Affiliations of authors: I. Orlow, M. Drobnjak, J. M. Woodruff, C. Cordon-Cardo (Department of Pathology), J. Lewis, M. F. Brennan (Department of Surgery), Memorial Sloan-Kettering Cancer Center, New York, NY; Z.-F. Zhang, Department of Epidemiology, School of Public Health, University of California, Los Angeles.
Correspondence to: Carlos Cordon-Cardo, M.D., Ph.D., Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021 (e-mail: cordon-c{at}mskcc.org).
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ABSTRACT |
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INTRODUCTION |
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Soft tissue sarcomas have been classified histogenetically according to their morphologic resemblance to normal cognate tissues. The prognosis of patients with soft tissue sarcomas has traditionally been determined by clinical and histopathologic features of the tumor such as grade, size, and depth. The histologic type of a patient's sarcoma has not been shown, however, to be a consistent independent prognostic factor. Recent advances in the recognition and understanding of molecular genetic events occurring in the pathogenesis of human sarcomas have resulted in attempts to define new diagnostic and prognostic markers. Several sarcomas have been found to be characterized by recurrent chromosomal aberrations, mainly translocations, that are specific to particular histologic types. One example is the reciprocal translocation t(9;22) (q22;q12) that is reported in most extraskeletal myxoid chondrosarcomas (23). Deletions in 9p12-p22 occur nonrandomly in some soft tissue tumors (24). The INK4A and INK4B genes have been found to be mutated in many tumor cell lines and several primary tumors (13,25-29). In addition, methylation of the 5' CpG island located in the promoter region of the p16 gene has been reported to be a frequent mechanism of that gene's inactivation in certain neoplasms (30,31). More recently, independent studies reported the targeted deletion of the Ink4a and specific loci within p19ARF exon 1ß in murine models (32,33). Both Ink4a- and p19ARF-deficient mice were viable, but they developed spontaneous tumors at an early age. Soft tissue sarcomas were one of the most common tumor types observed in these knockout mice. The evidence that INK4A encodes two products that have an impact on different tumor suppressorsp16 acting through pRB and p19ARF preventing Mdm2 neutralization of p53positions this gene at the nexus of the two most critical tumor suppressor pathways controlling neoplasia (21-33).
The present study was undertaken to better delineate the frequency and potential clinical relevance of INK4A and INK4B gene alterations in a well-characterized cohort of adult soft tissue sarcomas. We have also evaluated alterations of pRB, p53, and Mdm2 in the same cohort of sarcomas and analyzed their association with INK4A and INK4B mutations.
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MATERIALS AND METHODS |
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Tumors from 46 patients with adult soft tissue sarcomas were analyzed. The tumor specimens corresponded to 12 liposarcomas, 11 leiomyosarcomas, seven malignant fibrous histiocytomas, five malignant peripheral nerve sheath tumors, four fibrosarcomas, four synovial sarcomas, one rhabdomyosarcoma, one desmoid tumor, and one unclassified sarcoma. Twenty-six tumors were confined to the extremities (19 primary, four recurrent, and three metastatic), four tumors were retroperitoneal (one primary and three recurrent), and 16 tumors were localized in other sites including the lung, liver, stomach, rectum, small bowel, prostate, and pelvis (seven primary, one recurrent, and eight metastatic). Ten tumors were classified as low grade; 36 were classified as high grade. In nine cases, the tumor size was less than or equal to 5 cm; in 37 cases, it was greater than 5 cm. Samples were embedded in cryopreservation compound O.C.T. (i.e., optimal cutting temperature compound; Miles Laboratories, Elkhart, IN), snap-frozen in isopentane that had been precooled in liquid nitrogen, and stored at -70 °C. Representative hematoxylin-eosin-stained sections of each frozen block were examined microscopically to confirm the presence of tumor, and only lesions with more than 50% neoplastic cells were included in the study. Normal tissues were obtained from all patients, either from a tumor-free area, such as skeletal muscle, or from peripheral blood.
Southern Blotting Analysis
A 0.5-kb complementary DNA (cDNA) fragment containing human p16
sequences and a 2-kb cDNA fragment containing human p15 sequences were
used as probes to assess deletion and rearrangement of the INK4A and
INK4B genes, respectively. A cDNA fragment containing glyceraldehyde
phosphate dehydrogenase (GAPDH) sequences was used as a control. In
general, Southern blot analysis was performed as described previously
(26). Briefly, DNA was extracted by use of a non-organic
method (Oncor, Inc., Gaithersburg, MD) from paired normal tissue and
tumor samples. Extracted DNA (7.5-µg aliquots) was digested with
TaqI restriction endonuclease, and the digested DNA was
subjected to electrophoresis in 0.7% agarose gels and blotted onto
nylon membranes. The membranes were prehybridized with Hybrisol I
(Oncor, Inc.) at 42 °C for 1 hour and then incubated
overnight at 42-43 °C with probes labeled to high specific
activity with the use of [-32P]deoxycytidine
triphosphate (dCTP) (Dupont NEN Research Products, Boston, MA).
Hybridized membranes were washed at high stringency with 0.1x
standard saline citrate-0.1% sodium dodecyl sulfate at
-70 °C and autoradiographed with the use of intensifying
screens at -70 °C for 24-72 hours. Densitometric
evaluation of autoradiographic band intensities was performed by use of
an Ultrascan XL Laser Densitometer (Pharmacia LKB Biotechnology,
Piscataway, NJ). Relative amounts of a given CKI gene present were
determined by the comparison of gene-specific hybridization signals
with those obtained with the use of the control probe (26).
In addition, a human MDM2 cDNA fragment probe of 1.6 kb, pHDM (EcoRI), was used in Southern blots to assess gene amplification, in conjunction with a ß-actin probe (EcoRI) used as control, following protocols previously described (34).
Polymerase Chain Reaction-Single-Strand Conformation Polymorphism and Multiplex Polymerase Chain Reaction Analyses
Polymerase chain reaction-single-strand conformation polymorphism
(PCR-SSCP) assays were performed in a subset of the tumors following
protocols described previously (26) (see below for
details). The primers used to amplify the INK4A and INK4B genes can
be summarized as follows: 1) p16, exon 15' GGG AGC AGC ATG
GAG CCG 3' (F) and 5' AGT CGC CCG CCA TCC CCT (R); 2) p16, exon
25' GGA AAT TGG AAA CTG GAA GC 3' (42F), 5' TCT
GAG CTT
TGG AAG CTC T 3' (551R), and 5' GGA TAG AGA ACT CAA GAA GG
3' (35F); 3) p19ARF, exon 1ß (fragment 1, 439
bp)5' TCC CAG TCT GCA GTT AAG G 3' (F) and 5' GTC
TAA GTC
GTT GTA ACC CG 3' (R); 4) p19ARF, exon 1ß (fragment
2, 160 bp)5' AAC ATG GTG CGC AGG TTC 3' (F) and 5'
AGT
AGC ATC AGC ACG AGG G 3' (R); 5) p15, exon 15' AAG AGT GTC
GTT AAG TTT ACG 3' (51F) and 5' ACA TCG GCG ATC TAG GTT CCA
3' (32F); and 6) p15, exon 25' GGC CGG CAT CTC CCA TAC CTG
3' (41F) and 5' TGT GGG CGG CTG GGG AAC CTG 3' (365R). DNA
was amplified in 30 cycles of PCR by use of a thermal cycler (The
Perkin-Elmer Corp., Foster City, CA) and conditions that have been
described previously (26). Briefly, these PCR reactions were
performed in 10-µL volumes containing 80-100 ng of template DNA,
2.2 µCi of [
-32P]dCTP (Dupont NEN Research
Products) or [
-33P]dCTP (Amersham Life Science Inc.,
Arlington Heights, IL), 3 mM MgCl2, 100
µM deoxynucleotide triphosphates (dNTPs), 3% dimethyl
sulfoxide (DMSO), 0.6 U of TaqI polymerase, and 1x PCR
buffer (Promega, Madison, WI). The annealing temperatures ranged from
55 °C to 68 °C. For SSCP analysis of exon 2 of
the INK4A, 4 µL of a PCR product was digested with the restriction
enzyme BanII. For the SSCP analysis of the exon 1ß
(fragment 1) of the p19ARF, the PCR product was digested with
NarI and EheI restriction enzymes for 2 hours at
37 °C. Both digested and nondigested products were analyzed
by SSCP. The PCR products were denatured and loaded onto a
nondenaturing 8% polyacrylamide gel containing 10% glycerol
and subjected to electrophoresis at room temperature for 12-16 hours
at 10-12 W. After electrophoresis, the gels were dried and exposed to
x-ray film at -70 °C for 4-16 hours.
An independent DNA amplification was performed for the comparative
multiplex PCR assay. A simultaneous amplification of genomic DNA was
performed by use of two sets of primers, one to the target gene
sequence under study and the other to an internal control gene
sequence. The GAPDH and the androgen receptor (ANDRR) genes were
utilized as internal controls for DNA quality and loading. The primers
used for the GAPDH and ANDRR genes can be summarized as follows: 1)
GAPDH5' TGG TAT CGT GGA AGG ACT CAT GAC 3' (F) and
5' ATG CCA GTG AGC TTC CCG TTC AGC 3' (R) (fragment: 189 bp); 2)
ANDRR5' GTG CGC GAA GTG ATC CAG AA 3' (F) and 5'
TCT GGG ACG CAA CCT CTC TC 3' (fragment: 296 bp). The concentration and the
quality of each DNA template were determined by densitometry and by
comparison with mass markers on an agarose gel (Life Technologies, Inc.
[GIBCO BRL], Gaithersburg, MD). Each PCR reaction tube contained
50-100 ng of genomic DNA, 1x PCR buffer (Promega), 3.2 mM
MgCl2, 130 µM dNTPs, 5% DMSO, 0.4
µM of INK4A exon 1ß primer, 0.4 µM of
each ANDRR primer, 0.5 U Taq polymerase (Promega), and 1
µCi of [-33P]dCTP. Samples were preheated at
95 °C for 5 minutes and amplified for 25 cycles with
annealing temperatures ranging from 59 °C to
53 °C, followed by an extension at 72 °C for 10
minutes. PCR products were run in nondenaturing 8% acrylamide gels
at 40-45 W for 3-4 hours. Gels were dried and exposed to sensitive
film and to a phosphoimage plate. The sensitized plate was scanned by a
phosphoimager (Bac 1000-Mac; Bio Imaging System Fujix, Fuji, Japan).
The presence of the exon 1ß was expressed as the following ratio:
(target-band signal)/(control-band signal). All experiments were
conducted at least twice, and samples were analyzed by use of primers
directed to a smaller exon 1ß fragment (fragment 2; see
above) to rule out false-negative results due to partial DNA
degradation. For the establishment of potential p19ARF
allelic losses in the tumor DNA samples, tumor DNA samples lacking p16
and p15 genes by Southern blot analysis were used as control DNAs,
validating the quantitative nature of the multiplex PCR method.
Briefly, varying mixtures of tumor DNA and normal genomic DNA were
coamplified. These tumor-to-normal DNA mixtures represented a range of
the p19ARF exon 1ß content, varying from 0% of target
(tumor-sample control) to 100% of target (normal DNA counterpart).
Samples presenting less than 10% of the control signal were
considered to be homozygously deleted, and those presenting less than
60% were considered to be heterozygously deleted for exon 1ß.
In addition, a TP53 mutation analysis of exons 4-8 was conducted by PCR-SSCP and sequencing, following protocols previously described (34).
Analysis of Methylation
The methylation status of the 5' CpG island in the promoter region of the p16 gene was determined with the CpG WizTM p16 Methylation Kit (Oncor, Inc.). Briefly, 0.5-1 µg of DNA was denatured with 3 M NaOH at 50 °C for 10 minutes and treated with sodium bisulfite following the manufacturer's protocol. After completion of the DNA modification, the DNA was purified by precipitation. The dissolved DNA was amplified by PCR, utilizing primers specific for the methylated (M) or unmethylated (U) sequences. A 2- to 3-µL aliquot of template (corresponding to treated DNA, positive control for methylated DNA, positive control for unmethylated sequences, and distilled water as negative control) was amplified in the presence of 10x Universal PCR Buffer, 2.5 mM dNTP mix, U or M primers, and AmpliTaq GoldTM (The Perkin-Elmer Corp.), under the following conditions: preheating (95 °C, 12 minutes), followed by 35 cycles (95 °C, 45 seconds; 66 °C, 45 second; and 72 °C, 1 minute). The PCR product was analyzed on a 2% agarose gel. The presence of DNA methylation was determined by the identification of a 145-bp fragment in those samples amplified with the M primers. All the cases were evaluated for the presence of an unmethylated specific fragment (154 bp), which served as internal control for the quality of the treated DNA.
Monoclonal Antibodies and Immunohistochemistry
Well-characterized mouse monoclonal antibodies to p53, Mdm2, and pRB proteins were used for this study. Antibody PAbl80l reacts with both wild-type and mutant p53 products, recognizing a denaturation-resistant epitope that expands between amino acids 32 to 79 (34). A mouse monoclonal antibody identifying an epitope in the central portion of Mdm2, clone 2A10, was also used in the study (34). Antibody Rb-PMG3-245 was generated by immunizing mice with the TrpE/Rb fusion protein, and it has been shown to specifically recognize the 110-kd RB gene product (35). A class-matched mouse monoclonal antibody (MIgS-Kp-1; Pharmingen, San Diego, CA) was used at the same working concentrations as the primary antibodies, serving as a negative control.
Immunohistochemistry was performed on 5-µm frozen sections by use of the avidin-biotin complex immunoperoxidase technique (34,35). Sections were incubated with appropriately diluted primary antibodies (PAbl80l200 ng/mL; 2A101 : 1000 dilution from tissue culture supernatant; Rb-PMG3-24510 µg/mL) for 1 hour at room temperature. Secondary, biotinylated horse anti-mouse antibodies (Vector Laboratories, Inc., Burlingame, CA) were used at 1 : 100 dilution, and avidin-biotin peroxidase complexes (Vector Laboratories, Inc.) were used at a dilution of 1 : 25. Diaminobenzidine was used as the final chromogen (0.06%), and hematoxylin was used as the nuclear counterstain.
Immunohistochemical evaluation was performed by scoring the estimated percentage of tumor cells that showed nuclear staining. Nuclear immunoreactivities of p53 and Mdm2 were considered positive when at least 20% of the tumor cells showed nuclear staining (34). pRB was considered undetectable when nuclear staining was observed in normal endothelial cell and/or inflammatory elements but not in tumor cells (35). The immunohistochemical analysis was done in a blinded fashion (i.e., without knowledge of the molecular data or clinical information).
Statistical Analyses
Patients were categorized according to the stage, grade, histologic
type, site, and presentation of their tumors as defined in Table
1. INK4A/INK4B deletions included those patients with
homozygous or hemizygous deletions of the INK4A and the INK4B genes.
INK4A/INK4B alterations included patients with gene deletions and
rearrangements. A cut point of 20% of tumor cells stained
(<20% staining as negative and
20% staining as
positive) was used for immunophenotypic variables, including altered
patterns of p53 and Mdm2. TP53 mutations were coded as "yes" or
"no" in the data analysis. A two-tailed Fisher's exact test
(36) was used to assess the associations between INK4A and
INK4B deletions or alterations and clinicopathologic parameters
(including tumor grade, size, site, and presentation) and to explore
the association between INK4A and INK4B alterations and other
variables, including TP53 mutations and immunophenotypic patterns. The
FREQ procedure in SAS was used (37). In the analysis of
disease-specific survival, patients who died of soft tissue sarcomas
were classified as dead of disease, whereas patients who were still
alive, had died of unrelated causes, or were lost to follow-up during
the study period were coded as censored. Survival time was defined as
the time from date of surgery to the endpoint (death or censoring).
Disease-specific survivals were evaluated with the use of the
Kaplan-Meier method (38) and logrank test (39).
The LIFETEST procedure in SAS was used for analysis of these data
(37). Proportional hazards analysis was used to obtain maximum
likelihood estimates of relative risks (RRs) and their 95%
confidence intervals (CIs) in a multivariate analysis (40,41).
The data were consistent with the assumptions of Cox proportional
hazards analysis.
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RESULTS |
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The overall frequency of alterations in this cohort of soft tissue
sarcomas was approximately 15% for the INK4A and INK4B genes, with
theses changes affecting high-grade sarcomas exclusively. Statistically
significant associations were observed between INK4A and INK4B gene
deletions and poor survival (P = .036; Fig. 3,
A) and between INK4A/INK4B gene alterations and poor
survival (P = .005; Fig. 3, B).
In a multivariate analysis
that used the Cox proportional hazards model, the calculated RRs after
controlling for tumor grade and size were 2.29 (95% CI =
0.80-6.58; P = .12) for INK4A/INK4B deletions and 2.98
(95% CI = 1.09-8.18; P = .03) for INK4A/B alterations.
None of the RRs, calculated from the proportional hazards model, for
either tumor grade or size were statistically significant (data not shown).
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DISCUSSION |
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Genotypic or phenotypic alterations of TP53 were detected in 16 cases, only two of which simultaneously displayed a homozygous deletion affecting both INK4A and INK4B genes. Alterations in the MDM2 gene and/or in Mdm2 protein expression were identified in 21 tumors. Of interest, a tumor in which both TP53 and MDM2 were altered also showed LOH at the INK4A/INK4B region, but the contralateral alleles, including INK4A exon 1ß, were found to be of the wild type. Similarly, only one tumor in which concomitant pRB and INK4A alterations were identified possessed an abnormal pattern of INK4A bands, suggestive of a rearrangement. Six tumors had p53 and pRB alterations; however, none of these cases showed mutations in either the INK4A or the INK4B genes. These results suggest that additional alterations in the p53 pathway (through changes in p19ARF) and the pRB pathway (through changes in p16 and p15) as part of tumorigenesis of sarcomas would be unnecessary and redundant.
Coordinate inactivation of p53 and pRB appears to be an essential requirement for the genesis of most human cancers (42-44). It has also been postulated that the loss of function of certain CKIs, mainly p16, might also lead to tumor development (32). Even though, at the protein sequence level, p16 and p15 share 82% homology, their activities are regulated differently. p15 is an effector of transforming growth factor-ß (TGF-ß)-induced cell cycle arrest (14). The regulatory pathway involving p16 has not yet been well characterized; however, membrane-signaling pathways, including those mediated by TGF-ß, do not appear to be involved. A report (45) has shown that p16 accumulates in cell lines devoid of pRB, which suggests that its expression may be influenced by a transcription factor modulated by pRB. Evidence suggests that the p16/CDK4/cyclin D1/RB pathway behaves as a single mutagenic target during tumorigenesis (1-3). Cell cycle arrest produced by p19ARF has been shown to be dependent on p53, because cells that lack p53 do not respond to the action of p19ARF (33). More recently, it has been reported that p19ARF interacts with Mdm2 protein and blocks the Mdm2-induced degradation and transactivational silencing of p53 (21,22).
Several studies (44,46,47) have reported that alterations of p53 and pRB are potentially synergistic on the proliferative activity of tumors and that they exert a cooperative negative effect on both progression and survival in certain human primary tumors. It is postulated that aberrant p53 and pRB expression deregulates cell cycle control at the G1 checkpoint, resulting in tumor cells with reduced response to programmed cell death. The imbalance produced by enhanced proliferative activity combined with decreased apoptosis may explain the aggressive clinical course of tumors harboring both p53 and pRB alterations. The recent evidence that INK4A encodes two products that have an impact on these two suppressor pathways, p16 through the pRB pathway and p19ARF through the p53 pathway, positions the INK4A gene at the nexus of the two most critical tumor-suppressor pathways governing neoplasia (21-33). Inactivation of p16 and p19ARF in primary tumors may not be functionally equivalent to alterations in p53 and pRB expression but still might produce aggressive biologic behavior. Data from our study confirm this prediction, because adult patients affected with high-grade soft tissue sarcomas that harbor homozygous deletions of the INK4A gene had a poor clinical outcome. It is of interest that, in the cohort of sarcomas analyzed by us, there were no point mutations or methylation changes that could silence p16 alone and consequently affect the pRB, but not the p53, pathway.
In summary, recent data (21,22,33) provide for a "one gene/two products/two pathways" hypothesis that can explain the high rate of INK4A alterations in human primary cancers, as well as in a wide variety of tumor cell lines. These data also offer an answer for the tumor-prone phenotype observed in the Ink4a (32) and p19ARF (33) knockout models, as compared with other CKIs such as p21 (48) and p27 (49-51). Our study shows that the alteration of the INK4A and INK4B genes, particularly a coincident homozygous deletion that eliminates three important cell-growth inhibitors (p16, p19ARF, and p15), appears to be a frequent event in adult soft tissue sarcomas. Inactivation of p16 and p19ARF in primary tumors may not be functionally equivalent to alterations of p53 and pRB, yet it appears to produce cancers with aggressive biologic behavior. The statistically significant reduction in survival of patients bearing tumors with INK4A and INK4B gene alterations suggests that these genes may provide prognostic molecular parameters for the evaluation of patients affected by soft tissue sarcomas.
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NOTES |
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Manuscript received July 24, 1998; revised October 19, 1998; accepted October 29, 1998.
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