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Human Cancer Biology |
Authors' Affiliations: 1 Unit of Experimental Molecular Pathology, 2 Department of Pathology; 3 Head and Neck Cancer Medical Oncology Unit, and Departments of 4 Head and Neck Surgery and 5 Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori; and 6 Istituto Fondazione Italiana Ricerca Cancro di Oncologia Molecolare, Fondazione Italiana Ricerca Cancro, Institute of Molecular Oncology, Milan, Italy
Requests for reprints: Silvana Pilotti, Department of Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milan, Italy. Phone: 39-2-2390-2293; Fax: 39-2-2390-2198; E-mail: silvana.pilotti{at}istitutotumori.mi.it.
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Abstract |
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Experimental Design: Cell cycle regulators and epidermal growth factor receptor (EGFR) and BRAF genes were analyzed in a series of 90 oropharyngeal SCCs of a cohort of surgically treated patients from a single institution, and the results were matched with the presence of high-risk human papillomavirus (HR-HPV) DNA and the TP53 status.
Results: At least four distinct groups of tumors were identified sharing a common histology but displaying different molecular/cytogenetic patterns: (a) 19% were HPV-positive SCCs whose lack of alterations of the investigated genes could explain their particular natural history, which requires less aggressive treatment; (b) 37% were HPV-negative SCCs carrying TP53 mutations, which may be more effectively treated by drugs acting through p53-independent apoptosis; (c) 34% were HPV-negative SCCs carrying wild-type TP53 and loss of 9p21 (p16INK4a and p15INK4b) and/or cyclin D1 overexpression that justify treatment with DNA-damaging drugs followed by cell cycle inhibitors; and (d) 10% were HPV-negative lacking tumor suppressor genes and cell cycle alterations. The second, third, and fourth groups also showed an increased copy number of EGFR and chromosome 7 (43%) that might justify the additional or alternative use of EGFR inhibitors.
Conclusions: Our findings suggest that assessing HPV, TP53, 9p21, and EGFR status may be crucial to finding more tailored and beneficial treatments for oropharyngeal SCCs.
Despite their shared histology, the presence or absence of high-risk human papillomavirus (HR-HPV) identifies two distinct HNSCC entities with different clinical properties and genetic-molecular patterns. Clinically, the tumors with integrated viral DNA show a more favorable outcome than those that are HR-HPV negative (1). At molecular level, the HR-HPV–positive HNSCCs are characterized by the lack of p16INK4a gene deletion coupled with p16 protein expression (2) and a unique gene expression profile (3), with a decreased occurrence of TP53 mutations, cyclin D up-regulation/amplification, 14-3-3
and RASSF1A promoter methylation (4), and loss of heterozygosity at 17p, 9p, and 3p (5, 6). These latter are common alterations in patients with HPV-negative HNSCCs.
Clinical behavior of HPV-negative HNSCCs is heterogeneous, and it is warranted to better characterized this gray zone. The most frequent molecular alteration carried by patients with HPV-negative HNSCCs is the TP53 mutation, which seems to correlate with a poor response to both radiotherapy (7) and chemotherapy (8, 9) as well as with a poor prognosis (10). Few information is available regard the role of cell cycle regulators and epidermal growth factor receptor (EGFR) gene status in HPV-negative HNSCCs (11).
The aim of this study was to acquire further insights into the pathogenetic pathways of the subsets of HR-HPV–positive and HR-HPV–negative oropharyngeal SCCs that may be useful for identifying major new biomarkers and help in the development of more tailored treatment approaches. To this end, we undertook molecular/cytogenetic analyses of tumor suppressor genes/cell cycle regulators, as well as receptor tyrosine kinase and serine/threonine kinase in a series of 90 patients with surgically resected oropharyngeal SCCs who had been previously characterized in terms of HR-HPV DNA and TP53 status.
Our findings highlight the heterogeneous biology underlying oropharyngeal SCCs, and the fact that the frequently overlapping cell cycle protein and receptor tyrosine kinase alterations are mainly restricted to HPV-negative tumors. These findings may have therapeutic implications that warrant further investigation in prospective studies.
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Materials and Methods |
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All of the formalin-fixed, paraffin-embedded tumors had been previously investigated for HR-HPV16 DNA and physical status by means of real-time PCR, for the TP53 mutation, p16 immunophenotyping, and homozygous deletion (2).
Detection and relative quantification of HPV16 E6 and E7 expression
Total RNA from microdissected samples was isolated by means of the TRIzol method (Life Technologies, Carlsbad, CA) and reverse transcribed with Superscript reverse transcriptase (Life Technologies) using both oligo dT and random hexamers. The HPV16 DNA–positive samples were investigated for E6 and E7 mRNA expression by means of real-time quantitative PCR (ABIPRISM 5700 PCR Sequence Detection Systems, Applied Biosystems, Foster City, CA) using a Taqman-based analysis (12). Formalin-fixed, paraffin-embedded CaSki cells were used as a positive control.
Immunohistochemistry
p53, cyclin D1, and cyclin-dependent kinase 4. Immunohistochemistry was done on 2-µm cut formalin-fixed and paraffin-embedded tumoral sections using the peroxidase-streptavidin method (1:300 in PBS, DAKO, Carpinteria, CA). All of the stains were developed using 3,3-diaminobenzidine (Sigma, St. Louis, MO). The primary antibodies were a p53 mouse monoclonal antibody diluted 1:400 (YLEM, Rome, Italy) and cyclin D1 rabbit monoclonal antibody diluted 1:100 (Clone SP4, Lab Vision, Fremont, CA), with antigen retrieval using 5 mmol/L citrate buffer (pH 6) in an autoclave at 95°C for 6 to 15 minutes; and cyclin-dependent kinase 4 (Cdk4) rabbit polyclonal antibody diluted 1:400 (C-22, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
The positive controls were a serous ovarian carcinoma with a known TP53 mutation for p53, a mantle cell lymphoma for cyclin D1, and a WD/DD liposarcoma for Cdk4.
For p53, a cutoff value of 
50% positive nuclei was used as a tentative marker of predicted mutation (13).
EGFR. EGFR was immunostained using the EGFR-DAKO kit. A ready-to-use monoclonal mouse anti-human EGFR antibody and CAMA-1 and HT-29 cell lines (negative and positive controls) were provided. EGFR-immunostained cells were quantitatively evaluated, and the staining intensity was scored as detailed in Table 1 . A final score obtained by combining the two score values was calculated.
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For the 9p21 locus, the cosmid C5, spanning the chromosomal region from the p16INK4a to the p15INK4b gene (about 50 kb), and the specific probe BAC clone RP11-149I2 (kindly provided by Prof. M. Rocchi, University of Bari, Bari, Italy), spanning 100 bp and directly labeled with Spectrum Orange dUTP by the Vysis Nick Translation kit were used associated with the Spectrum Green-labeled control probe CEP9 (Vysis). The 9p21 hybridization pattern was evaluated on the basis of previously described criteria (14).
The LSI EGFR dual-colour probe (Vysis) was used for EGFR. The EGFR copy number in each nucleus was assessed in relation to the chromosome 7 copy number, with the presence of an "amplification cluster" of EGFR indicating gene amplification. The numerical status of chromosome 7 was defined as being balanced polysomic when the cancer cell population showed multiple (>3) 7 centromere signals associated with the same number of EGFR signals. A distinction was made between low (trisomic and tetrasomic hybridization patterns) and high polysomy, when the chromosome 7 copy number was 5.
Homozygous deletions of p14ARF and p15INK4b genes
DNA was extracted from 7-µm serial sections of the most representative tumoral block as previously described (15), and its quality was tested in each sample by amplifying the human ß-globin gene fragment (200 bp).
The homozygous deletion assays were done using 30 cycles of comparative duplex PCR to investigate p14ARF exon 1ß and p15INK4b exons 1 and 2. Each exon was coamplified with a fragment of human ß-globin as previously described (2). The primers and annealing temperature used for each PCR are shown in Table 2 . The negative control was the K562 cell line, which has a homozygous deletion at the INK4A locus.
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BRAF. Mutational analysis was restricted to exon 15 of the BRAF gene. The DNA was amplified using previously described specific primers (19).
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Results |
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Tumor suppressor genes and cell cycle regulators
P53
TP53 mutation analysis. Overall, as previously reported (2), 35 of 90 (39%) cases harbored TP53 mutations (TP53 mut), and 55 cases (61%) carried TP53 wild type (TP53 wt). The frequency of TP53 mutations was significantly lower (P < 0.025) in the HPV+ SCCs (2 of 17, 12%; Table 3) than in the HPV– tumors (33 of 73, 45%; Tables 3 and 4
).
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50% positive nuclei for p53 immunoreactivity was used as a tentative marker of predicted mutation (13). The fact that p53 immunoreactivity was found in 27 of 35 (77%) TP53 mut cases (Tables 3 and 4), whereas none of 55 TP53 wt tumors was p53 immunopositive (P < 0.001; Tables 3 and 5
), indicates that 50% nuclear immunoreactivity may make a good surrogate of TP53 mutations.
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0.025) in the HPV+ SCCs (1 of 17, 6%; Table 3) than in the HPV– tumors (27 of 73, 37%).
9p21 locus
Homozygous deletion of p16INK4a, p14ARF, and p15INK4b tumor suppressor genes. We have previously shown that 100% of the HPV+ cases carried a normal p16INK4a gene in keeping with p16 overexpression, whereas a p16INK4a homozygous deletion was found in 47% of the HPV– cases, similarly distributed among the TP53 mut (48%) and TP53 wt tumors (46%; ref. 2).
To characterize the DNA status of the two other tumor suppressor genes (TSG) mapping in tandem to the 9p21 locus (p14ARF and p15INK4b), we assayed homozygous deletions by means of comparative duplex PCR. A homozygous deletion of one or more of the three TSGs was found in 43 of 76 SSCs (56%), in 23 of which (53%) it encompassed all three TSGs.
The frequency of an homozygous deletion of one or more TSGs at the 9p21 locus was significantly lower (P < 0.001) in the HPV+ (3 of 15, 20%) than the HPV– tumors (40 of 61, 65%; Fig. 1 ), both TP53 mut (21 of 32) and TP53 wt (19 of 29; Tables 4 and 5, respectively). In the HPV+ tumors, the homozygous deletion involved both p14ARF and p15INK4b in one case and only p14ARF or p15INK4b in the remaining two (Table 3).
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All 17 HPV+ cases showed a normal hybridization pattern (Table 3), whereas 62% of the HPV– cases carried a 9p21 loss (P 0.01; Fig. 2
): 20 of 32 TP53 mut (Table 4) and 23 of 37 TP53 wt (Table 5).
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The frequency of 9p21 loss was significantly lower (P < 0.001) in HPV+ tumors (3 of 17, 18%) than HPV– tumors (58 of 71, 82%).
Cyclin D1 and Cdk4 immunohistochemical analysis
Cyclin D1. Eighty-eight cases were suitable for cyclin D1 analysis. Taking a cutoff of 70% positive cells, no immunoreactivity was found in 16 HPV+ cases (one was not evaluable; Table 3), whereas 11 of 72 (15%) HPV– cases were positive. Cyclin D1 overexpression was coupled with 9p21 loss in 9 of 11 cases (82%). In terms of TP53 status, cyclin D1 immunoreactivity involved 7 of 33 TP53 mut cases (21%) and 4 of 39 TP53 wt cases (10%; Tables 4 and 5).
Cdk4. None of the 89 analyzed cases showed any Cdk4 immunoreactivity (data not shown).
Cumulative evidence of TSG and cell cycle alterations. Despite their similar phenotype, we identified four different molecular groups by means of HR-HPV, TP53, 9p21, and cyclin D1 analyses:
Groups II and III showed additional homozygous deletion of the p14ARF gene in 22 and 23 cases, respectively.
The concurrent abnormalities of TP53 and p14ARF and p16INK4b, p15INK4b, and cyclin D1 provide further evidence of the possible collaborative role of multiple components of the same pathway (6, 20).
Kinases
Receptor thyrosine kinase: EGFR
Immunohistochemistry. The EGFR immunohistochemical analysis was done in 87 SCC cases, the majority of which (77 of 87, 88%) had an intermediate or high expression score. No significant difference in EGFR expression was found between the HPV+ and HPV– cases (100% versus 86%) or between the TP53 mut and TP53 wt cases (90% versus 82%; Tables 3, 4, and 5).
FISH analysis. To investigate EGFR gene and chromosome 7 copy numbers, 86 SCCs were successfully analyzed by means of FISH, 38 (44%) of which showed an abnormal hybridization pattern: 5 of EGFR amplification, 26 of balanced polysomy, 3 of monosomy, 3 of hemizygous deletions, and 1 of homozygous deletion of chromosome 7.
There was a significant difference in the copy numbers of EGFR and chromosome 7 between the HPV+ and HPV– cases (P < 0.01; Fig. 3 ). The 17 HPV+ tumors showed no EGFR amplification; however, a single case carried high polysomy (6%; Table 3); whereas 30 of 69 HPV– cases (43%) had an increased EGFR or chromosome 7 copy number: 16 of 33 TP53 mut (48%) and 14 of 36 TP53 wt (39%; Tables 4 and 5, respectively). EGFR amplification occurred in 5 of 69 HPV– SCCs (7%), coupled with TP53 mutations in all cases and with low polysomy in three cases. High polysomy was found in 9 (13%) and low polysomy (trisomy and tetrasomy) in 16 (23%) of the 69 cases. Interestingly, EGFR amplification or chromosome 7 polysomy was coupled with cyclin D1 overexpression in six cases.
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Mutational analysis. Forty of the 90 SCCs underwent EGFR mutational analysis restricted to exon 19: 15 HPV+ and 25 HPV– cases (14 TP53 mut and 11 TP53 wt).
No EGFR mutation was found in the HPV+ cases (Table 3), whereas 1 of 25 HPV– cases (4%) carried the Gly719Glu (GGA > GAA) mutation (Table 4).
Cumulative molecular evidence of EGFR alterations. Gene amplification and chromosome 7 polysomy and mutation accounted for 32 (37%) of the 86 oropharynx SCCs, divided into the following four previously defined groups:
There was a significant difference in the rate of EGFR alterations between the HPV+ and HPV– cases (6% versus 45%; P < 0.01). Among HPV-negative cases, EGFR alterations did not segregate with a specific subgroup.
Serine/threonine kinase: BRAF
Mutational analysis. Thirty of the 90 SCCs were analyzed for BRAF mutations: 12 HPV+, 8 HPV–/TP53 mut, and 10 HPV–/TP53 wt. We sequenced exon 15 of the BRAF gene, in which the classic V599E mutation is located, which occurs in >90% of BRAF-altered neoplastic diseases. No BRAF mutations were found, regardless of HPV or TP53 status (data not shown).
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Discussion |
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The first group is represented by 17 (19%) HPV-positive oropharyngeal SCC, where the molecular/cytogenetic analyses here reported confirm our previous preliminary evidence that HPV-positive tumors are a distinct molecular entity (2). Consistently, in addition to the presence of a significant low occurrence of TP53 mutations (12%; P < 0.025), HPV-positive SCCs show a significant reduced p14ARF-p15INK4b deletion (20%; P < 0.001) and lack of cyclin D1 overexpression in comparison with HPV-negative tumors. Our 9p21 data are in line with the occasional allelic loss of chromosome arm 9p found in HPV-positive HNSCC by microsatellite analysis (5, 6). Moreover, the lack of EGFR-activating mutation and EGFR amplification and the unusual chromosome 7 polysomy (6%; P < 0.01) in comparison with HPV-negative tumors corroborate the peculiar simple profile of HPV-positive SCC characterized by low rate of genotypic alterations. Remarkably, despite of a normal disomic EGFR/chromosome 7 cytogenetic pattern in HPV-positive SCC and an amplified polysomic pattern in 43% of HPV-negative tumors, EGFR protein resulted similarly overexpressed in both tumor groups (100% versus 86%), supporting the notion that EGFR immunophenotyping does not mirror EGFR gene status. Cumulatively, our data strongly support that HPV-positive SCCs deserve an individualized less aggressive treatment (21), and that the neoplastic process is likely mainly due and supported by the oncogenic viral genes.
The second group (37%) consisted of 33 HPV-negative oropharyngeal SCCs carrying TP53 mutations. The correlation between TP53 status and the response to cisplatin/fluorouracil–based chemotherapy in HNSCC (9, 10) supports the idea that the response to cytotoxic DNA-damaging action requires an efficient p53-dependent apoptotic pathway. In this light, drugs acting through p53-independent apoptosis may be more effective in oropharyngeal SCCs carrying TP53 mutations (22). Although the patients who may benefit from p53-independent treatment are best selected by means of mutational analysis, immunohistochemical assessments of increasing half-lives of p53 may represent a good surrogate if the cutoff point is 50% of tumoral cells with strong nuclear immunoreactivity (13). Using this cutoff level in our series, we found a significant correlation between protein stabilization and the presence of TP53 mutations (P < 0.001). In this group, TP53 alterations might represent the main oncogenic driving force.
The third group (34%) consisted of 31 HPV-negative cases carrying TP53 wt, 9p21 (p16INK4a and p15INK4b) loss, and/or cyclin D1 overexpression. The presence of these alterations offers a rationale for therapeutically target the cell cycle by means of CDK inhibition (23). Because of loss of checkpoint integrity due to p16INK4a-p15INK4b inactivation or cyclin D1 overexpression, tumor cells are unable to stop at predetermined points of the cell cycle, favoring an uncontrolled proliferation. CDK inhibitors are promising new antitumor agents that suppress cell growth and then facilitate the induction of apoptosis. As this third group harbors the TP53 wt, treatment with cytotoxic DNA-damaging drugs inducing p53-dependent apoptosis would seem to be sound. However, tumor cells treated with DNA-damaging drugs may undergo cell cycle arrest and DNA repair but not apoptosis (24), leading to a cell cycle–mediated drug resistance that may limit the effectiveness of chemotherapy. In this light, clinical observations suggest that the activity of DNA-damaging drugs in the presence of a TP53 wt could be improved by sequentially following them with the administration of CDK inhibitors (25), which can convert a cell from cell cycle arrest to cell death by modulating the expression of antiapoptotic proteins, such as p21 (26) and cyclin D1 (27). In this group, a synergy between loss of TSGs and activation of cyclins seems to play the main role in the neoplastic process.
Cell cycle–mediated chemotherapy may also be an alternative for treating SCCs carrying TP53 mutations (group II) because, despite the lack of G1 arrest due a nonfunctional p53 protein, the pharmacologically induced DNA damage may counteract apoptosis by means of Chk1-mediated G2 cell cycle arrest. Preclinical evidence suggests that chemotherapy followed by inhibitors of Chk1 or of hsp90, of which Chk1 is a client, enhances apoptosis in cells with TP53 mutations (24). Clinical trials of these agent are currently being carried out.
It is known that p14ARF plays a role in the biology of oropharyngeal SCCs (28), and the tumors in our groups II and III showed evidence of p14ARF deletion. However, this seems to be less relevant in therapeutic terms because, in group II, the deletion simply worsens the p53 function that is already impaired by the TP53 mutation and, in group III, the effect of the single p14ARF deletion on p53 function is not regarded as equivalent to a TP53 mutation.
Because of its frequent overexpression in HNSCCs, it has recently been suggested that EGFR may be a potentially useful therapeutic target. The addition of cetuximab to radiotherapy is a new therapeutic option able to increase both the locoregional control and the survival in advanced HNSCC (29), although the mechanism underlying the selective sensitivity of HNSCC to EGFR inhibitors is still unknown. We observed EGFR overexpression in almost all of our SCCs, thus confirming that alone it is insufficient to predict the response to EGFR inhibitors (30), and, in line with previously published findings (16, 17, 31), we also found very few somatic EGFR mutations (4%). On the contrary, EGFR amplification (7%) and chromosome 7 polysomy (36%), which have previously been reported in HNSCC (31–33), accounted for 43% of our HPV-negative SCCs (groups II and III). Given the published data concerning other tumors, such as non–small cell lung carcinoma (34, 35) and colorectal cancer (36), it could be very interesting to verify the effect of these EGFR alterations on the response of oropharyngeal SCCs to EGFR inhibitors. The only study (to the best of our knowledge) correlating EGFR amplification and the response to EGFR inhibitors in HNSCC (31) indicates that responsive cases do not carry the amplification; however, the number of cases was very small. Moreover, we found that the EGFR alterations in our SCCs were generally coupled with TP53 mutations and/or cell cycle alterations (groups II and III), and so the interaction of different pathways has to be considered when using EGFR inhibitors. In line with this, it has been found at preclinical level that cyclin D1 overexpression (which we found to be coupled with chromosome 7 polysomy) may be associated with the decreased efficacy of EGFR inhibitors in HNSCC (37).
In the fourth group, representing a minority of the cases, no specific alterations of the biomarkers investigated were found, with the exception of 3 cases carrying chromosome 7 polysomy; thus, further investigations are needed.
CDK4 overexpression and BRAF mutation do not seem to play any role at all.
In conclusion, the results of this comprehensive analysis of a cohort of surgically treated patients from a single institution support the notion that oropharyngeal SCCs are generated by at least three different mechanisms, although the inactivation of pathways regulated by TSGs like p53 and Rb through viral gene interaction or alterations of molecular key players seems to be a common theme in the neoplastic process of these tumors. Because these three pathways may benefit from an appropriate drug combination, assessing HR-HPV status (by means of real-time PCR), TP53 (by means of mutation analysis or immunohistochemistry), and 9p21 and EGFR (by means of FISH) seems to be crucial to try to individuate tumor subgroups to be treated in a more specific way.
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Footnotes |
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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.
Note: M.A. Pierotti and S. Pilotti are senior coauthors.
Received 7/19/06; revised 9/ 1/06; accepted 9/ 8/06.
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in HPV– case 68 (lane 1) and retention in HPV+ case 5 (lane 2). C+, positive control, normal genomic DNA; C–, negative control, DNA from K562 cell lines carrying the 9p21 locus homozygous deletion; M, 1-kb molecular marker.
