| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/ULP, F-67404 Illkirch cedex, C. U. de Strasbourg [R. R., K. C., K. W., S. d. M.], and Laboratoire de Biologie Tumorale, Centre Paul Strauss, F-67085 Strasbourg cedex [D. M., J. A.], France
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Considerable efforts by allelotyping, cytogenetics, and CGH have been dedicated to pinpoint genetic aberrations associated with the natural history of HNSCC or with clinical outcome. It emerges that HNSCC karyotypes belong to the most complex in solid tumors (2 , 3) . Among the numerous recurrent sites of aberrations, chromosome 3 comes out as the most prominent (present in more than 50% of the CGH compiled cases), independent of the clinical and pathological characteristics (3) .
By LOH studies at specific sites, Califano et al. (Ref. 4 and references therein) correlated common lost loci with tumor natural history, building a genetic progression model of HNSCC. In premalignant lesions, 3p, 9p21, and 17p13 deletions were identified as early genetic events.
Whole-genome exploration of genetic imbalances at early disease was sparse (nine cases found in the literature; Refs. 5, 6, 7 ), certainly because of frequent advanced stage at the time of diagnosis. Interestingly, using CGH, Bockmühl et al. (5) reported 3q gain, 3p loss, and 9p loss in three low-grade HNSCC (G1-well differentiated).
3q gain was also shown as an early event in cervical squamous cell carcinoma development (8) . High prevalence of chromosome 3 alterations is a common theme in squamous cell carcinomas (5, 6, 7, 8, 9, 10, 11, 12, 13) , with an overlapping area of gain at 3q26-qter but variable minimal regions. These chromosomal bands certainly harbor genes, gained and overexpressed, involved in the progression of squamous cell carcinomas. Search for positional and functional candidate genes within 3q26-qter has begun and led to two outstanding proponents.
The PIK3CA gene, located at 3q26, encodes for the catalytic subunit (p110
) of a class IA PI3-K, one component of a lipid-signaling pathway involved in multiple cancer-related functions: cell survival, proliferation, cell migration, vesicle trafficking, and vesicle budding (14
, 15)
. In cervical squamous cell carcinoma cell lines as in ovarian cancer cell lines, even low-level increased PIK3CA copy number (i.e., three copies) results in higher PI3-K activity (16
, 17)
. Furthermore, in these cell lines, in contrast with normal PIK3CA copy number cells, treatment with a PI3-K inhibitor decreases proliferation and increases apoptosis. Consequently, PIK3CA was proposed for the role of oncogene in cervical and ovarian cancer, but its involvement in HNSCC with 3q26-qter gain remains to be investigated.
p63, a gene located at 3q28 and originally identified as homologous to p53, encodes for different isoforms with distinct transcriptional activities (18) . The TAp63 isoforms contain one NH2-terminal transcriptional activation domain like p53 and could act as tumor suppressor gene. The DNp63 isoforms, which are the major transcripts in tumor and normal epithelial tissues, lack this domain and are able to suppress p53 transactivation. Thus, these dominant negative p63 variants are bonafide candidates for oncogenic functions. Indeed, frequent increased p63 gene copy number was reported in squamous cell carcinomas of head and neck and of lung (19) , as well as RNA and protein overexpression of a DNp63 variant (p68AIS). A correlation between DNp63 expression level and gene copy number would support its contribution in the pathogenesis of HNSCC with 3q26-qter gain.
In this study, we examined chromosomal aberrations in well-differentiated (low-grade) tumors without lymph node involvement (N0) or distant metastasis (M0), considered as an early stage in HNSCC development. We found a specific and simple pattern of aberrations for T1-2N0M0 tumors with predominant implication of the long arm of chromosome 3. By combining genome and expression approaches, we showed that PIK3CA, in contrast to p63, may contribute, consequent to increased gene dosage, to the HNSCC pathogenesis.
Recently, 3q21-q29 gain was identified as a prognostic marker in HNSCC by CGH analysis of 113 tumors with variable grades and stages (20) . Accordingly, in our population of 21 early disease HNSCC, PIK3CA gene copy number, at 3q26, is a potential marker for clinical outcome. This remains to be evaluated in larger populations.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The cell lines Cal27 (ATCC CRL-2095), Cal33 (established at the Center Antoine Lacassagne, Nice, France), and FaDu (ATCC HTB-43) are derived from human HNSCC. Metaphase chromosomes, interphase nuclei, and DNA were prepared from these cell lines using standard protocols.
Tumor DNA Extraction and Interphase Nuclei Isolation.
One 5-µm-tissue section from each specimen, stained using an H&E standard protocol, was used to mark out the tumor. Soft tissue was removed using syringe needles under a binocular lens. For DNA extraction, 10 paraffin-embedded 10-µm sections were dissected, dewaxed in Histo-Clear (National Diagnostics, Atlanta, GA), washed in 100% ethanol, and incubated overnight in 1 ml of sodium sulfocyanate (1 M) at 37°C. Then cells were digested in a proteinase K solution (0.2 mg/ml) during 2 days at 55°C, and DNA was extracted using a phenol-chloroform standard protocol. For nuclei isolation, dissected tissue from two 40-µm sections was dewaxed and digested with pepsin [Sigma Chemical Co.; 5 mg/ml; NaCl 0.9% (pH 1.5)] for 2 h at 37°C, mixed between. The digest was filtered through a 40-µm nylon mesh. Isolated nuclei were washed in PBS and then spotted on polylysine-coated slides in PBS. Then slides were air dried at 45°C, rinsed in PBS, dehydrated, and stored at -20°C.
Comparative Genomic Hybridization.
Tumor and control DNA were labeled by nick translation with biotin-16-dUTP and digoxigenine-11-dUTP, respectively. Quality of the DNA extracted from archival material was variable. When DNA showed high fragmentation, DOP-PCR was performed, using universal primer (UN1-primer; 5'-CCG ACT CGA GNN NNN NAT GTG G-3'; for details, see Ref. 23
). CGH was done as described previously (24)
. Briefly, control and tumor DNA probes were ethanol coprecipitated in the presence of Cot-1 fraction of normal DNA (Life Technologies, Inc.) and salmon sperm DNA (Sigma Chemical Co.). They were cohybridized at 37°C to human normal metaphase chromosomes for 2 days and then detected using fluorescein conjugated to avidin and biotinylated anti-avidin antibody (Vector Laboratories) for the tumor probe and mouse anti-digoxin, rabbit antimouse conjugated to tetramethylrhodamine isothiocyanate and goat antirabbit conjugated to tetramethylrhodamine isothiocyanate antibodies (Sigma Chemical Co.) for the control probe. Chromosomes were counterstained with 4,6-diamidino-2-phenylindole. Ratio images and profiles were determined as described (24)
.
FISH.
FISH analysis was performed using BAC or YAC probes. The BAC clone 245_C_23 (RPCI-11 library) that contains the PIK3CA gene (3q26) was obtained from the Roswell Park Cancer Institute. The YAC clones, purchased by the Fondation Jean Dausset-CEPH (Paris, France), were 970_H_11, containing the p63 gene (3q28), 819_C_1 (6p12), 928_A_7 (3q13), and 967_H_1 (3q22-q24).
Before hybridization, nuclei on slides were treated 60 min at 37°C with a proteinase K solution (0.05 mg/ml), rinsed in 2 x SSC, incubated 5 min in 2 x SSC/NP40 0.1%, and washed in 2 x SSC. Nuclei on slides were fixed in formaldehyde (1% in PBS/MgCl2 50 mM) before dehydration. Then nuclei were denatured in formamide 70%/2 x SSC during 10 min, dehydrated in ice-cold ethanol, and air-dried.
YACs were amplified by Alu-PCR using primers CL1 and CL2 (5'-TCC CAA AGT GCT GGG ATT ACA G-3' and 5'-CTG CAC TCC AGC CTG GG-3', respectively) as described (25) . BAC clone and Alu-PCR products were labeled by nick translation with biotin-16-dUTP or digoxigenine-11-dUTP (Roche Diagnostics). Probes were then ethanol coprecipitated, and FISH was performed as described for CGH, except for the detection steps: avidin was conjugated to FITC (Vector Laboratories) for the biotin-labeled clone, and mouse anti-digoxin (Sigma Chemical Co.) and goat antimouse were conjugated to Cy3 (Jackson ImmunoResearch Laboratories) antibodies for the digoxigenin-labeled clone.
DNA sequence copy number was evaluated for each clone by counting spots in at least 100 nuclei. Then a ratio was calculated between the average copy numbers of the PIK3CA or p63 gene and of the 6p12 sequence. One tumor was considered as presenting an increased PIK3CA or p63 copy number when this ratio was superior to 1.25. A ratio of 1.25 corresponds to the presence of a relative trisomy for the locus in 50% of cells. This ratio was used as the threshold of sensitivity for CGH analysis too.
Quantitative RT-PCR.
Total RNA was extracted from human tissue samples using RNeasy kit (Qiagen, France). First-strand cDNA synthesis was performed on 1 µg of RNA in 20 µl of a reaction mixture with 0.3 µg of hexanucleotide used as primer (Boehringer-Mannheim), 200 units of SuperScript II RNase H- reverse transcriptase (Life Technologies, Inc.), and 40 units of RNasin (Promega). The mixture was incubated at 42°C for 1 h and then heated to 70°C for 15 min to inactivate reverse transcriptase. PCR was performed with 0.1 µl of template DNA (RT product) on the Roche Lightcycler machine. Oligonucleotides used as specific primers (annealing at 60°C) were: (a) 5'-CAA TGC CCA GAC TCA ATT TAG TGA-3' and 5'-GCG CCG TGA CGC TGT T-3' for the DNp63 isoforms; (b) 5'-TCA AAG GAT TGG GCA CTT TT-3' and 5'-GCC TCG ACT TGC CTA TTC AG-3' for the PIK3CA transcripts; and (c) 5'-TTG CCC TCA ACG ACC ACT TT-3' and 5'-TGG TGG TCC AGG GGT CTT AC-3' for the GAPDH transcripts. Continuous fluorescence observation of amplifying DNA was possible by using SYBR Green I (Light Cycler DNA master SYBR Green I; Roche Diagnostics). Amplification specificity was controlled by production of a melting curve. After the validation of primer pair efficiency by construction of a standard curve, expression level of each gene was measured three times independently in every sample.
Survival Analysis.
Crude survival was calculated from the time of surgery to the date of death or the date of the last follow-up for those alive at the date of analysis. Survival analysis was undertaken using the Kaplan-Meier method (26)
, and curves were compared by the log-rank test. Statistical analysis was performed using the BMDP statistical software.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Stratification according to tumor size (pT1-2 versus pT3) provides a refinement of this description. pT1-2 tumors presented a simple pattern of recurrent aberrations involving three chromosomes (Fig. 1A)
: 3q gain (10 of 15; 67%), 3p loss (7 of 15; 47%), 8q gain (7 of 15; 47%), and, to a lesser extent, 11q13 gain (4 of 15; 27%). pT3 tumors contained more chromosomal changes. The average number of aberrations/tumor was significantly higher for pT3 than for pT1-2 (11.8 and 4.3, respectively; P = 0.006; Fig. 2
). Some additional aberrations occur frequently in the pT3 tumors (Fig. 1B)
: 9p loss and 18q loss. These two chromosomal deletions were more frequent in pT3 than in pT1-2 tumors (9p, like 18q, in 4 of 6 pT3 versus 1 of 15 pT1-2).
|
|
. Interestingly, in metaphase spreads of all of the three cell lines, an isochromosome 3q, or a structure evolved from i(3q), was found. These results suggest that i(3q) are recurrent in HNSCC. An isochromosome 3 can be visualized using FISH on interphase tumor nuclei with a 3q para-centromeric probe. The systematic association of two signals in nuclei indicates two physically linked chromosome 3 long arms. Indeed, by using such approach in two tumors selected for entire 3q gain, we detected in one tumor (314; Fig. 3B
, bottom) the presence of two i(3q) (or derivatives), indicated by two pairs of green spots confined in two small areas of the nuclei. Thus, the formation of an isochromosome 3q may be one intermediate figure leading to gain of the long arm and loss of the short arm of chromosome 3 in head and neck tumors and cell lines.
|
, top). Whereas most of the tumors showed a gain for the entire consensus region, four individual CGH profiles exhibited localized gains or narrow peaks visualizing amplifications (Fig. 3C
, bottom). These restricted gains suggested two alternative sites of amplification, 3q26 (tumors 445 and 267) and 3q28-qter (tumors 326 and 434). In fact, 3q26 and 3q28 regions harbor two strong candidates for the function of oncogene (see "Introduction"), respectively, PIK3CA and p63. We hypothesized that these two genes could be alternately targeted by amplifications in the 3q26-qter region. Thus, we decided to confirm the presence of the two distinct amplification sites and to evaluate their relative occurrence in tumors by measuring PIK3CA and p63 relative gene copy numbers by FISH on interphase nuclei for the tumors 267, 445, and 326 and for 24 new selected cases.
FISH results for eight tumors with high-level gains or with differential gains are listed in Table 1
(tumor 434, nuclei not available) and illustrated in Fig. 3D
. A high-level gain (relative gene copy number >2 or high-level copy gain by CGH) was found in six tumors (326, 440, 445, 527, 594, and 776). For four tumors, both genes were gained equally, whereas p63/PIK3CA differential gains were detected in four cases: more PIK3CA copies in two cases and more p63 copies in two cases. Concerning the cell lines, both representations of the genes were normal for Cal27, whereas PIK3CA copy number was significantly higher for FaDu and Cal33 (P < 0.01).
|
. p63 or PIK3CA were in few cases preferentially gained or amplified (four tumors and two cell lines), but in most of the tumors, the 3q26-qter region was gained at low level. We concluded that genomic analysis alone does not delimit p63 and PIK3CA relative involvement in low-grade HNSCC progression and then decided to pursue with expression analysis of these genes.
|
DNp63, the putative oncogenic isoforms of p63, were highly predominant when compared with TAp63 transcripts in all of the normal and tumor samples (10–1000 times; data not shown). Striking variations of DNp63 level were observed (range, 1–45). DNp63 were more expressed in tumors than in normal tissues (ANOVA; P < 0.05; Fig. 4A
). However, DNp63 was equally represented in tumors with and without 3q28 gain (Fig. 4A)
. We concluded that the expression of DNp63 in tumors is altered by other mechanisms than gene dosage effect.
|
), whereas tumors with a normal 3q26 copy number displayed an intermediate average transcription level (no statistical differences with normal tissues and with 3q26-gained tumors). These results favor the hypothesis that PIK3CA expression is dependent of gene copy number and could act as an overexpressed oncogene in low-grade HNSCC.
3q26 Impact on Clinical Outcome.
We attempted to correlate the 3q26 and 3q28 gains detected by CGH with the clinical evolution of the patients for 21 low-grade HNSCC. The Kaplan-Meier representations for the overall survival are presented on Fig. 5
. Interestingly, the 5-year overall survival rate for patients with 3q26 (the PIK3CA locus) gain was 58% against 89% for those without gain. For 3q28 (the p63 locus), it was 69% against 75%. Nevertheless, no statistically significant difference was found (log rank, 3q26: P = 0.20; 3q28: P = 0.71). Likewise, no significant difference was found using the number of chromosomal aberrations (stratified in two groups; n 
6 versus n > 6: log rank, P = 0.37) or the T stage (two groups; T1-2 versus T3: P = 0.73).
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CGH analysis of 15 well-differentiated T1–2N0M0 tumors resulted in the depiction of a simple and specific pattern of genetic aberrations at early disease in HNSCC, in particular 3p loss, 3q gain, and 8q gain and, to a lesser extent, 11q13 gain or amplification. pT3 cases exhibit more complex karyotypes and carry additional recurrent aberrations, in particular 18q and 9p loss. The small number of listed low-grade cases and the diversity of grading systems impair comparison with published CGH data. By compilation of genetic changes for three well-differentiated (G1) HNSCCs of unknown TNM stage, Bockmühl et al. (5) associated 3p loss, 3q gain, and 9p loss with early tumor development. Incidentally, 3p, 9p, and 18q losses and 3q, 8q, and 11q13 gains occurred in other G1 (well-differentiated) HNSCCs (six cases reported of unknown or variable TNM status; Refs. 6 , 7 ). These reports are compatible with our results for 21 tumors.
LOH studies led to a genetic progression model for head and neck cancer (4) . 3p, 9p21, and 17p13 were listed as the most consistent losses at the earliest tumor stages. Both LOH and CGH analyses underline the high occurrence of 3p loss, whereas 9p21 and 17p13 (the respective INK4a and p53 loci) deletions appear underestimated by CGH. This method, the spatial resolution of which was evaluated to about 10 megabases, overlooks small interstitial deletions (28) .
Taken together, CGH and LOH data suggest a general pattern of genetic alterations at HNSCC early natural history or clinical stages. Frequent LOH at 3p, 9p21 (INK4a), and 17p13 (p53) arise in premalignant lesions and are present in the majority of early squamous cell carcinomas. For low-grade T1–2N0M0 HNSCC, our cytogenetic analysis pinpoints recurrent 3p loss in accordance with LOH data and additional 3q, 8q, and 11q13 gains. Later on, genetic changes accumulate while tumor sizes increase.
In addition to INK4a (9p21) and p53 (17p13), the roles of which are established in HNSCC development, cyclin D1, one of the oncogenes in the region 11q13, was clearly demonstrated to activate cell proliferation (29) . This cytogenetic band is amplified frequently in HNSCC (30) with homogeneously staining region structures (31) . On chromosome 8, the common region of gain, 8q23-q24, encompasses c-myc, but its amplification is relatively infrequent in HNSCC (30) . Other genes located in these two cytogenetic bands could be the targets of these extended low-level gains as well (32) .
The chromosome 3 is the preponderant site of chromosomal imbalances in our tumor population with, in most cases, concomitant gains on the long arm and losses on the short arm. In three head and neck cancer cell lines, we showed the presence of an isochromosome 3q, even when the CGH chromosome 3 profile was not immediately suggestive of such structure. In fact, isochromosomes are common rearrangements in solid tumors, found in 10% of cases (and in 19% of HNSCC; Ref. 33 ) in a survey of about 18,000 neoplasms. Furthermore, cytogenetic analysis of 11 oral squamous cell carcinomas underlined that most of chromosome breakpoints (60%) occur in centromeric regions and often result in isochromosome formation [in particular i(8q) and i(3q); Ref. 34 ]. Consistently, in our series, 39% of the breakpoints detected on CGH profiles clustered near centromeres. These data suggest that centromeric breakage is an important mechanism in the development of HNSCC. We propose that i(3q) is one of the intermediary cytogenetic figures resulting in simultaneous long-arm gains and short-arm losses in HNSCC.
The short arm of chromosome 3 is widely known as one of the most altered sites very early in the natural history of squamous cell carcinomas of the head and neck, but also of the lung and, to a lesser extent, of the uterine cervix. At least three distinct common regions of loss were defined: 3p13-p21.1, 3p21.3-p25, and 3p25 (35) . Several putative tumor suppressor genes were described within these regions, i.e., FHIT at 3p14.2 (36) and RASSF1 at 3p21.3 (37) . However, their role in the development of head and neck tumors is not yet clearly established.
The long arm of the chromosome 3 was the location of total or partial 3q gains in 67% of our low-grade HNSCC population. This result is in accordance with previous data, which showed 3q over-representations in 50 to 87% of head and neck carcinomas (5
, 7
, 9)
. Refined analysis pinpointed that the common region of gain extends from 3q26 to 3qter, as in earlier reports (6
, 7) . Whereas the large majority of tumors carry a low-level gain of this entire wide area, differential gains were found in this genomic segment using CGH (4 of 21 cases), suggesting the presence of two distinct regions at 3q26 and at 3q28-qter (Fig. 3C)
.
Two outstanding candidate oncogenes, PIK3CA and p63, located respectively at 3q26 and 3q28, emerge from a survey of the literature. Genome analysis alone did not enable us to evaluate the respective roles of these two genes in the pathogenesis of HNSCC. Indeed, both PIK3CA and p63 were over-represented in 27 of 45 low-grade N0M0 HNSCCs analyzed by FISH on interphase nuclei or CGH, with twice a preferential gain in favor of PIK3CA and twice a preferential gain in favor of p63 (Tables 1
and 2
). Consequently, to test whether p63 and/or PIK3CA can contribute to the pathogenesis of 3q-gained tumors, we measured the transcription levels of these two genes in samples with normal or increased gene copy number.
DNp63 was already described as an oncogene in HNSCC, with an elevated transcription level in tumors exhibiting increased p63 gene copy number (19) . It is noteworthy in this study that DNp63 expression was higher in tumors than in normal tissues but was independent to gene copy number. Thus, DNp63 is not one of the oncogenes activated by increased gene dosage in HNSCC and does not contribute to the recurrent retention of 3q gain in these tumors. Accordingly, the cell line FaDu, which was reported with p63 gain and overexpression (19) , actually shows a maximal gain for PIK3CA that excludes p63 in our refined mapping.
Nevertheless, p63 was consistently overexpressed in our population of well-differentiated tumors compared with normal tissues. In a previous work (38) , p63 RNA level was shown related to the differentiation state. Additional investigations are necessary to explore p63 histological distribution with respect to differentiation degree or proliferative potential.
The PIK3CA gene encodes the p110 protein, the catalytic subunit of PI3-K . This type I PI3-K belongs to a large family of lipid kinases involved in a wide range of malignant functions (14
, 15) . Furthermore, this gene was reported as an oncogene in ovarian and cervical cancer cell lines, with a phenotype correlated to its gene dosage, even with slightly increased copy number (16
, 17)
. In our work, p110 transcription level was significantly higher in tumors with 3q26 gain, but not in tumors with normal 3q26 copy number, than in normal tissues. All of these data together suggest that PIK3CA is one oncogene overexpressed as a consequence of 3q gain.
Novel experimental strategies finally uncovered a PI3-K -specific function in cell survival (39)
. Furthermore, PI3-K -increased activity was detected at early stages of primary colon and bladder tumors (39)
. These data, associated with the high frequency of increased PIK3CA copy number in low-grade HNSCC (in this study), point to an essential function of this gene in tumor growth at early stages of HNSCC.
Recently, gain on 3q21–29 was identified as a prognostic marker in HNSCC by CGH analysis of 113 tumors from various sites and stages and particularly for a subgroup of 41 pN0 tumors (20) . We correlated the presence of 3q26 gain with patients’ overall survival for 21 well-differentiated N0M0 HNSCC. The 5-year cumulated survival was 89% for patients with tumors normal for 3q26 versus 58% for patients with tumors showing a gain (difference not significant). It is likely that 3q26 gain, the locus of PIK3CA, could predict clinical outcomes in well-differentiated N0M0 tumors; this remains to be demonstrated in larger populations.
To analyze 3q26-qter gains in low-grade HNSCC, we combined CGH on chromosomes, a whole-genome screening method for DNA imbalances with a limited spatial resolution (10 megabases; Ref. 28 ), and FISH on interphase nuclei for two selected loci, a more resolutive but nonexhaustive technique. Using these tools coupled with quantitative RT-PCR, we showed that PIK3CA (3q26) rather than p63 (3q28) is a likely target of 3p26-qter amplifications, submitted to gene dosage effect, that participate in head and neck cancer development. However, the fact that two tumors exhibit 3q26-qter amplifications excluding PIK3CA prompts the question of additional gene targets. Recent pioneering works of CGH on arrayed genomic clones (40 , 41) or cDNA chips (42) finally permit the extensive cartography of amplicons, without compromise on spatial resolution. Such chips, dedicated to the 3q26-qter region, are under construction in our laboratory in order to map 3q gains in head and neck tumors but also in a broad range of other tumor types (uterus, ovary, colon, and liver; Ref. 3 ). Furthermore, cDNA chips will provide the means to correlate DNA gains and gene expression and to isolate those submitted to DNA dosage effect as proposed by Pollack et al. (42) . This approach will enable a first sorting of the candidate genes toward the dissection of their respective roles in tumor pathogenesis.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by a Grant from the Comité Régional du Haut-Rhin de la Ligue contre le Cancer and by funds from Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and the Hôpital Universitaire de Strasbourg. The molecular cytogenetic equipment necessary to set up the laboratory was funded by the Comité Régional du Haut-Rhin de la Ligue contre le Cancer, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Association pour la Recherche sur le Cancer, and Fondation pour la Recherche Médicale. 
2 To whom requests for reprints should be addressed, at Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, B. P. 163, F-67404 Illkirch cedex, C. U. de Strasbourg, France. E-mail: dumanoir{at}igbmc.u-strasbg.fr
3 The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; CGH, comparative genomic hybridization; PI3-K, phosphatidylinositol 3-kinase; PIK3CA, PI3-K catalytic subunit ; FISH, fluorescence in situ hybridization; RT-PCR, reverse transcription-PCR; LOH, loss of heterozygosity; ANCA, average number of copy alterations; TNM, tumor-node-metastasis staging; BAC, bacterial artificial chromosome; YAC, yeast artificial chromosome; DOP-PCR, degenerate oligonucleotide-primed polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received 12/ 1/00. Accepted 3/19/01.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(p85-p110) in cell survival and for phosphatidylinositol 3-kinase ß (p85-p110ß) in de novo DNA synthesis of human colon carcinoma cells. Oncogene, 19: 5083-5090, 2000.[Medline]
This article has been cited by other articles:
|
|
 |
|
  G. B. van den Broek, V. B. Wreesmann, M. W.M. van den Brekel, C. R.N. Rasch, A. J.M. Balm, and P. H. Rao Genetic Abnormalities Associated with Chemoradiation Resistance of Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., August 1, 2007; 13(15): 4386 - 4391. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Pelosi, B. Del Curto, M. Trubia, A. G. Nicholson, M. Manzotti, G. Veronesi, L. Spaggiari, P. Maisonneuve, F. Pasini, A. Terzi, et al. 3q26 Amplification and Polysomy of Chromosome 3 in Squamous Cell Lesions of the Lung: A Fluorescence In situ Hybridization Study Clin. Cancer Res., April 1, 2007; 13(7): 1995 - 2004. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Sarkaria, P. O-charoenrat, S. G. Talbot, P. G. Reddy, I. Ngai, E. Maghami, K. N. Patel, B. Lee, Y. Yonekawa, M. Dudas, et al. Squamous Cell Carcinoma Related Oncogene/DCUN1D1 Is Highly Conserved and Activated by Amplification in Squamous Cell Carcinomas Cancer Res., October 1, 2006; 66(19): 9437 - 9444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J.C. Slebos, Y. Yi, K. Ely, J. Carter, A. Evjen, X. Zhang, Y. Shyr, B. M. Murphy, A. J. Cmelak, B. B. Burkey, et al. Gene Expression Differences Associated with Human Papillomavirus Status in Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., February 1, 2006; 12(3): 701 - 709. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Isakoff, J. A. Engelman, H. Y. Irie, J. Luo, S. M. Brachmann, R. V. Pearline, L. C. Cantley, and J. S. Brugge Breast Cancer-Associated PIK3CA Mutations Are Oncogenic in Mammary Epithelial Cells Cancer Res., December 1, 2005; 65(23): 10992 - 11000. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Amornphimoltham, V. Patel, A. Sodhi, N. G. Nikitakis, J. J. Sauk, E. A. Sausville, A. A. Molinolo, and J. S. Gutkind Mammalian Target of Rapamycin, a Molecular Target in Squamous Cell Carcinomas of the Head and Neck Cancer Res., November 1, 2005; 65(21): 9953 - 9961. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. S. Patmore, L. Cawkwell, N. D. Stafford, and J. Greenman Unraveling the Chromosomal Aberrations of Head and Neck Squamous Cell Carcinoma: A Review Ann. Surg. Oncol., October 1, 2005; 12(10): 831 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. B. Wreesmann, W. Shi, H. T. Thaler, A. Poluri, D. H. Kraus, D. Pfister, A. R. Shaha, J. P. Shah, P. H. Rao, and B. Singh Identification of Novel Prognosticators of Outcome in Squamous Cell Carcinoma of the Head and Neck J. Clin. Oncol., October 1, 2004; 22(19): 3965 - 3972. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-Y. Guan, J. M-W. Fung, N.-F. Ma, S.-H. Lau, L.-S. Tai, D. Xie, Y. Zhang, L. Hu, Q.-L. Wu, Y. Fang, et al. Oncogenic Role of eIF-5A2 in the Development of Ovarian Cancer Cancer Res., June 15, 2004; 64(12): 4197 - 4200. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. G. Masayesva, P. Ha, E. Garrett-Mayer, T. Pilkington, R. Mao, J. Pevsner, T. Speed, N. Benoit, C.-S. Moon, D. Sidransky, et al. Gene expression alterations over large chromosomal regions in cancers include multiple genes unrelated to malignant progression PNAS, June 8, 2004; 101(23): 8715 - 8720. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. P. Massion, P. M. Taflan, S. M. Jamshedur Rahman, P. Yildiz, Y. Shyr, M. E. Edgerton, M. D. Westfall, J. R. Roberts, J. A. Pietenpol, D. P. Carbone, et al. Significance of p63 Amplification and Overexpression in Lung Cancer Development and Prognosis Cancer Res., November 1, 2003; 63(21): 7113 - 7121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Imoto, Y. Yuki, I. Sonoda, T. Ito, Y. Shimada, M. Imamura, and J. Inazawa Identification of ZASC1 Encoding a Kruppel-like Zinc Finger Protein as a Novel Target for 3q26 Amplification in Esophageal Squamous Cell Carcinomas Cancer Res., September 15, 2003; 63(18): 5691 - 5696. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhang, N. Yang, D. Katsaros, W. Huang, J.-W. Park, S. Fracchioli, C. Vezzani, I. A. Rigault de la Longrais, W. Yao, S. C. Rubin, et al. The Oncogene Phosphatidylinositol 3'-Kinase Catalytic Subunit {alpha} Promotes Angiogenesis via Vascular Endothelial Growth Factor in Ovarian Carcinoma Cancer Res., July 15, 2003; 63(14): 4225 - 4231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Worsham, G. Pals, J. P. Schouten, R. M. L. van Spaendonk, A. Concus, T. E. Carey, and M. S. Benninger Delineating Genetic Pathways of Disease Progression in Head and Neck Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg, July 1, 2003; 129(7): 702 - 708. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Estilo, P. O-charoenrat, I. Ngai, S. G. Patel, P. G. Reddy, S. Dao, A. R. Shaha, D. H. Kraus, J. O. Boyle, R. J. Wong, et al. The Role of Novel Oncogenes Squamous Cell Carcinoma-related Oncogene and Phosphatidylinositol 3-Kinase p110{alpha} in Squamous Cell Carcinoma of the Oral Tongue Clin. Cancer Res., June 1, 2003; 9(6): 2300 - 2306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Sawyer, J. Sturge, D. C. Bennett, M. J. O'Hare, W. E. Allen, J. Bain, G. E. Jones, and B. Vanhaesebroeck Regulation of Breast Cancer Cell Chemotaxis by the Phosphoinositide 3-Kinase p110{delta} Cancer Res., April 1, 2003; 63(7): 1667 - 1675. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Redon, T. Hussenet, G. Bour, K. Caulee, B. Jost, D. Muller, J. Abecassis, and S. d. Manoir Amplicon Mapping and Transcriptional Analysis Pinpoint Cyclin L as a Candidate Oncogene in Head and Neck Cancer Cancer Res., November 1, 2002; 62(21): 6211 - 6217. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. P. Massion, W.-L. Kuo, D. Stokoe, A. B. Olshen, P. A. Treseler, K. Chin, C. Chen, D. Polikoff, A. N. Jain, D. Pinkel, et al. Genomic Copy Number Analysis of Non-small Cell Lung Cancer Using Array Comparative Genomic Hybridization: Implications of the Phosphatidylinositol 3-Kinase Pathway Cancer Res., July 1, 2002; 62(13): 3636 - 3640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Fujii, T. Dracheva, A. Player, S. Chacko, R. Clifford, R. L. Strausberg, K. Buetow, N. Azumi, W. D. Travis, and J. Jen A Preliminary Transcriptome Map of Non-Small Cell Lung Cancer Cancer Res., June 1, 2002; 62(12): 3340 - 3346. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
) and T2 (•) tumors show significantly fewer chromosomal changes than T3 (
) cases (Mann-Whitney U test; P = 0.006). For each group, mean ± 1 SE is given next to individual data points.
