Purpose: Malignant peripheral nerve sheath tumor (MPNST) can arise sporadically or in association with neurofibromatosis type 1. Deletions at the 9p21 locus have been reported in these tumors. To additionally characterize the status of this chromosomal region, in this study we performed a comprehensive, mostly PCR-based molecular analysis of the three tumor suppressor genes p15INK4b, p14ARF and p16INK4a located at the 9p21 locus in 26 cryopreserved MPNSTs.

Experimental Design: Fourteen neurofibromatosis type 1-related and 12 sporadic cases were investigated for homozygous deletion coupled with fluorescent in situ hybridization, promoter methylation, and mutational analysis, as well as m-RNA expression.

Results: The results showed that an inactivation of one or more genes occurred in 77% of MPNSTs and was mainly achieved through homozygous deletion (46%), which, in turn, encompassed all of the three tandemly linked genes in 83% of the deleted cases. Promoter methylation was at a less extent involved in gene silencing (18%), and no mutations were found. Loss of function at DNA level strongly correlated with loss of mRNA expression accounting for 80% of the cases. Because of the close relationship between p14ARF and TP53 and between p15INK4b/p16INK4a and Rb, these results support a model of a coinactivation of TP53 and Rb pathways in 75% of MPNSTs, with functional consequences on cell growth control and apoptosis.

Conclusions: The inactivation of the 9p21 locus is a frequent and peculiar hallmark of MPNST genetic profile leading also to an impaired apoptosis that could be taken into account in treatment planning of these tumors.

MPNSTs4 are uncommon soft tissue neoplasms carrying a poor prognosis (1). They may occur as sporadic or associated with NF1.

Different molecular and genetic alterations have been reported in these tumors, such as TP53 mutation, mainly in sporadic MPNSTs (2), and loss of chromosome regions 9p, 17q, 17p, and 22q, encoding p16INK4a, NF1, TP53, and NF2 genes, respectively, in both sporadic and NF1-related MPNSTs (2, 3, 4, 5, 6).

In addition, there was evidence of loss of heterozygosity at the short arm of chromosome 9 (2, 7) where the INΚ4α/ARF locus is located (9p21). The INΚ4α/ARF locus encodes the two distinct genes p14ARF and p16INK4a, the products of which derive from two alternative first exons, 1β and 1α, respectively, complemented by the identical exons 2 and 3 (8). HDs of exon 2 (9), exons 1β and 2 (10), and exons 1α, 2, and 3 (2) were reported in MPNST, the last three equally distributed into NF1-related and sporadic groups (2). Moreover, aberrations of p15INK4b gene, also mapping on 9p21 chromosomal band, were reported in both groups of MPNSTs (7).

The two TSGs p16INK4a and p15INK4b belong to the Rb pathway. They are members of INK4 family of cyclin-dependent kinase inhibitors and specifically inhibit the cyclin D-CDK4 or 6, preventing phosphorylation-mediated Rb inactivation, and leading to a G1 cell cycle block (11, 12). p15INK4b suppresses cell growth in response to extracellular stimuli such as transforming growth factor β, in contrast with p16INK4a, which is activated by intracellular stimuli.

The TSG p14ARF protects the cell from uncontrolled growth induced by hyperproliferative stimuli (13) and plays its functional role interacting with TP53 pathway (14). p14ARF sequesters mdm2 in nucleoli, preventing the mdm2-mediated p53 degradation and, thus, stabilizing a transcriptionally active p53 in the nucleoplasm (15, 16).

Here we performed a comprehensive molecular analysis of 9p21chromosome region, where the TSGs p15INK4b, p14ARF, and p16INK4a map in tandem, in a series of 26 MPNSTs of which cryopreserved material was available, subdivided into 14 NF1-related and 12 NF1-unrelated cases. All three of the TSGs were analyzed for HD coupled with FISH, promoter methylation, and gene mutation, and also investigated for the expression of their relative transcripts. The results showed that evidence of inactivation of one or more of the three TSGs, mainly achieved by HD, occurs in 77% of MPNSTs with a good correlation between loss of function at DNA level and loss of mRNA expression.

Samples and Patients.

Thirty cryopreserved consecutive samples from 26 cases that had been diagnosed as MPNST and treated at the National Cancer Institute (Milan) from 1989 to 2000 were analyzed. The medical records were evaluated by a medical geneticist, who classified the cases according to officially coded NIH Neurofibromatosis Consensus Development Conference criteria (17). Fourteen patients were classified as NF1 with an associated MPNST (Table 1), whereas 12 subjects were classified as sporadic tumor patients, because no clinical signs or family history of NF1 were present (Table 2).

The formalin-fixed, paraffin-embedded tissues of 15 cases have been analyzed previously for p16INK4a gene HD by using a comparative duplex seminested PCR (2). In detail, the first 8 cases listed in Table 1 correspond to cases number 1, 2, 3, 4, 6, 7, 8, and 12 of our previous study, and the first 7 cases listed in Table 2 correspond to cases number 16, 19, 23, 24, 25, 26, and 28 of our previous study.

For the diagnosis of MPNST we applied morphological (presence of spindle cells with indistinct cytoplasm margins and wavy or S-shaped nuclei, arranged in fascicles with alternating cellular and myxoid areas), immunophenotypic (reactivity for at least one of the neural markers S-100, CD56, NGFR, or CD 57, in absence of staining for cytokeratines, topographic (clear evidence of origin from a nerve in sporadic cases), and molecular (lack of SYT-SSX fusion transcripts) criteria, as described previously (18).

Overall, we analyzed 7 NF1-associated and 6 sporadic primary tumors, 10 NF1-associated and 6 sporadic recurrences, and 1 NF1-related metastasis.

DNA and RNA Extraction.

DNA from frozen tissues was digested by proteinase K and submitted to phenol extractions following standard procedures.

Total RNA was isolated with the RNA extraction kit by homogenizing the tissue in 1 ml of RNAzol B (TEL-TEST INC., Friendswood, Texas), according to the manufacturer’s conditions. The RNA pellets were reconstituted in sterile water and stored at −80°C.

HD of p15INK4b, p14ARF, and p16INK4a Genes.

HD assay was performed by 25 cycles of comparative duplex PCR. Each TSG was coamplified with a fragment of human β-globin gene (19), by optimizing the reaction conditions for p15INK4b, p14ARF, and p16INK4a gene amplification advantage. To evaluate the occurrence of HD we based on the criteria described previously (2). Primer sequences are indicated in Table 3.

FISH.

Imprints from frozen samples were prepared on precleaned slides, placed under running water for a few seconds, air dried, and then fixed for 10 min in Carnoy fixative (methanol:acetic acid 3:1).

Each case was subjected to a dual-color hybridization with the probe pairing CEP9/p16 (9p21; Vysis, Downers Grove, IL). Probe p16 spans 190 Kb of genomic DNA on chromosome 9p21 including D9S1749, D9S1747, p16INK4a, p14ARF, D9S1748, p15INK4b, and D9S1752.

After treatment with RNase (1 mg/ml at 37°C for 45 min) followed by pepsin (50 μg/ml in 0.01 n HCl at 37°C for 10 min), slides were fixed in 50 mm MgCl2 in 1× PBS and finally dehydrated.

Ten μl of mixture, containing alphoide 9 Spectrum Green-labeled probe (CEP9) and p16 (9p21) Spectrum Orange-labeled probe, were placed on each target area, covered immediately with 18 × 18 coverslip, and sealed with rubber cement. Denaturation and hybridization were performed in Hybraite Thermoblock (Vysis) at 75°C for 1 min and at 37°C overnight, respectively. Coverslips were gently removed by dipping slides in 2× SSC. After one wash in 2× SSC plus 0.3% NP40 at 73°C for 2 min, slides were air-dried and mounted with 4′,6-diamidino-2-phenylindole II Antifade (Vysis).

Slides were observed using a Zeiss Axioscope fluorescent microscope (Zeiss) equipped with a 100-W mercury lamp and 4′,6-diamidino-2-phenylindole, Spectrum Green, and Spectrum Orange filters (Vysis). Slides were scored at 160× magnification two localized fields with nonoverlapping, well-defined tumor cell nuclei. Signals count was performed at ×630 to ×1000 magnification in at least 100 nuclei/slides by two independent operators. Image acquisition and enhancement was performed using a CCD camera (COHU 4912; Applied Imaging) and the Mc Probe software (Powergene).

A determination of homozygous p16INK4a deletion required the presence of only CEP9 signals in neoplastic cells with retained p16 signals in adjacent non-neoplastic elements.

A determination of p16INK4a hemizygous deletion required the presence of a CEP9:p16 ratio ≥2. In disomic cells the typical hybridization pattern corresponded to one p16INK4a red signal coupled with two centromeric green signals.

Polysomy was defined as >5% nuclei with >2 signals for both probes.

Hybridizations were considered noninformative if the FISH signals were too weak to interpret.

Analysis of p15INK4b, p14ARF, and p16INK4a Promoter Methylation Status.

DNA methylation patterns in the CpG islands of the p15INK4b, p14AR, and p16INK4a genes were determined by MSP. Bisulfite modification of DNA (1 μg) was performed as described by Herman et al.(20). Taq Gold polymerase (Applied Biosystems) was used in a final volume of 10 μl PCR reaction. Both specific primers for methylated and unmethylated promoters, and annealing temperature applied are described in Table 3. Five μl of each PCR reaction were loaded into nondenaturing 6% polyacrylamide or 2% agarose gels, stained with ethidium bromide, and visualized under UV illumination. We used the cells lines Raji as positive control for methylation reaction for p16INK4a and p15INK4b, and LoVo for p14ARFgene, respectively.

For p16INK4promoter methylation we also performed a nested PCR; the stage-1 PCR products were diluted 10-fold, and 2 μl were subjected to a stage-2 PCR in which primers specific to methylated and unmethylated template were used.

Mutation Analysis.

Mutation analysis was performed on all exons of p15INK4b, p14ARF, and p16INK4a genes. The primers used for PCR amplification are described in Table 3. After purification, the PCR products were subject to automated DNA sequencing (ABIprism 377; Applied Biosystems), and each sequence reaction was performed at least twice.

RT-PCR.

One μg of total RNA was reverse-transcribed with Superscript reverse transcriptase (Life Technologies, Inc.) using both oligodeoxythymidylic acid and random examers following the manufacturer’s conditions. Thirty μl of sterile 10 mm Tris (pH 8)-1 mm EDTA was added to 20 μl of total volume of cDNA obtained from each sample. All of the samples were tested for cDNA integrity by the amplification of HPRT housekeeping gene (18), and subsequently analyzed for the expression of p15INK4b, p14ARF, and p16INK4a transcript, respectively.

All of the PCR reactions were performed using Klen TaqI (Ab peptides Inc.) and specific primers, which are described in Table 3. The PCR results were confirmed from two to four repeated amplification procedures.

HD of p15INK4b, p14ARF, and p16INK4a TSGs by Comparative Duplex PCR.

HD of one or more of the three TSGs was observed in 7 of 14 (50%) NF1 and in 5 of 12 (42%) sporadic cases, overall in 12 of 26 (46%) MPNSTs. In 10 of 12 (83%) of the deleted MPNSTs HD encompassed all three of the TSGs (Fig. 1 A).

In detail, a similar occurrence of p15INK4b HD, mainly involving both p15INK4b exons, was found in NF1 (Table 1) and sporadic (Table 2) MPNSTs. In all but one of the cases (case 7 NF1-related) p15INK4b HD paralleled the loss of p14ARF and p16INK4a TSGs.

Data consistent with both p14ARF and p16INK4a HD, mainly involving all of the exons, were obtained in 11 of 26 (42%) cases, equally distributed into the two tumor groups (Tables 1 and 2). In 1 NF1 MPNST (case 3) the deletion affecting both p14ARF and p16INK4a TSGs was restricted to the exon 2.

Considering the sample type, similar findings were detected in primary tumors (33%) and recurrences (50%). In the NF1 group, 2 cases (cases 3 and 6) presented the same molecular profile in primary tumor and recurrence, whereas in 1 case (case 1) HD was detected in the recurrence only. The NF1-related metastasis (case 7) showed exclusively p15INK4b HD (Table 1).

FISH Analysis.

FISH analysis was performed in 10 MPNSTs showing HD by PCR analysis and with available residual material.

Overall, p16INK4a deletions were found in 7 of 10 (70%) MPNSTs, as homozygous (Fig. 2,A) and hemizygous (Fig. 2 B) deletion in 50% and 20% of the cases, respectively. Among the remaining 3 cases in which FISH did not reveal p16INK4a deletion, 1 NF1-related case (case 7) presented polysomy 9 and multiple scattered p16INK4a signals without evidence of association with centromeric signals; the second, a sporadic MPNST (case 9) showed most nuclei with 4 p16INK4a signals and 2 signals for the centromeric probe, a pattern suggestive of chromosome 9 rearrangement. Both of these cases showed chromosome 9 polisomy/rearrangement(s) by a subsequent hybridization with a painting probe (data not shown). The third case, NF1-related (case 3b), showed a normal hybridization pattern in all of the tumor cells.

Methylation Analysis of p15INK4b, p14ARF, and p16INK4a TSG Promoters by MSP.

Methylation of one or more gene promoters occurred in 1 of 10 (10%) NF1 and in 2 of 7 (28%) sporadic cases, overall in 3 of 17 (18%) MPNSTs.

p15INK4b MSP analysis was performed only in MPNSTs lacking p15INK4b exon 1 HD (Fig. 3,A), and all of the analyzed cases were unmethylated in both of the groups (Tables 1 and 2).

p14ARF and p16INK4a MSP analysis was performed only in MPNSTs lacking HD of 1β and 1α exons, respectively (Fig. 3, B and C). For both of the genes, promoter methylation was found in 2 of 16 (12%) cases, 1 NF1 and 1 sporadic (Tables 1 and 2).

Simultaneous p14ARF and p16INK4a promoter methylation was observed in 1 NF1 sample (case 6c; Table 1).

p16INK4a promoter methylation has also been investigated by using nested PCR, and the results showed methylation in 5 of 16 (31%) MPNSTs.

Considering the sample type, 2 primary tumors and 1 recurrence showed promoter methylation. p14ARF and p16INK4a methylation was present in only the second primary tumor in 1 NF1 case (case 6).

Mutation Analysis of p15INK4b, p14ARF, and p16INK4a Genes.

Mutation analysis was performed in 11 of 14 samples lacking HDs and promoter methylation. Nine MPNSTs (numbers 4, 6a, 6b, 9, 11, and 14 NF1-related, and 6, 7, and 8 sporadic) were investigated for p15INK4b, p14ARF, and p16INK4a gene mutation. Two MPNSTs (numbers 10 and 11 NF1-related) were analyzed for p14ARF gene mutation only.

No mutations were found in any case (data not shown).

In 1 NF1 case (case 6) both primary tumor and recurrence presented a polymorphism in p16INK4a gene exon 2 (G→A, codon 148) that resulted in the replacement of Ala→Thr (21), and a polymorphism in 3′-noncoding region of p16INK4a gene exon 3 (540C→G) described previously (22).

p15INK4b, p14ARF, and p16INK4a mRNA Expression by RT-PCR.

We successfully performed RT-PCR in all but 1 of the cases, using HPRT housekeeping gene as control for cDNA integrity (Tables 1 and 2). We evaluated possible interferences of minimal (10%) normal tissue infiltration using cDNA obtained from normal samples. We found that these normal samples always showed a low transcript level (data not shown); thus, we interpreted a low transcript as no signal.

The transcript of one or more of the three analyzed genes was undetectable or very low in 11 of 13 (85%) NF1 and in 9 of 12 (75%) of sporadic cases, overall in 20 of 25 (80%) MPNSTs.

In detail, the p15INK4b transcript was undetectable or very low in 14 of 24 (58%) MPNSTs, with a similar occurrence into the two groups (Tables 1 and 2). The lack of a detectable transcript correlated with p15INK4b HD in 10 of 14 (71%) cases (Fig. 1 B).

The p14ARF transcript was undetectable or very low 15 of 25 (60%) MPNSTs (Fig. 1 B). The loss of expression correlated with p14ARF HD in 11 of 15 cases (73%) and with p14ARF promoter methylation in 1 (case 6c) of 15 (7%) cases, respectively. In 1 sporadic case (case 3) p14ARF expression was coupled with p14ARF promoter methylation.

The p16INK4a transcript was undetectable in 16 of 25 (64%) MPNSTs (Fig. 1 B). The lack of a detectable transcript correlated with p16INK4a HD in 11 of 16 (69%) cases and with p16INK4a promoter methylation in 2 of 16 (12%) cases.

Considering the sample type, similar findings were observed in primary tumors (67%) and recurrences (81%). In 2 NF1 MPNSTs (cases 1 and 6) the lack of detectable transcripts was found in the recurrence and not in the relative primary tumor.

HD, Promoter Methylation, and mRNA Expression.

Cumulatively, an inactivation of one or more genes were found in 11 of 14 (78%) NF1-related and in 9 of 12 (75%) sporadic cases, overall in 20 of 26 (77%) MPNSTs.

A simultaneous inactivation of p14ARF, p15INK4b, and p16INK4a genes was detected in 9 of 11 (82%) NF1 and in 6 of 9 (67%) sporadic cases, overall in 15 of 20 (75%) MPNSTs.

The 9p21 chromosomal region harbors a gene cluster consisting of three TSGs, p15INK4b, p14ARF, and p16INK4a, which are altered frequently in human cancers. An involvement of this gene cluster has been reported previously in MPNSTs (2, 3, 5, 6, 7, 9, 10). However, except for cytogenetic analyses, previous studies have been performed in limited series where NF1-related and sporadic cases were not equally represented. The occurrence of HD of p16INK4a gene has been investigated by us in a series of NF1-related and sporadic MPNSTs on formalin-fixed, paraffin-embedded material (2). Here we have complemented our previous study carrying out a comprehensive molecular analysis of p15INK4b, p14ARF, and p16INK4a genes in 26 MPNST cases of which frozen material was available. Fourteen NF1-related and 12 sporadic MPNSTs were investigated for HD coupled with FISH analysis, promoter methylation, and mutational analysis, as well as mRNA expression.

Overall, the results showed an inactivation of one or more of these TSGs in 77% of MPNSTs equally distributed into NF1-related (78%) and sporadic (75%) groups. The loss of one or more of the three TSGs was found in 46% of cases and involved both NF1 (50%) and sporadic tumors (42%) with a similar occurrence. This finding strongly supports the notion that HD is the predominant inactivation mechanism of 9p21 chromosomal region in MPNST in keeping with previous molecular studies (2, 7, 9, 10). Moreover, in all but 2 of the deleted cases (83%) a complete deletion of the 9p21 gene cluster was found. Such a finding suggests that there is not a preferential target of deletion among p14ARF, p15INK4b, and p16INK4a TSGs, each of one encoding an important cell “keeper” protein. Because the loss of p14ARF gene reflects a deregulation of TP53 pathway and the loss of p16INK4a-p15INK4b an alteration of Rb pathway, the deletion of 9p21 gene cluster strongly suggests a coinactivation of both TP53 and Rb pathways in MPNSTs.

Considering the sample type, HD occurred similarly in primary tumors (33%) and recurrences (50%). Two cases presented the same molecular profile in primary and recurrent tumor, whereas 1 case showed HD in the recurrence only.

The FISH analysis confirmed the loss of 9p21 chromosome region in 70% of the cases showing deletions by duplex-PCR. This finding does not contrast with previous FISH analyses performed on unselected MPNSTs (5, 6) considering that only homozygous cases were analyzed here.

The apparent discrepancy between FISH and PCR analyses, showing hemizygous deletion by the former and HD by the latter procedure, was because of the large size of commercial 9p21 probe (190 Kb).

In the 2 cases carrying chromosome 9 polisomy/rearrangement(s) the pattern of hybridization was suggestive of extensive chromosome 9 rearrangements accompanying or after gene deletion (23). In both occurrences, p16INK4a-specific signal may be because of inactive p16INK4a loci either carrying cryptic deletion(s) or involved into chromosome translocation(s). The presence of chromosome 9 polisomy/rearrangement(s) was confirmed by the painting profile (data not shown). Finally in case 3b, showing a p16INK4a deletion restricted to exon 2 and a normal hybridization pattern by FISH, we can assume the presence of a cryptic p16INK4a deletion involving both the alleles.

In conclusion, FISH assay revealed an aberrant hybridization pattern (deletion or rearrangement) in all but 1 of the cases showing HD by PCR analysis.

The MSP analysis revealed the absence of p15INK4b promoter methylation and an occurrence of p14ARF and/or p16INK4a methylation in 18% of the cases, indicating that this mechanism of gene silencing occurs less frequently that HD in MPNSTs. One NF1-related case (case 6c) showed methylation of both promoters. To our knowledge, this is the first time that p15INK4b and p14ARF methylation analysis was performed in MPNSTs. Regarding p16INK4a methylation, previous investigations reported a complete absence of this epigenetic alteration in this tumor type (7, 9, 10). In our hands, p16INK4a promoter methylation involved 12% of the MPNSTs. We cannot rule out that the discrepancy might be because of a limited number of cases analyzed in the published series in comparison with the present one. Interestingly, the p16INK4a methylation occurrence rose up to 31% when we applied the nested-PCR, which increases the reaction sensitivity. Hence, it is tempting to suspect that also p15INK4b and p14ARF methylation occurrence could have been underestimated.

Considering the sample type, promoter methylation was present in the first primary tumor and its recurrence, and absent in the second primary tumor in 1 NF1 case (case 6). This different molecular profile could be ascribed to a different origin of the primary tumors from two independent clones.

Although the inactivation of TSGs is mostly achieved by HD or promoter methylation, to exclude alterations because of gene mutations we performed a mutational analysis of the three TSGs in MPNST cases where they were not found deleted or methylated. No mutations were identified in the 11 cases analyzed.

To complement the DNA analyses and additionally support the observed data, we assessed p15INK4b, p14ARF, and p16INK4a mRNA expression by RT-PCR. The results showed loss of one or more transcripts in 80% of the MPNSTs equally distributed in NF1-related (75%) and sporadic (85%) cases. We found a good correlation between gene expression and gene deletion or methylation. With the exception of 1 sporadic MPNST (case 3), in all of the cases showing HD or promoter methylation the relative transcripts were undetectable or very low. In case 3 the expression of p14ARF transcript paralleled the methylation of the relative promoters, suggesting an ineffective methylation perhaps affecting only one of the two alleles of this gene. The loss of expression of p14ARF and p16INK4a-p15INK4b genes in 75% of the cases additionally reinforces the notion of the already mentioned simultaneous inactivation of TP53 and Rb pathways in MPNSTs.

In 8 cases showing normal gene status we observed loss or very low amount of mRNA expression. Because we have excluded mutational events, other still undefined molecular mechanisms could be responsible for gene silencing in these cases, such as mutations in noncoding sequences or loss of heterozygosity at microsatellites located near these TSGs.

In conclusion, our PCR analysis indicates that the inactivation of 9p21 gene cluster (77%) represents the most frequent hallmark of NF1-related and sporadic MPNST gene profile. This inactivation is mainly achieved through HD (46%) and to a lesser extent through promoter methylation (18%), and is additionally supported by loss of expression (80%) of one or more of the three genes p15INK4b, p14ARF, and p16INK4a located in the 9p21 locus. Because of the close relationship between p14ARF and TP53, and between p15INK4b - p16INK4a and Rb, the DNA analysis results as well as the mRNA expression data strongly support a coinactivation of TP53 and Rb pathways in these tumors (75%). A failure of a balanced and coordinated function of p53 and pRb in cell growth control and apoptosis is, thus, expected in mostly MPNSTs and, in particular, in TP53 mutation carrying sporadic subtype we have reported previously (2). Therefore, the presence of an impaired apoptosis should be taken into account in treatment planning of these tumors. In fact, they are currently treated, as the majority of soft tissue tumors, by DNA-damaging drug-based schemes, disregarding the alterations of their genetic profile that could make ineffective their apoptosis-mediated cell killing mechanisms.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by Grant 420.198.122: Associazione Italiana Ricerca sul Cancro/Associazione and Fondazione Italiana per la Ricerca sul Cancro.

4

The abbreviations used are: MPNST, malignant peripheral nerve sheath tumor; NF1, neurofibromatosis type 1; TSG, tumor suppressor gene; FISH, fluorescence in situ hybridization; HD, homozygous deletion; MSP, methylation-specific-PCR; RT-PCR, reverse transcription-PCR.

Fig. 1.

p15INK4b, p14ARF, and p16INK4a gene molecular analysis. A, HD analysis by using comparative duplex PCR of p15INK4b exon 1, p14ARF exon 1β, and p16INK4a exon 1α. Upper band corresponds to β-globin fragment gene, coamplified with the gene of interest (lower band). Absence of p15INK4b exon 1 (Lanes 1 and 2), p14ARF exon 1β (Lanes 5 and 6), and p16INK4a exon 1α (Lanes 9 and 10) in sporadic #5 and NF1 #2 cases. Retention of p15INK4b exon 1 (Lanes 3 and 4), p14ARF exon 1β (Lanes 7 and 8), and p16INK4a exon 1α (Lanes 11 and 12) in sporadic #7 and 12 cases. B, m-RNA expression analysis by RT-PCR. Absence of p15INK4b (Lanes 1 and 2), p14ARF (Lanes 5–7) and p16INK4a (Lanes 9–11) transcripts in sporadic #5, 7, 12, and NF1 #2 cases. Sp., sporadic.

Fig. 1.

p15INK4b, p14ARF, and p16INK4a gene molecular analysis. A, HD analysis by using comparative duplex PCR of p15INK4b exon 1, p14ARF exon 1β, and p16INK4a exon 1α. Upper band corresponds to β-globin fragment gene, coamplified with the gene of interest (lower band). Absence of p15INK4b exon 1 (Lanes 1 and 2), p14ARF exon 1β (Lanes 5 and 6), and p16INK4a exon 1α (Lanes 9 and 10) in sporadic #5 and NF1 #2 cases. Retention of p15INK4b exon 1 (Lanes 3 and 4), p14ARF exon 1β (Lanes 7 and 8), and p16INK4a exon 1α (Lanes 11 and 12) in sporadic #7 and 12 cases. B, m-RNA expression analysis by RT-PCR. Absence of p15INK4b (Lanes 1 and 2), p14ARF (Lanes 5–7) and p16INK4a (Lanes 9–11) transcripts in sporadic #5, 7, 12, and NF1 #2 cases. Sp., sporadic.

Close modal
Fig. 2.

FISH analysis with CEP9/p16 probe to interphasic nuclei. Fig. 1,A, p16INK4a HD: tumor cell nuclei showed two centromere 9 signals (green spots) and no p16INK4a signal (red spots), compared with normal cell nucleus in which two centromere 9 signals were coupled with two p16INK4a signals. Fig. 1B, p16INK4a hemizygous deletion: tumor cell nuclei showed two centromere 9 signals (green spots) coupled with a single p16INK4a signal (red spot), compared with normal cell nucleus. Note close association between centromeric and p16INK4a signals.

Fig. 2.

FISH analysis with CEP9/p16 probe to interphasic nuclei. Fig. 1,A, p16INK4a HD: tumor cell nuclei showed two centromere 9 signals (green spots) and no p16INK4a signal (red spots), compared with normal cell nucleus in which two centromere 9 signals were coupled with two p16INK4a signals. Fig. 1B, p16INK4a hemizygous deletion: tumor cell nuclei showed two centromere 9 signals (green spots) coupled with a single p16INK4a signal (red spot), compared with normal cell nucleus. Note close association between centromeric and p16INK4a signals.

Close modal
Fig. 3.

Analysis of p15INK4b (Fig. 2,A), p14ARF (Fig. 2,B), and p16INK4a (Fig. 2 C) promoter methylation by MSP in normal control (Lanes 1 and 2), sporadic case 7 (Lanes 3 and 4), and NF1 case 6c (Lanes 5 and 6). A visible PCR product in lanes marked u and m indicates the presence of unmethylated and methylated promoters, respectively. M, marker (Φ X-174 or 1-Kb ladder).

Fig. 3.

Analysis of p15INK4b (Fig. 2,A), p14ARF (Fig. 2,B), and p16INK4a (Fig. 2 C) promoter methylation by MSP in normal control (Lanes 1 and 2), sporadic case 7 (Lanes 3 and 4), and NF1 case 6c (Lanes 5 and 6). A visible PCR product in lanes marked u and m indicates the presence of unmethylated and methylated promoters, respectively. M, marker (Φ X-174 or 1-Kb ladder).

Close modal
Table 1

NF1-related case data

CaseSex/ageTumor location/sample typeHomozygous deletionMSPRT-PCR
p15                  INK4bp14                  ARF                  /p16                  INK4a
ex1ex2ex1βex1αex2aex2bex3p15                  INK4bp14                  ARFp16                  INK4aHPRTp15                  INK4bp14                  ARFp16                  INK4a
1aa m/16b Pelvic region/Pr n.e. n.e. n.e. n.e. n.e. n.e. 
1ba  Sacral region/Re HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
2a f/9 Left shoulder/Re HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
3aa f/26 Left thigh/Pr HD n.e. − low − 
3ba  Left thigh/Re HD − − 
4a f/39 Paravertebral region/Pr n.e. n.e. n.e. n.e. 
5a f/32 Right thigh/Re HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
6aa m/29 Popliteal region/1 Pr 
6ba  Popliteal region/Re − − 
6ca  Paravertebral region/II Pr n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. − − 
7a f/19 Lung/Met HD HD n.e. n.d. low 
8a f/31 Pelvic region/Re HD HD n.e. HD HD HD HD n.d. n.d. low − − 
m/27 Right gluteus/Pr − − 
10 f/13 Right arm/Re 
11 m/40 Popliteal region/Re − − 
12 m/23 Omentum/Re 
13 m/23 Axillary region/Pr HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
14 f/8 Left gluteus/Re − 
   43%  43%     0% 10% 11%  61% 69% 69% 
CaseSex/ageTumor location/sample typeHomozygous deletionMSPRT-PCR
p15                  INK4bp14                  ARF                  /p16                  INK4a
ex1ex2ex1βex1αex2aex2bex3p15                  INK4bp14                  ARFp16                  INK4aHPRTp15                  INK4bp14                  ARFp16                  INK4a
1aa m/16b Pelvic region/Pr n.e. n.e. n.e. n.e. n.e. n.e. 
1ba  Sacral region/Re HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
2a f/9 Left shoulder/Re HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
3aa f/26 Left thigh/Pr HD n.e. − low − 
3ba  Left thigh/Re HD − − 
4a f/39 Paravertebral region/Pr n.e. n.e. n.e. n.e. 
5a f/32 Right thigh/Re HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
6aa m/29 Popliteal region/1 Pr 
6ba  Popliteal region/Re − − 
6ca  Paravertebral region/II Pr n.e. n.e. n.e. n.e. n.e. n.e. n.e. n.e. − − 
7a f/19 Lung/Met HD HD n.e. n.d. low 
8a f/31 Pelvic region/Re HD HD n.e. HD HD HD HD n.d. n.d. low − − 
m/27 Right gluteus/Pr − − 
10 f/13 Right arm/Re 
11 m/40 Popliteal region/Re − − 
12 m/23 Omentum/Re 
13 m/23 Axillary region/Pr HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
14 f/8 Left gluteus/Re − 
   43%  43%     0% 10% 11%  61% 69% 69% 
a

Case previously investigated.

b

m, male; f, female; Re, recurrence; Pr, primary; Met, metastasis; ex, exon; HD, homozygous deletion; N, normal; n.d., not done; n.e., not evaluable; U, unmethylated; M, methylated; +, positive; −, negative.

Table 2

Sporadic case data

CaseSex/ageTumor location/sample typeHomozygous deletionMSPRT-PCR
p15                  INK4bp14/                  ARF                  /p16                  INK4a
ex1ex2ex1βex1αex2aex2bex3p15                  INK4bp14                  ARFp16                  INK4aHPRTp15                  INK4bp14                  ARFp16                  INK4a
1a f/61 Chest region/Re HD HD HD HD HD HD HD n.d. n.d. n.d. low − − 
2a m/52 Right arm/Re low 
3a m/42 Paravertebral region/Pr 
4a f/55 Submandibular region/Pr HD HD HD HD HD HD HD n.d. n.d. n.d. low − − 
5a f/72 Shoulder/Pr HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
6a m/66 Right forearm/Pr n.e. low 
7a f/69 Left shoulder/Pr − − 
f/22 Axillary region/Re − 
f/24 Pelvic region/Re HD HD HD HD HD HD n.e. n.d. n.d. n.e. − − 
10 f/46 Abdominal region/Re n.e. 
11 m/31 Left foot/Re HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
12 f/69 Left thigh/Pr 
   42%  42%     0% 17% 14%  54% 50% 58% 
CaseSex/ageTumor location/sample typeHomozygous deletionMSPRT-PCR
p15                  INK4bp14/                  ARF                  /p16                  INK4a
ex1ex2ex1βex1αex2aex2bex3p15                  INK4bp14                  ARFp16                  INK4aHPRTp15                  INK4bp14                  ARFp16                  INK4a
1a f/61 Chest region/Re HD HD HD HD HD HD HD n.d. n.d. n.d. low − − 
2a m/52 Right arm/Re low 
3a m/42 Paravertebral region/Pr 
4a f/55 Submandibular region/Pr HD HD HD HD HD HD HD n.d. n.d. n.d. low − − 
5a f/72 Shoulder/Pr HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
6a m/66 Right forearm/Pr n.e. low 
7a f/69 Left shoulder/Pr − − 
f/22 Axillary region/Re − 
f/24 Pelvic region/Re HD HD HD HD HD HD n.e. n.d. n.d. n.e. − − 
10 f/46 Abdominal region/Re n.e. 
11 m/31 Left foot/Re HD HD HD HD HD HD HD n.d. n.d. n.d. − − − 
12 f/69 Left thigh/Pr 
   42%  42%     0% 17% 14%  54% 50% 58% 
a

Case previously investigated.

b

m, male; f, female; Re, recurrence; Pr, primary; Met, metastasis; ex, exon; HD, homozygous deletion; N, normal; n.d., not done; n.e., not evaluable; U, unmethylated; M, methylated; +, positive; −, negative.

Table 3

Primers used for duplex PCR, mutation analysis, MSP, and RT-PCR

AnalysisPrimer sequence (5′→3′)Annealing temp. (°C)
SenseAntisense
Duplex PCR and mutational analysis    
p15INK4b exon 1 CTG CGC GTC TGG GGG CTG C CCT CCC GAA ACG GTT GAC TCC 62 
p15INK4b exon 2 ACC GGT GCA TGA TGC T TCA GTC CCC CGT GGC TGT 62 
p14ARF exon 1β AAC ATG GTG CGC AGG TTC AGT AGC ATG AGC ACG AGG G 60 
p16INK4a exon 1α ACG AGG CAG CAT GGA GCC CCA GGT CCA CGG GCA GA 62 (5 cycles)/58 (20 cycles) 
p16INK4a exon 2a CTG GCT CTG ACC ATT CTG T CCA GGT CCA CGG GCA GA 62 (5 cycles)/58 (20 cycles) 
p16INK4a exon 2b TGG ACG TGC GCG ATG CC TCT GAG CTT TGG AAG CTC T 60 
p16INK4a exon 3 CCG GTA GGG ACG GCA AGA GA GCA GTT GTG GCC CTG TAG GA 60 
MSP    
 M-p15INK4a GCG TTC GTA TTT TGC GGT T CGT ACA ATA ACC GAA CGA CCG A 60 
 U-p15INK4 TGT GAT GTG TTT GTA TTT TGT GGT T CCA TAC AAT AAC CAA ACA ACC AA 50 
 M-p14ARF GTG TTA AAG GGC GGC GTA GC AAA ACC CTC ACT CGC GAC GA 58.5 
 U-p14ARF TTT TTG GTG TTA AAG GGT GGT GTA GT CAC AAA AAC CCT CAC TCA CAA CAA 68 
p16INK4a stage-1 PCR GAA GAA AGA GGA GGG GTT GG CTA CAA ACC CTC TAC CCA CC 60 
 M-p16INK4a stage-2 PCR TTA TTA GAG GGT GGG GCG GAT CGC GAC CCC GAA CCG CGA CCG TAA 68 
 U-p16INK4a stage-2 PCR TTA TTA GAG GGT GGG GTG GAT TGT CAA CCC CAA ACC ACA ACC ATA A 68 
RT-PCR    
p15INK4b CGC TGC CCA TCA TCA TGA C CTA GTG GAG AAG GTG CGA CA 57 
p14ARF CCC TCG TGC TGA TGC TAC TGA ACC ACC AGC GTG TCC AGG AA 61 
p16INK4a GCT GCC CAA CGC ACC GAA TA ACC ACC AGC GTG TCC AGG AA 61 
AnalysisPrimer sequence (5′→3′)Annealing temp. (°C)
SenseAntisense
Duplex PCR and mutational analysis    
p15INK4b exon 1 CTG CGC GTC TGG GGG CTG C CCT CCC GAA ACG GTT GAC TCC 62 
p15INK4b exon 2 ACC GGT GCA TGA TGC T TCA GTC CCC CGT GGC TGT 62 
p14ARF exon 1β AAC ATG GTG CGC AGG TTC AGT AGC ATG AGC ACG AGG G 60 
p16INK4a exon 1α ACG AGG CAG CAT GGA GCC CCA GGT CCA CGG GCA GA 62 (5 cycles)/58 (20 cycles) 
p16INK4a exon 2a CTG GCT CTG ACC ATT CTG T CCA GGT CCA CGG GCA GA 62 (5 cycles)/58 (20 cycles) 
p16INK4a exon 2b TGG ACG TGC GCG ATG CC TCT GAG CTT TGG AAG CTC T 60 
p16INK4a exon 3 CCG GTA GGG ACG GCA AGA GA GCA GTT GTG GCC CTG TAG GA 60 
MSP    
 M-p15INK4a GCG TTC GTA TTT TGC GGT T CGT ACA ATA ACC GAA CGA CCG A 60 
 U-p15INK4 TGT GAT GTG TTT GTA TTT TGT GGT T CCA TAC AAT AAC CAA ACA ACC AA 50 
 M-p14ARF GTG TTA AAG GGC GGC GTA GC AAA ACC CTC ACT CGC GAC GA 58.5 
 U-p14ARF TTT TTG GTG TTA AAG GGT GGT GTA GT CAC AAA AAC CCT CAC TCA CAA CAA 68 
p16INK4a stage-1 PCR GAA GAA AGA GGA GGG GTT GG CTA CAA ACC CTC TAC CCA CC 60 
 M-p16INK4a stage-2 PCR TTA TTA GAG GGT GGG GCG GAT CGC GAC CCC GAA CCG CGA CCG TAA 68 
 U-p16INK4a stage-2 PCR TTA TTA GAG GGT GGG GTG GAT TGT CAA CCC CAA ACC ACA ACC ATA A 68 
RT-PCR    
p15INK4b CGC TGC CCA TCA TCA TGA C CTA GTG GAG AAG GTG CGA CA 57 
p14ARF CCC TCG TGC TGA TGC TAC TGA ACC ACC AGC GTG TCC AGG AA 61 
p16INK4a GCT GCC CAA CGC ACC GAA TA ACC ACC AGC GTG TCC AGG AA 61 
a

M, specific primers for methylated promoter; U, specific primers for unmethylated promoter.

We thank Gianni Roncato for photographic assistance.

1
Ducataman B. S., Scheithauer B. W., Piepgras D. G., Reiman H. M., Ilstrup D. M. Malignant peripheral nerve sheath tumors.
Cancer (Phila.)
,
57
:
2006
-2021,  
1986
.
2
Birindelli S., Perrone F., Oggionni M., Lavarino C., Pasini B., Vergani B., Ranzani G. N., Pierotti M. A., Pilotti S. Rb and TP53 pathway alterations in sporadic and NF1-related malignant peripheral nerve sheath tumors (MPNSTs).
Lab. Investig.
,
81
:
833
-844,  
2001
.
3
Fletcher C. D. M., Dal Cin P., de Wever I., Mandahl N., Mertens F., Mitelman F., Rosai J., Rydholm A., Sciot R., Tallini G., van den Berghe H., Vanni R., Willén H. Correlation between clinicopathological features and karyotype in spindle cell sarcomas.
Am. J. Pathol.
,
154
:
1841
-1847,  
1999
.
4
Koga T., Iwasaki H., Ishiguro M., Matsuzaki A., Kikuchi M. Frequent genomic imbalances in chromosome 17, 19, and 22q in peripheral nerve sheath tumours detected by comparative genomic hybridization analysis.
J. Pathol.
,
197
:
98
-107,  
2002
.
5
Plaat B. E. C., Molenaar W. M., Mastik M. F., Hoekstra H. J., te Meerman G. J., van der Berg E. Computer-assisted cytogenetic analysis of 51 malignant peripheral-nerve-sheath tumors: sporadic vs. neurofibromatosis-type-1-associated malignant schwannomas.
Int. J. Cancer
,
83
:
171
-178,  
1999
.
6
Perry A., Kunz S. N., Fuller C. E., Banerjee R., Marley E. F., Liapis H., Watson M. A., Gutmann D. H. Differential NF1, p16, and EGFR patterns by interphase cytogenetics (FISH) in malignant peripheral nerve sheath tumor (MPNST) and morphologically similar spindle cell neoplasms.
J. Neuropathol. Exp. Neurol.
,
61
:
702
-709,  
2002
.
7
Berner J. M., Sorlie T., Mertens F., Henriksen J., Sæter G., Mandahl N., Brogger A., Myklebost O., Lothe R. A. Chromosome band 9p21 is frequently altered in malignant peripheral nerve sheath tumors: studies of CDKN2A and other genes of the pRB pathway.
Genes Chromosome Cancer
,
26
:
151
-160,  
1999
.
8
Quelle D. E., Zindy F., Ashmun R. A., Sherr C. J. Alternative reading frame of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest.
Cell
,
83
:
993
-1000,  
1995
.
9
Nielsen G. P., Stemmer-Rachamimov A. O., Ino Y., Moller M. B., Rosenberg A. E., Louis D. N. Malignant transformation of neurofibromas in neurofibromatosis 1 is associated with CDKN2A/p16 inactivation.
Am. J. Pathol.
,
155
:
1879
-1884,  
1999
.
10
Kourea H. P., Orlow I., Scheithauer B. W., Cordon-Cardo C., Woodruff J. M. Deletions of the INK4A gene occur in malignant peripheral nerve sheath tumors but not in neurofibromas.
Am. J. Pathol.
,
155
:
1855
-1860,  
1999
.
11
Serrano M., Hannon G. J., Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4.
Nature (Lond.)
,
16
:
704
-707,  
1993
.
12
Hannon G., Beach D. p15INK4b is a potential effector of TGF-β-induced cell cycle arrest.
Nature (Lond.)
,
371
:
257
-261,  
1994
.
13
Sher C. J. The Pezcoller lecture: cancer cell cycles revisited.
Cancer Res.
,
60
:
3689
-3695,  
2000
.
14
Kamijo T., Weber J. D., Zambetti G., Zindy F., Roussel M. F., Sherr C. J. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2.
Proc. Natl. Acad. Sci. USA
,
95
:
8292
-8297,  
1998
.
15
Weber J. D., Taylor L. J., Roussel M. F., Sherr C. J., Bar-Sagi D. Nucleolar Arf sequesters Mdm2 and activates p53.
Nat. Cell Biol.
,
1
:
20
-26,  
1999
.
16
Tao W., Levine A. J. p19ARF stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2.
Proc. Natl. Acad. Sci. USA
,
96
:
6937
-6941,  
1999
.
17
National Institute of Health. Neurofibromatosis. Conference statement. National Institutes of Health Consensus Development Conference.
Arch. Neurol.
,
45
:
575
-578,  
1998
.
18
Tamborini E., Agus V., Perrone F., Papini D., Romano’ R., Pasini B., Gronchi A., Colecchia M., Rosai J., Pierotti M. A., Pilotti S. Lack of SYT-SSX fusion transcripts in malignant peripheral nerve sheath tumors on RT-PCR analysis of 34 archival cases.
Lab. Investig.
,
82
:
609
-618,  
2002
.
19
Lawn R. M., Efstratiadis A., O’Connell C., Maniatis T. The nucleotide sequence of the Human β-globin gene.
Cell
,
21
:
647
-651,  
1980
.
20
Herman J. G., Graff J. R., Myohanen S., Barry, Nelkin D., Baylin S. B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.
Proc. Natl. Acad. Sci. USA
,
93
:
9821
-9826,  
1996
.
21
Gu K., Mes-Masson A. M., Gauthier J., Saad F. Analysis of the p16 tumor suppressor gene in early-stage prostate cancer.
Mol. Carcinog.
,
21
:
164
-170,  
1998
.
22
Sauroja I., Smeds J., Vlaykova T., Kumar R., Talve L., Hahka-Kemppinen M., Punnonen K., Jansèn C. T., Hemminki K., Pyrhönen S. Analysis of G1/S checkpoint regulators in metastatic melanoma.
Genes Chromosomes Cancer
,
28
:
404
-414,  
2000
.
23
Lòpez-Guerrero J. A., Pellìn A., Noguera R., Carda C., Llombart-Bosch A. Molecular analysis of the 9p21 locus and p53 genes in Ewing family tumors.
Lab. Investig.
,
81
:
803
-814,  
2001
.