Full text of DOI:10.1038/labinvest.3780405

0023-6837/02/8202-123$03.00/0
LABORATORY INVESTIGATION
Vol. 82, No. 2, p. 123, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
p16INK4a Is Selectively Silenced in the Tumoral
Progression of Mycosis Fungoides
Inmaculada C. Navas, Patrocinio Algara, Marisol Mateo, Pedro Martínez,
Carmen García, Jose L. Rodriguez, Francisco Vanaclocha, Nuria Barrientos,
Luis Iglesias, Lydia Sánchez, Miguel A. Piris, and Pablo Ortiz-Romero
Departments of Genetics (ICN, PA, MM, PM) and Dermatology (CG), Virgen de la Salud Hospital, Toledo;
Departments of Dermatology (FV, NB, LI, PO-R) and Pathology (JLR), 12 de Octubre Hospital, Madrid; and Centro
Nacional de Investigaciones Oncológicas (LS, MAP), Madrid, Spain
SUMMARY: Knowledge about the molecular mechanisms involved in the pathogenesis of tumoral progression in mycosis
fungoides (MF) is still scarce. Because the 9p21 locus seems to be a good target for a detailed study in MF, this prompted us
to compare the mechanisms of inactivation of the p16INK4a, p15INK4b, and p14ARF genes in aggressive and stable forms of MF,
performing microsatellite analysis, methylation-specific polymerase chain reaction, direct sequencing, and p16INK4a protein
expression by immunohistochemistry. Additionally, the p53 gene was also sequenced in tumoral lesions. Thirty-nine patients with
stable MF were studied. Alterations in p16INK4a and p15INK4b genes were detected in 18% and 5% of the cases, respectively.
None of the cases analyzed showed alterations of the p14ARF gene. In contrast with these findings, in the 11 patients with
aggressive MF, alterations of the p16INK4a, p15INK4b, or p14ARF genes were found in 8 (73%), 3 (27%), and 2 (18%) cases,
respectively. A significant proportion (4/11) of these alterations were already present in the p16INK4a gene in the initial plaque
lesions in these aggressive forms of MF. Alterations in the p16INK4a gene, either methylation or loss of heterozygosity, were clearly
more frequent than those in the p15INK4b and p14ARF genes. These p16INK4A alterations were confirmed using immunohisto-
chemistry. None of the nine tumoral lesions analyzed showed mutations in exons 1-2 of the p16INK4a gene or in exons 5-8 of the
p53 gene. These results seem to suggest that 9p21 alterations, and selectively p16INK4a silencing, could be a characteristic
phenomenon in MF progression. (Lab Invest 2002, 82:123–132).
ycosis fungoides (MF) is an indolent subtype of
cutaneous T-cell lymphoma that slowly evolves
the 9p21 region, play key roles in the maintenance of
the Rb and p53 tumor suppressor pathways, respec-
tively (Ching et al, 1998), whereas p15INK4b is a medi-
ator of extracellular growth inhibition signals (Hannon
and Beach, 1994).
M
through patch, plaque, and tumor stages before the
lymph nodes and visceral organs become involved.
The evolution from a disease restricted to the skin
towards a widespread disseminated disease may take
years or even decades. The timing of this aggressive
transformation varies from case to case, thus whereas
some patients may show an aggressive course from
the beginning, in others the disease follows a pro-
tracted course, which suggests that there is some
variability in the biologic characteristics of these neo-
plasms that decides the moment at which the aggres-
sive transformation takes place (Diamandidou et al,
1996).
INK4 proteins (p16INK4a and p15INK4b) specifically
bind to and inhibit the cyclin D-dependent kinases
(CDKs), CDK4 and CDK6, thus preventing pRb phos-
phorylation and exit from the G1 phase (Serrano,
1997). In contrast with this, p14ARF does not bind to
CDKs but interacts and sequesters Mdm2 (Kamijo et
al, 1998; Lohrum et al, 2000; Zhang et al, 1998), a
protein that has the capacity of promoting p53 degra-
dation and also functionally inactivating Rb, leading to
cell cycle arrest (Carnero et al, 2000; Pomeranntz et al,
1998; Weber et al, 1999).
The 9p21 region encodes the growth suppressor
genes p16INK4a, p15INK4b, and p14ARF. Alterations in
this region are one of the most frequent findings in
human cancer (Sherr, 1996). It has been shown that
p16INK4a and p14ARF, the two alternative products of
Genetic and epigenetic alterations of the INK4/ARF
locus on 9p21 are one of the most frequent molecular
alterations observed in human tumorigenesis (Cairns
et al, 1995; Kamb et al, 1994). Different mechanisms of
inactivation of these genes have been described in
different types of human cancer. Homozygous dele-
tions (HD), methylation, and mutation of the INK4/ARF
locus have been reported in lymphoid malignancies
(Drexler, 1998; Gombart et al, 1995; Herman et al,
1996b; Koduru et al, 1995; Ogawa et al, 1995; Quelle
et al, 1997; Siebert et al, 1995), such as lymphoblastic
lymphoma/acute lymphoblastic leukemia (Gardie et al,
Received July 3, 2001.
This work was supported by grants 98/005 and 98/0993 from the Fondo de
Investigaciones Sanitarias, Ministerio de Sanidad y Consumo, and the
Comision Interministerial de Ciencia y Tecnologia (1FD97-0431), Spain.
Address reprint requests to: Dr. Patrocinio Algara, Department of Genetics,
Hospital Nacional de Parapléjicos, Finca La Peraleda s/n, 45071 Toledo,
Spain. E-mail: palgara@cht.insalud.es
Laboratory Investigation • February 2002 • Volume 82 • Number 2 123

Navas et al
1998; Okuda et al, 1995), and in B-cell lymphomas
(Baur et al, 1999; Fernandez-Piqueras et al, 1997;
Herman et al, 1997; Klangby et al, 1998; Martínez-
Delgado et al, 1998; Ng et al, 1997), where it has been
shown to be involved in the transformation of low-
grade to high-grade tumors (Elenitoba-Johnson et al,
1998; Pinyol et al, 1998; Villuendas et al, 1998).
instability in advanced cutaneous stages of MF, sug-
gesting that a tumor suppressor gene or genes in this
region may be associated with disease progression. A
recent report by Marrogi et al (1999) described a high
frequency of p53 mutations in tumoral MF samples,
but this has not been confirmed in other series (Garatti
et al, 1995).
Methylation of the different genes present in 9p21
seems to be a random phenomenon, which may affect
only one of the genes or combinations of them, in a
pattern that has been linked to specific types of
neoplasms. Thus, concomitant hypermethylation of
p16INK4a/p15INK4b genes, which is unusual in other
malignancies (such as myeloblastic leukemia, lympho-
blastic leukemia, and glioma) (Aggerholm et al, 1999;
Cameron et al, 1999; Herman et al, 1996b), has often
recently been detected in multiple myeloma (Ng et al,
1999). The p14ARF promoter demonstrates selective
epigenetic silencing in a subset of colorectal tumors,
with a hypermethylated promoter (p14ARF) between
Concerning the role of the 9p21 locus, Peris et al
(1999) have shown a lack of p16INK4a protein expres-
sion in patients with MF, suggesting that alterations in
this gene might play a pathogenic role in this type of
cutaneous lymphoma. Moreover, a previous report by
our group (Navas et al, 2000) showed that alterations
of p16INK4a are frequent in MF cases with aggressive
transformation, but we were unable to determine
whether p16INK4a silencing is a frequent and early
finding in all cases of MF or only in the subset of cases
showing progression. In the first instance, p16INK4a
alterations could be a characteristic finding of the
disease, whereas in the second, p16INK4a silencing
would be a risk factor for disease progression. On the
other hand, information about p14ARF and p15INK4b
status in MF is scarce.
two unmethylated promoters (p16INK4a and p15INK4b
)
that in contrast are frequently methylated in other
tumor types (Esteller et al, 2000). Recent studies (Baur
et al, 1999) have observed the absence of p14ARF
gene mutations or methylation in all lymphomas ana-
lyzed, which suggests that p14ARF inactivation is not
implicated in human lymphomagenesis or could be
dependent on additional and varied mechanisms.
The molecular mechanisms underlying the progres-
sion of MF into tumoral stage, a phenomenon usually
associated with large cell transformation and aggres-
sive behavior, are not well known. Several articles
have shown a low frequency of genetic alterations
associated with p53, lyt10, c-myc, bcl-1, and bcl-2
genes in patients with MF (de Misa et al, 1995; Garatti
et al, 1995). Recently, Scarisbrick et al (2000) found
loss of heterozygosity (LOH) on 10q and microsatellite
The aim of this study was to investigate the hypo-
thetical pathogenic role of p16INK4a, p15INK4b, and
p14ARF inactivation in a group of stable MF patients
(patch/plaque lesions that had not progressed after a
minimum of 30 months of follow-up), comparing them
with aggressive MF before and after progression.
Results
Clinical Data
The clinical data of the patients with stable and
aggressive MF are summarized in Table 1.
There were 11 patients with aggressive MF. All of
them developed tumoral lesions mostly evidenced by
Table 1. Summary of the Clinical Data of our Patients
Stable MF
Aggressive MF
n ϭ 11
Number of cases
Sex distribution
Mean age at beginning (range)
n ϭ 39
M/F ϭ 20/19
41.3 yr
M/F ϭ 6/5
42.6 yr
(4–91)
(27–70)
Mean time until biopsy sample analyzed (range)a
Maximum stage
108.6 mo
(3–360)
T1 ϭ 13
T2 ϭ 26
Plaque: 111.6 mo (6–288)
Tumors: 121.5 mo (6–298)
IIb ϭ 6
IVa ϭ 3
IVb ϭ 2
Status (January 2001)b
Complete remission
T1N0M0
11
18
9
3
0
T2N0/1M0
4
3
T3anyNanyM
0
Dead of lymphoma
Dead not of lymphoma
0
1
1c
1d
Stable MF, patients with patch/plaque lesions without tumoral transformation; Aggressive MF, patients that have suffered tumoral transformation.
a Months from the beginning of the disease until biopsy of the analyzed sample.
b Number of cases.
c Small cell lung carcinoma.
d Brain tumor.
124 Laboratory Investigation • February 2002 • Volume 82 • Number 2

9p21 Region Alterations in MF
large tumoral cells. Sex distribution was M/F:6/5.
Mean age at the beginning of MF was 42.6 (range,
27–70) years; mean delay to diagnosis was 42 (range,
3–168) months. The mean time until the appearance of
tumors was 94.4 (range 0–298) months. Two patients
developed tumors and patch/plaques simultaneously.
In another patient, tumors appeared within 6 months
of the beginning of plaque lesions.
In four patients the patch/plaque and tumoral sam-
ples were taken simultaneously. In five patients,
patch/plaque samples were taken 10 to 94 months
before sampling of tumoral lesions. In two of them,
samples of tumoral lesions were taken 7 and 24
months before the plaque lesion. Patient MF90 died of
lymphoma. Patient MF122 died of a pulmonary small
cell cancer. The rest of the patients are still alive. Three
of the patients (MF36, MF82, and MF79) achieved
complete remission (CR), and they remain free of
disease after 41, 101, and 16 months of follow–up,
respectively. Only three remaining patients (MF73,
MF78, and MF91) underwent CR during their medical
history, at 6, 11, and 8 months, respectively.
Allelic Loss Studies
In 21 cases of patients with stable MF, DNA was
available from nontumoral tissue for the analysis of
microsatellites surrounding the p16INK4a, p15INK4b
,
and p14ARF genes in the 9p21 region. None of the 21
cases analyzed showed allelic loss.
In contrast with this finding, in patients with aggres-
sive MF, 3 of 11 samples taken in the tumoral phase
showed genetic loss, 1 showed HD, and 2 showed
LOH. In one of these cases (MF39), LOH was detected
in only two contiguous markers, without including the
p16INK4a, p15INK4b, and p14ARF genes. Some of these
data have been previously published (Navas et al,
2000) (Fig. 1; Table 3).
Aberrant Methylation at the 5'-CpG Islands in 9p21
Gene Cluster
The methylation status of the p16INK4a, p15INK4b, and
p14ARF CpG islands was investigated in 58 MF sam-
ples (Fig. 2).
Methylation of the p16INK4a gene was found in only
7 (18%) of 39 lesions from cases of stable MF,
whereas it was observed in 6 (60%) of 10 tumoral
samples from cases of aggressive MF. When ana-
lyzed, plaque lesions of aggressive forms of MF
showed an intermediate value, and 3 (33%) of 9 of
cases were positive. Some of these data have been
previously published (Navas et al, 2000).
Molecular Studies
A table showing the results obtained by analysis of the
p16INK4a, p15INK4b, and p14ARF genes in stable and
aggressive MF is included (Table 2).
In patients with stable MF, alterations of the
p16INK4a gene were detected in 7 (18%) of 39 and
alterations of the p15INK4b gene were detected in 2
(5%) of 39 of the lesions. None of the 39 cases
analyzed showed alterations of the p14ARF gene. Only
methylation of some of these genes was observed,
and no genetic loss was detected.
In contrast with these results, in patients with ag-
gressive MF (11 cases), alterations of the p16INK4a
gene were found in 8 (73%) of 11 tumoral samples (2
cases of allelic loss [MF36, MF113] and 6 of methyl-
ation); alterations of the p15INK4b gene were found in 3
(27%) of 11 samples (2 cases of allelic loss [MF36,
MF113] and 1 of methylation); and alterations of the
p14ARF gene were found in 2 (18%) of 11 samples (2
cases of allelic loss [MF36 and MF113]). All of these
alterations were present in the tumoral phase. When
analyzing the plaque stage of these aggressive MF,
alterations of the p16INK4a gene were found in 4 (36%)
of 11 samples (1 allelic loss and 3 methylation);
alterations of the p15INK4b gene were found in 2 (20%)
of 10 samples (1 case of allelic loss and 1 of methyl-
ation); and alterations of the p14ARF gene were found
in 1 (10%) of 10 samples (1 allelic loss) (Table 3).
Methylation of the p15INK4b gene was found in 2
(5%) of 39 cases of stable MF. In aggressive MF,
p15INK4b hypermethylation was observed in 2 (20%) of
10 cases, associated in both with p16 methylation. No
case showed p14ARF gene methylation (Table 3).
All of the 35 negative controls included (skin, lym-
phoid tissue in different reactive conditions, and pe-
ripheral blood lymphocytes) showed amplification with
unmethylated-specific primers (U) but not with
methylated-specific primers (M).
Mutational Analysis
To determine whether mutations of the p16INK4a and
p53 gene were present, exons 1 and 2 and 5 to 8 of
these genes, respectively, were analyzed by direct
sequencing. None of the nine cases studied, all of
which corresponded to tumoral lesions, showed mu-
tations in any of the genes.
Immunohistochemical Studies
Immunohistochemical studies for p16INK4a were per-
formed on paraffin-embedded tissue samples from 39
Table 2. Summary of p16INK4a, p15INK4b, and p14ARF Gene Alterations in MF
Total alterations
p15INK4b
Number of
samples
p16INK4a
p14ARF
Aggressive MF
Stable MF
11
39
8 (73%)
7 (18%)
3 (27%)
2 (5%)
2 (18%)
0
Includes both methylation and deletion analysis.
Laboratory Investigation • February 2002 • Volume 82 • Number 2 125

Navas et al
Table 3. Summary of 9p21 Region Alterations in the Plaque and Tumoral Stages of Aggressive MF
Methylation
p16INK4a
p15INK4b
p14ARF
Allelic loss 9p21
Mutation p16INK4a
Tumor
Cases
Plaque
Tumor
Plaque
Tumor
Plaque
Tumor
Plaque
Tumor
MF36a
ND
ϩ
Ϫ
Ϫ
Ϫ
Ϫ
ϩ
ϩ
ϩ
ϩ
ND
ϩ
ND
Ϫ
Ϫ
ND
Ϫ
Ϫ
Ϫ
ND
Ϫ
ND
Ϫ
Ϫ
ND
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
ND
ND
Ϫ
HD
LOHc
Ϫ
Ϫ
Ϫ
wtb
ND
wt
wt
wt
MF39a
MF73a
MF78a
MF79
Ϫ
Ϫ
MF91a
MF113a
MF122a
MF82a
MF90a
MF172
Alterations
ϩ
Ϫ
ϩ
Ϫ
ND
Ϫ
3/9
ϩ
Ϫ
ϩ
Ϫ
ND
Ϫ
6/10
Ϫ
Ϫ
Ϫ
ND
ND
Ϫ
ϩ
Ϫ
Ϫ
Ϫ
ND
Ϫ
1/9
Ϫ
Ϫ
Ϫ
ND
ND
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
ND
Ϫ
0/9
Ϫ
LOH
Ϫ
Ϫ
Ϫ
Ϫ
1/9
Ϫ
LOH
Ϫ
Ϫ
Ϫ
Ϫ
3/11
wt
wt
wt
wt
ND
wt
0/9
1/7
0/7
HD, homozygous deletion; LOH, loss of heterozygosity; ND, not done; ϩ, positive; Ϫ, negative.
a The results corresponding to the p16INK4a gene of these patients have been published previously (including deletion, mutation, and methylation analysis) (Navas
et al, 2000).
b wt due to reactive cells associated with the tumor as it presents HD.
c In case MF39, LOH was detected in two markers, without including p16INK4a, p15INK4b, and p14ARF genes (Navas et al, 2000).
Figure 1.
Summary of microsatellite analysis of locus 9p21 in aggressive mycosis
fungoides (MF). Three samples that showed deletions at one or more loci are
presented. All of them are illustrated in plaque (PL) and tumoral (T) stages. The
status of each chromosomal locus is indicated as follows: ꢀ, homozygous
Figure 2.
Methylation-specific PCR assay of the p16INK4a (A), p15INK4b (B), and p14ARF
(C) gene. Agarose gel electrophoresis of some representative cases with stable
MF is shown. Primer sets used for amplification are designated as unmethyl-
ated (U) or methylated (p16-M2, p15-M, and p14-M). The Raji cell line and
peripheral blood lymphocytes (PBL) serve as positive controls for methylated
deletion; u, loss of heterozygosity; , retention of both alleles; □, noninfor-
mative; ^, noninformative loci with a signal markedly diminished in compar-
ison with DNA normal.
patients. Loss of p16INK4a expression was observed in
12 of 39 cases, 7 of them characterized by p16INK4a
promoter methylation and 5 cases, scored as p16INK4a
negative in the Table 4, and lacking molecular alter-
ations (2/5 cases showed a mixture of positive and
negative cells). In 27 cases, intraepithelial and atypical
lymphocytes showed a nuclear distinct expression,
comparable to that seen in the epithelial cells. Repre-
sentative cases are illustrated in Figure 3.
(M2/M) and unmethylated (U) p16INK4a,p15INK4b
respectively.
,
and p14ARF alleles,
Correlation Between Clinical and Molecular Data
We statistically compared the 39 patients with stable
MF and the 11 patients with aggressive MF (Wilcoxon
and median score tests). We could find no significant
differences between the two populations in terms of
126 Laboratory Investigation • February 2002 • Volume 82 • Number 2

9p21 Region Alterations in MF
Table 4. Relationship Between the Results of IHC of p16
Protein and p16INK4a Gene Status in Samples from
Patients with MF
plaque lesions (7 [18%] of 39 cases). This was not
statistically significant.
p16INK4a gene status
Status of Stable MF with 9p21 Alterations
p16INK4a IHC
Altered
Nonaltered
There were no statistical differences in the epidemio-
logic data of the patients with stable MF, whether
p16INK4a was hypermethylated or not.
Negative
Positive
7
0
5
27
Two patients presented p15INK4b hypermethylation.
One patient then was under RePUVA treatment when
she underwent biopsy, whereas the other was not
receiving any treatment. They remain alive in the IIa
and Ia stages, respectively.
IHC, Immunohistochemistry.
Seven patients presented p16INK4a hypermethyl-
ation. Three of them are in Stage Ia, one is in Stage Ib,
and three are in complete remission. One patient had
associated lymphomatoid papulosis. Three patients
were receiving topical corticosteroids when they un-
derwent biopsy. The rest were not being treated.
Discussion
MF is a cutaneous T-cell lymphoma whose clinical and
morphologic characteristics have been thoroughly de-
scribed. However, despite the high frequency of ag-
gressive transformation in this disease, knowledge of
the molecular mechanisms involved in pathogenesis
of the tumoral progression is still scarce. The 9p21
locus seems to be a good target for a detailed study in
MF, because some previous studies on cutaneous
T-cell lymphoma have already pointed out some of the
genes present in this chromosomal region. Thus de-
letions in this area have been characterized by Ogawa
et al (1995) and Stranks et al (1995) in two cases of
Sézary syndrome; Peris et al (1999) showed a reduced
expression of p16INK4a in MF lesions, and our own
group (Navas et al, 2000) has shown a relatively high
frequency of p16INK4a inactivation when only aggres-
sive forms of MF were analyzed. Nevertheless, so far
no study has compared the frequency of inactivation
of the different genes codified in 9p21 between ag-
gressive versus quiescent forms of cutaneous T-cell
lymphoma, which could eventually point out key tar-
gets for the prediction of progression and therapeutic
intervention.
Figure 3.
p16INK4a immunohistochemistry showing three positive cases (A–C) and three
negative cases (D–F).
sex distribution, age at onset, the mean time interval
between the first clinical manifestation of MF and the
date of the biopsy, time of follow-up after the biopsy
specimen was analyzed, or treatments performed
before the biopsy specimen was analyzed. Both pop-
ulations were homogeneous.
We then analyzed the frequency of inactivation of
the different genes, when comparing stable and ag-
gressive forms of MF. When we investigated p16INK4a
inactivation (secondary to both genetic loss and/or
hypermethylation), we found that the frequency of
p16INK4a inactivation was significantly higher in tu-
moral lesions corresponding to aggressive MF (8/11,
73%). In comparison with this, in the biopsy speci-
mens taken from the stable form of MF, p16INK4a
inactivation was present in 7 (18%) of 39 cases. The
comparison between the tumoral lesions of aggres-
sive MF and the stable MF yielded statistically signif-
icant differences (p ϭ 0.001, Fisher’s exact test). This
difference was also present when only hypermethyl-
ation was analyzed (6/10 vs. 7/38; 60% vs. 18%) (p ϭ
0.01, Fisher’s exact test).
This study had the aim of supplying this information,
by comparing the status of p16INK4a, p15INK4b, and
p14ARF genes in a large series of aggressive MF with
a group of 39 cases of stable MF, as defined by the
absence of progression in a minimum follow-up of 30
months.
Alterations in the p16INK4a gene were clearly more
frequent than in the p15INK4b and p14ARF genes.
Inactivation of the p16INK4a gene (hypermethylation or
deletion) was found in 18% (7/39) of the lesions of
stable MF versus 73% (8/11) of the tumoral lesions of
aggressive MF. Statistical analysis was able to detect
significant differences when comparing plaque lesions
of stable MF with tumoral lesions of aggressive MF (p
ϭ 0.001, Fisher’s exact test). This finding is parallel
with those obtained in B-cell lymphomas, where si-
lencing of p16INK4a has been found to play a critical
Plaque lesions of those patients who developed an
aggressive form of MF also showed more frequent
p16INK4a inactivation (4 [36%] of 11 cases) than stable
Laboratory Investigation • February 2002 • Volume 82 • Number 2 127

Navas et al
role in tumoral progression (Elenitoba-Johnson et al,
1998; Pinyol et al, 1998; Villuendas et al, 1998).
The frequency of p16INK4a alterations in the plaque
lesions of those cases that go on to show tumoral
transformation (36%) was also higher than in the
plaque lesions of stable MF (18%), although these
differences did not attain statistical significance. Sim-
ilarly, the frequency of p16INK4a alterations was higher
in tumoral lesions (73%) than it was in plaque lesions
of aggressive MF (36%), but these differences were
not significantly different either. The relatively small
size of our series can make proportions that practically
double each other statistically insignificant. These
data are compatible with the hypothesis that p16INK4a
inactivation could be a necessary step for tumoral
progression of MF, but additional changes are re-
quired to allow or accelerate the progression, because
as shown, not all cases with p16INK4a inactivation
necessarily progress to aggressive forms during the
length of this study.
1999; Drexler, 1998; Elenitoba-Johnson et al, 1998;
Pinyol et al, 1998; Villuendas et al, 1998). It is, never-
theless, noteworthy that the two cases with double
inactivation of both p16/Rb and ARF/p53 pathways
showed an aggressive form of the disease, as could
be predicted.
Generally speaking, these results seem to support
that tumoral progression in MF is associated with a
very high frequency of p16/Rb pathway silencing and
preservation of the ARF/p53 pathway, as is shown by
the rarity of p53 mutation or p14ARF silencing. Strik-
ingly, p53 alterations were not observed in any case in
this series, which contrasts with data published pre-
viously by Marrogi et al (1999). No explanation is
suggested for this difference, because all cases in our
series were sequenced from exon 5 to 8, the same
area where p53 mutations were observed in the study
by Marrogi et al (1999).
The tumoral progression of MF may be related to
alternative additional mechanisms. Cytokines are be-
lieved to play an important role in the pathogenesis of
cutaneous T-cell lymphoma (Rook et al, 1997). MF
progression has been associated with significantly
higher IL-10 and lower IFN-␥ mRNA expression (As-
adullah et al, 1996) suppressing normal T-cell activity
and giving the malignant T cells a growth advantage.
Several mechanisms may therefore contribute to tu-
mor progression, and the likelihood of a single factor
being solely responsible is probably low.
Unfortunately, the results of this study do not make
it possible to conclusively predict whether a de novo
patient with MF will show an accelerated phase, using
only examination of p16INK4a status. However, be-
cause the hypermethylation of the p16INK4a promoter
was the preferred mechanism of inactivation of the
p16INK4a gene in our patients, it suggests that efforts
aimed at reversing the methylation status of p16INK4a
could have a positive impact on the evolution of the
disease. In the future, demethylating agents—such as
decitabine, or others—could be used to design clini-
cal assays in this group of patients (Wijermans et al,
2000).
We have also taken into account the possibility that
the lower frequency of p16INK4a alterations in plaque
lesions may only reflect a smaller population of tu-
moral cells within lesions of this kind. We therefore
performed sensitivity assays that showed that the
methylation-specific PCR (MSP) technique used was
able to detect methylation when the amount of tu-
moral cells was 0.5% for p16INK4A, 10% for p15INK4b
,
and 2.5% for p14ARF (data not shown). The samples
analyzed here included the necessary amount of tu-
moral cells, as assessed by morphologic and immu-
nohistochemical staining. Thereafter, the use of immu-
nohistochemical staining for p16INK4A has made it
possible to confirm the results obtained by LOH and
MSP, with only a minor exception, two cases with a
mixture of p16INK4A-positive and -negative cells. Ad-
ditionally, the percentage of tumoral cells in plaque
stage MF was previously found to exceed the thresh-
old of sensitivity of a PCR technique for detection of
TCR-␥ rearrangements, located in 10% of tumoral
cells (Algara et al, 1994).
A relatively low frequency of changes involving
p15INK4B genes were observed in this study, and
additionally in aggressive MF no case was observed to
harbor changes exclusively in the p15INK4b gene,
because all cases showed concomitant alteration of
the p16INK4a gene. Thus alterations of the p15INK4b
gene (deletion and methylation) were observed in 2
(5%) of 39 patients with stable MF and in 3 (27%) of 11
of tumoral samples. Consistent with these results, and
in concordance with previous observations in which
HDs at 9p21 generally occur at a low frequency in
non-Hodgkin’s lymphomas (Drexler, 1998; Gombart et
al, 1995; Koduru et al, 1995; Ogawa et al, 1995;
Siebert et al, 1995; Uchida et al, 1995), only one case
Material and Methods
Case Selection
A group of patients with MF was selected from the
medical records of the 12 de Octubre Hospital, Ma-
drid, and the Virgen de la Salud Hospital, Toledo (both
of which are in the center of Spain). Diagnosis was
based on generally accepted clinicopathologic crite-
ria. Patch or plaque lesions were erythematous,
slightly scaling macules or plaque lesions with well-
defined borders. Histopathologically they presented a
characteristic infiltrate of epidermotropic atypical T
cells with convoluted cerebriform nuclear contours.
Tumors were defined as nodules, whether or not they
were ulcerated, with a majority of large cells on
histopathologic examination.
in this series (MF36) showed HD in the p16INK4a
,
p15INK4b, and p14ARF genes. It seems that at least in
this tumoral model, the concurrent inactivation of the
p16INK4a and p15INK4b genes just gives a relatively
small additional advantage on tumoral cells, thus
confirming the diversity of situations observed in lym-
phomas when analyzing the 9p21 genes (Baur et al,
We defined “stable MF” as cases that had not
progressed into tumoral stages in at least 30 months
128 Laboratory Investigation • February 2002 • Volume 82 • Number 2

9p21 Region Alterations in MF
of follow-up after the biopsy. We could obtain fresh-
frozen material from 39 stable MF cases with patches/
plaques (13 T1, 26 T2). Until January 2001 the mini-
mum follow-up without progression into tumoral stage
was 34 months. “Aggressive MF” was defined as
cases that progressed into tumoral stage. We identi-
fied 11 MF cases from which we could obtain
formalin-fixed paraffin-embedded tissue or fresh-
frozen material from patch/plaque as well as tumoral
lesions. Some patients had patch/plaque and tumoral
lesions present simultaneously on different areas on
their skin. Some of the findings concerning nine of
these cases have been published previously (Navas et
al, 2000). All of the samples analyzed were studied in
parallel for routine hematoxylin-eosin examination.
Complete remission is defined as no evidence of
residual disease after a follow-up of at least 3 months.
The TNM staging system was used, as defined by the
MF Co-operative Group of Mycosis Fungoides/Sézary
Syndrome.
different samples of reactive lymphoid tissue (25
cases of peripheral blood lymphocytes from healthy
donors, 3 reactive lymph nodes, and 1 normal thymus)
and 6 cases of inflammatory dermatosis (pytiriasis
lichenoides) was used as negative control. The Raji
cell line was used as the positive control for the
p16INK4a and p15INK4b genes, and the L540 cell line for
the p14ARF gene. For analysis of allelic loss, nontu-
moral DNA was obtained from peripheral blood neu-
trophils (24 cases), oral swabs (7 cases), or nontu-
moral paraffin-embedded tissue (1 case).
Allelic Loss Assays at the 9p21 Locus. Tumoral and
normal DNA was analyzed for LOH or HD by amplifi-
cation of dinucleotide repeats containing sequence
microsatellite markers, under conditions previously
described by Villuendas et al (1998). These markers
are at the 9p21 region, surrounding the p16INK4a
,
p15INK4b
,
and p14ARF genes (IFN-␣, D9S736,
D9S1749, D9S1747, D9S974, D9S1748, D9S1752,
D9S171). Amplification conditions were as described
by Navas et al (2000) in a previously published report.
LOH was determined if the intensity of signal from
any one allele was significantly reduced in the tumor
DNA when compared with normal DNA by direct
visualization. The presence of HD was also assessed
in all cases, by comparative multiplex PCR assay
using three primer sets from loci outside the 9p21
region (D9S934, D7S1824, and D7S460, in chromo-
somes 9q, 7q, and 7p, respectively).
Immunohistochemistry
Sections were cut to a thickness of 3 ␮m, dried for 16
hours at 56° C before being dewaxed in xylene and
rehydrated through a graded ethanol series to PBS.
Antigen retrieval was heat mediated in a pressure
cooker treated for 2 minutes in 10 mM citrate buffer
(pH 6.5). Before staining the sections, endogenous
peroxidase was blocked.
Methylation Studies. DNA methylation patterns in
the CpG islands of the first exon of p15INK4b, exon 1␣
of p16INK4a, and exon 1␤ of p14ARF were determined
by MSP assays (Herman et al, 1996a). DNA was
modified with sodium-bisulfite as described in a pre-
vious report of our group (Navas et al, 2000). Bisulfite-
Immunohistochemical staining of p16INK4a was per-
formed with p16INK4a (F12) monoclonal antibody (San-
ta Cruz Biochemicals, Santa Cruz, California) diluted
at 1:50. Immunodetection was performed with biotin-
ylated antimouse immunoglobulins, followed by
peroxidase-labeled streptavidin, LSAB-2 (DAKO,
Glostrup, Denmark) with diaminobenzidine chromo-
gen as substrate. Sections were counterstained with
hematoxylin. Incubation omitting the specific anti-
body, as well as with unrelated antibodies, was used
as a control of the technique. A positive control was
included with each batch of staining to ensure consis-
tency between consecutive runs.
Results were interpreted according to established
criteria (Geradts et al, 1995, 1998; Kratzke et al, 1996),
and cases were considered negative (abnormal ex-
pression) if a large majority of intraepithelial T cells
were p16INK4a negative, contrasting with the positive
staining observed in epithelial cells. These criteria
reproduce those previously used in the assessment of
p16INK4a reactivity in lymphoma and other tumors
(Cohen and Geradts, 1997; Gulley et al, 1998; Reed et
al, 1996; Villuendas et al, 1998), and closely reflect the
results obtained using a quantitative measurement
system in this series (data not shown).
modified DNA was amplified using p16INK4a, p15INK4b
,
and p14ARF unmethylated-specific primers (U),
methylated-specific primers (M), and unmodified or
wild-type primers (W) (Herman et al, 1996a; Esteller et
al, 2000).
Controls without DNA and positive controls for U
and M reactions were performed for each set of PCRs.
The PCR product was visualized in agarose gels
stained with ethidium bromide under ultraviolet illumi-
nation. If a methylation-specific PCR product was
detected, the whole procedure using sodium bisulfite
and MSP was performed again to minimize the pos-
sible influence of contamination or incomplete bisulfite
treatment.
DNA methylation was determined by the presence
of a 148-bp and 122-bp fragment in those samples
amplified with the p15-M and p14-M primers, respec-
tively (Herman et al, 1996a; Esteller et al, 2000).
However, to confirm the findings with the initial p16-M
primer set (150 bp) and to allow analysis of additional
CpG sites in the promoter region, one additional
primer set specific for the methylated sequence,
p16-M2 (234 bp), was used in all cases (Herman et al,
1996a). DNA from the Raji and L540 cell lines was
used as a positive control for the amplification reaction
with the p16-M/M2 and p15-M primers, and from the
L540 cell line for the p14ARF gene.
Molecular Studies
DNA Extraction. Genomic DNA from the cases se-
lected was obtained from frozen or paraffin-
embedded tissue from patch/plaque and tumoral le-
sions. For methylation studies, DNA obtained from
Laboratory Investigation • February 2002 • Volume 82 • Number 2 129

Navas et al
Mutational Analysis of p16INK4a and p53 Genes.
Cameron E, Baylin SB, and Herman JG (1999). P15 INK4b
CpG island methylation in primary acute leukemia is hetero-
geneous and suggests density as a critical factor for tran-
scriptional silencing. Blood 94:2445–2451.
DNA from nine frozen tumoral stage samples was
analyzed for mutations in exons 5 to 8 of the p53 gene
and exons 1 and 2 of the p16INK4a gene, comprising
97% of the coding sequences, with primers and
amplification conditions described previously (Villuen-
das et al, 1993, 1998). In both cases, direct sequenc-
ing of amplified products was performed with an
Automated DNA Sequencer ABI PRISM 310 Genetic
Analyzer (PE Applied Biosystems, Foster City, Califor-
nia) according to the manufacturer’s procedures.
Carnero A, Hudson JD, Price CM, and Beach DH (2000).
P16INK4a and p19ARF act in overlapping pathways in cel-
lular immortalization. Nat Cell Biol 2:148–155.
Ching L, Pomerantz J, and DePinho RA (1998). The INKAa/
ARF tumour suppresser: One gene-two products-two path-
ways. Trends Biochem Sci 23:291–296.
Cohen JA and Geradts J (1997). Loss of Rb and MTS1/
CDKN2 (p16) expression in human sarcomas. Human Pathol
28:893–898.
Data Analysis
de Misa RF, Azana JM, Harto A, Bellas C, and Ledo A (1995).
Infrequent expression of protein p53 in epidermotropic vari-
ants of cutaneous T-cell lymphomas. J Dermatol 22:524–
526.
Statistical analysis was performed by means of EpiInfo
6.02 or SAS software. The Wilcoxon and median score
tests were used to compare the homogeneity of the
epidemiologic data of the group of patients with stable
or aggressive MF. The same tests were used to
evaluate the influence of the epidemiologic data over
the existence and type of p16INK4a, p15INK4b, or
p14ARF genetic alteration.
Diamandidou E, Cohen PR, and Kurzrock R (1996). Mycosis
fungoides and Sézary syndrome. Blood 88:2385–2409.
Drexler HG (1998). Review of alterations of the cyclin depen-
dent kinase inhibitor INK4 family genes p15, p16, p18 and
p19 in human leukemia-lymphoma cells. Leukemia 12:845–
849.
The following characteristics were evaluated using
2
Fisher’s exact text or the ␹ test, with Yates’ correc-
tion when necessary: treatment performed before the
Elenitoba-Johnson KS, Gascoyne RD, Lim MS, Chhanabai
M, Jaffe ES, and Raffelfd M (1998). Homozygous deletions at
chromosome 9p21 involving p16 and p15 are associated
with histologic progression in follicle centre lymphoma.
Blood 91:4677–4685.
biopsy versus genetic alteration of the p16INK4a
,
p15INK4b, or p14ARF genes; and differences in p16INK4a
,
p15INK4b, or p14ARF genetic alteration among the three
groups of lesions (stable MF and aggressive MF before
and after tumoral transformation).
Esteller M, Tortola S, Toyota M, Capella G, Peinado MA,
Baylin SB, and Herman
J (2000). Hypermethylation-
Acknowledgements
associated inactivation of p14ARF is independent of
p16INK4a methylation and p53 mutational status. Cancer
Res 60:129–133.
We would like to thank Dr. Agustín Gómez for his
useful help with statistical analysis and Dr. Pablo
Morales for his kind contribution of patient samples.
Fernandez-Piqueras J, Santos J, Perez de Castro I, Melén-
dez B, Martínez B, Robledo M, Rivas C, and Benítez J (1997).
Frequent allelic losses of 9p21 markers and low incidence of
mutations at p16 (CDKN2) gene in NHL of B-cell lineage.
Cancer Genet Cytogenet 98:63–68.
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