The ARF-p16 gene locus in carcinogenesis and therapy of head and neck squamous cell carcinoma

OBJECTIVES/HYPOTHESIS: We have identified families with a high incidence of tumors including head and neck squamous cell carcinoma (HNSCC). The occurrence of melanoma in these kindreds suggested that the ARF-p16 gene may be involved in carcinogenesis. We wished to determine the gene defect associated with the familial predisposition to HNSCC and to determine whether restoration of the gene may have therapeutic benefit. STUDY DESIGN: Translational molecular research. METHODS: Molecular techniques were used to identify mutations of the ARF-p16 gene from the affected families and to test the activity of p16 and ARF mutants. In additional, HNSCC tumor tissue was analyzed to determine whether the wild-type p16 allele was lost or maintained. ARF-expressing adenoviruses were created, and their effect on HNSCC cell lines and normal head and neck epithelial cells was determined. RESULTS: Mutation of the ARF-p16 gene was found in two families with predisposition to develop HNSCC. Independent mutations detected in the germline DNA of both families inactivated p16, but not ARF, and the inactive mutant p16 allele segregated with disease within both families. The wild-type p16 allele was lost in HNSCC tumor tissue from both families. The efficacy of ARF in treatment of HNSCC was found to depend on retention of p53 activity within HNSCC tumor cells. Remarkably, ARF expression was found to kill cells, depending on loss of retinoblastoma activity. Because loss of retinoblastoma activity is nearly universal in tumors, ARF killed tumor cells that retained p53, but ARF spared normal cells. CONCLUSIONS: Our results support the recognition of a new clinical entity of familial head and neck cancer. We have shown that this syndrome is associated with inactivating mutations of the p16 gene that these mutations segregate with disease in two described families. Loss of the wild-type p16 allele in HNSCC tissue from both families strongly supports the role of the mutant p16 in carcinogenesis. We have also investigated the therapeutic utility of the alternate reading frame product of the p16 gene, ARF. The finding that ARF kills cells depending on loss of retinoblastoma activity and retention of p53 suggests that ARF may be effective in treatment of roughly 50% of head and neck cancers while sparing normal cells. Recognition of p16 mutations as an etiological factor in familial HNSCC provides an accessible tool for diagnosis of this syndrome. Clinical acceptance of familial head and neck cancer will ensure that patients are appropriately diagnosed and managed.     

p53 codon 72 polymorphism association with head and neck squamous cell carcinoma

p53 tumoral suppressor gene harbors a functional polymorphism which codes either arginine (Arg) or proline (Pro) in the protein p53 of codon 72. Such polymorphism has been associated with the development or prognosis of head and neck squamous cell carcinoma (HNSCC).Aim: we assessed codon 72 p53 allelic frequencies and genotypes in HNSCC Iranian patients.Study design: Case Study.Materials and Methods: a total of 132 HNSCC patients and 123 healthy controls were genotyped. DNA source was from mononuclear cells of the peripheral blood. DNA amplification was done by means of the allele-specific polymerase chain reaction.Results: genotypes and allele distribution were not significantly different between patients and controls. Moreover, no statistically significant association was found between the 72 and p53 codon tumor location, gender or age at the time of diagnosis. However, the Pro/Pro genotype was significantly increase in stage IV patients (30.8%) when compared to stages I-III of the disease (11.1%) (p=0.03), and a significantly higher percentage of patients with the Pro allele had and a risk increase in stage IV disease (OR=2.2, 95% CI=1.2-4.2, p=0.01).Conclusion: data revealed that the p53 polymorphism do not impact the risk of HNSCC in Iranians, nonetheless, it can affect tumor progression to a higher tumor stage.     

Biallelic inactivation of the p16-Gen in a metachronous triple carcinoma in the oropharyngeal region

AIMS: Recent studies have shown that most Dutch families with atypical multiple-mole melanoma (FAMMM) have a 19-bp deletion (p16-Leiden) in exon 2 of the p16 gene. Apart from reports on metachronous pancreatic tumors, other cancer types have never been described in such families. Due to heterozygous p16-Leiden constitution, our proband with multiple head and neck carcinomas was a suitable model for studying the type of p16 inactivation according to the Knudson-two-hit model. METHODS: p16 mutations in exons 1 and 2 were determined using PCR-SSCP-Sequencing analysis. p16 methylation was assessed by methylation-specific PCR. RESULTS: All three metachronous (larynx, pharynx, oral cavity) tumors had a methylated p16 promotor. The p16 protein loss detected by immunohistochemistry clearly confirmed a complete loss of p16 tumor suppressor function. Thus, all three tumors exhibited biallelic inactivation of p16, caused by aberrant methylation of the p16 promotor. CONCLUSIONS: This is the first report on p16-Leiden mutation in head and neck cancer. We provide evidence that the somatic methylation of p16 promotor is associated with the germline transmission of p16-Leiden mutation. This is an example for the rare event of in which aberrant methylation acting as the 'second hit' in a familial cancer syndrome. Our results show that this epigenetic event is equivalent to genetic alterations (mutation/LOH) confirming the Knudson's hypothesis for tumor suppressor gene inactivation.     

Delineating genetic pathways of disease progression in head and neck squamous cell carcinoma

OBJECTIVE: To identify altered gene targets that characterize disease progression in squamous cell carcinoma (SCC) of the head and neck (HNSCC). Genetic alterations in HNSCC cell lines reflect the tumor in vivo and can serve as valuable tools to study the development and progression of HNSCC. Identification of key molecular events may be useful for more accurate distinction of prognostic groups for selection and targeting of therapy. DESIGN: Individual gene loci were analyzed for genetic alterations using a novel genomewide strategy. SUBJECTS: Head and neck squamous cell carcinoma primary (A) and recurrent or metastatic (B) cell lines UMSCC-11A/11B, UMSCC-17A/17B (previously karyotyped), and UMSCC-81A/81B are described. RESULTS: At the genome level, loss and gain of genetic loci concurred with tumor karyotypes. Several abnormal gene loci not apparent by cytogenetics were also identified. All except 11B indicated loss of CDKN2A (encodes p14 and p16), with concomitant loss of CDKN2B (encodes p15) in 11A, 17B, and 81A. All 6 cell lines showed gain of PIK3CA (encodes a PI3 kinase) located at 3q26.3. CONCLUSIONS: We provide evidence for the role of 3 critical pathways in the development and progression of HNSCC. The CDKN2A/B genes encode various components of the Rb and p53 pathways, and the PIK3CA gene makes a catalytic subunit of the protein phosphatidylinositol 3-OH kinase (PI3K), which is known to be involved in the PI3K/ATK signaling pathways. Molecular events may ultimately serve to achieve genomic alterations that set off an interplay among key gene loci along discrete genetic pathways used by tumor cells in HNSCC.     

Cervical paragangliomas: is SDH genetic analysis systematically required?

To evaluate the prevalence of succinate dehydrogenase (SDH) B, C, and D germline mutations in a surgical series of cervical paragangliomas and to precise the characteristics of patients presenting with familial form. Among 29 patients operated on cervical paragangliomas (carotid or vagal body) at our institution between 1994 and 2007, 23 could be asked for a genetic analysis and a familial study. Clinical characteristics of patients harboring a germline SDH mutation were studied and compared with those presenting without mutation. Mutations were found in 8/23 (35%) patients, mostly in SDHD gene (6 cases), and in SDHB and SDHC gene, respectively, in one case each. Mean age at onset was significantly lower for patients with mutation (34 vs. 51.5 years, P = 0.01). In patients presenting with a mutation, 50% had a family history of paraganglioma compared with 0% for others (P = 0.008) and 87.5% had a multifocal form of paragangliomas versus 0% for others (P = 0.001). No difference was found concerning malignant forms between the two groups (12.5 vs. 13.3%). In the 16 patients who had an apparently sporadic paraganglioma, 6% had mutations in the SDH gene. A positive family history of paraganglioma and/or the presence of bilateral or multiple paragangliomas and/or an early age of onset are the main parameters associated with SDH mutations. Genetic testing should be considered for all patients with a cervical paraganglioma, even for those presenting with an apparently sporadic tumor as familial form may be such identified in 6% of cases.     

Positive correlation of tissue inhibitor of metalloproteinase-3 and death-associated protein kinase hypermethylation in head and neck squamous cell carcinoma

OBJECTIVES/HYPOTHESIS: Promoter hypermethylation of tumor suppressor genes is common in head and neck cancer as well as other primary cancers resulting in epigenetic gene silencing. Tissue inhibitor of metalloproteinase-3 (TIMP-3) has been shown to have promoter hypermethylation in several solid tumors, but has not been identified in head and neck squamous cell carcinoma (HNSCC). Our objective was to determine if TIMP-3 promoter was hypermethylated in HNSCC, if there was any correlation with death associated protein kinase (DAPK), a tumor suppressor whose promoter has been hypermethylated at high levels in HNSCC, and if any clinical factors influence hypermethylation of either of these genes. STUDY DESIGN: Prospective study. METHODS: Tumor samples from 124 patients with HNSCC were evaluated for promoter hypermethylation for TIMP-3 and DAPK using quantitative methylation specific polymerase chain reaction (qMSP). We compared both TIMP-3 and DAPK hypermethylation in HNSCC with each other as well as with other clinical variables. RESULTS: We found that TIMP-3 was hypermethylated in approximately 71.8% of the tumor samples and DAPK was hypermethylated in 74.2%. The presence of TIMP-3 and DAPK promoter hypermethylation was significantly higher than in control specimens. More importantly, TIMP-3 and DAPK hypermethylations in these samples were highly correlated with a concordance of 78% (P < .001). DAPK was also correlated with current alcohol consumption (P < .028), but neither TIMP-3 nor DAPK hypermethylation was significantly correlated with other clinical variables or with survival. CONCLUSION: TIMP-3 promoter hypermethylation is elevated in HNSCC and is highly correlated with DAPK hypermethylation, implying a functional relationship between these genes.     

Compound heterozygous GJB2 mutations associated to a consanguineous Han family with autosomal recessive non-syndromic hearing loss

Conclusion: This study demonstrates that the gap junction protein beta-2 gene (GJB2) p.R32C and p.L79Cfs*3 variants are associated to a consanguineous family with autosomal recessive non-syndromic hearing loss (ARNSHL). The p.R32C variant is found for the first time in the NSHL patients of Han Chinese origin. The finding sheds new light on the accurate genetic diagnosis and counseling for the family. Objective: ARNSHL is a highly heterogeneous genetic disease. ARNSHL usually displays non-progressive congenital or pre-lingual deafness. In this study, the aim is to detect the disease-causing mutation(s) in a Han family with ARNSHL. Methods: A consanguineous Han family with ARNSHL was enrolled. Two hundred ethnicity-matched unrelated subjects without any hearing impairments were used as normal controls. Exome sequencing and Sanger sequencing were applied to identify the causative mutation in the ARNSHL family. Results: Compound heterozygous variants c.94C?>?T (p.R32C) and c.235delC (p.L79Cfs*3) in the GJB2 gene were identified in the two patients of the ARNSHL family, and the heterozygous GJB2 c.94C?>?T and c.235delC variants were identified in his unaffected father and mother, respectively. The two variants in the GJB2 gene were absent in the 200 unrelated controls.     

Intraoperative molecular margin analysis in head and neck cancer

BACKGROUND: Tumor-specific molecular alterations in surgical margins have been shown to predict risk of local recurrence. However, assays used for these analyses are time-consuming and therefore cannot be used in the intraoperative setting. OBJECTIVE: To detect and quantify tumor-specific methylated promoter sequences in surgical margins in a time frame suitable for intraoperative use. DESIGN: A novel quantitative methylation-specific polymerase chain reaction (QMSP) protocol. METHODS: A total of 13 patients with head and neck squamous cell carcinoma (HNSCC) were initially characterized for molecular alterations in their tumor at the time of biopsy. Six primary tumors were found to harbor promoter hypermethylation for p16 and O6-methylguanine-DNA-methyltransferase (MGMT) genes. Rapid QMSP was then used to identify promoter hypermethylation of these genes in the surgical margins. Results were compared with standard intraoperative histologic frozen section analysis and with conventional QMSP. RESULTS: Using our rapid QMSP assay, we found that 3 patients had methylation-positive margins. Tumor margins from 2 patients were methylated for p16 alone, and margins from 1 patient were methylated for p16 and MGMT simultaneously. Molecular margin analysis was completed in less than 5 hours, a time frame appropriate for selected major HNSCC resections that require combined primary tumor resection, cervical lymphadenectomy, and complex reconstruction. This technique was comparable in sensitivity to conventional QMSP. CONCLUSION: Rapid molecular margin analysis using QMSP is feasible and may be performed intraoperatively in selected patients with HNSCC that requires extensive resection.     

p16基因突变与喉癌生物学行为的相关性

目的 :研究 p16基因突变与喉癌生物学行为及预后的关系。方法 :应用 PCR- SSCP对 2 4例喉癌组织及 10例喉部良性病变组织中 p16基因 (exon2 )进行检测。结果 :喉癌突变检出率为 6 2 .5 % (15 / 2 4) ,而且随着组织分化程度、浸润范围和淋巴结转移的不同而不同 ;喉部良性病变无一例突变。结论 :p16基因突变与喉癌的多种生物学行为有明显关系 (P≤ 0 .0 5 ) ;这种关系有助于判断喉癌的恶性程度及预后。 PCR- SSCP检测喉癌突变 ,具有敏感、简便、重复性好 ,快速而经济等优点 ,是一种适用于临床样本筛选的方法。     

A de novo PABPN1 germline mutation in a patient with oculopharyngeal muscular dystrophy

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominantly inherited disorder characterized by dysphagia, ptosis, and proximal limb weakness and is caused by germline mutations (triplet repeat expansions) in the polyadenylate binding protein nuclear 1 (PABPN1) gene. OBJECTIVE: To describe a 70-year-old female patient with OPMD on the clinical and molecular genetic level and to develop a rapid and efficient molecular genetic screening method to study large patient groups. METHODS: Detailed family history and clinical assessment of the OPMD patient were followed by mutation analysis of the PABPN1 gene by direct DNA sequencing and by our newly developed method, fluorescent PABPN1 polymerase chain reaction (PCR) product (flPPP) method. A cohort of 50 healthy Swiss probands was screened using the flPPP to assess the frequency of the (GCG)7 allele in the Swiss population. Cricopharyngeal myotomy was performed as treatment for dysphagia. RESULTS: A heterozygous (GCG)9 triplet repeat expansion in PABPN1 was identified. Since the family history proved to be negative, the mutation is likely to have occurred de novo. The frequency of the (GCG)7 allele among healthy Swiss controls amounted to 1%. The flPPP method showed a sensitivity and specificity of 100%. Two years after cricopharyngeal myotomy, the patient is still relieved of dysphagia. CONCLUSIONS: An otolaryngologist should include OPMD in the differential diagnosis of a patient presenting with dysphagia, as this symptom can be the first sign of the disease and family history can be negative. Molecular genetic testing represents a highly accurate and rapid way to confirm the clinical diagnosis of OPMD. Cricopharyngeal myotomy relieves the patient of dysphagia in the majority of cases.     

Transforming growth factor beta 1 genotype and p16 as prognostic factors in head and neck squamous cell carcinoma

Abstract Conclusion: Transforming growth factor β1 gene (TGFβ1) genotype is a potential p16 independent prognostic factor predicting response to chemoradiotherapy in head and neck squamous cell carcinoma (HNSCC). Objectives: Expression of p16 and epidermal growth factor receptor (EGFR) has been reported to be associated with survival in HNSCC. We have previously reported that genetic polymorphism of TGFβ1 is linked with survival in HNSCC patients who have undergone chemoradiotherapy. We evaluate here whether TGFB1 genotype can serve as a prognostic factor independent of tumor p16 and EGFR expression. Methods: Expression of p16 and EGFR was studied by immunohistochemistry in tumors from 130 HNSCC patients. Peripheral blood DNA was used to genotype 95 patients for single nucleotide polymorphism rs1800470 within the TGFβ1 gene. The minimum follow-up time was 31 months. Results: p16 overexpression was associated with an improved disease-free survival (hazard ratio (HR) = 0.39, 95% CI 0.19-0.78), whereas no evident association was observed between EGFR expression and disease-free survival (HR = 0.90, 95% CI 0.68-1.19). Among the 37 patients who had received chemoradiotherapy, TGFβ1 genotype was associated with disease-free (HR = 0.44, 95% CI 0.19-1.02) and overall survival (HR = 0.31, 95% CI 0.12-0.80) independent of tumor p16 expression.     

Expression of p16, nm23-H1, E-cadherin, and CD44 gene products and their significance in nasopharyngeal carcinoma

OBJECTIVE: The present study was aimed to determine whether p16/MTS1, nm23-H1, E-cadherin, and CD44 proteins were expressed in nasopharyngeal carcinoma (NPC) and whether those expressions were pathologically significant in the progress of NPC. METHOD: We examined non-cancerous nasopharyngeal mucosa (20 cases) and NPC (80 cases) using immunohistochemistry with six different types of monoclonal antibodies against p16, nm23-H1, E-cadherin, CD44H, CD44v3, and CD44v6 proteins. RESULTS: The results showed that 1) the rates of positive p16 protein expression and of preserved E-cadherin protein expression in NPC were significantly lower than those in non-cancerous tissue (P <.01); 2) no significant difference in the rate of positive expression of nm23-H1, CD44H, CD44v3, and CD44v6 proteins were observed between non-cancerous nasopharyngeal mucosa and NPC; 3) no significant difference in the expression of those proteins were found by respective correlation analyses of sex, stage, and size of primary tumor in NPC; and 4) no significant difference in the rates of positive expression of CD44H, CD44v3, and CD44v6 proteins were observed in NPC between with and without lymph node metastasis, indicating that those gene products did not correlate with lymph node metastasis in NPC. However, there were inverse correlations between the expression of p16, nm23-H1, or E-cadherin protein and lymph node metastasis (P <.05), indicating that the expression of p16, nm23-H1, and E-cadherin gene were related to the carcinogenesis and tumor progression of NPC. CONCLUSION: Detecting the expressions of those gene products may provide clinically valuable information for therapeutic strategy and for predicting the prognosis of patients with NPC.     

Detection of p53 protein in advanced head and neck cancer

P53 protein expression, synonymous to p53 gene mutation, is a common event in head and neck squamous cell carcinoma (HNSCC). However, the correlation between p53 expression and clinico-pathologic parameters remains unclear. Primary tumors for analysis were obtained from 99 patients with HNSCC. The expression of p53 was analyzed with monoclonal D07 antibody (Dako Corp., Glostrup, Denmark). Seventy five percent of 79 valid cases expressed the protein. A correlation was found between alcohol intake and p53 expression. None of the other clinico-pathologic factors were associated to p53 expression. Our results suggest that p53 may be involved in the development of HNSCC. However its clinical significance is still not completely understood.     

A Comparative Study of Eya1 and Eya4 Protein Function and Its Implication in Branchio-oto-renal Syndrome and DFNA10

Allele variants of EYA1 and EYA4, two members of the vertebrate Eya gene family, underlie two types of inherited human deafness, branchio-oto-renal (BOR) syndrome and DFNA10, respectively. To clarify how mutations in these two genes and their encoded proteins impact the normal biology of hearing, we completed a number of functional studies using the yeast-two-hybrid system. We verified that bait constructs of the homologous region (Eya1HR and Eya4HR) interact with Six1 prey constructs, although no interaction with Dach1 prey was demonstrable. To compare interaction affinities, we evaluated -galactosidase activity after cotransformation of Eya1HR/Six1 and Eya4HR/Six1 and found that the latter interaction was weaker. By immunofluorescence staining, we showed Eya4HR localization to the cytoplasm. After coexpression of Six1, Eya4HR was translocated to the nucleus. Results with Eya1HR were similar. Translation of mutant constructs (Eya4HR _R564X and Eya1HR _R539X) could not be demonstrated. Using dual Eya-containing constructs (with two wild-type alleles or wild-type and mutant alleles), we confirmed no translation of the mutant allele, even if the mutation was nontruncating. These results are consistent with clinical data and implicate haploinsufficiency as the cause of BOR syndrome and DFNA10.     

The clinical impact of p16 status in fine-needle aspirates of cervical lymph node metastasis of head and neck squamous cell carcinomas

Lymph node involvement is prognostically the most determinant clinical factor for patients with head and neck squamous cell carcinomas (HNSCCs). Ultrasound of the neck and fine-needle aspiration (FNA) cytology is one of the first diagnostic procedures and the most accurate diagnostic staging tool for the neck. Patients with HPV-positive oropharyngeal carcinomas (OPSCC) show a significantly better prognosis when compared with HPV-negative OPSCC. P16 overexpression is accepted as surrogate marker for HPV-positive in OPSCC. These HPV/p16-positive OPSCC are localized either in the palatal tonsils or the base of tongue and frequently present with lymph node metastases. We analyzed the correlation and reliability of p16 expression of the FNA of the lymph node metastasis with the immunohistochemical expression of p16 of the same lymph node metastasis and its corresponding primary tumor, as it could be of importance for determining the localization and different prognosis of the primary tumor. 54 HNSCC patients were evaluated, p16 expression of the primary tumors and their lymph node metastases correlated precisely. In 25 of the 54 HNSCC patients, a FNA of the lymph node metastases was taken before the treatment. The positive cytological and immunohistochemical p16 staining correlated exactly. Of the 17 histologically p16-negative lymph node metastases 15 FNA were p16-negative, whereas two samples were p16-positive. In our view, a cytological p16 analysis of cervical lymph node metastasis can facilitate the correct localization of the primary tumor and discriminate reliably HPV-positive OPSCC from HPV-negative HNSCC with their significantly diverse prognosis.     

Cyclin D1 amplification and p16(MTS1/CDK4I) deletion correlate with poor prognosis in head and neck tumors

OBJECTIVES/HYPOTHESIS: Cyclin D1, a cell cycle regulator localized to chromosome 11q13, is amplified in several human tumors including head and neck squamous cell carcinoma (HNSCC). Amplification and/or overexpression of cyclin D1 have been correlated to a poor prognosis. Deletion of the p16 gene, localized to 9p21, has also been observed in a significant proportion of HNSCC. The p16 gene regulates cyclin D1-CDK4 activity and prevents retinoblastoma tumor suppressor gene phosphorylation, thereby downregulating cellular proliferation. Detection of cyclin D1 amplification and p16 deletion using a simple and sensitive method will be valuable for the development of effective treatment modalities for head and neck cancer. STUDY DESIGN: We have used fluorescence in situ hybridization (FISH) to study cyclin D1 amplification and p16 gene deletion in head and neck tumors. Both single- and dual-color FISH were performed. METHODS: Paraffin-embedded tissues from 103 patients with HNSCC were analyzed using genomic DNA probes for cyclin D1 and p16. Dual-color FISH was performed with chromosome 11 or 9 centromeric probes as a control. Twenty-eight of these samples were analyzed for p16 expression by immunohistochemistry. RESULTS: Cyclin D1 amplification was observed in 30% (31/103) of patients, and p16 deletion in 52% (54/103). Lack of p16 expression was observed in 64% (18/28) of patients. There was a good correlation between the deletion of p16 sequences and the loss of p16 expression (P = .008). Amplification of cyclin D1 had a statistically significant association with recurrence, distant metastasis, and survival at 36 months. There was a significant association between p16 deletion and the development of distant metastases. Cyclin D1 amplification and p16 deletion together correlated with recurrence, distant metastasis, and survival. CONCLUSIONS: We demonstrate that FISH is a simple and sensitive method for detecting cyclin D1 amplification and p16 deletion in head and neck cancer. Our results suggest that these two genetic aberrations together portend a poorer outcome than either of the abnormalities alone in head and neck cancer.     

Homozygous EDNRB mutation in a patient with Waardenburg syndrome type 1

Objective

To examine and expand the genetic spectrum of Waardenburg syndrome type 1 (WS1).

Overexpression of p53-related proteins predicts rapid growth rate of head and neck cancer

OBJECTIVES/HYPOTHESIS: The p53 tumor suppressor gene plays an important role for cell cycle regulation and is the most frequent mutated gene in head and neck cancer. Controversy remains regarding the biological and clinical value of immunohistochemical identification of the proteins accumulated in association with inactivation of the p53 gene and increased tumor growth. Therefore, the objective of the present study was to perform a cell kinetic analysis of cases with untreated squamous cell carcinoma and to compare the result with immunostaining for p53-related proteins in the tumor cells. STUDY DESIGN: A prospective series of 32 patients presenting with various stages of untreated squamous cell carcinoma of the head and neck were included. Bromodeoxyuridine (BrdU) was injected as a tracer dose before tumor biopsy for cell kinetic analysis, and p53 protein accumulation was detected using two antibodies (DO7 and PAb 1801). RESULTS: Antibody DO7 showed the highest and the optimal immunoreactivity. Diploid tumors were found in 27 cases (84%), and the mean potential doubling time (Tpot) was 55 +/- 7 hours for these tumors. Positivity of DO7 (>1%) was demonstrated in 85% of the cases. However, a discrimination level exceeding 20% was required to obtain a significant negative relationship (Spearman's rank correlation coefficient test, P < or = .03) between Tpot and DO7 positivity. At that level, 33% of the tumors remained DO7-positive. The corresponding Tpot was not significantly different from the overall mean. The rates of metastatic disease and survival were not dependent on DO7 immunoreactivity or cancer cell kinetics. CONCLUSION: Accumulation of p53-related proteins is associated with an unrestrained growth of head and neck cancer.