Microsatellite instability in squamous cell carcinoma of the head and neck.

Genomic instability or microsatellite instability (MI) in simple repeated sequences was initially recognised in colonic carcinomas and subsequently in other tumours. MI has been associated with mutations in genes concerned with replication and DNA repair. We investigated 34 microsatellite markers in squamous cell carcinoma of the head and neck (SCCHN). Fifty-six tumours, were studied, of which 25 were investigated with ten or more microsatellite markers. In this study we consider two or more microsatellite alterations in a tumour to be diagnostic of MI. We demonstrated that 7/25 (28%) of the tumours had MI at two or more loci and three of these tumours exhibited evidence of 20 or more loci with MI. No correlations were found between MI and previous treatment, site, histological differentiation, positive nodes at pathology, a history of alcohol intake or survival. MI has been demonstrated in T1N0 stage tumours, indicating that these changes may occur early in the disease process. A negative correlation was found between MI and a history of smoking (P = 0.02). Two or more markers of MI were found in three of four non-smokers compared with one of 13 in the smoking group of patients, which suggests a novel mechanism of carcinogenesis in non-smokers.     

Microsatellite instability and loss of heterozygosity in oral squamous cell carcinoma in Malaysian population

Loss of heterozygosity (LOH) and microsatellite instability (MSI) have been documented as important events in oral squamous cell carcinoma (OSCC). Five microsatellite markers D3S192, D3S966, D3S647, D3S1228 and D3S659 were selected on chromosome 3p because of high frequency of alterations reported in head and neck squamous cell carcinoma and the involvement of von Hippel Lindau (VHL) at 3p25-26 and the fragile histidine triad (FHIT) at 3p14.2 genes proven in many tumour types. A total of 50 archival tissue samples of OSCC and corresponding normal samples were analyzed for LOH and MSI status. The overall LOH for the markers selected on 3p was 56 out of 189 informative cases (29.6%). The most frequent LOH was identified for the marker D3S966 which was 18/42 (42.8%) of informative cases suggesting the presence of putative tumour suppressor genes (TSGs) in this loci. In this study, high frequency of microsatellite instability was found in D3S966 which was 28.6% of informative cases; this reveals the possibility of mutations of MMR genes in this region. Frequent microsatellite alterations (MA) were observed in 3 markers D3S966 (71.4%), D3S1228 (56.7%) and D3S192 (41.0%). There was no significant association between LOH with gender, tumour stages and differentiation grades. However, there was a significant association between tumour stage and differentiation grades with MSI status in OSCC in Malaysian population with p values of 0.002 and 0.035, respectively. There was also a significant association between MA and differentiation grades (p=0.041).     

Cervical metastases of head and neck squamous cell carcinoma correlate with loss of heterozygosity on chromosome 16q

To assess the potential involvement of putative tumor suppressors or metastasis suppressors on chromosome 16q in head and neck squamous cell carcinoma (HNSCC), we have examined 42 primary HNSCCs for loss of heterozygosity (LOH) at 16q and correlated these findings with the occurrence of cervical nodal metastases and other clinical parameters. Seven of the 42 (17%) HNSCCs examined displayed LOH at chromosome 16q24. Three of the seven HNSCCs showed LOH at all of the informative loci analyzed along the chromosome arm, whereas the other four showed only loss of a subset of markers. When LOH at 16q was correlated with clinical parameters, there was no significant correlation with age, sex, clinical stage, T stage, N stage or survival. However, there was a correlation between LOH at chromosome 16q24 and involvement of cervical lymph nodes. Of the seven HNSCCs that had lost heterozygosity at 16q24, six had local metastases to lymph nodes indicating that LOH at 16q24 may have predictive value for the metastatic potential of HNSCCs.     

Microsatellite instability in squamous cell carcinoma of head and neck from the Indian patient population

Genomic instability in simple repeated sequences has been observed in several human cancers. We have analyzed 50 squamous cell carcinomas of the head and neck (SCCHN) and 5 pre-malignant severe dysplastic tissues from Indian patient populations for microsatellite instability in 18 different loci spread over eight different chromosomes. Among the tumors analyzed, 45% exhibited instability at two or more loci, and 15% exhibited instability at 40% of the markers tested. Similar analysis of SCCHN tumors from other populations (British, American and French) showed much less frequency of instability. SCCHN tumors in the present study did not show any instability in the mononucleotide repeat sequences. There is also a clear distinction in the nature of the instability in these tumors in comparison with colorectal tumors. These results suggest that the underlying mechanism generating this type of instability is different from those reported for colorectal tumors.     

A favorable subset of AJCC stage IV squamous cell carcinoma of the head and neck

AJCC (American Joint Committee on Cancer) Stage IV is subdivided into a relatively favorable subset, IVA, and an unfavorable subset IVB.     

Involvement of EphA2 in head and neck squamous cell carcinoma: mRNA expression, loss of heterozygosity and immunohistochemical studies

EphA2 is a 130-kDa transmembrane protein primarily found in adult human epithelial cells and is a member of one of the largest receptor tyrosine kinases. It is located on 1p36.1, a genetic hot spot in cancer. EphA2 overexpression has been observed in aggressive solid tumors and its potential role in tumorigenesis, which includes cell growth, survival, migration and angiogenesis have been reported. However, the role of EphA2 remains unknown in head and neck cancer. In this study, we investigated the genetic profile of EphA2 in primary head and neck squamous cell carcinoma (HNSCC) by determining mRNA level, status of loss of heterozygosity and protein expression. mRNA expression was also correlated with clinicopathological data. Infrequent loss of heterozygosity (20%) was observed, though a 10-fold increase of mRNA expression in tumors compared to normal tissues was noted. A significant number of samples with normal to high mRNA expression was observed among patients with regional metastasis, with T3-T4 tumor size and with moderate to poor differentiation. However, statistical studies did not show any correlation between mRNA expression and any of the clinicopathological parameters. Tumor cells expressed EphA2 protein, but only weakly. These results suggest that EphA2 might be involved in the early development of HNSCC although not directly responsible for its progression.     

Prognostic implications of loss of heterozygosity at 8p21 and 9p21 in head and neck squamous cell carcinoma

Loss of heterozygosity (LOH) in chromosomal regions that harbor tumor suppressor genes from tumor tissue may lead to decreased survival time in cancer patients with squamous cell carcinoma of the head and neck (HNSCC). We studied 8 regions frequently lost in HNSCC in 150 patients having a primary diagnosis of HNSCC. Tumor and normal tissue DNA were genotyped for microsatellite repeat markers in 8 unlinked chromosomal regions. The association between LOH and death from HNSCC was investigated, weighted by number of informative markers per region and adjusted for age at diagnosis, self-reported race, tumor stage and current smoking status. LOH at 3 chromosomal regions were independently associated with reduced survival. A greater risk for cancer mortality was observed for LOH at chromosomal regions 3p24.3-p14.3 (p = 0.02), 8p21.3-p11.21 (p = 0.02) and 9p24.2-p21.2 (p = 0.03). In these regions, LOH at one or more markers was observed in 66.9%, 43.3% and 60.6% of patients, respectively. Survival times were significantly shorter for those with LOH at marker NEFL on 8p21.2 (relative risk = 6.15; p = 0.0002) and at D9S126 on 9p21.2 (relative risk = 5.96; p = 0.0003). Our results indicate that LOH at several chromosomal sites may offer additional independent prognostic information beyond traditional indicators such as tumor stage and age.     

p16 gene alterations in locally advanced squamous cell carcinoma of the head and neck

We previously documented the presence of mutations/deletions in the tumor suppressor gene p16 in squamous cell carcinoma of the head and neck (SCCHN). However, the association of these p16 alterations with clinical outcome is unknown. In this study, RNA was isolated from 19 frozen SCCHN from 19 patients who were previously enrolled in the OSU intensification regimen 2. Quantitative real-time RT-PCR and direct sequencing analysis was then performed on the specimens to detect p16 gene alterations. Clinical outcome for each patient was updated and correlated with the p16 alterations found. Five tumor specimens were found to have no or very low expression of p16 when compared with normal tissue. The remaining 14 tumor samples demonstrated overexpression of p16 relative to the level of expression in normal tissue. Sequence analysis of the p16 RT-PCR product from these specimens allowed identification of mutational changes in the coding sequence of p16 in four of the SCCHN specimens. Subsequent analysis of clinical outcome associated with locoregional/distant failure demonstrated no correlation with either altered expression of p16 or mutational status of p16. Results from this study indicate that p16 alterations are frequently found in this cohort of SCCHN. However, p16 alterations alone do not appear to be associated with clinical outcome.     

Genetic expression profiles and biologic pathway alterations in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is associated with considerable mortality and morbidity and is a major public health concern worldwide. To date, > 20 studies incorporating DNA microarray analyses have examined genomewide genetic expression changes associated with the development of HNSCC. The authors identified published reports of genetic expression profiles of HNSCC by Medline database search. They performed a review of the reports to identify genes that have been found repeatedly to exhibit substantially altered expression in HNSCC. Genes with altered expression were subsequently examined in the context of defined biologic systems with the use of GenMapp 2.0 pathway analysis software. Genes most commonly found to exhibit altered expression were those encoding for cytoskeletal and extracellular matrix proteins, inflammatory mediators, proteins involved in epidermal differentiation, and cell adhesion molecules. Results of GenMapp 2.0 analysis suggested global down-regulation of genes that encode for ribosomal proteins and enzymes in the cholesterol biosynthesis pathway; and up-regulation of genes that encode for matrix metalloproteinases and genes that bear on the inflammatory response. The review indicated that there are several genes and pathways that exhibit substantially altered expression in cancerous versus noncancerous states across studies. Further investigation into the genomic, proteomic, and functional consequences of these gene expression alterations may provide insight into the pathophysiology of HNSCC.     

Microsatellite alterations in head and neck squamous cell carcinoma and relation to expression of pimonidazole, CA IX and GLUT-1.

BACKGROUND AND PURPOSE: Because the locoregional control for HNSCC is still disappointing, research efforts focus on the exploration of new molecular markers located in both tumour and microenvironment, which could help stratify patients. The aim of the present work was therefore first to assess microsatellite alterations and hypoxia in HNSCC as possible molecular markers. Second, a relation between both was investigated, as hypoxia is known to select for genetic alterations. MATERIAL AND METHODS: Forty-eight patients with advanced HNSCC treated by surgery+/-radiotherapy were included. MSI and LOH were investigated with microsatellite markers using automatic fragment analysis. The presence of hypoxia was assessed by immunohistochemistry for pimonidazole, CA IX and GLUT-1. The mutual relationship between MSI/LOH and hypoxia was evaluated. RESULTS: No MSI was detected in this patient group. LOH occurred mostly on chromosomal arms 3p, 5q, 9p, 17p and 17q. Patients with LOH at D17S799, located in the near environment of p53, showed a higher CA IX expression (p=0.01). CONCLUSIONS: LOH is a possible molecular marker in HNSCC. The positive correlation between LOH at D17S799 and CA IX is in full concordance with previous publications linking hypoxia to selective pressure on the p53 gene.     

Genomic alterations in head and neck squamous cell carcinoma determined by cancer gene-targeted sequencing

BackgroundTo determine genomic alterations in head and neck squamous cell carcinoma (HNSCC) using formalin-fixed, paraffin-embedded (FFPE) tumors obtained through routine clinical practice, selected cancer-related genes were evaluated and compared with alterations seen in frozen tumors obtained through research studies.Patients and methodsDNA samples obtained from 252 FFPE HNSCC were analyzed using next-generation sequencing-based (NGS) clinical assay to determine sequence and copy number variations in 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. Human papillomavirus (HPV) status was determined by presence of the HPV DNA sequence in all samples and corroborated with high-risk HPV in situ hybridization (ISH) and p16 immunohistochemical (IHC) staining in a subset of tumors. Sequencing data from 399 frozen tumors in The Cancer Genome Atlas and University of Chicago public datasets were analyzed for comparison.ResultsAmong 252 FFPE HNSCC, 84 (33%) were HPV positive and 168 (67%) were HPV negative by sequencing. A subset of 40 tumors with HPV ISH and p16 IHC results showed complete concordance with NGS-derived HPV status. The most common genes with genomic alterations were PIK3CA and PTEN in HPV-positive tumors and TP53 and CDKN2A/B in HPV-negative tumors. In the pathway analysis, the PI3K pathway in HPV-positive tumors and DNA repair-p53 and cell cycle pathways in HPV-negative tumors were frequently altered. The HPV-positive oropharynx and HPV-positive nasal cavity/paranasal sinus carcinoma shared similar mutational profiles.ConclusionThe genomic profile of FFPE HNSCC tumors obtained through routine clinical practice is comparable with frozen tumors studied in research setting, demonstrating the feasibility of comprehensive genomic profiling in a clinical setting. However, the clinical significance of these genomic alterations requires further investigation through application of these genomic profiles as integral biomarkers in clinical trials.     

Genetic alterations between primary head and neck squamous cell carcinoma and recurrence after radiotherapy

BACKGROUND:

In the attempt to characterize the genetic bases of recurrent head and neck squamous cell carcinoma (HNSCC) after radiotherapy (RT), the authors compared the molecular profiles of primary tumors and recurrences.

METHODS:

TP53 gene status and instability at 10 microsatellite markers were determined in pre‐RT lesions and corresponding local recurrences in a series of 16 HNSCCs.

RESULTS:

Eight (50%) HNSCCs showed both TP53 and microsatellite instability (MSI) status concordance in pre‐ and postirradiation biopsies; 3 (18.7%) showed discordance of both TP53 and MSI status; and finally 5 (31.2%) had discordance at only 1 genetic test. Accordingly, the authors interpreted as true recurrence the 8 concordant cases, and as true second primary malignancies the 3 discordant ones. In the remaining 5 cases with partial DNA correspondence, the exact nature of the new lesion only partially related to the original cancer is a matter of discussion. Patients showing the same mutations among pre‐ and post‐RT HNSCCs had a longer disease‐free interval (DFI) and better survival than those showing discordant genetic features (log‐rank test, P = .0045).

CONCLUSIONS:

Post‐RT recurrent HNSCCs are genetically heterogeneous. The genetic characterization of the recurrence, especially in those cases with a particularly short DFI showing partially discordant mutations, might have a useful clinical relevance in the restaging process. Cancer 2010. © 2010 American Cancer Society.     

Fractional allele loss indicates distinct genetic populations in the development of squamous cell carcinoma of the head and neck (SCCHN)

Loss of heterozygosity (LOH) had been widely used to assess genetic instability in tumours and a high LOH on chromosome arms 3p, 9p and 17p has been considered to be a common event in squamous cell carcinoma of the head and neck (SCCHN). We have investigated LOH in 52 SCCHN using a range of microsatellite markers. LOH was observed in 69% of individuals on 17p using seven markers, in 64% of individuals on 3p using 17 markers and in 61% of individuals on 9p using 11 markers. Fractional allele loss (FAL) has been calculated for each tumour (FAL is the number of chromosomal arms showing LOH divided by the number of informative chromosomal arms) and a median FAL value of 0.25 was obtained in the 52 SCCHN studied. The LOH data were examined on the basis of FAL scores: low FAL (LFAL), 0.00-0.19; medium FAL (MFAL), 0.20-0.32; high FAL (HFAL), 0.33-0.88. HFAL tumours demonstrated a significantly higher LOH on chromosome arms 3p, 9p and 17p, with 94% LOH on 3p, 94% on 9p and 100% on 17p compared with LFAL tumours. Six of the 16 patients in the LFAL group were found to have no LOH on 3p, 9p or 17p and of these four had LOH at other sites, on chromosomes 2p25-p24, 5q21-22, 7pter-p22, 8q13-q22.1, 11q23.3, 13q32, 17q, 18p11.21, 18q21.31 and 19q12-q13.1. These results indicate that LFAL patients form a subset of SCCHN tumours with distinct molecular initiating events which may represent a discrete genetic population.     

Nivolumab in squamous cell carcinoma of the head and neck

Introduction: The prognosis of recurrent/metastatic (R/M) squamous cell carcinoma of the head and neck (HNSCC) after failure of first line chemotherapy is dismal. Until the publication of the results of CheckMate 141, not a single agent provided any survival benefit as a second line treatment for R/M HNSCC.

Areas covered: A comprehensive review of the literature was conducted on the role of nivolumab in HNSCC.

Expert commentary: Nivolumab is approved by the Food and Drug Administration for the treatment of patients based on the results of CheckMate 141 showing an overall survival benefit as compared to standard care (single agent docetaxel, methotrexate, or cetuximab). Of particular interest are immune-related adverse events which should be managed according to published guidelines.     

Cetuximab in squamous cell carcinoma of the head and neck

Surgery and radiotherapy are the standard treatment options for patients with squamous cell carcinoma of the head and neck (SCCHN). Chemotherapy and chemoradiotherapy are new alternatives for locally advanced disease, particularly induction chemotherapy for patients with unresectable tumors. In recurrent/metastatic disease and after progression to platin-based regimens, no treatments other than best supportive care are currently available. Most SCCHN tumors overexpress the epidermal growth factor receptor (EGFR). This is a tyrosine kinase membrane receptor and has a clear implication in angiogenesis, tumor progression and resistance to different cancer treatments. Cetuximab is a monoclonal antibody that binds to EGFR and alters the tyrosine kinase-mediated signal transduction pathway. The drug is active in colon cancer and is currently being tested in SCCHN patients. For locally advanced disease, cetuximab/radiotherapy combination has demonstrated a benefit in survival when compared with radiotherapy alone as radical treatment. Cetuximab is an active treatment in platin-refractory patients with recurrent/metastatic disease.     

Re-irradiation for head and neck squamous cell carcinoma

Introduction

Local recurrences after curative treatment have a potential for cure with salvage surgery or with re-irradiation.

Docetaxel and squamous cell carcinoma of the head and neck

Chemotherapy in head and neck carcinoma is used as palliative treatment but also as induction treatment or combined treatment with concurrent radiation therapy. Platinum and 5-fluorouracil are the most commonly used cytotoxic agents. Docetaxel is an active drug for treating head and neck carcinoma. For patients with recurrent or metastatic disease, docetaxel could be used either as a second line chemotherapy or a first line for patients who received previously platinum or 5FU. In combination with platinum and 5FU, used as induction chemotherapy the TPF regimen is a very active treatment with an overall response rate of 85 to 90% with a manageable acute toxicity rate. This approach is under investigation in terms of ability to obtain more larynx preservation compared to the standard approach with platinum and 5FU. Docetaxel is a radiosensitizer. Concurrent radiochemotherapy using docetaxel alone is feasible, Trials are needed to define the optimal regimen for combining radiation, platinum and docetaxel.