Establishment and characterization of an orthotopic sinonasal squamous cell carcinoma mouse model.

BACKGROUND: Despite therapeutic improvements, patients with sinonasal squamous cell carcinoma (SCC) still face an unfavorable prognosis and there is great need for alternative treatments. METHODS: SCCNC4 cells, originally derived from a T2N1M0 primary and untreated sinonasal SCC, were inoculated in the maxillary sinus of immunodeficient mice. Histology, invasive behavior, and genetic features were evaluated and compared with the original primary tumor. RESULTS: The mice developed tumors that invaded bone, surrounding tissues, and brain, showing the same poor differentiation as the original primary tumor. Genetic analysis revealed an almost identical pattern of copy number alterations, except for the deletion and loss of expression of the genes CDKN2A and PTEN. CONCLUSION: This article shows the feasibility of an orthotopic mouse model of SCC of the maxillary sinus. Completed by genome-wide genetic profiling data, this model will be useful for preclinical testing of specific gene-targeted anticancer drugs.

Hypomethylation of LINE-1, and not centromeric SAT-α, is associated with centromeric instability in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a tumour type that generally carries very complex chromosomal aberrations. An interesting feature is the elevated occurrence (58 %) of whole arm translocations and isochromosomes, resulting from breakage and illegitimate recombination in centromeric or pericentromeric regions. We hypothesized that alterations in DNA methylation may play a role in the breakage of centromeric repeat sequences in these tumours. METHODS: We studied the DNA methylation status of global repeats (LINE-1), subtelomeric repeats (D4Z4) and centromeric repeats (SAT-α) in relation to centromeric instability in a series of HNSCC cancer cell lines and primary tumours. We analysed the methylation status by pyrosequencing and the chromosomal aberrations by microarray CGH. RESULTS: We found a significant association between centromeric instability and hypomethylation of LINE-1, but not D4Z4 and SAT-α. CONCLUSION: These data suggest that centromeric instability is associated with genomic DNA hypomethylation only when occurring at specific DNA repeat sequences.

Wood dust-related mutational profile of TP53 in intestinal-type sinonasal adenocarcinoma.

Intestinal-type sinonasal adenocarcinoma represents 8% to 25% of all malignant sinonasal cancer and is etiologically related to occupational exposure to wood dust. Despite its clear etiology, the mechanisms behind the carcinogenic effects of wood dust are unclear. Because it is known that carcinogens can leave specific mutational fingerprints, we aimed to analyze the spectrum of TP53 mutations and to relate the findings to the wood dust etiology of the patients. Forty-four primary tumors were examined for TP53 mutations by direct sequencing. In addition, p53 protein expression was analyzed by immunohistochemistry using a tissue microarray consisting of 92 tumors. We report a frequency of 41% (18/44) TP53 mutations and 72% (66/92) p53 immunopositivity in intestinal-type sinonasal adenocarcinoma, significantly related to wood dust, but not to tobacco etiology. G→A transition (50%, 9/18 cases) was the most common alteration detected, almost exclusively found in nonsmokers, whereas G→T (27%, 5/18 cases) was detected in smokers only. These data point to wood dust exposure as the causal factor in the mutagenesis of TP53, possibly caused by reactive nitrogen species generated through a chronic inflammatory process.

Genetic profile of second primary tumors and recurrences in head and neck squamous cell carcinomas.

BACKGROUND: Second primary tumors and recurrences are an important problem in patients with head and neck squamous cell carcinoma. The purpose of this study was to determine the genetic changes in tumor samples to improve knowledge of tumor progression. METHODS: Copy number changes of 37 genes were analyzed by multiplex ligation-dependent probe amplification (MLPA) in 36 primary tumors and their corresponding 21 second primary tumors and 15 recurrences. RESULTS: CCND1 and EMS1 amplifications and gain of BCL2L1 were the most common genetic alterations in the primary tumor, second primary tumor, and recurrence samples. Gains of ERBB2 and PTPN1 were associated with recurrences. CONCLUSION: Specific genetic profiles for each group have been found. Similarities between primary tumor and second primary tumor and dissimilarity between primary tumor and recurrence suggest that clinicopathological criteria do not always accurately differentiate these entities. Genetic profiling may aid in the diagnosis and prognosis of these difficult cases.

Gene amplification and protein overexpression of EGFR and ERBB2 in sinonasal squamous cell carcinoma.

BACKGROUND: Sinonasal squamous cell carcinomas (SNSCCs) are rare tumors with no etiologic link to tobacco or alcohol, as opposed to other squamous cell carcinomas of the head and neck. Despite improvements in the field of surgery and radiotherapy, patients with these tumors still face a very unfavorable prognosis, partly because of their localization in a complex anatomic area, which has special relevance for surgery and postoperative treatment. Therefore, there is a need for new therapeutic possibilities for patients with these tumors. METHODS: Gene copy numbers of epidermal growth factor receptor (EGFR) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2) were analyzed by fluorescence in situ hybridization and multiplex ligand-dependent probe amplification, and protein expression was evaluated by immunohistochemistry in 54 SNSCC specimens. The results were correlated with clinicopathologic and follow-up data. RESULTS: EGFR gene copy number increases were observed in 20 of 45 tumors (44%), and 21 of 54 tumors (39%) had EGFR protein overexpression. Eight of 38 tumors (21%) had ERBB2 copy number increases, and 4 of 54 tumors (7%) exhibited elevated protein expression levels. Both copy number increases and protein overexpression of EGFR and ERBB2 were mutually exclusive. v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were absent in 37 tumors that were analyzed. CONCLUSIONS: A substantial proportion of SNSCCs carried alterations in EGFR or ERBB2. Together with the absence of KRAS mutations, these findings indicate that therapies targeting these molecules may be promising additions to the therapeutic options for patients with SNSCC.

Genetic model of transformation and neoplastic progression in laryngeal epithelium.

BACKGROUND: The aim of this study was to analyze genetic alterations in the transformation-progression model of laryngeal tumors. METHODS: Copy number changes of 37 genes were analyzed by multiple ligation-dependent probe amplification (MLPA) in 94 tissue samples. RESULTS: In the smoker normal mucosa group TP53 loss was predominant, whereas in the precursor lesions CDKN2A loss and CDKN2D gain were most frequent. Precursor lesions with progression presented CTNNB1 loss. In the carcinoma group the most common changes were CDKN2A, MLH1, CTNNB1, and CASP6 losses and RECQL4, CCND1, and EMS1 gains. Positive lymph node primary tumors were related to TP53, IL1A, and RB1 losses and STK11 gain. The lymph node metastases differed from their corresponding primary tumor in LMNA, RECQL4, and IGF1R losses, and N33 and CDKN2D gains. CONCLUSIONS: Genetic changes and new key genes were found to be associated with specific steps. We included new steps, not presented in the classic models: normal mucosa tobacco exposed, positive lymph node primary tumor, and corresponding lymph node metastases.

The usefulness of new serum tumor markers in head and neck squamous cell carcinoma.

OBJECTIVE: To determine the usefulness of specific and reliable serum biomarkers to predict cervical lymph node metastasis. METHODS: A cross-sectional study of cases and controls. Thirty-nine serum samples of head and neck squamous cell carcinoma were collected from patients during neoplasm resection. Another 10 serum samples were collected from healthy individuals as a control group. Selected serum biomarkers were E-cadherin, MMP-2, MMP-9, active MMP-13, and p53 autoantibodies. RESULTS: We found a correlation between active MMP-13 (>685 pg/mL; ROC curve analysis 95% CI for sensitivity 79.6-99.3; specificity 49.2-95.1; positive predictive value 65-100; and negative predictive value 36-100) as well as the presence of p53 autoantibodies and lymph node metastasis. Multimarker analysis using MMP-13 and p53 autoantibodies together provided better sensitivity (76%) and specificity (100%). CONCLUSIONS: The combined determination of active MMP-13 and p53 autoantibodies could improve diagnosis of lymphatic metastasis in head and neck squamous cell carcinoma and aid therapeutic decision making.

Deletions of N33, STK11 and TP53 are involved in the development of lymph node metastasis in larynx and pharynx carcinomas.

BACKGROUND: Lymph node metastasis is the mayor cause of mortality in patients with head and neck squamous cell carcinomas (45%). The genetic changes underlying metastasis are still largely unknown and genetic markers to predict lymph node positivity still need to be found. The aim of this study was to search such markers by using Multiplex Ligation-dependent Probe Amplification (MLPA), a semi-quantitative PCR technique to detect gene copy number alterations. METHODS: Thirty-seven genes were analysed by MLPA in 34 larynx and 22 pharynx carcinomas. RESULTS: Losses of CDKN2A (9p21) and MLH1 (3p22) and gains of CCND1, EMS1 (both at 11q13), RECQL4 and PTP4A3 (both at 8q24) were the most frequent aberrations in both larynx and pharynx carcinomas. Amplifications were detected at EMS1, CCND1 and ERBB2 (17q21). A correlation between loss of N33 (8p22) and poor survival was found (p=0.02). Gain of EMS1 had the same relation with survival but not significant (p=0.08). Lymph node positive tumors presented a specific pattern of genetic alterations, with losses of N33, STK11 (19p13) and TP53 (17p13), the latter especially in larynx tumors. CONCLUSION: We propose that these 3 genes might play a role in the development of metastasis in larynx and pharynx squamous cell carcinomas.

Chromosomal changes in relation to clinical outcome in larynx and pharynx squamous cell carcinoma.

Invasive head and neck squamous carcinomas are among the cytogenetically most complex tumors. Perhaps for this reason, there is little consensus on the prognostic value of specific chromosomal aberrations. Here we present results of CGH analysis of 56 clinically well-characterized set of head and neck cancers, consisting of larynx and pharynx only. The aim was to find possible associations with clinical outcome. The major chromosome arms showing gains were (in decreasing order): 3q, 7q, 8q, 5p, 11q13, 17q and 18p, and losses occurred at 3p, 11qter, 4p, 18q, and 5q. The segments most frequently amplified were 3q26-qter, 11q13, 11q22, 3q12-13, 18p11.3, 18q11.2 and 8q24.3. Tumors with stages III and IV, and lymph node positive tumors had a worse clinical outcome. Surprisingly, no specific chromosomal abnormality correlated with disease-free survival. The only aberration that correlated to one of the clinico-pathological parameters was amplification 11q13, that occurred solely in lymph node positive, stage IV tumors. However 11q13 amplification did not correlate with disease-free survival. These results seem to indicate that genetic alterations at the level of chromosomes have limited prognostic value in patients with invasive larynx and pharynx squamous cell carcinomas.