Amplification of the 3q26.3 locus is associated with progression to invasive cancer and is a negative prognostic factor in head and neck squamous cell carcinomas

Abstract

Amplification of the 3q26-q27 has a high prevalence in squamous cell carcinomas of mucosal origin, including those originating in the head and neck region. To elucidate its role as a prognostic tool in head and neck squamous cell carcinoma, a yeast artificial chromosome (YAC) contig spanning the entire 3q26-27 region was constructed. The minimal region of amplification was refined within a 1- to 2-Mb genomic segment contained within three overlapping, nonchimeric YAC clones using sequential fluorescent in situ hybridization analysis. These YAC clones containing the apex of amplification were used to develop a two-color fluorescence in situ hybridization assay and applied to the detection of 3q copy numbers in interphase nuclei on archival tumor tissue from 29 cases of normal mucosa, 20 of premalignant mucosa, and 50 of invasive head and neck squamous cell carcinomas. The presence of 3q amplification increased from 3% in normal mucosa to 25% in premalignant mucosa and 56% in invasive cancers (P < 0.01). In invasive tumors, low-level 3q amplification (3 to 4 X copy number) was identified in 18 of 50 primary head and neck cancers and high-level amplification (>4 X copy number) in 10 of 50 cases. With a median follow-up of 82.5 months, an increasing proportion of recurrences (32%, 72%, and 90%; P = 0.003) and cancer-related deaths (14%, 44%, and 70%; P = 0.006) were seen in patients with normal 3q copy number, low-level amplification, and high-level amplification, respectively. The 3-year disease-free (69%, 56%, and 10%; P = 0.001) and cause-specific (94%, 83%, and 40%; P = 0.01) survivals also decreased from normal copy number to low-level and high-level amplification. Only high-level amplification at 3q remained a significant prognostic variable on multivariate analysis including common prognostic predictors for both disease-free (relative risk, 5.1; 95% confidence interval = 1.9 to 13.9) and cause-specific survival (relative risk, 7.6; 95% confidence interval = 1.9 to 29.6). The findings suggest that the 3q copy number status is an important marker for tumor progression and prognostication in patients with head and neck squamous cell carcinoma.

Figure 1.

Analysis of 3q amplification by FISH. A: Yeast artificial chromosome (YAC) coverage of the 3q26-27 region. B: Relative copy numbers based on sequential two-color fluorescent analysis (using YAC clones) of MDA886, the cell line with the smallest minimal common region of amplification identified by CGH.

Figure 2.

3q copy number status determined by FISH for normal, premalignant, and invasive cancer tissue. A: 3q copy number status determined by FISH on normal (two copies from 3q26.3 and 3q centromeric probe), in situ carcinoma (three copies from 3q26.3 and two copies from 3q centromeric probe), and invasive squamous cell carcinoma (six copies from 3q26.3 and two copies from 3q centromeric probe). DAPI-stained interphase nuclei are shown. Red signals represent a 3q centromeric probe and green signals represent the pooled YAC probe. B: Results of FISH analysis of paraffin-embedded tissue. Overall, amplification at 3q26.3 was significantly more common in invasive cancers (56%), than preinvasive lesions (25%) or normal mucosa (4%; P < 0.001). Note that amplification at 3q26.3 was only detected in normal and preinvasive mucosal samples obtained for margins adjacent to the invasive cancer.

Figure 3.

Survival outcomes based on 3q status. Curves were generated by the Kaplan-Meier method and compared using the log rank test. All P values are two-sided and considered statistically significant if less than or equal to 0.05. A: Probability of disease-free survival was significantly poorer in patients with 3q26.3 amplification identified by FISH than in those with normal copy number (P = 0.001). Multivariate analysis, including 3q copy number status, TNM stage, and treatment type, revealed that only high-level amplification (>4 X copy number) remained an independent predictor of disease-free survival with a relative risk of 5.1 (95% confidence interval, 1.9 to 13.9). B: Probability of cause-specific survival was significantly poorer for patients with 3q amplification (P = 0.006). Multivariate analysis, including 3q copy number status, TNM stage, and treatment type, revealed that only high-level amplification (>4 X copy number) remained an independent predictor of disease-free survival with a relative risk of 7.6 (95% confidence interval, 1.9 to 24.6).