Differential expression profiling of head and neck squamous cell carcinoma (HNSCC)

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer in men with an incidence of about 780000 new cases per year worldwide and a poor rate of survival. There is a need for a better understanding of HNSCC, for the development of rational targeted interventions and to define new prognostic or diagnostic markers. To address these needs, we performed a large-scale differential display comparison of hypopharyngeal HNSCCs against histologically normal tissue from the same patients. We have identified 70 genes that exhibit a striking difference in expression between tumours and normal tissues. There is only a limited overlap with other HNSCC gene expression studies that have used other techniques and more heterogeneous tumour samples. Our results provide new insights into the understanding of HNSCC. At the genome level, a series of differentially expressed genes cluster at 12p12-13 and 1q21, two hotspots of genome disruption. The known genes share functional relationships in keratinocyte differentiation, angiogenesis, immunology, detoxification, and cell surface receptors. Of particular interest are the 13 'unknown' genes that exist only in EST, theoretical cDNA and protein databases, or as chromosomal locations. The differentially expressed genes that we have identified are potential new markers and therapeutic targets.

Figure 1

Flowchart outlining the study. The flowchart indicates how the tumour samples were selected and processed, the PCR-DD primers that were used and the number of bands isolated, the number of clones tested by reverse Northern, the resulting number of genes identified, the types of confirmation used to validate the results, and the bioinformatics analysis to analyse the results.

Figure 2

Differential display gel comparing the three stages of tumours (T) with their corresponding normal (N) samples. E=early; NM=no metastatic potential; M=metastatic propensity. Highlighted are the three types of profiles (A, overexpressed in tumour; B, tumour-specific profiles; C, underexpressed in tumour), and the percentages give the overall proportions in these categories.

Figure 3

Reverse Northerns using tumour (T) or normal (N) tissue probes. The genes shown, which are overexpressed (A) or underexpressed (B) in tumours, are the first four in Tables 3A and B, respectively.

Figure 4

Classical Northerns: tumour (T), lymph node (LN) and normal (N) samples from the same patients were analysed. The RPLP0 control is shown under each lane. (A) Genes overexpressed in tumours: (a) DRG1, (b) APOL2. (B) Genes underexpressed in tumours: (a) PIGR, (b) LPRP. The lines separate the samples from particular patients, and comparisons should be made between the samples from each patient.

Figure 5

Virtual Northerns: the lanes 1–3 (E), 6,7 (NM) and 8,9 correspond to individual patients who were pooled for the PCR-DD. Tumour (T) and normal (N) samples from the same patient were compared. (A) Genes overexpressed in tumours: (a) HSPCB, (b) TRA1, and (c) LTBP1. (B) Genes underexpressed in tumours: (a) PIGR, (b) LPRP, and (c) PLUNC. (C) RPLP0 is the internal control.

Figure 6

Real-time quantitative PCR. (A) Genes overexpressed in tumours (T): (a) DRG1, (b) APOL2, (c) PON2, (d) HSPC150, (e) PSMB8, and (f) RP1-68D18. (B) Genes underexpressed in tumours: (a) PIGR and (b) EMP1. The values for the tumours (black columns) and matched normal (N) tissue (white columns) were adjusted according to RPLP0, the internal control. The median of the N values was set to 1. The patients indicated with a star have a matched T/N ratio greater or equal to 2. The P-values of t-tests between the tumour and normal tissues are indicated.