| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Department of Pathology,*
Department of
Medicine,
and Urology
Service,¶
Memorial Sloan-Kettering Cancer Center, New
York, New York and the Laboratoire d'Uro-Oncologie
Expérimentale
and Groupe de Recherche en
Épidémiologie,§
Centre de Recherche
en Cancérologie de l'Université Laval,
Québec, Canada
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Strong functional and genetic evidence supports an important role for INK4A as a tumor suppressor gene in a variety of tumors. The INK4A is altered in many tumor-derived cell lines and primary tumors, including bladder transitional cell carcinomas.13-17 In addition, germ line mutations of the INK4A gene are frequently found in patients with familial melanoma and pancreatic adenocarcinoma.18-20 More recently, independent studies reported the targeted deletion of the Ink4a and the specific p19ARF exon 1ß loci in murine models.21,22 Both Ink4a- and p19ARF-deficient mice were viable but developed spontaneous tumors at an early age.21,22
We have previously reported that deletions of INK4A and INK4B genes are found frequently in bladder tumors.15 The present study was conducted to further determine the frequency and potential clinical significance of detecting INK4A and INK4B mutations in superficial bladder cancer. We also assessed potential alterations affecting INK4A exon 1ß. Methylation of the 5' CpG island, located in the promoter region of p16, has been reported to be a common mechanism of p16 inactivation in certain neoplasms.23,24 Therefore, we investigated the presence of de novo methylation in the promoter region of p16. We also assessed p16 phenotype in normal and tumor tissues by means of immunohistochemical assays employing a p16-specific monoclonal antibody.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The patients included in this study had newly diagnosed superficial bladder tumors and were admitted for the first transurethral resection between September, 1990 and April, 1992 in hospitals of the province of Quebec, Canada. Criteria for patient eligibility included presence of histologically confirmed Ta or T1 transitional cell carcinoma, as previously described.25 The deletion and mutational status of the INK4A and INK4B genes were initially assessed on a group of 64 paired normal and tumor DNA samples. The analysis of the methylation condition of the p16 promoter was then analyzed on cases with nondeleted INK4A alleles (47 of 55 available cases), and tissue sections from 58 cases were also available for immunohistochemical assays. We then analyzed INK4A gene losses on 57 additional cases of paired normal and tumor DNA samples. The median follow-up period was 38.5 months (range, 2.1–51.7 months, n = 120). No follow-up was available in one of the cases. Ninety-six lesions were classified as superficial papillary tumors (Ta), and 23 lesions as invasive tumors into the lamina propria (T1). Thirty-eight tumors were classified as grade 1, 72 as grade 2, and 11 as grade 3. In 68 cases the tumor size was <3 cm, and in 47 cases it was >3 cm. In all cases with multiple tumors, the size of the largest tumor was the one reported. Eighty-six cases had single tumors and 35 cases presented multiple tumors. Samples were embedded in a cryopreservative solution (OCT Compound, Miles Laboratories, Elkhart, IN), snap-frozen in isopentane 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 >90% neoplastic cells were included in the study. Normal DNA samples from peripheral blood were obtained for all patients.
DNA Extraction
Tumor and normal DNA was extracted by methods previously described.26 Briefly, tumor DNA was extracted by proteinase K digestion from consecutive 20-µm frozen tissue sections or cell suspension. For the normal DNA, 200 µl of blood were resuspended with 20 mmol/L Tris, 5 mmol/L EDTA (pH 8.0), and NP40 0.5%, vortexed, and incubated at room temperature for 20 minutes. After centrifuging, the pellet was resuspended with 10 µl of sodium dodecyl sulfate (10%) and 10 µl of proteinase K (2.5 mg/ml) and incubated at 65°C for 2 hours. The DNA was precipitated with 100 µl NH4Ac 7.5 mol/L and 500 µl ethanol (100%) at -20°C, and resuspended in 20 mmol/L Tris and 1 mmol/L EDTA (pH 8.0).
Polymerase Chain Reaction-Single Stranded Conformation Polymorphism (PCR-SSCP) and Multiplex PCR Analyses
PCR-SSCP assays were performed in a subset of the tumors following
protocols previously described.15
The primer sets used to
amplify exons 1
(one fragment) and 2 (three overlapping fragments)
of the INK4A gene were published by Hussussian et al.18
For the exon 1ß of the INK4A gene and the INK4B gene, the following
primer pairs were used: 1) INK4A, exon 1ß (fragment 1, 439 bp): 5'
TCC CAG TCT GCA GTT AAG G 3' (forward) and 5' GTC TAA GTC GTT
GTA ACC CG 3' (reverse); 2) INK4A, exon 1ß (fragment 2, 160 bp): 5'
AAC ATG GTG CGC AGG TTC 3' (forward) and 5' AGT AGC ATC AGC ACG AGG G
3' (reverse); 3) INK4B, exon 1: 5' ATT ATC CGG GCC GCT GCG
C 3` (forward) and 5' TGC CGG CGA GGC CCT GG 3' (reverse); 4)
INK4B, exon 2 set A: 5' CCC GGC CGG CAT CTC CCA TA 3' (forward) and 5'
ACC ACC AGC GTG TCC AGG AA 3' (reverse); 5) INK4B, exon 2 set B: 5' CAC
CCG ACC GGT GCA TGA TG 3' (forward) and 5' GTG GGC GGC TGG GGA ACC TG
3' (reverse). DNA was amplified with 28 cycles of PCR using a thermal
cycler (Perkin-Elmer Cetus, Foster City, CA). Briefly, the 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, Boston, MA) or
[33P]dCTP (Amersham, Arlington Heights, IL),
3 mmol/L MgCl2, 100 µmol/L deoxynucleoside
triphosphates, 3% DMSO, 0.6 U of TaqI polymerase, and 1x
PCR buffer (Promega, Madison, WI). Annealing temperatures ranged from
55–65°C. 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. In general, 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. In those cases in which
mobility shifts were identified by PCR-SSCP, DNA was amplified and
sequenced with the Sequenase PCR product sequencing kit (Amersham,
Cleveland, OH).
An independent DNA amplification was performed for the comparative
multiplex PCR assay.27
A simultaneous amplification of
genomic DNA was performed using two sets of primers, one to the target
gene sequence under study and the other to an internal control gene
sequence. The following sets of primers were used as internal controls
for DNA quality and loading: 1) GADPH gene: 5' TGG TAT CGT GGA AGG ACT
CAT GAC 3' (forward) and 5' ATG CCA GTG AGC TTC CCG TTC AGC 3'
(reverse) (PCR product 189 bp); 2) ANDRR gene: 5' GTG CGC GAA GTG ATC
CAG AA 3' (forward) and 5' TCT GGG ACG CAA CCT CTC TC 3' (PCR product
296 bp) (reverse); 3) D9S196 (260 bp): Genome Data Base
Accession ID 62901. Each PCR reaction tube contained 50–100 ng of
genomic DNA, 1x PCR buffer (Promega), 3.2 mmol/L
MgCl2, 130 µmol/L dNTP, 5% DMSO, 0.4 µmol/L
of each target and control set of primers, 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 55–63°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 (Bas 1000-Mac, Bio Imaging System Fujix, Fuji). The
presence of INK4A or INK4B specific fragments was expressed as the
ratio [target-band signal]/[control-band signal].27
For the INK4A-exon 1ß, samples were also analyzed using primers
directed to a smaller exon 1ß fragment (fragment 2, see above) to
rule out false negative results due to partial DNA degradation. To
establish potential allelic losses in the tumor DNA samples, tumor DNA
samples lacking INK4A (p16) and INK4B genes by Southern blot analysis
were used as control DNA, 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 target content, varying from 0% (tumor
sample control) to 100% (normal DNA counterpart). Samples presenting
<25% of the control signal were considered homozygously deleted, and
those presenting <65% as hemizygously deleted for the
INK4A/B-specific fragments.
Antibodies and Immunohistochemistry
An avidin-biotin immunoperoxidase assay was performed on formalin-fixed, paraffin-embedded tissue sections. Deparaffinized sections were treated with 1% H2O2 to block endogenous peroxidase activity. Sections were subsequently immersed in boiling 0.01% citric acid (pH 6.0) in a microwave oven for 15 minutes to enhance antigen retrieval, allowed to cool, and incubated with 10% normal horse serum (Organon Teknika, West Chester, PA), to block nonspecific tissue immunoreactivities. A well characterized antibody to p16 (Ab-1, Oncogene Research Products, Cambridge, MA; 2 µg/ml final concentration) was then incubated overnight at 4°C. Biotinylated horse anti-mouse IgG antibodies (Vector Laboratories, Burlingame, CA, 1:500 final dilution) were used as the secondary reagent. This was followed by avidin-biotin immunoperoxidase complexes (Vector Laboratories, 1:25) for 30 minutes. Diaminobenzidine (Sigma Chemical, St. Louis, MO) was used as the final chromogen and hematoxylin was used as the nuclear counterstain. Sections were counterstained with hematoxylin. An esophageal adenocarcinoma case overexpressing p16 was always included as a positive control. Immunoreactivities were classified as a continuum of data (undetectable levels or 0% to 100% homogenous staining).
Analysis of Methylation
The methylation status of the 5' CpG island in the promoter region of the p16 gene was determined with the CpG Wiz p16 Methylation kit (Oncor, Gaithersburg, MD), based on the method developed by Herman et al,28 and following manufacturer's protocol. Briefly, 0.5–1 µg of DNA was denatured with 3 mol/L of sodium hydroxide at 50°C for 10 minutes and then treated with sodium bisulfite. After completion of the DNA modification, the DNA was purified by precipitation. The dissolved DNA was amplified by PCR using primers specific for the methylated (M) or unmethylated (U) sequences. Two to 3 µl of template (corresponding to treated DNA, positive control for methylated DNA, positive control for unmethylated sequences, and distilled water as negative control) were amplified in presence of 1x Universal PCR buffer, 2.5 mmol/L dNTP mix, U or M primers, and AmpliTaq Gold (Perkin-Elmer), under the following conditions: preheating at 95°C for 12 minutes, followed by 35 cycles (95°C for 45 seconds, 66°C for 45 seconds, and 72°C for 1 minute). The PCR product was analyzed on a 2% agarose gel. The DNA methylation was determined by the presence of a 145-bp fragment in those samples amplified with the M primers.
Statistical Methods
Statistical analysis was performed to evaluate possible
associations between INK4A/B deletions, prognostic variables measured
at diagnosis, and disease recurrence. Results of the
immunohistochemical analysis were analyzed as a continuous variable. A
two-tailed Fisher's exact test29
was used to assess the
associations between INK4A and INK4B status and clinicopathological
parameters. The latter included tumor stage (Ta versus T1),
tumor grade (G1 versus G2/G3), tumor size (
3 cm
versus >3 cm), and number of tumors (one versus
multiple). Tumor recurrence was defined as a new tumor detected during
follow-up cystoscopy. In the analysis of disease recurrence, patients
who recurred were classified as failures, whereas patients who did not
or were lost to follow-up during the study period were coded as
censored. The follow-up time was calculated as the time elapsed between
diagnosis by (transurethral resection) and first recurrence, or
the date of the last cytoscopic evaluation. Differences in
recurrence-free survival between the two groups were evaluated with
survival curves using the Kaplan-Meier method30
and the
log rank test.31
All statistical procedures were performed
using SAS software.32
All reported P values are
two-sided. Proportional hazards analysis was used to obtain maximum
likelihood estimates of relative risks (RR) and their 95% confidence
intervals in a multivariate analysis.33,34
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
summarize the clinicopathological
parameters at the time of diagnosis, correlating these data with the
status of the INK4A gene, as well as the results from the univariate
and multivariate analyses. Figures 1, 2, and 3
illustrate alterations occurring at the INK4A and INK4B genes, as
detected by the comparative multiplex PCR, PCR-SSCP and sequencing,
methylation-specific PCR, and immunohistochemistry.
|
|
|
|
|
. Among the 9 homozygously
deleted cases available for further analysis of INK4A exon 1ß and
INK4B gene, we found that all cases had a concomitant deletion of these
loci (Figure 1)
. Among the INK4A heterozygous cases, one case presented
simultaneous deletion of the INK4B gene, two cases presented no
additional alterations, and in one case the status of the INK4B gene
could not be evaluated. In addition, one case showed deletion of the
INK4A exon1ß with no involvement of the surrounding loci (data not
shown).
Two of 64 (3.1%) cases evaluated by PCR-SSCP showed extra bands or
mobility band shifts in tumor DNA. Direct sequencing of normal and
shifted bands demonstrated point mutations that affected both reading
frames (p16 and p19ARF) of the INK4A gene. In one
case, the change consisted of a transition, GAT
AAT (AspAsn),
that affected codon 108 of the p16. In the p19ARF
this mutation involved codon 121, CGA CAA (Arg Gln) (data not
shown). The second mutation was also a transition, GTG ATG (Val
Met), that affected codon 59 of the p16 (Case 59, Figure 2
). In the
p19ARF this nucleotide change affected codon 72,
AGA AAT (Ser Asn). This case also displayed a deletion of the
contralateral INK4A allele. In addition, mobility shifts were detected
in INK4A gene in both normal and tumor DNA in 4 cases corresponding to
a polymorphism at codon 148 (AlaThr). In 11 cases we observed a
transversion (C A) at nucleotide (-27) of the intron 1 of the INK4B
gene, corresponding also to a polymorphism at that site. In cases where
no signal was obtained after amplification by PCR, homozygous deletions
of the INK4A and the INK4B genes were confirmed by comparative
multiplex PCR.
Samples presenting one or more alleles of the INK4A gene were analyzed
for the methylation status of the 5'CpG island of p16. Methylated and
unmethylated control DNAs showed the corresponding fragment (Figure 3A)
. Seven of 47 (14.8%) informative tumors revealed the presence of a
methylation-specific fragment (Figure 3B)
. In cases without the
methylation-specific band, an unmethylated fragment was present,
indicating the existence of good quality bisulfite-modified DNA.
Therefore, the results were informative and the absence of methylated
bands reflected absence of methylation in the p16 promoter.
Immunohistochemical evaluation of p16 expression was performed
recording the estimated percentage of tumor cells that showed nuclear
staining as continuum data. The immunohistochemical analysis was done
in a blinded fashion, without knowledge of the molecular data or
clinicopathological information. Normal urothelium did not show
detectable nuclear or cytoplasmic p16 expression (Figure 1C)
. However,
different patterns were observed in tumors (Figures 1C and 3C)
.
Undetectable p16 levels (0% immunoreactive tumor cells) were observed
in 24 cases; 1 to 20%, in 14 cases; 21 to 70%, in 15 cases; and 71 to
100%, in 3 cases. None of the cases with homozygous INK4A deletions
had detectable p16 expression (Figure 1C)
. Similarly, those cases
that had a methylated p16 promoter did not present detectable p16
immunostaining (Figure 3C)
. However, certain unmethylated cases
revealed p16 nuclear overexpression (Figure 3C)
.
Overall, 19 of 55 (34.6%) cases in which the deletion, point mutation,
and methylation analyses were informative presented one or more
alterations in the INK4A and INK4B genes. Among the tumors with
homozygous deletions of the INK4A gene, 11 of 13 (84.6%) cases
corresponded to Ta lesions, while the 2 (15.4%) remaining cases were
T1 lesions. A significant correlation between homozygous deletions of
the INK4A gene and certain clinicopathological parameters was observed.
Tumors with INK4A deletions had larger diameters: 10 of 47 (21.3%)
tumors >3 cm versus 2 of 68 (2.9%) tumors 3 cm
(P = 0.003). In addition, 12 of 13 INK4A
deleted cases corresponded to high grade tumors and, although this
association was statistically non-significant (P
= 0.061), it revealed a trend.
In the prospective analysis, patients with homozygous deletion of the
INK4A gene had greater risk of recurrence than those with normal
genotype (RR = 1.95, 95% CI = 1.02–3.66) (Table 2)
. The
comparison of the recurrence-free survival data of patients with normal
or deleted INK4A alleles is presented in Figure 4
. The median time to survival was 7.1
months for the group of patients without INK4A deletions and 6.4 months
for the group of patients with deletions. Tumor size and multiplicity
were the only additional prognostic variables that affected the risk of
tumor recurrence. In the multivariate analysis of risk factors by the
proportional hazards model, the relative risks for recurrence were 1.6
(95% CI: 0.77–3.26, P = 0.216) for INK4A deletions
and 1.9 (95% CI: 1.13–3.02, P = 0.014) for the number
of tumors, after controlling for stage, grade, and tumor size. The
number of patients in this analysis was 112, due to missing values for
certain prognostic variables in nine cases. No correlation was found
between INK4A deletions and number of tumors. No associations were
observed between alterations of the INK4A, INK4B gene, and the
clinicopathological parameters at the time of diagnosis (stage, grade,
number of tumors, diameter) or recurrence. Only two cases presented
disease progression during the follow-up period: one at 35.9 months and
another at 39.5 months.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
or exon 1ß, two
proteins involved in cell cycle arrest: p16 and
p19ARF.7-10
The present study was
undertaken to analyze the status of INK4A and INK4B genes in a cohort
of 121 patients first diagnosed with superficial bladder tumors. We found deletions in 17 of 121 (14.1%) cases, most corresponding to homozygous deletions of the INK4A gene (n = 13). These 13 cases were also deleted at the telomeric exon 1ß and INK4B loci. Among the four cases with heterozygous INK4A deletions, only one case showed LOH of INK4B gene. In this study we identified only two cases in which the INK4A gene had tumor-specific point mutations. This low frequency of point mutations has also been found in other studies.14,15,35 It is of interest that in one case (case 59) the inactivation of the INK4A gene followed the "two hit" model, displaying deletion of one allele and a point mutation of the contralateral allele.36
In addition to tumor-specific mutations, p16 can be inactivated by
de novo methylation of the CpG dinucleotides located in the
promoter region.23,24
We found de novo
methylation in 14.8% of the evaluable cases. The percentage of
cases with this alteration is lower than that reported by other
investigators.24
This difference in results could be due
to several factors. Certain previous studies reported using
methylation-sensitive restriction enzymes. Although this method is
simple, an incomplete digestion can be interpreted as a false positive
result. The method used in the present study is more precise, because
it is based on the amplification of methylated or unmethylated DNA
sequences. In addition, it is possible that advanced stage lesions
(T2-T4) have an increased methylation rate of p16. To further evaluate
the expression of p16, we conducted immunohistochemical analysis using
a monoclonal antibody specific to exon 1. Normal uroepithelial cells
revealed undetectable p16 levels. However, 12 of 24 tumors in which p16
was not detected had normal p16 alleles. The remaining 12 cases had
either homozygous deletions (n = 7) or p16
methylation (n = 5). Even though other groups
have reported that absence of positive staining reflects p16
inactivation,16
it appears that the negative phenotype in
bladder cancer may reflect the normal physiological levels. Other
studies have reported that p16 overexpression is associated with pRB
inactivation, probably reflecting accumulation of otherwise inactive
p16 products.16,37,38
The comparative analysis of INK4A alterations and clinicopathological parameters revealed that homozygous deletions were the only mutations significantly associated with poor prognosis, including outcome. Known clinicopathological parameters associated with poor outcome include detection of multiple tumors and tumors presenting with large diameter. Specifically, homozygous deletions of the INK4A gene were significantly associated with tumors displaying large diameter (>3 cm). In this setting, loss of functional p16 appears to participate in an unrestricted cell growth. In addition, we observed a significant association between INK4A deletions and a greater risk of recurrence. After controlling for tumor stage, tumor grade, tumor size, and number of tumors, the risk of recurrence among patients with INK4A deletions was 60% higher than that of patients without INK4A deletions (RR = 1.58, 95% CI: 0.77–3.26).
An unusual genetic complexity is found on 9p21, where the INK4A and the INK4B genes encode three proteins involved in cell cycle arrest: p16, p15, and p19ARF.3-10 The p16 and p15 proteins form binary complexes exclusively with Cdk4 and Cdk6, inhibiting their function and, by doing so, inhibiting pRB phosphorylation during G1.3-6,39 It has been recently reported that p19ARF interacts with mdm2 and blocks the mdm2-induced p53 degradation and transactivational silencing.11,12 Thus, the INK4A gene encodes two products impacting on the two most critical tumor suppressor pathways controlling neoplasia, p16 through pRB and p19ARF preventing neutralization of p53 by mdm2.11,12,22 The pRB pathway could be linearly depicted as p16/CycD1/Cdk4/pRb/E2F1, and it appears to be involved in most tumors.35 The p53 pathway includes p19ARF/mdm2/p53/p21-bax, and it has been also documented to be altered in most neoplasms.40-42 The mechanistic basis for the inactivation of both pathways during cellular transformation stems, in part, from the deactivation of a p53-dependent cell suicide program that would normally be brought about as a response to unchecked cellular proliferation resulting from pRB deficiency. This cooperative effect of pRB and p53 has been documented in cultured cells as well as primary tumors.43-45 Homozygous deletions of the INK4A gene disrupt these pathways and, even though not functionally equivalent to direct inactivation of p53 and pRB, could generate tumors with an aggressive behavior. Data from this study support this hypothesis, because patients with superficial bladder tumors harboring homozygous deletions of the INK4A gene had a lower recurrence-free survival rate. It is of interest that other INK4A alterations, such as point mutations or de novo methylation, did not impact on clinical outcome. Although homozygous deletions of the INK4A completely inactivate both p16 and p19ARF proteins, other mutations or altered expression probably have an impact on a single protein product. Similarly, INK4B deletions do not seem to play a key role in the biology of the disease. However, based on functional differences, such as the activation by TGFß of p15 but not p16,6 codeletion of INK4A and INK4B genes could provide an additional selective growth.
In conclusion, the present study reports that de novo methylation of the p16 promoter region and deletions of the INK4A/B genes are common alterations in superficial bladder tumors. Whereas point mutations and methylation affect one protein product, the complete allelic loss of the INK4A includes both p16 and p19ARF, affecting the pRb and p53 suppressor pathways. The significant association of homozygous INK4A gene deletions with tumors of large size, as well as increased risk of disease recurrence, suggests that they may provide prognostic molecular parameters for the evaluation of patients affected with superficial bladder neoplasms.
|
Footnotes |
|---|
Supported in part by National Cancer Institute grants NCI-CA-47538 (to C. C. C.) and NCI-CA-47526 (to Y. F.) as part of the Cooperative Network for Evaluation of Markers of Urinary Bladder Cancer.
Accepted for publication March 20, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  W.-J. Kim, E.-J. Kim, P. Jeong, C. Quan, J. Kim, Q.-L. Li, J.-O. Yang, Y. Ito, and S.-C. Bae RUNX3 Inactivation by Point Mutations and Aberrant DNA Methylation in Bladder Tumors Cancer Res., October 15, 2005; 65(20): 9347 - 9354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Chapman, P. Harnden, P. Chambers, C. Johnston, and M. A. Knowles Comprehensive Analysis of CDKN2A Status in Microdissected Urothelial Cell Carcinoma Reveals Potential Haploinsufficiency, a High Frequency of Homozygous Co-deletion and Associations with Clinical Phenotype Clin. Cancer Res., August 15, 2005; 11(16): 5740 - 5747. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Aveyard and M. A. Knowles Measurement of Relative Copy Number of CDKN2A/ARF and CDKN2B in Bladder Cancer by Real-Time Quantitative PCR and Multiplex Ligation-Dependent Probe Amplification J. Mol. Diagn., November 1, 2004; 6(4): 356 - 365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Berggren, R. Kumar, S. Sakano, L. Hemminki, T. Wada, G. Steineck, J. Adolfsson, P. Larsson, U. Norming, H. Wijkstrom, et al. Detecting Homozygous Deletions in the CDKN2A(p16INK4a)/ARF(p14ARF) Gene in Urinary Bladder Cancer Using Real-Time Quantitative PCR Clin. Cancer Res., January 1, 2003; 9(1): 235 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-F. Hwang, C.-L. Cho, C.-C. Huang, J.-S. Wang, Y.-L. Shih, C.-Y. Su, and H.-W. Chang Loss of cyclin D1 and p16 expression correlates with local recurrence in nasopharyngeal carcinoma following radiotherapy Ann. Onc., August 1, 2002; 13(8): 1246 - 1251. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Tada, M. Wada, K.-i. Taguchi, Y. Mochida, N. Kinugawa, M. Tsuneyoshi, S. Naito, and M. Kuwano The Association of Death-associated Protein Kinase Hypermethylation with Early Recurrence in Superficial Bladder Cancers Cancer Res., July 15, 2002; 62(14): 4048 - 4053. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Dominguez, J. Carballido, J. Silva, J. M. Silva, J. M. Garcia, J. Menendez, M. Provencio, P. Espana, and F. Bonilla p14ARF Promoter Hypermethylation in Plasma DNA as an Indicator of Disease Recurrence in Bladder Cancer Patients Clin. Cancer Res., April 1, 2002; 8(4): 980 - 985. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Konishi, M. Nakamura, M. Kishi, M. Nishimine, E. Ishida, and K. Shimada Heterogeneous Methylation and Deletion Patterns of the INK4a/ARF Locus Within Prostate Carcinomas Am. J. Pathol., April 1, 2002; 160(4): 1207 - 1214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W. Y. Chan, L. W. Chan, N. L. S. Tang, J. H. M. Tong, K. W. Lo, T. L. Lee, H. Y. Cheung, W. S. Wong, P. S. F. Chan, F. M. M. Lai, et al. Hypermethylation of Multiple Genes in Tumor Tissues and Voided Urine in Urinary Bladder Cancer Patients Clin. Cancer Res., February 1, 2002; 8(2): 464 - 470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Maruyama, S. Toyooka, K. O. Toyooka, K. Harada, A. K. Virmani, S. Zochbauer-Muller, A. J. Farinas, F. Vakar-Lopez, J. D. Minna, A. Sagalowsky, et al. Aberrant Promoter Methylation Profile of Bladder Cancer and Its Relationship to Clinicopathological Features Cancer Res., December 1, 2001; 61(24): 8659 - 8663. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Orlow, P. Roy, A. Barz, R. Canchola, Y. Song, and M. Berwick Validation of Denaturing High Performance Liquid Chromatography as a Rapid Detection Method for the Identification of Human INK4A Gene Mutations J. Mol. Diagn., November 1, 2001; 3(4): 158 - 163. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
