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From the Departments of Genetics,*
Dermatology,
and
Pathology,¶
Virgen de la Salud Hospital, Toledo;
and the Departments of Dermatology
and
Pathology,§
12 de Octubre Hospital,
Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histopathologically it consists of epidermotropic band-like infiltrates containing small or medium-sized mononuclear cells with cerebriform nuclei. Colonization of the lower layers of the epidermis by simple or small groups of neoplastic cells is a characteristic finding. Epidermotropism is usually lost with progression to tumor stage, when the dermal infiltrate becomes more diffuse and the tumor cells increase in number and size, while there is a concomitant decrease in the number of admixed reactive inflammatory cells.
In lymphomas, as in other tumors, tumoral progression has been found to be the consequence of multiple cell cycle alterations, with loss of some of the negative regulatory pathways. Cell cycle progression is regulated by complexes formed between cyclins and cyclin-dependent kinases (CDKs). CDK4 and CDK6 bind to D-type cyclins in the G1 phase of the cell cycle and control G1/S transition through the phosphorylation of retinoblastoma protein (pRb). The activity of cyclin D-CDK4/6 complexes is subject to additional levels of regulation, including the association with CDK inhibitors (CDKIs). The p16INK4a gene is located in the short arm of chromosome 9, region 9p21, encoding for a nuclear protein that can block cell cycle progression by effectively inhibiting the kinase activity of CDK4/6, thereby exerting a negative control on cell proliferation.3
The p16INK4a gene has been found to be altered in a high percentage of cell lines (75%) and various primary human tumors,4-6 such as breast and colon cancer, lymphoma, and leukemia. In these tumors, the loss of protein expression, secondary to 5'CpG island methylation, homozygous deletion, and, less frequently, p16INK4a gene mutations, is usually associated with tumoral progression.7-15
The molecular mechanisms by which advanced cases of MF undergo large cell transformation and develop an aggressive behavior are essentially unknown. Several studies have failed to identify the genes involved in this transformation, and thus the data obtained in the analysis of p53, lyt10, c-myc, bcl-1, and bcl-2 genes have shown a low frequency of genetic alterations associated with MF.16-20 A large body of evidence has recently suggested that p16INK4a gene inactivation is a key event in the development and progression of hematological malignancies.21 However, the data on p16INK4a status in MF remain sparse and are less than compelling.22-27
The aim of this study was to investigate the hypothetical pathogenic role of p16INK4a inactivation in a representative panel of MF with patch/plaque and tumoral sequential samples. To determine the possible implication of p16INK4a gene alterations in the development and progression of MF, we analyzed the three most frequent mechanisms of inactivation of the p16INK4a gene: deletion, promoter hypermethylation, and mutation. As previous data obtained in the study of other lymphoma types have shown that p16INK4a inactivation is characteristically associated with advanced tumoral stages, we restricted analysis to cases concurrently showing both early and advanced stages of MF, thus making it possible to compare the results in biopsies that are representative of both phases of tumoral progression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A group of MF patients was selected from the medical records of
the 12 de Octubre Hospital, Madrid, and the Virgen de la Salud
Hospital, Toledo (both of which are in the center of Spain). Diagnosis
was based on generally accepted clinical-pathological
criteria.2
Patch or plaque lesions were erythematous,
slightly scaling macules or plaques 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 they were ulcerated
or not, when histopathological examination showed a majority of large
cells (Figure 1)
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Complete remission (CR) 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 Cooperative Group for MF/SS.2
Molecular Studies
DNA Extraction
Genomic DNA from the cases selected was obtained from frozen or paraffin-embedded tissue from patch/plaque and tumoral lesions. To provide a negative control in methylation studies, DNA was obtained from different samples of reactive lymphoid tissue (12 cases of peripheral blood lymphocytes from healthy donors, three reactive lymph nodes, and one normal thymus) and six cases of inflammatory dermatosis (pytiriasis lichenoides). The Raji cell line was used as the positive control. For analysis of allelic loss nontumoral DNA was obtained from neutrophils (seven cases), oral swabs (one case), or nontumoral paraffin-embedded tissue (one case).
Allelic Loss Assays at the 9p21 Locus
Tumoral and normal DNA was analyzed for loss of heterozygosity
(LOH) or homozygous deletion (HD) by amplification of dinucleotide
repeats containing sequence microsatellite markers,28
under conditions previously described by Villuendas et
al.9
These markers are at the 9p21 region, surrounding
the p16INK4a gene (IFN
, D9S736,
D9S1749, D9S1747, D9S974, D9S1748, D9S1752, D9S171). Briefly, 100 ng of
DNA was amplified in a volume of 25 µl with 1x polymerase chain
reaction (PCR) buffer, 200 µmol/L deoxynucleoside triphosphates, 10
pmol of each primer, 1 µCi of [32P]dCTP, and
1 U of Taq polymerase. Amplified products were separated by
electrophoresis by denaturing 7 mol/L urea on 6% acrylamide gels,
followed by autoradiography. Allelic loss was determined if the
intensity of the signal from any one allele was significantly reduced
in the tumor DNA when compared with normal DNA by direct visualization.
The presence of homozygous deletions was also assessed in all cases by comparative multiplex PCR assay, using two primer sets from loci outside the 9p21 region (D9S934 and D7S1824, in chromosomes 9q and 7q, respectively).
Methylation Studies
Methylation-specific PCR (MSP) assays were performed to analyze the methylation status of CpG islands of the first exon of p16INK4a, as described by Herman et al..29 Briefly, 1 µg of denatured genomic DNA was modified by reaction with sodium bisulfite under conditions that convert all unmethylated cytosine residues, except 5-methylcytosine, to uracil by deamination. Modified DNA was purified using the Wizard DNA clean-up system (Promega, Madison, WI). Modification was completed by NaOH 0.3 mol/L treatment for 5 minutes at room temperature, followed by ethanol precipitation.
Bisulfite-modified DNA was amplified using p16INK4a unmethylated-specific primers (U), methylated-specific primers (M), and unmodified or wild-type primers (W). One hundred nanograms of bisulfite-modified DNA was amplified using 1 U of AmpliTaq Gold (PE Applied Biosystems, Weiterstadt, Germany) under the following conditions: 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C, for 35 cycles. 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 UV illumination.
If a methylation-specific PCR product was detected, the whole procedure using sodium bisulfite and MSP was performed again to minimize the possible influence of contamination or incomplete bisulfite treatment.
DNA methylation was determined by the presence of a 150-pb fragment in those samples amplified with the M primers.
The PCR products obtained with p16-M and p16-U primers of two cases were sequenced with an Automated DNA Sequencer ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems) according to the manufacturer’s instructions.
Mutation Study
DNA from seven frozen tumoral stage samples were analyzed for mutations in exons 1 and 2 of the p16INK4a gene, comprising 97% of the coding sequences, with primers described previously.30 Briefly, 200 ng of DNA was amplified with 25 pmol of each primer, 200 µmol/L of each deoxynucleoside triphosphate, 5% dimethylsulfoxide, and 1 U Taq polymerase, under amplification conditions described previously by Villuendas et al.9 Direct sequencing of amplified products was performed with an Automated DNA Sequencer ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems) according to the manufacturer’s procedures.
Data Analysis
Statistical analysis was performed by means of Epilnfo6.0 or
SAS2.0 software. The following characteristics were evaluated using
Fisher’s exact test or the 
2
test, with Yates
correction when necessary: treatment previous to the biopsy (with
-interferon, psolaven and ultraviolet A light (PUVA),
sunbathing, radiotherapy, chemotherapy, topical
bis-chlorethylnitrosourea (BCNU), topical mechloretamine,
or topical corticosteroids) versus genetic alteration
(p16INK4a hypermethylation or
allelic loss or both). The same tests were used to analyze the
relationship between clinical outcome (long complete remission or not)
versus genetic alteration
(p16INK4a hypermethylation, allelic
loss, or both). The Wilcoxon and median score tests were used to
evaluate the influence of the variable interval of time between the
onset of the disease and biopsy examination and the existence and type
of p16INK4a genetic alteration.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clinical data of our patients are summarized in Table 1
. Sex distribution was 2:1, M/F; mean
age at the beginning of MF was 44.7 years (range 21–70 years); mean
delay to diagnosis was 75 months (range 3–288 months). The mean time
until the appearance of tumors was 93.1 months (range 0–298 months).
Two patients developed tumors and patch/plaques simultaneously. In
another patient tumors appeared within 6 months of the beginning of
patch/plaque lesions.
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Two of the patients achieved complete remission, and they remain free of disease after 39 and 99 months of follow-up, respectively. Only two of the seven remaining patients underwent complete remission during their medical history: MF73 and MF78, 6 and 11 months, respectively, early in the course of their disease.
Molecular Studies
A table showing the results obtained in the analysis of
p16INK4a is included (Table 2)
. Alterations of the
p16INK4a gene were found in four of nine
samples in plaque lesions and in seven of nine samples in tumoral
samples (including both deletion and methylation analysis).
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Nine cases of MF for which DNA from nontumoral tissue was
available were studied using markers for microsatellites surrounding
the p16INK4a gene in the 9p21 region. The
results of allelic loss analysis are shown in Figure 2
, and representative cases are
illustrated in Figure 3
.
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. Deletions were most commonly
observed at marker D9S1749.
Case MF36 (Figure 3)
showed LOH in one informative marker in the sample
corresponding to the tumoral phase. Homozygous deletion was identified
by multiplex PCR at the D9S974 marker, close to the
p16INK4a gene. Noninformative loci D9S1748
and D9S1752 also showed a band in tumoral DNA that was markedly
diminished in comparison with normal DNA. This case also showed
retention of heterozygosity at the D9S1747 locus. This apparent
retention of heterozygosity at a single locus within a large deletion
supports the hypothesis of the existence of a homozygous deletion,
including the p16INK4a gene, rather than
two separate regions of loss at 9p, as the signal from the deleted
alleles in the tumor lane probably arises from normal cell
contamination of the tumor biopsy.
In case MF39 (Figure 3)
, LOH was detected in only two markers.
Case MF113 displayed LOH, involving four different markers in plaque and tumoral samples. The existence of LOH in markers D9S974 and D9S1748, which flank the region occupied by the p16INK4a gene, indicates hemizygous deletion of this gene.
Methylation at the CpG Island of the p16INK4a Gene Assays
The MSP technique was used to identify the hypermethylation status
of the CpG island in the promoter region of
p16INK4a gene. Hypermethylation of the
p16INK4a gene was found in five of seven
tumoral lesions and three of six plaque lesions (Table 2)
. In these
three early cases, methylation of the
p16INK4a gene was also detected in the
sample corresponding to the tumoral phase. Two cases only presented
hypermethylation of the p16INK4a gene in
advanced-stage biopsy. Representative cases are illustrated in Figure 4
.
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All 22 controls included (skin, lymphoid tissue in different reactive
conditions, and peripheral blood lymphocytes) showed amplification with
unmethylated-specific primers (U) but not with methylated-specific
primers (M). DNA from the Raji cell line was used as a positive control
for the amplification reaction with p16-M primers, and no amplification
was seen in the PCR reaction with p16-U primers (Figure 4)
. PCR
reactions with wild-type primers (W) were used as a control of
efficiency of chemical modification.
p16-M and p16-U amplification products from the plaque and tumoral
stages of cases MF91 and MF122 were sequenced. The sequence of p16-M
products did not show any change from C to T in any of the CpG sites,
in the plaque or the tumoral samples, thus confirming the presence of
methylation in all of the CpG sites (Figure 5A)
. However, changes from C to T
(indicating the absence of methylation) were observed in the p16-U
product sequence (Figure 5B)
. p16-W PCR was performed using
non-bisulfite-treated DNA.
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. Mutational Analysis of the p16INK4a Gene
To determine whether mutations of the p16INK4a gene were present, exons 1 and 2 of the gene were analyzed by direct sequencing. None of the seven cases analyzed showed mutations.
Correlation between Clinical and Molecular Data
The type of molecular alteration identified in this study (methylation or gene deletion) was independent of previous treatment received by the patient (P > 0.05).
The existence of molecular alteration was also independent of the length of the evolution of the disease previous to the biopsy analyzed. For plaque lesions, the mean times were 147 months for hypermethylated and 74.5 months for nonhypermethylated plaques. The difference between these two groups is not significant (Wilcoxon and Median score tests). For tumoral lesions, the mean time interval between the first clinical manifestations of the disease and the date of the biopsy on which molecular study was performed was 124.5 months for the cases with hypermethylation or allelic loss and 74 months for cases with none. These results were not significantly different.
To determine whether clinical outcome is related to p16INK4a alteration or to some specific molecular abnormality (methylation or gene deletion), a comparison was made between patients in terms of their response to treatment. Five of the patients in whom complete remission was not achieved had p16INK4a hypermethylation in either the tumor or both the tumor and plaque stages, whereas neither of the two patients (MF36 and MF82) with long complete remission had p16INK4a hypermethylation in plaque or tumor lesions. However, patient MF36 showed homozygous deletion, thus making methylation unnecessary.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This series allowed us to investigate some molecular mechanisms underlying tumoral progression, which in other lymphoma types have been found to be associated with inactivation of some of the more common suppresser pathways, p14/ARF-p53 and p16INK4a-Rb. We decided to focus on p16INK4a, because previous works have shown there is a low frequency of p53 inactivation in advanced forms of MF.18-19
The tumor suppresser gene p16INK4a is now recognized as the one that is most often subject to genetic alteration in different types of tumor. The frequency and type of alterations depend on tumoral type, because it has been established that gene deletions, promoter hypermethylation, or point mutation may lead to the silencing of the p16INK4a gene.4-8,31 In non-Hodgkin’s lymphomas, p16INK4a inactivation has been found to be very characteristically associated with tumoral progression, this being caused most often by promoter hypermethylation and gene deletion. The highest frequencies of deletion of the p16INK4a gene are found in acute lymphoblastic leukemias (ALLs), mainly of the T-cell precursor type.13,24 Rates of hypermethylation of the 5' CpG island of p16INK4a are more strongly associated with a higher rate of tumor progression in Burkitt’s lymphoma, multiple myeloma,32-33 and large B-cell lymphoma than they are in stable forms of indolent B-cell lymphomas, such as B-chronic lymphocytic leukemia, follicle center lymphoma, mantle cell lymphoma, and mucosa-associated lymphoid tissue lymphoma.9-12 Information about the frequency and mechanisms of p16INK4a inactivation in MF is scarce, although recently the loss of p16INK4a expression in a significant proportion of cases of MF has been demonstrated.27
This study showed that p16INK4a alterations take place in the MF lesions of patients, at both the plaque and tumoral stages. Tumoral samples of MF showed a higher frequency (seven of nine cases) of p16INK4a gene alterations (including both deletion and methylation analysis), in contrast with a lower frequency (four of nine samples) in plaque samples. Comparing these data with those obtained in the analysis of B-cell lymphomas, the relatively higher frequency of p16INK4a alterations in early phases of the disease in this series of MF is remarkable. The higher frequency of p16INK4a alterations in tumoral lesions could suggest a role for p16INK4a in the progression of MF from the plaque to the tumoral stage. Screening for these abnormalities could hypothetically identify patients with a higher probability of transformation to aggressive forms of the disease, although we cannot exclude the possibility that the p16INK4a alteration could be a hallmark of MF that is frequently present from the beginning of the disease. A more comprehensive study of MF cases without progression would be needed to confirm the significance of this alteration. If additional studies confirm that p16INK4a inactivation is a marker of tumoral progression in MF, this could be used in the routine evaluation of these patients, thus making it possible to identify cases that would be candidates for different therapeutic schemes. Only one case (MF39), which shows methylation in both the plaque and tumoral phases of the disease, additionally acquired LOH in the tumoral sample, suggesting that in at least this case gene deletion is subsequent to methylation. Indeed, comparative analysis of the molecular results of this series suggests that the differences between plaques and tumors seem to be more closely related to allelic loss than they are to p16INK4a hypermethylation. However, we cannot discount the possibility that these differences could be due to technical difficulties associated with samples having a low percentage of tumoral cells, which may lead to a significant underestimation of allelic loss.
None of our cases presented mutations in tumoral DNA. Consistent with other investigations,27 our findings suggest that mutations of the p16INK4a gene are not an important mechanism in the tumoral progression of MF.
Furthermore, the clinical implications of the molecular data obtained in this study have been analyzed. Because most of our patients had undergone different forms of treatment over long periods of time, we hypothesized that some of these treatments could have selected cell subpopulations with molecular alterations that would give a survival advantage. None of the treatments that our patients had received before the biopsies analyzed seemed to influence the molecular results.
Second, we attempted to determine whether the acquisition of p16INK4a alterations could be a result of a longer period of disease development before biopsy. Although the mean duration of the disease until biopsy of the lesions without molecular alterations was lower than that in the case of samples with p16INK4a alterations, the small size of the series made it impossible for us to reach significant conclusions on this point. Finally, although p16INK4a hypermethylation was more frequent in those cases that failed to achieve complete remission, the small size of our series and the existence of a homozygous deletion in one of the cases without methylation forced us to make a cautious interpretation of this finding.
If these results were reproduced in a larger series, the existence of p16INK4a hypermethylation could be used as a predictor of aggressive disease and would be useful in identifying patients who could benefit from different treatments.
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Acknowledgements |
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Footnotes |
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Drs. Navas and Ortiz-Romero contributed equally to this work.
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