GSTM1 copy number and promoter haplotype as predictors for risk of recurrence and/or second primary tumor in patients with head and neck cancer

Figures & data

Figure 1 Level of GSTM1 expression in human liver tissue. (A) Samples have been arranged in order of increasing expression. (B) levels of GSTM1 protein expression in liver tissue by GSTM1 copy number.

Note: *Expression level among two copies genotype was significantly higher than the one copy genotype (P < 0.01).
Abbreviations: CNV, copy number variant; GST, glutathione S-transferase; GSTM1, glutathione S-transferase Mu class 1.

Table 1 List of genetic variants in the promoter of GSTM1 identified in Caucasians

Genetic variants	Position*	rs number	MAF (%)
1. INS	−1542	NA	40.0
2. C/G	−1529	rs36210087	7.3
3. A/G	−1490	rs36209763	19.3
4. A/G	−1143	rs36209754	24.5
5. A/T	−888	NA	11.0
6. C/G	−498	rs412543	14.6
7. C/G	−486	rs3815029	9.9
8. C/T	−471	NA	12.8
9. A/G	−426	rs412302	26.7
10. C/T	−344	rs4147561	28.5
11. A/T	−343	rs4147562	25.8
12. C/T	−341	NA	15.9
13. C/T	−339	rs4147563	47.3
14. G/A	−304	rs28529287	18.5
15. T/C	−164	rs36208869	22.5

Note:

* Upstream of the ATG start site of the GSTM1 gene.

Abbreviations: GSTM1, glutathione S-transferase Mu class 1; MAF, minor allele frequency; NA, not assigned; INS, insertion; CNV, copy number variant.

Figure 2 Kaplan–Meier plots of time (months) and REC/SPT-free survival in HNC patients according to GSTM1 CNV.

Abbreviations: CNV, copy number variant; GSTM1, glutathione S-transferase Mu class 1; HNC, head and neck cancer; REC, recurrence; SPT, second primary tumor.

Figure 3 Kaplan–Meier estimates of time (months) and REC/SPT-free survival in HNC patients by GSTM1 promoter SNPs. (A) GSTM1–498C>G SNP and REC/SPT-free survival. (B) GSTM1 −426A>G SNP and REC/SPT-free survival. (C) GSTM1–339C>T SNP and REC/SPT-free survival.

Abbreviations: GSTM1, glutathione S-transferase Mu class 1; HNC, head and neck cancer; REC, recurrence; SNP, single nucleotide polymorphism; SPT, second primary tumor.

Table 2 Copy number frequencies of GSTM1 and risk of SPT/REC in head and neck cancer

CNV	No SPT/REC, N (%)	SPT/REC, N (%)	HR (95% CI)*	P^#	Logrank test^$
0	167 (54.2)	85 (63.9)			0.07
1	76 (24.7)	29 (21.8)	0.73 (0.48–1.12)	0.15
2+	65 (21.1)	19 (14.3)	0.57 (0.35–0.95)	0.03

Notes:

* Adjusted for age, sex, race, smoking status, alcohol consumption, tumor site, tumor stage, and randomization arm;

# P values of multivariate Cox regression;

$ P values of logrank test.

Abbreviations: CI, confidence interval; GSTM1, glutathione S-transferase Mu class 1; HR, hazard ratio; REC, recurrence; SPT, second primary tumor; CNV, copy number variant.

Table 3 Allele frequencies of GSTM1 promoter SNPs and the risk of SPT/REC in head and neck cancer

SNPs	No SPT/REC, N (%)	SPT/REC, N (%)	HR (95% CI)*	P^#	Logrank test^$
−498 C>G
Allele C	99 (69.72)	43 (30.28)	1.00 (Ref)
Allele G	26 (96.30)	1 (3.70)	0.11 (0.02–0.85)	0.04	0.004
−426 A>G
Allele A	79 (67.52)	38 (32.48)	1.00 (Ref)
Allele G	46 (88.46)	6 (11.54)	0.28 (0.11–0.74)	0.01	0.002
−339 C>T
Allele C	82 (80.39)	20 (19.61)	1.00 (Ref)
Allele T	43 (64.18)	24 (35.82)	2.02 (1.07–3.82)	0.03	0.03

Notes:

* Adjusted for age, sex, race, smoking status, alcohol consumption, tumor site, tumor stage, and randomization arm;

# P values of multivariate Cox regression;

$ P values of logrank test.

Abbreviations: CI, confidence interval; GSTM1, glutathione S-transferase Mu class 1; HR, hazard ratio; REC, recurrence; SNP, single nucleotide polymorphism; SPT, second primary tumor.

Figure 4 Kaplan–Meier estimates of time (months) and REC/SPT-free survival in HNC patients by in haplotype P^{-498G−-426G−-339C} and P^{498C−-426A−-339T}.

Abbreviations: HNC, head and neck cancer; REC, recurrence; SPT, second primary tumor.

Table 4 GSTM1 haplotype frequencies and risk of SPT/REC in head and neck cancer

Haplotype	No SPT/REC, N (%)	SPT/REC, N (%)	HR* (95% CI)	P^#
P^-498C−-426A−-339T	39 (62.9)	23 (37.1)	1.00 (ref)
P^-498C−-426A−-339C	40 (72.7)	15 (27.3)	0.72 (0.37–1.42)	0.35
P^-498C−-426G−-339T	4 (80.0)	1 (20.0)	0.50 (0.02–10.54)	0.66
P^-498C−-426G−-339C	16 (80.0)	4 (20.0)	0.41 (0.14–1.47)	0.13
P^-498G−-426G−-339C	26 (96.3)	1 (3.7)	0.09 (0.01–0.69)	0.02

Notes:

* Adjusted for age, sex, race, smoking status, alcohol consumption, tumor site, tumor stage, and randomization arm;

# P values of multivariate Cox regression.

Abbreviations: CI, confidence interval; GSTM1, glutathione S-transferase Mu class 1; HR, hazard ratio; REC, recurrence; SPT, second primary tumor.

Figure 5 Transient reporter gene expression assays with constructs containing full-length GSTM1 promoter. Luciferase expression of the eight constructs in T409 cells co-transfected with pRL-SV40 to standardize transfection efficiency.

Note: *denotes significantly higher luciferase activity compared to other constructs.

Abbreviation: GSTM1, glutathione S-transferase Mu class 1; RLU, relative luciferase units.