aminoacidch

ange,-:notpresent.

3Low

peaksin

sequence

analysisdueto

lowertumorpercentage.4Confirm

ationin

primary

tumornotpossible

because

of

lowerquality

DNA.

Figure 1. (a,b): negative p16 immunostaining in metastasis and

primary tumor respectively, of Patient 4 (magnification �200). Note

the internal positive control in inflammatory cells (arrows). (c,d):

sequence analysis of CDKN2A of Patient 4, Exon 2 forward: (p16)

c.238C > T, p.Arg80X in metastasis (c) and primary tumor (d). (e,f):

positive p53 immunostaining in metastasis and primary tumor

respectively, of patient 35 (magnification �200). (g,h): sequence

analysis of TP53 in patient 35, Exon 7 reverse: c.700T > C,

p.Tyr234His, in metastasis (g) and primary tumor (h). (i,j): positive

p14 immunostaining in metastasis and primary tumor respectively,

of Patient 15 (magnification �200 and �400). In j, the inset

shows nucleolar staining pattern.

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between p16 or p14 expression and mutation status or typeof mutation (Table 4). About 23 of 35 metastases (66%)showed positive p53 staining (Figs. 1e and 1f). Positive p53staining proved to be significantly associated with the pres-ence of missense mutations (missense versus nonsense andframe shift) (p ¼ 0.002) (Table 4).

HPV detection and genotyping

Both the metastases and primary tumors were tested. Alpha-PV DNA was detected in 4 of 35 primary tumors (11%), ofwhich 1 could be genotyped as HPV 18. In the correspond-ing metastases, no HPV DNA was detected (Table 5). Beta-PV DNA was present in 9 primary tumors (26%) and in 3metastases (9%) (Table 5). Genotyping revealed Types 20, 24and 15 in the primary tumors and Types 22, 23, 24 and 36in the metastases. In 6 primary tumors and 1 metastasisHPV DNA was detected but could not be genotyped (HPVX). In 1 metastasis HPV DNA was detected while the corre-sponding primary tumor contained no HPV DNA. Combin-ing the results of both methods, we found an overall fre-quency of HPV DNA of 31.4% (11/35) in the primarytumors. For the metastases, this overall frequency of HPVDNA was 9% (3/35). About 3 out of 4 immunesuppressedindividuals contained either beta-PV or alpha-PV DNA inthe primary tumor (Table 5).

Clinicopathological correlations, including survival

analyses

For both the CDKN2A and TP53 genes no significant associa-tion was present between mutation status nor protein expres-sion with any of the following clinicopathological variables:sex, differentiation grade, location of primary tumor (sun-exposed skin site), immune status nor HPV status (Fisher’sexact test). No significant differences were present in mean age,tumor size and invasion depth between tumors containing ei-ther a CDKN2A or a TP53 mutation and tumors without thesemutations (Student’s t test). This was also the case for p16, p14or p53 positive versus negative tumors. We could not detect asignificant association between HPV status and any of the clini-copathological variables (sex, differentiation grade, sun-exposedskin site, immune status). For 25 patients follow-up data wereavailable (Table 6). At the time of this report 9 patients werealive, 8 patients died of metastatic disease and 8 patients diedof non-disease related causes. With Fisher’s exact test CDKN2A

mutations were significantly associated with disease-specificdeath (p ¼ 0.001), while this was not the case for TP53 muta-tions, differentiation grade, immune status nor HPV status(data not further shown). CDKN2A mutation status had a neg-ative effect on mean survival time (p ¼ 0.040) (ANOVA). Atunivariate Cox’s regression analysis tumor size (p ¼ 0.010),invasion depth (p ¼ 0.030) and CDKN2A mutations (p ¼0.040) were significantly related to shorter disease-specific sur-vival. At multivariate Cox’s regression analysis only tumor sizewas significantly associated with shorter disease-specific survival(p ¼ 0.002). With the Kaplan–Meier method a significant dif-ference was observed in disease-specific survival betweenpatients with or without a CDKN2A mutation (p ¼ 0.010, log

Table 3. Immunohistochemical results for the metastases

P531 (%) P532 (%) Total (%)

P16þ/p14þ 5 (14) 3 (9) 8 (23)

P16þ/p14� 5 (14) 2 (6) 7 (20)

P16-/p14� 11 (31) 7 (20) 18 (51)

P16-/p14þ 2 (6) 0 2 (6)

Total 23 (66) 12 (34) 35 (100)

Table 4. Immunohistochemistry versus mutation status in themetastases

Immunohistochemistry

Positive Negative p1

Mutation status

n ¼ no. of metastases

TP53

Wild type (n ¼ 17) 12 5

Mutation (n ¼ 18) 11 7

Type of TP53 mutation

Missense (n ¼ 11)2 10 1

Nonsense (n ¼ 2)3 0 2 0.002

Frame shift (n ¼ 5) 1 4

P16

Wild type (n ¼ 24) 11 13

Mutation (n ¼ 11) 4 7

Type of p16 mutation

Missense (n ¼ 3) 2 1

Nonsense (n ¼ 4)4 0 4

Splice site (intron A/B)(n ¼ 3)

1 2

Nonsense þ missense(n ¼ 1)

1 0

P14

Wild type (n ¼ 24) 8 16

Mutation (n ¼ 11) 2 9

Type of p14 mutation

Missense (n ¼ 6)5 1 5

Splice site (n ¼ 3) 1 2

Silent (n ¼ 2) 0 2

1Fisher’s exact test. 22 metastases contained a double missensemutation and one metastasis contained 3 missense mutations (all withpositive p53 immunostaining). 3one metastasis contained anadditional silent mutation which is unlikely to affect the protein, andone metastasis contained an additional splice site mutation. 4onemetastasis also contained a mutation in intron 1A which is unlikely toaffect the protein. 5one metastasis contained an additional silentmutation (case with positive p14 immunostaining).

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rank), while this was not the case for TP53 mutations (p ¼0.60, log rank) (Table 6). Kaplan–Meier survival plots forCDKN2A and TP53 are presented in Figure 2, with p valuesfrom log rank test.

DiscussionPrognostic factors for recurrence and metastasis in CSCCs arewell investigated and include mainly histological variables suchas tumor size, depth of invasion and differentiation grade.27

Genetic alterations in CSCCs which might serve as prognosticbiomarkers are not well investigated. CDKN2A and TP53 muta-tions have been shown to be of prognostic value in differenttumor types.13–16 For instance, in head and neck SCCs, muta-tions in the CDKN2A and TP53 gene are shown to have a neg-ative effect on survival.13,14,28 In CSCCs, the prognostic effectof these genes has not been explored yet.

One of the purposes of this study was to investigate themutation status of the CDKN2A and TP53 gene in a series ofmetastatic CSCCs in relation to survival. We detected aCDKN2A mutation frequency of 31% (11/35), which isslightly higher than frequencies reported in the literature forprimary sporadic CSCCs (up to 24%).8,10 Soufir et al.reported similar higher frequencies of CDKN2A mutations(33%) in Xeroderma pigmentosa patients, who have a geneticdefect in DNA repair and are particularly prone to developaggressive CSCCs.9 In our study, CDKN2A mutations weresignificantly associated with disease-specific death (p ¼0.001). Furthermore, a significant difference was observed indisease-specific survival between patients with or without aCDKN2A mutation (p ¼ 0.010). These data indicate that theCDKN2A mutation status might be of prognostic value inpatients with metastatic CSCCs. At univariate Cox’s regres-sion analysis, CDKN2A mutations together with tumor sizeand invasion depth had a significant adverse effect on

Table 5. Prevalence of HPV in metastases and primary tumors in relation to immune status

Patient

Alpha-PV Beta-PV

Immune statusPrimary tumor Metastasis Primary tumor Metastasis

3 – – X1 – ISI2

5 X – 24 – ICI3

6 18 – – – ISI

7 – – X – ICI

14 X – – – ICI

16 – – X 22, 23, 24, 36 ICI

19 – – 15 – ICI

23 – – X – ICI

24 – – X – ICI

25 – – X 24 n.a.

26 X – 20, 24 – ISI

27 – – – X n.a.

Total 4/35 (11%) 0 9/35 (26%) 3/35 (9%)

–: no HPV DNA detected.1undetermined HPV type. 2ISI, immunesuppressed individual. 3ICI, immunocompetent individual, n.a.: data not available.

Table 6. Survival data in relation to CDKN2A and TP53 mutation status

No. of patients

Mean survivaltime (months) p2

Death ofdisease

Death ofnon-cancerrelated cause Alive

Lost tofollow-up1

With CDKN2A mutation (n ¼ 11) 6 1 0 4 34.1 6 8.1 0.010

No CDKN2A mutation (n ¼ 24) 2 7 9 6 52.1 6 19.5

With TP53 mutation (n ¼ 18) 3 4 6 5 45.1 6 20.1 0.60

No TP53 mutation (n ¼ 17) 5 4 3 5 43.6 6 16.9

Total group (n ¼ 35) 8 8 9 10 44.3 6 17.7

1Not included in the statistical analyses. 2p values from log rank test. For CDKN2A a significant difference in disease-specific survival was observedbetween patients with or without mutations (Kaplan–Meier method with log rank test).

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disease-specific survival. However, at multivariate analysis,only tumor size was significantly associated with shorter dis-ease-specific survival. To further define the prognostic effectof the CDKN2A gene in metastatic CSCCs, larger studieswith complete follow-up are needed. All CDKN2A mutationsdetected in the metastases were also present in the primary

tumors. This implicates that these mutations are early eventsin CSCC tumorigenesis, before metastases occur, and seem toremain stable during the process of metastasis.

The TP53 gene was mutated in 51% of the metastases,which is comparable to frequencies in the literature for pri-mary CSCCs.29,30 We could not detect any prognostic effectof the TP53 gene on disease-specific survival. Therefore,our data do not support a prognostic role for the TP53gene in metastatic CSCCs. Only 1 new TP53 mutation wasdetected in a metastasis and not in the corresponding pri-mary tumor. As we cannot exclude the presence of anotherprimary tumor in this patient, we cannot draw any firmconclusion about this TP53 mutation being a late eventduring tumorigenesis. Similar low percentages of potentiallynew mutations at the metastasis sites are reported in headand neck SCCs.31,32

In 5 patients a mutation was present in both the TP53gene and CDKN2A gene (14%), which is higher than fre-quencies in the literature for sporadic CSCCs (5%).9 Higherfrequencies of these combined mutations are again seen inXeroderma pigmentosum-associated tumors.9 Because of lim-ited follow-up we could not evaluate the effect of these com-bined mutations on survival.

Mutation status was correlated with immunohistochemicalresults. No significant association was found between p16/p14expression and mutation status or type of mutation. It isknown that other mechanisms than mutations can cause aber-rant expression of p16 and p14, such as loss of parts or theentire short arm of chromosome 933–35 or epigenetic eventslike hypermethylation.36 However, for p53, a significant associa-tion was present between p53 expression and type of mutation,with missense mutations being associated with positive staining,and nonsense and frame shift mutations with negative staining(p ¼ 0.002). Missense mutations can lead to a structurallyaltered protein that is more stable than the wild type, resultingin higher levels of protein detectable by antibody.37,38 Nonsensemutations resulting in a stop codon and frame shift mutations,often giving rise to a stop codon downstream, can lead to non-sense-mediated decay of the mRNA or to the production of atruncated protein, which is likely to be more unstable.39

Remarkably, 12 of 17 metastases with positive p53 staininglacked an underlying TP53 mutation. An explanation can bethat mutations were present outside the analyzed ‘‘hot-spot’’region (Exon 5–8). The few studies in nonmelanoma skin can-cer including Exons 2 through 11 have shown mutations in upto 25% outside this ‘‘hot-spot’’ region.40,41 Furthermore, it isknown that accumulation of the nonmutated protein can occurdue to the formation of complexes between p53 and other cel-lular proteins such as MDM2 or viral proteins such as the viraloncoprotein E6.42 In conclusion, positive p53 stainings are notsynonymous with TP53 mutations.

Up to now, only few studies have included HPV testing inskin tumors and their metastasis.20,21 We found an overallfrequency of HPV DNA of 31.4% in the primary tumors,which is in the range reported in the literature for sporadic

Figure 2. Kaplan–Meier survival plots. (a): a significant difference

in disease-specific survival was present between patients with or

without a CDKN2A mutation (p ¼ 0.010). (b): for TP53 mutations

no significant difference in disease-specific survival was observed

(p ¼ 0.60). p values from log rank test.

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CSCCs (between 27% and 60%).18 In the metastases an over-all frequency of HPV of 9% was detected, which is low incomparison to the primary tumors. In 2 of 3 metastases,HPV Type 24 was present but no further comparisons canbe made with the corresponding primary tumors, as the exactHPV type remained undetermined. These undeterminedtypes can either be 1 of the 25 established beta-PV types,present in such a low concentration that only a reaction withthe universal probe is seen, or these can be novel types notyet characterized.43 There could be several explanations forthe lower detection rate of HPV in the metastases comparedto the primary tumors. First, HPV DNA could be lost duringtumor progression which can be expected since in squamousskin tumors HPV integration in the host genome is rare.44

Second, HPV might just play a coincidental role in skin car-cinogenesis, supported by studies that show the presence ofHPV in normal skin samples of healthy individuals.45 Sur-prisingly, in 1 metastasis we detected beta-PV DNA but notin the primary tumor. This might be the result of samplingeffects, assuming that HPV is heterogeneously distributedthroughout a lesion. In 1 primary tumor HPV Type 18 waspresent. Interestingly, there are some recent studies reportingHPV Type 18 in up to 75% of HPV-positive skin tumors,suggesting a role for HPV Type 18 in skin carcinogenesis.46,47

With respect to immune status, in 3 out of 4 immunesup-pressed patients HPV DNA was present in the primary tu-mor, possibly indicating a higher incidence of HPV infections

in immunesuppressed patients. In the literature, the presenceof HPV in metastatic CSCCs is relatively unexplored. Ostrowet al. reported the presence of HPV Type 5 in both a primaryCSCC and its metastasis in 2 epidermodysplasia-verrucifor-mis patients.21 Nindl et al. reported the presence of cutane-ous HPV DNA in a primary CSCC and its metastasis in animmunocompetent and immunocompromised patient.20 Inour study however, we could not detect any persistent HPVinfection. Further studies are required to investigate the pos-sible role of HPV in metastatic CSCCs.

In conclusion, our study indicates that the CDKN2Amutation status might be of prognostic value in metastaticCSCCs. In most cases, both CDKN2A and TP53 mutationsare early genetic events in CSCC tumorigenesis, which occurbefore metastasis. The possible role of HPV in metastaticCSCCs needs to be further explored. Future studies for mo-lecular prognostic markers in CSCC remain importantbecause the incidence of non-melanoma skin cancer is stillrising, as is the number of patients with increased risk formetastases such as immunesuppressed patients.

AcknowledgementsThe authors thank the PALGA foundation, the national histopathology andcytopathology databank in the Netherlands, for providing them with patientinformation. Prof. J.H. van Krieken, Department of Pathology, RadboudUniversity Nijmegen Medical Centre, has reviewed and commented on themanuscript.

References

1. Alam M, Ratner D. Cutaneous squamous-cell carcinoma. N Engl J Med 2001;344:975–83.

2. Dreno B, Mansat E, Legoux B, Litoux P.Skin cancers in transplant patients. NephrolDial Transplant 1998;13:1374–9.

3. Epstein E, Epstein NN, Bragg K, Linden G.Metastases from squamous cell carcinomasof the skin. Arch Dermatol 1968;97:245–51.

4. Veness MJ, Morgan GJ, Palme CE, GebskiV. Surgery and adjuvant radiotherapy inpatients with cutaneous head and necksquamous cell carcinoma metastatic tolymph nodes: combined treatment shouldbe considered best practice. Laryngoscope2005;115:870–5.

5. Boukamp P. Non-melanoma skin cancer:what drives tumor development andprogression? Carcinogenesis 2005;26:1657–67.

6. McGregor JM, Berkhout RJ, Rozycka M,Ter SJ, Bouwes Bavinck JN, Brooks L,Crook T. p53 mutations implicate sunlightin post-transplant skin cancer irrespectiveof human papillomavirus status. Oncogene1997;15:1737–40.

7. Nelson MA, Einspahr JG, Alberts DS,Balfour CA, Wymer JA, Welch KL,Salasche SJ, Bangert JL, Grogan TM, BozzoPO. Analysis of the p53 gene in human

precancerous actinic keratosis lesions andsquamous cell cancers. Cancer Lett 1994;85:23–9.

8. Soufir N, Moles JP, Vilmer C, Moch C,Verola O, Rivet J, Tesniere A, Dubertret L,Basset-Seguin N. P16 UV mutations inhuman skin epithelial tumors. Oncogene1999;18:5477–81.

9. Soufir N, ya-Grosjean L, de la SP, MolesJP, Dubertret L, Sarasin A, Basset-SeguinN. Association between INK4a-ARF andp53 mutations in skin carcinomas ofxeroderma pigmentosum patients. J NatlCancer Inst 2000;92:1841–7.

10. Saridaki Z, Liloglou T, Zafiropoulos A,Koumantaki E, Zoras O, Spandidos DA.Mutational analysis of CDKN2A genes inpatients with squamous cell carcinoma ofthe skin. Br J Dermatol 2003;148:638–48.

11. Brown VL, Harwood CA, Crook T, CroninJG, Kelsell DP, Proby CM. p16INK4a andp14ARF tumor suppressor genes arecommonly inactivated in cutaneoussquamous cell carcinoma. J Invest Dermatol2004;122:1284–92.

12. Blokx WA, Ruiter DJ, Verdijk MA, deWilde PC, Willems RW, de Jong EM,Ligtenberg MJ. INK4-ARF and p53mutations in metastatic cutaneous

squamous cell carcinoma: case report andarchival study on the use of Ink4a-ARFand p53 mutation analysis in identificationof the corresponding primary tumor. Am JSurg Pathol 2005;29:125–30.

13. Bazan V, Zanna I, Migliavacca M, Sanz-Casla MT, Maestro ML, Corsale S,Macaluso M, Dardanoni G, Restivo S,Quintela PL, Bernaldez R, Salerno S, et al.Prognostic significance of p16INK4aalterations and 9p21 loss of heterozygosityin locally advanced laryngeal squamous cellcarcinoma. J Cell Physiol 2002;192:286–93.

14. Poeta ML, Manola J, Goldwasser MA,Forastiere A, Benoit N, Califano JA, RidgeJA, Goodwin J, Kenady D, Saunders J,Westra W, Sidransky D, et al. TP53mutations and survival in squamous-cellcarcinoma of the head and neck. N Engl JMed 2007;357:2552–61.

15. Salinas-Sanchez AS, Lorenzo-Romero JG,Gimenez-Bachs JM, Sanchez-Sanchez F,Donate-Moreno MJ, Rubio-Del-Campo A,Hernandez-Millan IR, Segura-Martin M,Tienzar-Tobarra M, Escribano-Martinez J.Implications of p53 gene mutations onpatient survival in transitional cellcarcinoma of the bladder: a long-termstudy. Urol Oncol 2008;26:620–6.

Can

cerGenetics

Kusters-Vandevelde et al. 2131

Int. J. Cancer: 126, 2123–2132 (2010) VC 2009 UICC

16. Vidaurreta M, Maestro ML, Sanz-CaslaMT, Rafael S, Veganzones S, de lO V,Cerdan J, Arroyo M, Torres A. Colorectalcarcinoma prognosis can be predicted byalterations in gene p53 exons 5 and 8. Int JColorectal Dis 2008;23:581–6.

17. Majewski S, Jablonska S. Doepidermodysplasia verruciformis humanpapillomaviruses contribute to malignantand benign epidermal proliferations? ArchDermatol 2002;138:649–54.

18. Iftner A, Klug SJ, Garbe C, Blum A, StancuA, Wilczynski SP, Iftner T. The prevalenceof human papillomavirus genotypes innonmelanoma skin cancers ofnonimmunosuppressed individualsidentifies high-risk genital types as possiblerisk factors. Cancer Res 2003;63:7515–9.

19. Harwood CA, Surentheran T, McGregorJM, Spink PJ, Leigh IM, Breuer J, ProbyCM. Human papillomavirus infection andnon-melanoma skin cancer inimmunosuppressed and immunocompetentindividuals. J Med Virol 2000;61:289–97.

20. Nindl I, Koehler A, Meyer T, Forschner T,Meijer CJ, Snijders PJ, Sterry W, StockflethE. Detection of human papillomavirusDNA in primary squamous cell carcinomaand metastases. Br J Dermatol 2006;154:797–9.

21. Ostrow RS, Bender M, Niimura M, Seki T,Kawashima M, Pass F, Faras AJ. Humanpapillomavirus DNA in cutaneous primaryand metastasized squamous cell carcinomasfrom patients with epidermodysplasiaverruciformis. Proc Natl Acad Sci USA1982;79:1634–8.

22. Casparie M, Tiebosch AT, Burger G,Blauwgeers H, van de PA, van Krieken JH,Meijer GA. Pathology databanking andbiobanking in The Netherlands, a centralrole for PALGA, the nationwidehistopathology and cytopathology datanetwork and archive. Cell Oncol 2007;29:19–24.

23. McKee PH. Pathology of the skin, 3rdedn., Vol. 2. London: Elsevier Mosby,2005. 1204.

24. Kusters-Vandevelde HV, de Koning MN,Melchers WJ, Quint WG, de Wilde PC, deJong EM, van de Kerkhof PC, Blokx WA.Expression of p14(ARF), p16(INK4A) andp53 in relation to HPV in (pre)malignantsquamous skin tumors. J Cell Mol Med2008; DOI 10.1111/j.1582–4934.2008.00452.x.

25. Kleter B, van Doorn LJ, Schrauwen L,Molijn A, Sastrowijoto S, Ter SJ, LindemanJ, Ter HB, Burger M, Quint W.Development and clinical evaluation of ahighly sensitive PCR-reverse hybridizationline probe assay for detection andidentification of anogenital humanpapillomavirus. J Clin Microbiol 1999;37:2508–17.

26. de Koning M, Quint W, Struijk L, KleterB, Wanningen P, van Doorn LJ,Weissenborn SJ, Feltkamp M, Ter SJ.Evaluation of a novel highly sensitive,broad-spectrum PCR-reverse hybridizationassay for detection and identification ofbeta-papillomavirus DNA. J Clin Microbiol2006;44:1792–800.

27. Rowe DE, Carroll RJ, Day CL, Jr.Prognostic factors for local recurrence,metastasis, and survival rates in squamouscell carcinoma of the skin, ear, and lip.Implications for treatment modalityselection. J Am Acad Dermatol 1992;26:976–90.

28. Sailasree R, Abhilash A, Sathyan KM,Nalinakumari KR, Thomas S, Kannan S.Differential roles of p16INK4A andp14ARF genes in prognosis of oralcarcinoma. Cancer Epidemiol BiomarkersPrev 2008;17:414–20.

29. Bolshakov S, Walker CM, Strom SS, SelvanMS, Clayman GL, El-Naggar A, LippmanSM, Kripke ML, Ananthaswamy HN. p53mutations in human aggressive andnonaggressive basal and squamous cellcarcinomas. Clin Cancer Res 2003;9:228–34.

30. Giglia-Mari G, Sarasin A. TP53 mutationsin human skin cancers. Hum Mutat 2003;21:217–28.

31. Burns JE, McFarlane R, Clark LJ, MitchellR, Robertson G, Soutar D, Parkinson EK.Maintenance of identical p53 mutationsthroughout progression of squamous cellcarcinomas of the tongue. Eur J Cancer BOral Oncol 1994;30B:335–7.

32. Tjebbes GW, Leppers vd Straat FG,Tilanus MG, Hordijk GJ, Slootweg PJ. p53tumor suppressor gene as a clonal markerin head and neck squamous cellcarcinoma: p53 mutations in primarytumor and matched lymph nodemetastases. Oral Oncol 1999;35:384–9.

33. Mortier L, Marchetti P, Delaporte E,Martin de LE, Thomas P, Piette F,Formstecher P, Polakowska R, Danze PM.Progression of actinic keratosis tosquamous cell carcinoma of the skincorrelates with deletion of the 9p21 regionencoding the p16(INK4a) tumorsuppressor. Cancer Lett 2002;176:205–14.

34. Popp S, Waltering S, Herbst C, Moll I,Boukamp P. UV-B-type mutations andchromosomal imbalances indicate commonpathways for the development of Merkeland skin squamous cell carcinomas. Int JCancer 2002;99:352–60.

35. Quinn AG, Sikkink S, Rees JL. Delineationof two distinct deleted regions onchromosome 9 in human non-melanomaskin cancers. Genes Chromosomes Cancer1994;11:222–5.

36. Murao K, Kubo Y, Ohtani N, Hara E,Arase S. Epigenetic abnormalities in

cutaneous squamous cell carcinomas:frequent inactivation of the RB1/p16 andp53 pathways. Br J Dermatol 2006;155:999–1005.

37. Finlay CA, Hinds PW, Tan TH, Eliyahu D,Oren M, Levine AJ. Activating mutationsfor transformation by p53 produce a geneproduct that forms an hsc70-p53 complexwith an altered half-life. Mol Cell Biol1988;8:531–9.

38. McNutt NS, Saenz-Santamaria C,Volkenandt M, Shea CR, Albino AP.Abnormalities of p53 protein expression incutaneous disorders. Arch Dermatol 1994;130:225–32.

39. Mendell JT, Dietz HC. When the messagegoes awry: disease-producing mutationsthat influence mRNA content andperformance. Cell 2001;107:411–4.

40. Ziegler A, Leffell DJ, Kunala S, SharmaHW, Gailani M, Simon JA, Halperin AJ,Baden HP, Shapiro PE, Bale AE. Mutationhotspots due to sunlight in the p53 gene ofnonmelanoma skin cancers. Proc Natl AcadSci USA 1993;90:4216–20.

41. Brash DE, Rudolph JA, Simon JA, Lin A,McKenna GJ, Baden HP, Halperin AJ,Ponten J. A role for sunlight in skincancer: UV-induced p53 mutations insquamous cell carcinoma. Proc Natl AcadSci USA 1991;88:10124–8.

42. Bazan V, Migliavacca M, Tubiolo C,Macaluso M, Zanna I, Corsale S, Amato A,Calo V, Dardanoni G, Morello V, La FM,Albanese I, et al. Have p53 gene mutationsand protein expression a differentbiological significance in colorectal cancer?J Cell Physiol 2002;191:237–46.

43. Pfister H. Chapter 8: Humanpapillomavirus and skin cancer. J NatlCancer Inst Monogr 2003;31:52–6.

44. Yabe Y, Tanimura Y, Sakai A, HitsumotoT, Nohara N. Molecular characteristics andphysical state of human papillomavirusDNA change with progressing malignancy:studies in a patient with epidermodysplasiaverruciformis. Int J Cancer 1989;43:1022–8.

45. de Koning MN, Struijk L, Bavinck JN,Kleter B, Ter SJ, Quint WG, Feltkamp MC.Betapapillomaviruses frequently persist inthe skin of healthy individuals. J Gen Virol2007;88:1489–95.

46. Biliris KA, Koumantakis E, DokianakisDN, Sourvinos G, Spandidos DA. Humanpapillomavirus infection of non-melanomaskin cancers in immunocompetent hosts.Cancer Lett 2000;161:83–8.

47. Shahmahmoudi S, Mahmoodi M, AzadTM, Rad KS, Tabatabaie H, Sarijlou M,Pour YY, Yousefi M, Ghasemi M, Far KJ,Nategh R. Prevalence of mucosal types ofhuman papillomavirus in skin lesions innorth part of Iran. Cancer Lett 2007;247:72–6.

Can

cerGenetics

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