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• A conference was held to foster collaboration on sinonasal cancer research. • New tumor and immumodels were presented. • Developments in surgical and radio-oncological treatments were discussed. • Initiatives for diagnostic/genetic core facilities and cancer registry were taken. Sinonasal cancer is a clinically and histologically heterogeneous group of rare tumors with generally poor clinical outcomes. Their low incidence hampers the advancement of clinical management as well as translational research, and calls for multicenter and multinational collaboration between physicians and researchers. This report describes the proceedings of a two-day conference organized by the European Network for Sinonasal Cancer Research (EUSICA) and COST Action 'IMMUNO-model', fostering such collaboration and focusing on preclinical tumor and immuno models, surgical and radio-oncological treatments, core facilities for genetic characterization and molecular tumor classification, and cancer registry. [ABSTRACT FROM AUTHOR]

Background: Recent studies support a role for human papillomavirus (HPV) in oropharyngeal squamous cell carcinomas (SCCs); however, the significance of HPV in non-oropharyngeal head and neck cancers is uncertain. The aim of this study was to determine the prevalence of HPV in a large cohort of laryngeal and hypopharyngeal SCCs in northern Spain. Materials and methods: Clinical records and paraffin-embedded tumor specimens of 124 consecutive patients surgically treated for laryngeal (62 cases) and hypopharyngeal (62 cases) SCCs between 2002 and 2007 were retrieved. All cases were histologically evaluated, and presence of HPV was assessed by p16-immunohistochemistry followed by GP5+/6+-PCR-based DNA detection. Samples positive in both assays were subjected to HPV genotyping and HPV E6 transcript analysis. Results: Seventeen cases (14%) were positive for p16 immunostaining, of which 2 (1 larynx, 1 hypopharynx, 1.6% of total series) were found positive for HPV DNA by subsequent GP5+6+-PCR. Both SCCs contained HPV type 16 and showed HPV16 E6 mRNA expression. Conclusions: HPV is only occasionally involved in laryngeal and hypopharyngeal SCC patients in northern Spain. [ABSTRACT FROM AUTHOR]

Objectives: Head and neck squamous cell carcinomas (HNSCCs) are characterized by marked heterogeneity in their biological behavior and response to treatment. Our goal was the identification of biomarkers that can be used to predict response to chemotherapy in these patients.Materials and Methods: The expression of EGFR, p53, Cyclin D1, p16, p21, p27, p-AKT, HIF-1α, Caspase 3 and BCL2 was analyzed by immunohistochemistry in 41 primary laryngeal/hypopharyngeal squamous cell carcinomas of patients that received induction chemotherapy (cisplatin and 5-fluorouracil) as part of their treatment.Results: Positive expression of p27 and BCL2 had a significant predictive value for chemotherapy response in univariate analysis. The combination of both proteins was not superior in predicting the response to chemotherapy. Furthermore, p27 expression was the only significant predictor of chemotherapy response in multivariate analysis (P=0.015).Conclusion: p27 Expression may serve as predictive biomarker of response to induction chemotherapy in HNSCC patients. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Summary: Annexins are a structurally related family of calcium- and phospholipid-binding proteins that have been implicated in a broad range of molecular and cellular processes. The altered expression of individual annexins has been implicated in tumor development and progression. In this study the expression of annexin A1 and annexin A2 was studied by immunohistochemistry in intestinal-type sinonasal adenocarcinoma using a study set of 57 intestinal-type sinonasal adenocarcinomas represented on an intestinal-type sinonasal adenocarcinoma tissue microarray to assess its potential role in the pathogenesis of these tumors. Our results showed that annexin A1 expression was consistently lost in 52 (91%) intestinal-type sinonasal adenocarcinomas, as compared with the normal epithelium. The expression of annexin A2 was more heterogeneous, and only 19 (33%) cases showed annexin A2 negative expression. Annexin A2 expression was correlated with the histopathological type, being lower in the mucinous type (P = .022). The loss of annexin A2 expression correlated with a reduced survival in univariate analysis (P = .004). However, the impact of annexin A2 expression on patient survival could be an indirect consequence of its association with the histopathological type, since annexin A2 expression was not found to be an independent predictor in multivariate analyses. These results suggest that annexin A1 expression is frequently and commonly lost in intestinal-type sinonasal adenocarcinoma development. Annexin A2 expression is also reduced in intestinal-type sinonasal adenocarcinoma, and this loss of expression is associated to the more aggressive histopathological types. [ABSTRACT FROM AUTHOR]

Abstract: A detailed analysis of the constitutional chromosomal changes in two pediatric patients was performed using high resolution genetic analysis techniques, microarray comparative genomic hybridization (array CGH) and multiplex ligation-dependent probe amplification (MLPA) as well as FISH. The aim was to come to a more precise characterization of the genotype/phenotype relationship. Case 1 was a girl of 25 months, showing areas of hypopigmentation along the lines of Blaschko and no other developmental abnormality. She carried a ring chromosome 19 which we found not to have resulted in loss of subtelomeric sequences, ruling out the possibility that a small subtelomeric loss was causally related to this patient''s phenotype. Case 2 was a 9-year-old girl with facial anomalies and mild growth and mental retardation carrying an unidentified addition on chromosome 2p. We found that the addition was duplicated 2q35-q37.3 and that the addition was not accompanied by loss of 2pter or any other chromosomal region. Together with literature data, we hypothesize that pediatric patients with ''pure'' trisomy 2q including bands 2q35-q37.1 may have a moderate clinical phenotype as opposed to patients with duplications proximal to 2q33 or patients with duplications 2q3 with accompanying distal deletion. These two examples illustrate the additonal value of new, high resolution genetic analysis techniques for a better characterization of the genotype/phenotype relationship in childhood chromosomal disorders. [Copyright &y& Elsevier]

Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal region. We provide a comprehensive analysis of this malignancy with molecular and clinical trial data on a subset of our cohort to report on the potential efficacy of somatostatin receptor 2 (SSTR2)-targeting imaging and therapy. We conducted a retrospective analysis of 404 primary, locally recurrent, and metastatic olfactory neuroblastoma (ONB) patients from 12 institutions in the United States of America, United Kingdom and Europe. Clinicopathological characteristics and treatment approach were evaluated. SSTR2 expression, SSTR2-targeted imaging and the efficacy of peptide receptor radionuclide therapy [PRRT](177Lu-DOTATATE) were reported in a subset of our cohort (LUTHREE trial; NCT03454763). Dural infiltration at presentation was a significant predictor of overall survival (OS) and disease-free survival (DFS) in primary cases (n = 278). Kadish-Morita staging and Dulguerov T-stage both had limitations regarding their prognostic value. Multivariable survival analysis demonstrated improved outcomes with lower stage and receipt of adjuvant radiotherapy. Prophylactic neck irradiation significantly reduces the rate of nodal recurrence. 82.4% of the cohort were positive for SSTR2; treatment of three metastatic cases with SSTR2-targeted peptide-radionuclide receptor therapy (PRRT) in the LUTHREE trial was well-tolerated and resulted in stable disease (SD). This study presents pertinent clinical data from the largest dataset, to date, on ONB. We identify key prognostic markers and integrate these into an updated staging system, highlight the importance of adjuvant radiotherapy across all disease stages, the utility of prophylactic neck irradiation and the potential efficacy of targeting SSTR2 to manage disease. • Dural infiltration is prognostic and integrated into the Kadish-INSICA staging system (International Network for Sinonasal Cancer and Skull Base Tumours (www.insica.org). • Adjuvant radiotherapy is beneficial across all stage groups, including early stage. • Prophylactic neck irradiation prevents neck nodal recurrence. • 82.4% express Somatostatin Receptor 2 (SSTR2) protein which can be used for SSTR2-targeted imaging. • SSTR2-targeted peptide receptor radionuclide therapy is useful in metastatic disease. [ABSTRACT FROM AUTHOR]

Summary: Objectives: Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay is a method that has rarely been exploited in DNA methylation profiling of laryngeal squamous cell carcinoma (LSCC). Material and methods: Methylation of the gene was investigated by MS-MLPA in a well-characterized series of 53 LSCC and 30 samples of healthy mucosa. Aberrant promoter hypermethylation was confirmed using bisulfite pyrosequencing, and methylation-specific. Results: Promoter hypermethylation was observed in 36 of the 53 patients (68%). CDKN2B (28%), APC (17%), RARβ (15%), DAPK1 (11%) and CHFR (11%) were most frequently hypermethylated. Aberrant methylation of CHFR was mainly a late-stage event. Methylation-specific polymerase chain reaction and bisulfite pyrosequencing confirmed aberrant methylation for CDKN2B, APC and DAPK1. Conclusion: Promoter methylation profiling of LSCC using MS-MLPA identified CDKN2B, DAPK1, RARβ, APC, and CHFR as frequent epigenetic events. The clinical implications of these genes as biomarkers are highly relevant as attractive targets for cancer therapy, given the reversible nature of epigenetic gene silencing. [Copyright &y& Elsevier]

Head and neck squamous cell carcinoma (HNSCC) have very complex karyotypes that show all types of structural rearrangements. The most frequent aberrations are whole-arm translocations, which appear to have their breakpoints in centromeric or pericentromeric regions. We aimed to pinpoint the exact location of the breakpoints of these marker chromosomes with high-resolution cytogenetic and genetic analyses using microarray comparative genomic hybridization (CGH), multiplex ligation-dependent probe amplification (MLPA), and fiber fluorescence in situ hybridization (FISH). Among the seven cell lines in this study, six (84%) harbored one or more centromeric breakpoints or whole-arm translocations. In total, microarray CGH identified 163 breakpoints, 47 (29%) of which were in centromeric regions. Microarray CGH and MLPA results indicated that the translocation breakpoints were localized between the microarray oligonucleotide clones and MLPA probes closest to the centromere. High-resolution fiber-FISH revealed adjacent or minimally overlapping signals of probes that recognize the pericentromeric sequences of the two participating chromosomes. This indicates that whole chromosome arm translocation breakpoints occur within the pericentromeric chromatin and not the centromere core sequences. [Copyright &y& Elsevier]

Summary: Objetives: Despite improvements in the field of surgery and radiotherapy, the overall prognosis of sinonasal carcinomas is poor, mainly due to the difficulty to resect the tumour completely in this anatomically complex region. Therefore, there is great need for alternative treatments. Knowledge of the KRAS and BRAF mutational status would become clinically important with regard to the possible use of anti-EGFR therapies. Material and methods: DNA was extracted from paraffin embedded tumour samples from 57 cases of sinonasal squamous cell carcinoma (SNSCC) and from fresh frozen tumour samples from 58 cases of intestinal-type sinonasal adenocarcinoma (ITAC). Point mutations were analysed for KRAS exon 2 (codons 12 and 13) and BRAF (exon 15, V600E) by direct sequencing. Results: Neither KRAS nor BRAF showed any mutations in the SNSCC, whereas 7/58 (12%) ITAC harboured KRAS mutations and no BRAF mutations. All seven cases with KRAS mutation concerned well-differentiated and less aggressive (papillary and colonic type) ITAC, all patients being woodworkers and 4/7 tobacco smokers. Conclusion: Neither of SNSCCs carried mutations in KRAS and BRAF and a low frequency of KRAS mutation was found in ITAC. This suggests that KRAS and BRAF mutations play a limited role in the development of sinonasal cancer and that mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy in sinonasal cancer. [ABSTRACT FROM AUTHOR]

Summary: Background: Paragangliomas and phaeochromocytomas are neuroendocrine tumours associated frequently with germline mutations of SDHD, SDHC, and SDHB. Previous studies have shown the imprinted SDHAF2 gene to be mutated in a large Dutch kindred with paragangliomas. We aimed to identify SDHAF2 mutation carriers, assess the clinical genetic significance of SDHAF2, and describe the associated clinical phenotype. Methods: We undertook a multicentre study in Spain and the Netherlands in 443 apparently sporadic patients with paragangliomas and phaeochromocytomas who did not have mutations in SDHD, SDHC, or SDHB. We analysed DNA of 315 patients for germline mutations of SDHAF2; a subset (n=200) was investigated for gross gene deletions. DNA from a group of 128 tumours was studied for somatic mutations. We also examined a Spanish family with head and neck paragangliomas with a young age of onset for the presence of SDHAF2 mutations, undertook haplotype analysis in this kindred, and assessed their clinical phenotype. Findings: We did not identify any germline or somatic mutations of SDHAF2, and no gross gene deletions were noted in the subset of apparently sporadic patients analysed. Investigation of the Spanish family identified a pathogenic germline DNA mutation of SDHAF2, 232G→A (Gly78Arg), identical to the Dutch kindred. Interpretation: SDHAF2 mutations do not have an important role in phaeochromocytoma and are rare in head and neck paraganglioma. Identification of a second family with the Gly78Arg mutation suggests that this is a crucial residue for the function of SDHAF2. We conclude that SDHAF2 mutation analysis is justified in very young patients with isolated head and neck paraganglioma without mutations in SDHD, SDHC, or SDHB, and in individuals with familial antecedents who are negative for mutations in all other risk genes. Funding: Dutch Cancer Society, European Union 6th Framework Program, Fondo Investigaciones Sanitarias, Fundación Mutua Madrileña, and Red Temática de Investigación Cooperativa en Cáncer. [Copyright &y& Elsevier]

Summary: Lymph nodes metastasis is a major risk factor related to poor survival in larynx and pharynx carcinomas. The aim of this study is to search for markers of lymph node involvement analyzing the genetic differences between primary larynx and pharynx squamous cell carcinomas and their corresponding lymph node metastases. Twenty-five primary tumors and their corresponding lymph node metastases were examined. DNA copy number changes of 37 genes were analyzed by multiplex ligation-dependent probe amplification (MLPA). Loss of CDKN2A (9p21) occurred in 14 out of 25 pairs (56%) of primary tumor and lymph node metastases. Loss of LMNA (1q21) was exclusively detected in 8 lymph node samples (32%). Loss of CTNNB1 (3p22) and gain of CDKN2D (19p13) were also significantly more frequent in lymph node metastases. Other aberrations related to lymph node metastases were loss of MFHAS1 (8p23), RECQL4 (8q24) and gain of N33 (8p22) and TP53 (17p13). Primary tumor and corresponding lymph node metastases showed common genetic changes. However, the lymph node metastases presented with a number of additional alterations. Acquisition of these alterations may play a role in lymphatic metastasis development. [Copyright &y& Elsevier]