TP53 mutation and human papilloma virus status of oral squamous cell carcinomas in young adult patients.

OBJECTIVE: Little is known about the molecular carcinogenesis of oral squamous cell carcinoma (OSCC) in young adult patients. The aim of this study was to investigate the detailed TP53 mutation and human papilloma virus (HPV) status of OSCC in patients, younger than 45 years. METHODS: TP53 mutations were determined with direct sequencing on paraffin-embedded carcinoma tissue from 31 young patients and compared with two older age OSCC reference groups: one from the same institute (N = 87) and an independent one (N = 675). Biologically active tumour HPV was detected by p16-immunohistochemistry followed by a HPV-DNA GP5 + /6 + -PCR. RESULTS: HPV16 was present in one OSCC (3%). TP53 mutations were found in 14 (45%) OSCC: five were missense and nine resulted in a truncated protein. Six of these latter were insertions or deletions of one or more nucleotides leading to frameshift, one was at a splice site and two resulted in a stop codon. The percentage of truncating mutations (64% of all mutations) was higher than that observed in the institute's reference group (44%, P = 0.23) and in the independent reference group (24%, P = 0.002). CONCLUSIONS: This study shows that TP53 mutations are common in OSCC of young adult patients; infection with biologically active HPV is rare.

Expression signature in peripheral blood cells for molecular diagnosis of head and neck squamous cell carcinoma.

Patients with head and neck squamous cell carcinoma (HNSCC) have a poor prognosis due to the development of locoregional recurrences, distant metastases, and second primary tumors. There is an urgent need for biomarkers that enable detection and monitoring of the disease to provide adequate therapeutic strategies. In this study, we have investigated markers in peripheral blood cells (PBC) of 28 HNSCC patients who underwent surgery by means of expression profiling. Our hypothesis is that nucleated blood cells circulate continuously, also pass the tumor, and change their expression profile in response to tumor cell factors. For comparison, we enrolled a control group of 11 patients who underwent surgery in the head and neck region for non-HNSCC reasons. A set of 2949 genes was found to be statistically different between the groups (P < 0.05, false discovery rate-corrected) and the most prominently different pathways were EIF2, EIF4, and mTOR signaling. These preliminary results are promising and warrant further studies on the definitive role of PBC gene expression as a biomarker for HNSCC detection and monitoring.

Effector memory T-cell frequencies in relation to tumour stage, location and HPV status in HNSCC patients.

BACKGROUND: The immune system plays an important role in tumour immune surveillance. Head and neck squamous cell carcinoma patients are often immune compromised. OBJECTIVE: To chart the baseline levels of T-cell subpopulation frequencies in patients with cancer prior to treatment. SUBJECTS AND METHODS: Blood samples of patients were taken at the time of diagnosis, analysed with flowcytometry and compared with blood samples of healthy donors. RESULTS: Compared to healthy donors, a significant shift from naive to effector memory T cells was observed. This effect was most prominent in stage II patients. A similar shift from naive to effector memory T cells was noted in patients with oropharynx or larynx squamous cell carcinomas. Furthermore, the percentage of effector memory and effector T cells was higher in the group of patients with human papillomavirus-positive oropharyngeal squamous cell carcinomas, compared with patients with human papillomavirus-negative tumours, suggestive of virus-induced T-cell activation. CONCLUSION: Here, we provide a simple and easily implementable tool to document T lymphocyte subsets in the peripheral blood of head and neck cancer patients, which might be useful for prognosis and/or therapy response prediction.

Treatment choice for locally advanced head and neck cancers on the basis of risk factors: biological risk factors.

Patients with locally advanced head and neck squamous cell carcinoma often experience relapse, the cause of poor survival statistics. Relapse occurs following the three main types of treatment, surgery with or without post-operative (chemo)radiotherapy, or chemoradiation (containing cisplatin). Cancer relapse can result from (i) outgrowth of residual tumour cells, sometimes with a number too small to be detected by routine histopathology or (ii) development of another carcinoma in a field of pre-neoplastic cells that has remained after treatment of the primary carcinoma. At this moment, clinical staging is not enough to identify patients who will develop relapse and who need tailored treatment. This review describes the latest knowledge of mechanisms of cancer relapse, addresses the biomarkers of potential interest detectable in the tissue of the tumour or its surgical margins and discusses three biomarkers, human papillomavirus, TP53 and epidermal growth receptor in more detail. Once a marker panel has been established, treatment should be focussed on the patients at risk of relapse by improved tailoring of existing treatment modalities. Also, the implementation of more targeting therapies based on the characteristics of the discovered markers should lead to better survival rates.

Genome-wide DNA copy number alterations in head and neck squamous cell carcinomas with or without oncogene-expressing human papillomavirus.

Oncogene-expressing human papillomavirus type 16 (HPV16) is found in a subset of head and neck squamous cell carcinomas (HNSCC). HPV16 drives carcinogenesis by inactivating p53 and pRb with the viral oncoproteins E6 and E7, paralleled by a low level of mutations in TP53 and allelic loss at 3p, 9p, and 17p, genetic changes frequently found in HNSCCs of nonviral etiology. We hypothesize that two pathways to HNSCC exist: one determined by HPV16 and the other by environmental carcinogens. To define the critical genetic events in these two pathways, we now present a detailed genome analysis of HNSCC with and without HPV16 involvement by employing high-resolution microarray comparative genomic hybridization. Four regions showed alterations in HPV-negative tumors that were absent in HPV-positive tumors: losses at 3p11.2-26.3, 5q11.2-35.2, and 9p21.1-24, and gains/amplifications at 11q12.1-13.4. Also, HPV16-negative tumors demonstrated loss at 18q12.1-23, in contrast to gain in HPV16-positive tumors. Seven regions were altered at high frequency (>33%) in both groups: gains at 3q22.2-qter, 5p15.2-pter, 8p11.2-qter, 9q22-34.1, and 20p-20q, and losses at 11q14.1-qter and 13q11-33. These data show that HNSCC arising by environmental carcinogens are characterized by genetic alterations that differ from those observed in HPV16-induced HNSCC, and most likely occur early in carcinogenesis. A number of genetic changes are shared in both tumor groups and can be considered crucial in the later stages of HNSCC progression.

Expression profiling and prediction of distant metastases in head and neck squamous cell carcinoma.

BACKGROUND: For breast and prostate cancer, a gene expression signature of the tumour is associated with the development of distant metastases. Regarding head and neck squamous cell carcinoma (HNSCC), the only known risk factor is the presence of > or =3 tumour-positive lymph nodes. AIM: To evaluate whether a HNSCC gene expression signature can discriminate between the patients with and without distant metastases. METHODS: Patients with HNSCC with and without distant metastases had >3 tumour-positive lymph nodes, and did not differ with respect to other risk factors. Statistical analysis was carried out using Student's t test, as well as statistical analysis of microarrays (SAM), to assess the false discovery rate for each gene. These analyses were supplemented with a newly developed method that computed deviations from gaussian-order statistics (DEGOS). To validate the platform, normal mucosa of the head and neck was included as control. RESULTS: 2963 genes were differently expressed between HNSCC and normal mucosa (t test; p<0.01). More rigorous statistical analysis with SAM confirmed the differential expression of most genes. The comparison of genes in HNSCC with and without metastases showed 150 differently expressed genes (t test; p<0.01), none of which, however, could be confirmed using SAM or DEGOS. CONCLUSIONS: No evidence for a metastasis signature is found, and gene expression profiling of HNSCC has seemingly no value in determining the risk of developing distant metastases. The absence of such a signature can be understood when it is realised that, for HNSCC in contrast with breast cancer, the lymph nodes are a necessary in-between station for haematogenous spread.

Enhanced success rate of transplantation with human tumors in cyclophosphamide-treated nude mice.

Immunosuppression was investigated to enhance the take rate of various human tumor types in nude BALB/c or B10.LP/Cpb mice. Cyclophosphamide (CY), known as an immunosuppressive agent, was injected ip at a dose of 100 mg/kg 24 hours prior to subcutaneous implantation of tumor fragments obtained from established xenograft lines or from patients. In 3 out of 8 ovarian adenocarcinoma tumor lines, with a take of 50% or less in untreated control animals, tumor take was significantly increased by CY treatment to values between 75 and 100%. No effect of CY treatment on tumor take was seen with squamous cell carcinomas of the head and neck region and with lung and prostatic carcinomas. The growth rate of these xenografts was not affected by CY pretreatment. These results indicate that immunologic mechanisms indeed can play a role in the inhibition of tumor growth in nude mice. CY pretreatment may enhance the success rate of transplantation, but this effect appears to be limited to certain tumors.

Inherited susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes.

BACKGROUND: Susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes may reflect the way a person deals with carcinogenic challenges. This susceptibility (also referred to as mutagen sensitivity) has been found to be increased in patients with environmentally related cancers, including cancers of the head and neck, lung, and colon, and, in combination with carcinogenic exposure, this susceptibility can greatly influence cancer risk. The purpose of this study was to assess the heritability of mutagen sensitivity. METHODS: Heritability was determined by use of a maximum likelihood method that employed the FISHER package of pedigree analysis. Bleomycin-induced breaks per cell values for 135 healthy volunteers without cancer were determined. These individuals were from 53 different pedigrees and included 25 monozygotic twin pairs (n = 50), 14 pairs of dizygotes (twin pairs and siblings, n = 28), and 14 families selected on the basis of a first-degree relative who was successfully treated for head and neck cancer and who had no sign of recurrence for at least 1 year. All data were analyzed simultaneously, and different models of familial resemblance were fitted to the data. All P values are two-sided. RESULTS: Our results showed no evidence for the influence of a shared family environment on bleomycin-induced chromatid breaks. Genetic influences, however, were statistically significant (P =. 036) and accounted for 75% of the total variance. CONCLUSIONS: The high heritability estimate of the susceptibility to bleomycin-induced chromatid breaks indicates a clear genetic basis. The findings of this study support the notion that a common genetic susceptibility to DNA damage--and thereby a susceptibility to cancer--may exist in the general population.

Genetic susceptibility to head and neck squamous cell carcinoma.

BACKGROUND: In addition to influences of exposure to carcinogenic compounds, the development of cancer may depend on an individual intrinsic cancer susceptibility. Biomarkers for cancer susceptibility can be powerful additions to epidemiologic analyses. PURPOSE: This multicenter, case-control analysis combines previously published data and new data to substantiate the value of mutagen sensitivity as a biomarker of susceptibility to head and neck squamous cell carcinoma and, more importantly, to gain insight into the interaction between susceptibility and exposure to carcinogens. METHODS: Mutagen sensitivity (mean number of chromatid breaks per cell of cultured lymphocytes treated with bleomycin in the late S-G2 phase of the cell cycle) was determined in 313 patients with head and neck cancer and in 334 control subjects at two major U.S. medical institutions and one European institution, yielding a unique study population. The ages of the case and control subjects, as well as their history of use of tobacco and alcohol, were also recorded. The relationships between variables were analyzed by use of Student's t tests, Spearman's rank correlations, and multiple linear regression. For estimation of cancer risk, crude odds rations (ORs) were measured and multiple logistic regression was performed. All P values were based on two-sided tests. RESULTS: There were no differences across institutions in the distribution of mutagen sensitivity (Kruskal-Wallis test) for both case subjects and control subjects. Values for case subjects were consistently and significantly (P<.0001) higher than values for control subjects in the overall analyses. Age and tobacco or alcohol use did not influence the outcome in terms of mutagen-sensitivity values for either the case or the control subjects. A mean number of breaks per cell dichotomized at 1.0 was found to be the best predictor of a hypersensitive phenotype. For nonsensitive, heavy smokers, the OR was 11.5 (95% confidence interval [CI] = 5.0-26.6). This risk increased dramatically in mutagen-hypersensitive, heavy smokers to 44.5 (95% CI = 17.4-114.0). Multiple logistic regression analysis confirmed these results, and a significant trend was found (P<.01) for the dose-dependent increase in cancer risk by smoking. The consumption of alcohol potentiated the effects of smoking, resulting in an OR of 57.5 (95% CI = 17.5-188.0) in hypersensitive persons. CONCLUSIONS: Mutagen sensitivity was found to be a biomarker of cancer susceptibility. This study underscores the importance of utilizing both susceptibility markers and the exposure data for the identification of persons at high risk of developing cancer. IMPLICATIONS: More accurate risk estimation can define susceptible subgroups who might be targeted for intensive behavioral interventions, surveillance through screening, and enrollment in chemoprevention programs.

Preclinical in vivo activity of 2',2'-difluorodeoxycytidine (Gemcitabine) against human head and neck cancer.

2',2'-Difluorodeoxycytidine (dFdCyd, Gemcitabine) is a new deoxycytidine analogue with striking preclinical antitumor activity in solid tumors from murine and human origin. In this study, dFdCyd was tested for its antitumor effect in human tumor xenografts derived from squamous cell carcinoma of the head and neck (SCCHN). NMRI nude mice bearing s.c. growing tumors with a volume of 50 to 150 mm3 were given i.p. injections of a maximum tolerated dose of 120 mg/kg dFdCyd, every 3 days for four injections. A significant antitumor effect was observed in all five tested SCCHN tumor lines; in four of these lines the median tumor volume doubling time increased more than a 3-fold upon dFdCyd treatment. In two lines dFdCyd was curative (no tumor regrowth 90 days after treatment) in one of six and two of eight xenografts, respectively. Schedule dependency was investigated in three SCCHN lines, showing, in the two most sensitive lines, that treatment with a 3-day interval was superior to the schedules with daily or weekly injections. At equitoxic doses, dFdCyd was more active in this model than the drugs that are clinically used in SCCHN, i.e., cisplatin, methotrexate, 5-fluorouracil, and cyclophosphamide. dFdCyd is a good candidate for clinical trials with SCCHN patients.

Phase II preclinical drug screening in human tumor xenografts: a first European multicenter collaborative study.

In a European joint project carried out in 6 laboratories a disease-oriented program was set up consisting of a panel of 7 tumor types, each represented by 4 to 8 different human tumor lines, for secondary screening of promising anticancer drugs. Human tumor lines were selected on the basis of differences in histology, growth rate, and sensitivity to conventional cytostatic agents. Xenografts were grown s.c. in nude mice, and treatment was started when tumors reached a mean diameter of 6 mm in groups of mice where at least 6 tumors were evaluable. Drugs were given at the maximum tolerated dose. For evaluation of drug efficacy, median tumor growth curves were drawn, and specific growth delay and treated/control x 100% were calculated. Doxorubicin (8 mg/kg i.v. days 1 and 8) was effective (treated/control < 50%, and specific growth delay > 1.0) in 0 of 2 breast cancers, 1 of 3 colorectal cancers, 2 of 5 head and neck cancers, 3 of 6 non-small cell lung cancers, 4 of 6 small cell lung cancers, 0 of 3 melanomas, and 3 of 6 ovarian cancer lines. Amsacrine (8 mg/kg i.v. days 1 and 8) was not effective, while datelliptium (35 mg/kg i.p. days 1 and 8) was active against 2 of 6 small cell lung cancer lines. Brequinar sodium (50 mg/kg i.p. days 1-5) showed efficacy in 4 of 5 head and neck cancers, 5 of 8 non-small cell lung cancers, and 4 of 5 small cell lung cancer lines. The project has been shown to be a feasible approach. Clinical activity for doxorubicin and inactivity for amsacrine against solid tumor types was confirmed in the human tumor xenograft panel. Additional anticancer drugs will be studied in the European joint project to further define the reliability of this novel, promising screening approach.

Expression of a folate binding protein in L1210 cells grown in low folate medium.

We have isolated variants of L1210 cells (L1210B) expressing, in addition to the "classical" high affinity/low capacity system for reduced folate uptake, high levels of a membrane-associated folate binding protein. This folate binding protein was expressed in L1210 cells grown at low physiological folate levels (less than 0.5 nM), but down-regulated after transfer in standard high folate (2 microM) medium. The binding capacity of L1210B cells for [3H]folic acid and [3H]-methotrexate was identical (5-11 pmol/10(6) cells) but affinities were different. The affinities relative to folic acid were 0.5 for 5-methyltetrahydrofolate, 0.25 for 5-formyltetrahydrofolate, 0.08 for 10-ethyl-10-deazaaminopterin, and 0.05 for methotrexate, respectively. L1210B cells exposed to low extracellular concentrations of [3H]folic acid (25 nM) accumulated 15 pmol [3H]folic acid/10(7) cells over a 5-h period. [3H]Folic acid accumulation by wild-type L1210 cells could not be demonstrated under these conditions. The folate binding protein in L1210B cells could be specifically and covalently labeled at 4 degrees C with a N-hydroxysuccinimide ester of [3H]-methotrexate or [3H]folic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of detergent-solubilized membrane proteins showed a major labeled band with Mr 42,000-44,000.

Moving towards personalised therapy in head and neck squamous cell carcinoma through analysis of next generation sequencing data.

Personalised medicine tumour boards, which leverage genomic data to improve clinical management, are becoming standard for the treatment of many cancers. This paper is designed as a primer to assist clinicians treating head and neck squamous cell carcinoma (HNSCC) patients with an understanding of the discovery and functional impact of recurrent genetic lesions that are likely to influence the management of this disease in the near future. This manuscript integrates genetic data from publicly available array comparative genome hybridization (aCGH) and next-generation sequencing genetics databases to identify the most common molecular alterations in HNSCC. The importance of these genetic discoveries is reviewed and how they may be incorporated into clinical care decisions is discussed. Considerations for the role of genetic stratification in the clinical management of head and neck cancer are maturing rapidly and can be improved by integrating data sets. This article is meant to summarise the discoveries made using multiple genomic platforms so that the head and neck cancer care provider can apply these discoveries to improve clinical care.

Considerations for in vitro retinoid experiments: importance of protein interaction.

Retinoids, natural and synthetic substances structurally related to vitamin A, are important modulators of cell proliferation and differentiation, and have proven activity in cancer therapy. Experiments to reveal the mechanism of action of retinoids are routinely performed in in vitro models. As retinoids are relatively hydrophobic and unstable, we hypothesized that the composition of culture media is of critical importance for the stability and bioavailability of these compounds. Various culture media were incubated with all-trans-, 13-cis- and 9-cis-retinoic acid (RA). Without fetal calf serum (FCS) or bovine serum albumin (BSA) in the medium, the concentration of these retinoids was found to decrease to considerably low levels. This excessive loss of retinoids was due to absorption to culture plates, reaction tubes and pipet tips. Binding of retinoids to BSA was demonstrated to have attenuating effects on uptake and metabolism of all-trans-RA, as studied in oral keratinocytes and head and neck cancer cells, indicating that a balance exists between the bioavailability and the aspecific loss of retinoids. In this study we demonstrate that the type of culture medium and especially the presence of protein in the medium is of paramount importance to perform reproducible experiments with retinoids.

Enhanced turnover of all-trans-retinoic acid and increased formation of polar metabolites in head and neck squamous cell carcinoma lines compared with normal oral keratinocytes.

Retinoids show promise in the treatment of various (pre)malignancies, including head and neck squamous cell carcinoma (HNSCC). Previous studies have shown that the metabolic pathways of retinoids are important in the anticancer effect of retinoids, and that these pathways may change during carcinogenesis. In the present study, we analyzed HNSCC cell lines (n = 11) and normal oral keratinocyte cultures (n = 11) by reverse-phase high-performance liquid chromatography and conducted growth inhibition assays. We demonstrate here that in contrast to normal oral keratinocytes, HNSCC cell lines: (a) had averaged a 17-fold greater turnover rate of all-trans-retinoic acid (RA); (b) had a 1.9-fold less RA-induced growth inhibition; (c) were able to form polar metabolites; and (d) were able to catabolize 4-oxo-RA. Furthermore, the mRNA expression of the RA-specific 4-hydroxylase, CYP26A1, was dramatically increased after RA-induction in the two HNSCC cell lines with the highest metabolism, was undetectable in normal keratinocytes, and was not inducible by RA. Next, introduction of CYP26A1 cDNA in a low-metabolizing HNSCC cell line resulted in an 11-fold higher turnover rate of RA and a 12-fold increase in the amount of polar metabolites, but it did not change sensitivity to RA. These observations point to fundamental changes in RA metabolism pathways during HNSCC carcinogenesis and may provide clues to a more rational approach for RA-mediated intervention.

Persistence of genetically altered fields in head and neck cancer patients: biological and clinical implications.

In 1953, Slaughter et al. [D. P. Slaughter et al., Cancer (Phila.), 6: 963-968, 1953] proposed the concept of field cancerization in patients with squamous cell carcinoma of the head and neck (HNSCC) and discussed its clinical significance for the development of second primary tumors and local recurrences. To define the process of field cancerization and its putative clinical implications, we analyzed genetic aberrations in HNSCC and the accompanying macroscopically normal mucosa. In 28 HNSCC patients, loss of heterozygosity was determined in tumor and five noncontiguous mucosal biopsies using eight microsatellite markers at 9p, 3p, and 17p. For patients who showed loss of heterozygosity in their mucosal biopsies, all margins of the surgical specimen were subsequently analyzed to determine the extension of the field. In these cases, additional markers at 8p, 13q, and 18q as well as p53 mutations were included to determine subclonal differences between field and tumor. Genetically altered fields were detected in 36% (10 of 28) of the HNSCC patients. The field varied in size between patients and consisted of genetically different subclones. In 7 of 10 cases, the field extended into the surgical margins. One particular patient with a genetically altered field in a surgical margin developed a local recurrence after 28 months of follow-up. Microsatellite analysis showed that this recurrence had more molecular markers in common with the nonresected premalignant field than with the original tumor, suggesting that this persistent field has progressed further into a new malignancy. Our data show that genetically altered mucosa remains after treatment in a significant proportion of HNSCC patients, which may explain in part the high frequency of local recurrences and second primary tumors. Adequate identification and risk assessment of these genetically altered fields may have profound implications for future patient management.

Quantitative diffusion-weighted MRI parameters and human papillomavirus status in oropharyngeal squamous cell carcinoma.

BACKGROUND AND PURPOSE: Patients with human papillomavirus-positive oropharyngeal squamous cell carcinomas have a better survival rate than those with human papillomavirus-negative oropharyngeal squamous cell carcinomas. DWI characterizes biologically relevant tumor features, and the generated ADC may also provide prognostic information. We explored whether human papillomavirus status and ADC values are independent tumor characteristics. MATERIALS AND METHODS: Forty-four patients with oropharyngeal squamous cell carcinomas underwent pretreatment DWI. ADC values for the primary tumors were determined by using 3 b-values in an ROI containing the largest area of solid tumor on a single section of an axial DWI image. Human papillomavirus status was determined with p16 immunostaining, followed by high-risk human papillomavirus DNA detection on the p16-positive cases. RESULTS: Twenty-two patients were human papillomavirus-positive (50.0%). ADC values were not significantly different between human papillomavirus-negative (ADC(mean) = 1.56 [1.18-2.18] × 10(3) mm(2)/s) and human papillomavirus-positive tumors (ADC(mean) = 1.46 [1.07-2.16] × 10(3) mm(2)/s). CONCLUSIONS: No significant association between ADC and human papillomavirus status was found in oropharyngeal squamous cell carcinomas. In our study population, differences in genetic and histologic features between human papillomavirus-positive and human papillomavirus-negative oropharyngeal squamous cell carcinomas did not translate into different ADC values. Long-term follow-up studies are needed to establish whether ADC has prognostic value and whether this is independent of the human papillomavirus status.

The relation between cancer incidence among relatives and the occurrence of multiple primary carcinomas following head and neck cancer.

Despite improvement in therapeutic modalities in head and neck squamous cell carcinoma (HNSCC) the overall survival rate has only marginally improved during the last decades. The occurrence of second primary tumors (SPTs) in the respiratory and upper digestive tract (RUDT) is the main cause of treatment failure in early stage HNSCC. Identification of risk factors for the development of SPT by epidemiological analysis may lead to better risk assessment in individual cases. Ninety-seven HNSCC patients who ultimately developed SPTs and 100 HNSCC patients who remained free of other carcinomas after treatment of the first for a minimal period of 6 years were interviewed about the incidence of RUDT carcinomas within parents and siblings. All questioned patients were smokers. Among the SPT-positive patients, 50 (8.9%) of the 562 family members were reported to have had cancer of the respiratory or upper digestive tract versus 16 (2.5%) of the 629 family members of the SPT-negative patients. This difference was statistically significant (P < 0.0001) with the stratified version of Fisher's exact test. All these 66 probands with RUDT cancer were smokers, and the percentages of smokers were similar in both proband groups. Neither age and sex of the patient, nor tumor stage influenced the occurrence of SPTs in this study. The percentages of probands with tumors outside the RUDTs were almost similar, 8.0 and 7.0% in the SPT-positive and -negative groups, respectively. Having one or more relatives with RUDT cancer was established as a risk factor (odds ratio, 3.8; 95% confidence interval, 2.0-7.6) for patients with initial HNSCC to develop an SPT. These findings suggest that, in addition to external carcinogens, an intrinsic susceptibility may influence the risk for the development of SPTs in HNSCC patients.

Lack of effect of daily N-acetylcysteine supplementation on mutagen sensitivity.

The European Organization for Research and Treatment of Cancer multicenter Euroscan trial was set up to prevent the occurrence of second primary tumors in the upper aerodigestive and respiratory tract in patients cured for early stage head and neck squamous cell carcinoma. One randomized group of patients receive daily N-acetylcysteine, an antioxidant that may be protective especially in the early steps of carcinogenesis. Mutagen sensitivity, measured as sensitivity to bleomycin in peripheral blood lymphocytes, has been found to be increased in head and neck squamous cell carcinoma and is hypothesized to reflect cancer susceptibility. The aim of this study was to investigate whether mutagen sensitivity is influenced by oral N-acetylcysteine supplementation and can therefore be used as intermediate end point in chemoprevention. Patients (n = 19) who had various periods of N-acetylcysteine supplementation (600 mg daily for 3-9 months) were analyzed. In addition, a patient group (n = 14) that did not receive N-acetylcysteine supplementation was analyzed for comparison. Our results show no evidence that administration of N-acetylcysteine did influence the mutagen sensitivity level. The only explanatory variable in the analysis of the difference between two samples of one person was the b/c value of the first measurement. Moreover, the variability in these repeated measurements (coefficient of variation of 14%) indicates that additional studies should be performed to minimize this variability and to optimize the testing of mutagen sensitivity to accurately identify individual patients at high risk for the development of multiple primary tumors.

Mutagen sensitivity as a biomarker for second primary tumors after head and neck squamous cell carcinoma.

The occurrence of second primary tumors after curative treatment of early stage head and neck squamous cell carcinoma negatively influences the overall survival. Our aim was to prospectively evaluate whether mutagen sensitivity (mean number of chromatid breaks per cell in cultured lymphocytes exposed to bleomycin) could be used as a biomarker to predict which patients will develop second malignancies in the respiratory or upper digestive tract. Patients treated for head and neck squamous cell carcinoma (n = 218) were followed for approximately 6 years. Nineteen patients developed a second primary tumor, and each of these patients was matched on age, gender, cumulative smoking, tumor site, and tumor stage to two patients who did not develop any second malignancy. No difference between the groups was found with respect to mutagen sensitivity. Smoking at the time of the index tumor had a significant influence on the occurrence of second primary tumors (log-rank, P = 0.019). There was a significantly (P = 0.005) higher mean breaks-per-cell value in those patients who had developed their second primary tumor > or = 3 years after the first tumor (0.97 +/- 0.24; n = 10) compared with early second primary tumor patients (0.69 +/- 0.09; n = 9). Conditional on a more than 3-year second primary tumor-free survival (n = 38), there is a significantly (log-rank, P = 0.036) higher probability of a second primary tumor for mutagen-sensitive patients [relative risk, 7.8 (95% confidence interval, 0.99-61.74; P = 0.05)]. Mutagen sensitivity is a potential biomarker for the occurrence of 'late' second malignancies (> 3 years between tumors), and additional studies on the inclusion of this biomarker in chemoprevention trials is commendable because it would greatly improve their efficiency.