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Abstract

Radiotherapy and cisplatin lead to cell killing in head and neck squamous cell carcinoma patients, but adverse events and response to treatment are not the same in patients with similar clinicopathological aspects. The aim of this prospective study was to evaluate the roles of TP53 c.215G > C, FAS c.-671A > G, FAS c.-1378G > A, FASL c.-844 C > T, CASP3 c.-1191A > G, and CASP3 c.-182-247G > T single nucleotide variants in toxicity, response rate, and survival of cisplatin chemoradiation-treated head and neck squamous cell carcinoma patients. Genomic DNA was analyzed by polymerase chain reaction for genotyping. Differences between groups of patients were analyzed by chi-square test or Fisher’s exact test, multiple logistic regression analysis, and Cox hazards model. One hundred nine patients with head and neck squamous cell carcinoma were enrolled in study. All patients were smokers and/or alcoholics. Patients with FAS c.-671GG genotype, FAS c.-671AG or GG genotype, and FASL c.-844CC genotype had 5.52 (95% confidence interval (CI): 1.42–21.43), 4.03 (95% CI: 1.51–10.79), and 5.77 (95% CI: 1.23–27.04) more chances of presenting chemoradiation-related anemia of grades 2–4, lymphopenia of grade 3 or 4, and ototoxicity of all grades, respectively, than those with the remaining genotypes. FAS c.-671GG genotype was also seen as an independent predictor of shorter event-free survival (hazard ratio (HR): 2.05; P = 0.007) and overall survival (HR: 1.83; P = 0.02) in our head and neck squamous cell carcinoma patients. These findings present, for the first time, preliminary evidence that inherited abnormalities in apoptosis pathway, related to FAS c.-671A > G and FASL c.-844 C > T single nucleotide variants, can alter toxicity and survival of tobacco- and alcohol-related head and neck squamous cell carcinoma patients homogeneously treated with cisplatin chemoradiation.

Keywords

Cisplatin head and neck squamous cell carcinoma single nucleotide variants

Introduction

Head and neck (HN) squamous cell carcinoma (SCC) accounts for only 5% of all new cancer cases reported worldwide,¹ but more than half of patients present advanced locoregional disease at diagnosis² and have 5 years overall survival (OS) rates lower than 50%.³ HNSCC has tobacco smoking and alcohol consumption or human papillomavirus type 16 (HPV16) infection as risk factors.⁴

Standard treatment for patients with locally advanced HNSCC, whose surgical resection is not amenable, is radiotherapy (RT) with cisplatin (CDDP).⁵ Ionizing radiation (IR) leads to DNA single- and double-strand breaks promoting cancer cell death.⁶ The cytotoxic effects of CDDP are mediated by binds with DNA forming interstrand and intrastrand cross-links.⁷ Response to IR⁸ and CDDP⁹ damages involves P53 activation that stimulates the transcription of FAS receptor, which binds to the FAS ligand (FASL), and this interaction results in formation of the death-inducing signaling complex (DISC) and triggers apoptosis by activation of effector caspase 3 (CASP3).

The variability in adverse events and tumor response induced by RT¹⁰ and CDDP¹¹ among patients receiving the same treatment can be attributed to genetic changes causing modifications in cellular phenotype. Functional studies revealed that variant alleles of TP53 c.215G > C,¹²FAS c.-671A > G,¹³FAS c.-1378G > A,¹³ and FASL c.-844 C > T¹⁴ single nucleotide variants (SNVs) reduce apoptosis capacity when compared with wild-type alleles. Reduced apoptosis was also associated with the variant alleles of CASP3 c.-1191A > G¹⁵ and CASP3 c.-182-247G > T¹⁶ SNVs, respectively, by in silico analysis.

TP53 c.215G > C was previously associated with myelotoxicity in ovarian cancer patients treated with CDDP-cyclophosphamide,¹⁷ nephrotoxicity in lung cancer patients treated with CDDP or carboplatin,¹⁸ gastrointestinal toxicity in elderly lung cancer patients treated with platinum plus navelbine, paclitaxel, docetaxel, or gemcitabine,¹⁹ response to therapy, disease-free survival (DFS) and OS in lung cancer patients treated with platinum plus vinorelbine, paclitaxel, docetaxel, or gemcitabine,²⁰ and DFS in stage I or II radiation-treated HNSCC patients.²¹FAS c.-671A > G and FASL c.-844 C > T modulated DFS in patients with SCC of oropharynx (SCCO) treated with RT, chemotherapy, and/or surgery,²² and FAS c.-1378G > A altered OS of chemoradiation-treated HPV16-positive SCCO patients;²³ it is important to note that the chemotherapeutic agents used in both studies were not specified.

The aim of this study was to evaluate the roles of TP53 c.215G > C, FAS c.-671A > G, FAS c.-1378G >A, FASL c.-844 C > T, CASP3 c.-1191A > G, and CASP3 c.-182-247G > T SNVs in toxicity, response rate, and survival of HNSCC patients treated with a homogeneous protocol consisting of RT and CDDP chemotherapy.

Materials and methods

Study population

In this prospective study, we evaluated 109 patients with HNSCC diagnosed at the Clinical Oncology Service of the General Hospital of University of Campinas between June 2011 and February 2014. The study was conducted according to the Declaration of Helsinki and was approved by the local Ethics Committee (number 274/2011), where all subjects provided written informed consent.

Patients were classified as smokers or non-smokers and drinkers or abstainers.²⁴ HNSCC was diagnosed²⁵ and staged²⁶ according to standard criteria.

The presence of HPV16 in tumor fragments embedded in paraffin was analyzed by immunohistochemical staining and in situ hybridization as previously described.^27,28

Patients were treated with chemoradiation as definitive treatment due to locoregional unresectable tumor, refusal of surgery related to expected functional or anatomic sequels, or an organ preservation protocol. The treatment consisted of 35 sessions of RT being 2 Gy per session and intravenous CDDP at dose of 80–100 mg/m² on days 1, 22, and 43 (100 mg/m² of CDDP was administered to patients with Karnofsky Performance Scale (KPS) 80%–100% and without comorbidities, and patients with KPS 60%–70% and without comorbidities or KPS higher than 70% with comorbidities received CDDP at dose of 80 mg/m²). Patients who presented adverse side effects of grades 3 or 4 received CDDP at lower dose in further administrations or had suspension of CDDP.^29,30 As antiemetic premedication, mannitol (125 ml of 20%), ondansetron (24 mg), and dexamethasone (20 mg) were dissolved in 3000 ml of saline 0.9% and infused before CDDP infusion; and oral dexamethasone (8 mg, every 12 h for 3 days) and metoclopramide (10 mg, every 6 h for 3 days) after each CDDP infusion.^31,32 Adherence to prescriptions was considered in study.³³

Vomiting was accessed after CDDP infusion and during the four following days. Cytopenias were evaluated by hematological exams performed after CDDP infusion. Nephrotoxicity and ototoxicity were analyzed using ⁵¹Cr-EDTA glomerular filtration rate and audiometric exam, respectively, performed pre and after chemoradiation. Toxicities were graded using the National Cancer Institute (NCI) criteria version 4.0,³⁴and the worst toxicity grade during treatment was considered for analysis.

The response to treatment was quantified by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1.³⁵ Patients who failed to respond to their initial treatment regimen or relapsed received intravenous methotrexate as palliative chemotherapy.³⁶ Patients were followed at 3-month intervals and the latest follow-up was recorded in December 2018.

Genetic polymorphism analysis

TP53 c.215G > C (rs1042522),³⁷FAS c.-671A > G (rs1800682),³⁸FAS c.-1378G > A (rs2234767) and FASL c.-844 C > T (rs763110),³⁹CASP3 c.-1191A > G (rs12108497),¹⁵ and CASP3 c.-182-247G > T (rs4647601)⁴⁰ genotypes were identified in genomic DNA by polymerase chain reaction and enzymatic digestion. Briefly, the regions of interest for each gene were amplified using the mix of sterile water, 10× buffer with (NH4)2SO4 (Thermo Scientific™, USA), 25 mM MgCl2 (Thermo Scientific™, USA), 40 mM dNTP (Thermo Scientific™, USA), 10 pmoles/µL of each primer (Integrated DNA Technologies™, USA), 2.5 U Taq DNA polymerase (Thermo Scientific™, USA), and 200 ng DNA. The amplification products were subjected to fragmentation with 5 U of each enzyme: BstUI (rs1042522 and rs2234767) (Thermo Scientific™, USA), MvaI (rs1800682) (Thermo Scientific™, USA), BsrDI (rs763110) (Thermo Scientific™, USA), StuI (rs12108497) (Thermo Scientific™, USA), and Hpych4 V (rs4647601) (New England Biolabs^®, USA). Subsequently, the fragments were visualized on agarose gel stained with ethidium bromide. Positive and negative controls were used in reactions. The amount of 15% of genotype determinations was carried out twice in independent experiments with 100% of concordance.

Statistical analysis

The Haploview 4.2 software (www.broad.mit.edu/mpg/haploview) was used to perform pairwise LD analyses of FAS and CASP3 haplotypes, and those with frequency higher than 10% were selected to analyses. Differences between groups were analyzed by chi-square or Fisher’s exact test. To obtain adjusted odds ratio (OR) values in associations among SNVs, toxicity, and response to treatment, the logistic regression model was used. Bonferroni correction was applied to correct for bias arising out of multiple comparisons. The statistical power of analysis (PA) was calculated using the Researcher’s Toolkit.⁴¹

Event-free survival (EFS) and OS were calculated from date of diagnosis to date of progression, relapse, death from disease or last follow-up, and date of diagnosis until date of death by any cause or lost to follow-up, respectively. EFS and OS were calculated using the Kaplan–Meier method. The prognostic impact of clinic pathological and genetic variants was examined using univariate Cox proportional hazard ratio (HR) regression. All variables with P-value ≤ 0.15 were included in multivariate Cox regression analysis. Significant results were internally validated conducting on the final model via the bootstrap method using 1000 replications.

Significance results were two-sided and achieved when P-value was < 0.05. All tests were done using the SPSS 24.0 software (SPSS Incorporation, IL, USA).

Results

The patients’ median age was 56 years old. Most patients were males, smokers, and drinkers; the three non-smoker patients referred accentuated alcohol consumption and the nine non-drinker patients were important smokers. About 60% of patients had tumor in oral cavity or oropharynx and more than 80% of patients showed well or moderately differentiated tumor and tumors of III or IV stage. All analyzed cases tested negative for HPV16. Vomiting was reported in about 60% of cases, anemia was identified in about 97% of cases, leukopenia in 72% of cases, neutropenia in 60% of cases, lymphopenia in 97% of cases, and almost 30% of patients presented thrombocytopenia during or after treatment. About one-quarter of the available patients obtained a complete response with chemoradiation, while the remainder presented partial or stable disease (Table 1).

Table 1.

Clinical and tumor aspects of 109 patients with head and neck squamous cell carcinoma.

Variable	Median (range) or N (%)
Age (years)	56 (27–74)
Gender
Male	101 (92.7)
Female	8 (7.3)
Tobacco consumption
Smokers	106 (97.2)
Non-smokers	3 (2.8)
Alcohol consumption
Drinkers	100 (91.7)
Abstainers	9 (8.3)
Tumor location
Oral cavity or oropharynx	64 (58.7)
Hypopharynx or larynx	45 (41.3)
Histological grade^a
Well or moderately	73 (82.0)
Poorly or undifferentiated	16 (18.0)
Tumor stage
I or II	6 (5.5)
III or IV	103 (94.5)
Human papillomavirus type 16^a
Positive	0 (0.0)
Negative	57 (100.0)
Toxicity^a,b
Vomiting	50 (56.8)
Anemia	95 (96.9)
Leukopenia	70 (71.4)
Neutropenia	58 (59.2)
Lymphopenia	95 (96.9)
Thrombocytopenia	36 (36.7)
Ototoxicity	68 (76.4)
Nephrotoxicity	73 (85.9)
Response rate^a
Complete	21 (23.9)
Partial	62 (70.4)
Stable disease	5 (5.7)

N: number of patients.

The number of patients differed from the total quoted in the study (N = 109) because it was not possible to obtain consistent information in some cases. ^bGrades 1–4 for myelotoxicity and ototoxicity and 1–5 for nephrotoxicity.

All patients received the total dose of RT (70 Gy). Eighty-five patients received three CDDP infusions and 24 patients were treated with two CDDP infusions due to the occurrence of myelotoxicity and/or nephrotoxicity. Median cumulative CDDP amount administered to patients was 260 mg/m² (range: 100–300 mg/m²). Medium or high adherence (97.7%) to antiemetics was observed among patients.

The median follow-up time of the 109 patients was 22 months (range: 3–89). The estimated probabilities of 24-month EFS and OS were 41.7% and 47.2%, respectively. At the study end, 34 patients were alive (6 of them were with HNSCC and 28 without HNSCC) and 75 patients died (67 of them by disease and 8 by others causes).

Association between genetic variants, toxicity, and response to therapy

When genotypes of SNVs were analyzed in patients stratified by bone marrow toxicity to chemoradiation, it was found an excess of FAS c.-671GG genotype in patients with anemia grades 2– 4 than in those without anemia or with anemia grade 1 (30.4% versus 14.3%, P = 0.01; PA: 45.8%) after Bonferroni correction (Figure 1(a)); patients with FAS c.-671GG genotype had 5.52 more chance of presenting anemia grades 2–4 than those with FAS c.-671AA or AG genotype. FAS c.-671AG or GG genotype was also more common in patients with lymphopenia grade 3 or 4 than in those without lymphopenia or with lymphopenia grade 1 or 2 (80.0% versus 56.3%; P = 0.005; PA: 71.9%) after Bonferroni correction (Figure 1(b)); patients with FAS c.-671AG or GG genotype had 4.03 more chance of presenting lymphopenia grade 3 or 4 than those with FAS c.-671AA genotype (Table 2).

Figure 1.

Bars of genotypes frequencies in (a) patients without anemia or with anemia grade 1 (gray bars) versus patients with anemia grades 2–4 (black bars), (b) patients without lymphopenia or with lymphopenia grade 1 or 2 (gray bars) versus patients with lymphopenia grade 3 or 4 (black bars), and (c) patients without ototoxicity (gray bars) versus with patients grades (1–4) ototoxicity (black bars). *FAS c.-671GG genotype was more common in patients with anemia grades 2–4 than in those without anemia or with anemia grade 1 (30.4% versus 14.3%, P = 0.01; PA: 45.8%), FAS c.-671AG or GG genotype was more common in patients with lymphopenia grade 3 or 4 than in those without lymphopenia or with lymphopenia grade 1 or 2 (80.0% versus 56.3%; P = 0.005; PA: 71.9%), and FASL c.-844CC genotype was more common in patients with ototoxicity (38.2% versus 9.5%, P = 0.02; power of analysis: 75.4%); patients with the respective genotypes had 5.52 more chance of presenting anemia grades 2–4, 4.03 more chance of presenting lymphopenia grade 3 or 4, and 5.77 more chance of presenting ototoxicity compared with patients with other genotypes. Multivariate Cox regression curve for (d) event-free and (e) overall survival in patients with FAS c.-671AA or AG genotype (N = 83) versus GG genotype (N = 26), (f) event-free and (g) overall survival in patients with FAS c.-1378 GG or GA genotype (N = 102) versus AA genotype (N = 7), and (h) event-free and (i) overall survival in patients with FASL c.-844CC or CT genotype (N = 82) versus TT genotype (N = 27), adjusted by gender and tumor stage.

Table 2.

TP53 c.215G > C, FAS c.-671A > G and c.-1378G > A, FASL c.-844 C > T, CASP3 c.-1191A > G and c.-182-247G > T single nucleotide variants and FAS and CASP3 haplotypes in 109 patients with head and neck squamous cell carcinoma stratified by myelotoxicity with treatment with chemoradiotherapy.

Variable	Anemia		Leukopenia		Neutropenia		Lymphopenia		Thrombocytopenia
	G0 + G1	G2–G4	G0–G2	G3 + G4	G0–G2	G3 + G4	G0–G2	G3 + G4	G0	G1–G4
	N (%)	N (%)	N (%)	N (%)	N (%)	N (%)	N (%)	N (%)	N (%)	N (%)
P53 c.215G > C
GG	23 (54.8)	22 (39.3)	39 (45.3)	6 (50.0)	37 (45.7)	8 (47.1)	24 (50.0)	21 (42.0)	25 (40.3)	20 (55.6)
GC or CC	19 (45.2)	34 (60.7)	47 (54.7)	6 (50.0)	44 (54.3)	9 (52.9)	24 (50.0)	29 (58.0)	37 (59.7)	16 (44.4)
P-value	0.254		0.762		0.917		0.386		0.147
OR (95% CI)	1.65 (0.69–3.92)		0.83 (0.24–2.77)		0.94 (0.33–2.69)		1.43 (0.63–3.25)		0.54 (0.23–1.24)
GG or GC	40 (95.2)	48 (85.7)	77 (89.5)	11 (91.7)	72 (88.9)	16 (94.1)	42 (87.5)	46 (92.0)	54 (87.1)	34 (94.4)
CC	2 (4.8)	8 (14.3)	9 (10.5)	1 (8.3)	9 (11.1)	1 (5.9)	6 (12.5)	4 (8.0)	8 (12.9)	2 (5.6)
P-value	0.315		0.820		0.525		0.424		0.260
OR (95% CI)	2.31 (0.45–11.91)		0.77 (0.09–6.74)		0.50 (0.05–4.23)		0.56 (0.14–2.27)		0.39 (0.08–1.98)
FAS c.-671A > G
AA	15 (35.7)	16 (28.6)	24 (27.9)	7 (58.3)	23 (28.4)	8 (47.1)	21 (43.8)	10 (20.0)	21 (33.9)	10 (27.8)
AG or GG	27 (64.3)	40(71.4)	62 (72.1)	5 (41.7)	58 (71.6)	9 (52.9)	27 (56.3)	40 (80.0)	41 (66.1)	26 (72.2)
P-value	0.485		0.042		0.138		0.005		0.532
OR (95% CI)	1.38 (0.55–3.45)		0.27 (0.08–0.95)^c		0.44 (0.15–1.29)		4.03 (1.51–10.79)^e		1.33 (0.54–3.27)
AA or AG	36 (85.7)	39 (69.6)	64 (74.4)	11 (91.7)	63 (77.8)	12 (70.6)	40 (86.3)	35 (70.0)	51 (82.3)	24 (66.7)
GG	6 (14.3)	17 (30.4)	22 (25.6)	1 (8.3)	18 (22.2)	5 (29.4)	8 (16.7)	15 (30.0)	11 (17.7)	12 (33.3)
P-value	0.013		0.215		0.526		0.120		0.083
OR (95% CI)	5.52 (1.42–21.43)^a		0.26 (0.03–2.16)		1.45 (0.45–4.68)		2.19 (0.81–5.89)		2.31 (0.89–6.00)
FAS c.-1378G > A
GG	31 (73.8)	39 (69.6)	62 (72.1)	8 (66.7)	59 (72.8)	11 (64.7)	4 (70.8)	36 (72.0)	42 (67.7)	28 (77.8)
GA or AA	11 (26.2)	17 (30.4)	24 (27.9)	4 (33.3)	22 (27.2)	6 (35.3)	14 (29.2)	14 (28.0)	20 (32.3)	8 (22.2)
P-value	0.999		0.697		0.501		0.918		0.292
OR (95% CI)	1.00 (0.38–2.60)		1.29 (0.35–4.69)		1.46 (0.48–4.43)		1.04 (0.42–2.56)		0.60 (0.23–1.55)
GG or GA	40 (95.2)	52 (92.9)	81 (94.2)	11 (91.7)	78 (96.3)	14 (82.4)	45 (93.8)	47 (94.0)	58 (93.5)	34 (94.4)
AA	2 (4.8)	4 (7.1)	5 (5.8)	1 (8.3)	3 (3.7)	3 (17.6)	3 (6.3)	3 (6.0)	4 (6.5)	2 (5.6)
P-value	0.827		0.735		0.047		0.910		0.859
OR (95% CI)	1.22 (0.20–7.43)		1.47 (0.15–13.79)		5.57 (1.01–30.44)^d		1.10 (0.21–5.76)		0.85 (0.14–4.90)
FAS + FAZ
GA	9 (21.4)	14 (25.0)	20 (23.3)	3 (25.0)	19 (23.5)	4 (23.5)	11 (22.9)	12 (24.0)	16 (25.8)	7 (19.4)
Other haplotypes	33 (78.6)	42 (75.0)	66 (76.7)	9 (75.0)	62 (76.5)	13 (76.5)	37 (77.1)	38 (76.0)	46 (74.2)	29(80.6)
P-value	0.848		0.894		0.995		0.625		0.475
OR (95% CI)	1.10 (0.39–3.06)		1.10 (0.27–4.45)		1.00 (0.29–3.44)		1.26 (0.49–3.27)		0.69 (0.25–1.89)
FASL c.-844 C > T
CC	10 (23.8)	20 (35.7)	26 (30.2)	4 (33.3)	24 (9.6)	6 (35.3)	18 (37.5)	12 (24.0)	17 (27.4)	13 (36.1)
CT or TT	32 (76.2)	36 (64.3)	60 (69.8)	8 (66.7)	57 (70.4)	11 (64.7)	30 (62.5)	38 (76.0)	45 (72.6)	23 (63.9)
P-value	0.225		0.827		0.646		0.164		0.369
OR (95% CI)	0.54 (0.20–1.45)		0.86 (0.24–3.13)		0.77 (0.25–2.32)		1.89 (0.77–4.63)		0.66 (0.27–1.61)
CC or CT	37 (88.1)	37 (66.1)	63 (73.3)	11 (91.7)	58 (71.6)	16 (94.1)	37 (77.1)	37 (74.0)	44 (71.0)	30 (83.3)
TT	5 (11.9)	19 (33.9)	23 (26.7)	1 (8.3)	23 (28.4)	1 (5.9)	11 (22.9)	13 (26.0)	18 (29.0)	6 (16.7)
P-value	0.020		0.195		0.081		0.868		0.175
OR (95% CI)	4.11 (1.24–13.57)^b		0.24 (0.03–2.03)		5.78 (0.71–47.09)		1.08 (0.41–2.81)		0.48 (0.17–1.37)
CASP3 c.-1191A > G
AA	15 (35.7)	25 (44.6)	36 (41.9)	4 (33.3)	32 (39.5)	8 (47.1)	17 (35.4)	23 (46.0)	30 (48.4)	10 (27.8)
AG or GG	7 (64.3)	31 (55.4)	50 (58.1)	8 (66.7)	49 (60.5)	9 (52.9)	31 (64.6)	27 (54.0)	32 (51.6)	26 (72.2)
P-value	0.410		0.575		0.565		0.519		0.048
OR (95% CI)	0.69 (0.28–1.66)		1.44 (0.40–5.15)		0.73 (0.25–2.10)		0.75 (0.32–1.75)		2.43 (1.00–5.89)^f
AA or AG	35 (83.3)	53 (94.6)	77 (89.5)	11 (91.7)	72 (88.9)	16 (94.1)	43 (89.6)	45 (90.0)	56 (90.3)	32 (88.9)
GG	7 (16.7)	3 (5.4)	9 (10.5)	1 (8.3)	9 (11.1)	1 (5.9)	5 (10.4)	5 (10.0)	6 (9.7)	4 (11.1)
P-value	0.115		0.820		0.525		0.881		0.821
OR (95% CI)	0.30 (0.06–1.33)		0.14 (0.09–6.74)		0.50 (0.05–4.23)		0.90 (0.24-.3.37)		1.16 (0.30–4.44)
CASP3 c.-182–247G > T
GG	15 (35.7)	18 (32.1)	30 (34.9)	3 (25.0)	25 (30.9)	8 (47.1)	17 (35.4)	16 (32.0)	22 (35.5)	11 (30.6)
GT or TT	27 (64.3)	38 (67.9)	56 (65.1)	9 (75.0)	56 (69.1)	9 (52.9)	31 (64.6)	34 (68.0)	40 (64.5)	25 (69.4)
P-value	0.545		0.500		0.204		0.919		0.619
OR (95% CI)	1.32 (0.53–3.29)		1.60 (0.40–6.38)		0.50 (0.17–1.45)		1.04 (0.44–2.46)		1.25 (0.51–3.01)
GG or GT	36 (85.7)	49 (87.5)	76 (88.4)	9 (75.0)	71 (87.7)	14 (82.4)	42 (87.5)	43 (86.0)	56 (90.3)	29 (80.6)
TT	6 (14.3)	7 (12.5)	10 (11.6)	3 (25.0)	10 (12.3)	3 (17.6)	6 (12.5)	7 (14.0)	6 (9.7)	7 (19.4)
P-value	0.568		0.213		0.560		0.997		0.177
OR (95% CI)	0.70 (0.21–2.34)		2.53 (0.58–10.94)		1.52 (0.37–6.24)		1.00 (0.29–3.40)		2.25 (0.69–7.32)
CASP3 + CASP3
GT	14 (33.3)	20 (35.7)	28 (32.6)	6 (50.0)	29 (35.8)	5 (29.4)	17 (35.4)	17 (34.0)	17 (27.4)	17 (47.2)
Other haplotypes	28 (66.7)	36 (64.3)	58 (67.4)	6 (50.0)	52 (64.2)	12 (70.6)	31 (64.6)	33 (66.0)	45 (72.6)	19 (52.8)
P-value	0.499		0.241		0.616		0.987		0.049
OR (95% CI)	1.38 (0.54–3.51)		2.07 (0.61–7.00)		0.15 (0.02–1.25)		1.00 (0.43–2.35)		2.36 (1.00–5.59)^g

G: grade of toxicity; N: number of patients; OR: odds ratio; CI: confidence interval; NE: not evaluated.

ORs were adjusted by gender to lymphopenia, and by age and tumor stage to anemia. The total number of patients differed from the total quoted in the study (N = 109) because it was not possible to obtain consistent information about toxicities in some cases.

Power of analysis (PA): 45.8%; ^bPA: 72.4%; ^cPA: 56.1%; ^dPA: 56.0%; ^ePA: 71.9%; ^fPA: 51.7%; ^gPA: 51.7%. ^a and ^eSignificant even after Bonferroni correction for multiple comparisons (corrected P-value = 0.01).

The SNVs analyzed in this study did not alter the occurrence of vomiting, response to CDDP chemoradiation, and nephrotoxicity (Supplementary Table 1). However, an excess of FASL c.-844CC genotype was found in patients with ototoxicity (all grades) than in those without ototoxicity (38.2% versus 9.5%, P = 0.02; PA = 75.4%) (Figure 1(c)); patients with FASL c.-844CC genotype had 5.77 more chance of presenting ototoxicity after therapy than those with FASL c.-844CT or TT genotype.

Association between genetic variants and survival

At 24 months of follow-up, lower EFS and OS were observed in patients with stage III or IV tumor (38.8% versus 83.3%; P = 0.02) and (44.1% versus 83.3%; P = 0.01), respectively, compared with those with tumor at stage I or II (Kaplan–Meier estimates). The variable mentioned above remained predictors of shorter EFS and OS in HNSCC patients in univariate analysis. Multivariate analysis showed that tumor at III or IV stage (HR: 9.77, P = 0.02; P_bootstrap = 0.02) and (HR: 9.74, P = 0.02; P_bootstrap = 0.01), and FAS c.-671GG genotype (HR: 2.05, P = 0.007, P_bootstrap = 0.02) and (HR: 1.83, P = 0.02, P_bootstrap = 0.045) were independent predictors of shorter EFS and OS, respectively (Table 3, Figure 1(d) and (e)). EFS and OS were not altered by TP53 c.215G > C, FAS c.-1378G > A (Figure 1(f) and (g)), FASL c.-844 C > T (Figure 1(h) and (i)), CASP3 c.-1191A > G, and CASP3 c.-182-247G > T SNVs.

Table 3.

Association of clinicopathological aspects and P53 c.215G > C, FAS c.-671A > G and c.-1378G > A, FASL c.-844 C > T, CASP3 c.-1191A > G and c.-182-247G > T genotypes and FAS and CASP3 haplotypes of 109 head and neck squamous cell carcinoma patients with event-free survival and overall survival.

Variable	Event-free survival					Overall survival
	N events/N total	Univariate		Multivariate		N events/N total	Univariate		Multivariate
	N events/N total	P-value	HR (95% CI)	P-value	HR (95% CI)	N events/N total	P-value	HR (95% CI)	P-value	HR (95% CI)
Age (years)
≤56	40/56	0.24	1.31 (0.82–2.08)	NE		38/56	0.79	1.06 (0.67–1.67)	NE
>56	34/53		Reference			37/53		Reference
Gender
Male	66/101	0.10	Reference	0.06	Reference	70/101	0.83	Reference	NE
Female	8/8		1.85 (0.88–3.87)		2.01 (0.96–4.22)	5/8		1.10 (0.44–2.74)
Ethnic origin
White	64/97	0.60	1.19 (0.61–2.33)	NE		67/97	0.73	1.13 (0.54–3.37)	NE
Non-white	10/12		Reference			8/12		Reference
Tobacco consumption
Smokers	71/106	0.23	2.03 (0.63–6.49)	NE		74/106	0.41	2.27 (0.31–16.36)	NE
Nonsmokers	3/3		Reference			1/3		Reference
Alcohol consumption
Drinkers	69/100	0.57	1.30 (0.52–3.24)	NE		70/100	0.39	1.48 (0.59–3.68)	NE
Abstainers	5/9		Reference			5/9		Reference
Tumor location
Oral cavity or oropharynx	44/64	0.90	Reference	NE		43/64	0.96	0.99 (0.62–1.56)	NE
Hypopharynx or larynx	30/45		1.03 (0.64–1.64)			32/45		Reference
Histological grade
Well or moderately	49/72	0.24	Reference	NE		48/73	0.21	Reference	NE
Poorly or undifferentiated	11/16		1.47 (0.76–2.84)			12/16		1.50 (0.79–2.83)
Tumor stage
I or II	1/6	0.04	Reference	0.02^a	Reference	1/6	0.04	Reference	0.02 ^c	Reference
III or IV	73/103		7.28 (1.00–52.58)		9.77 (1.33–71.41)	74/103		7.95 (1.10–57.46)		9.74 (1.33–70.92)
P53 c.215G > C
GG	35/51	0.85	Reference	NE		35/51	0.85	Reference
GC or CC	39/58		0.95 (0.60–1.51)			40/58		1.04 (0.66–1.64)
GG or GC	66/98	0.89	Reference	NE		68/98	0.73	Reference	NE
CC	8/11		0.95 (0.45–1.98)			7/11		0.87 (0.40–1.90)
FAS c.-671A > G
AA	23/34	0.77	Reference	NE		22/34	0.31	Reference	NE
AG or GG	51/75		1.07 (0.65–1.76)			53/75		1.29 (0.78–2.12)
AA or AG	54/83	0.07	Reference	0.007 ^b	Reference	56/83	0.11	Reference	0.02 ^d	Reference
GG	20/26		1.59 (0.95–2.67)		2.05 (1.21–3.45)	19/26		1.52 (0.90–2.56)		1.83 (1.08–3.10)
FAS c.-1378G > A
GG	53/80	0.87	Reference	NE		54/80	0.86	Reference	NE
GA or AA	21/29		0.96 (0.57–1.59)			21/29		0.95 (0.57–1.58)
GG or GA	69/102	0.99	Reference	NE		71/102	0.54	Reference	NE
AA	5/7		0.99 (0.40–2.47)			4/7		0.73 (0.26–2.01)
FAS + FAS
GA	14/24	0.93	0.97 (0.56–1.68)	NE		17/24	0.90	0.96 (0.56–1.66)	NE
Other haplotypes	57/85		Reference			58/85		Reference
FASL c.-844 C > T
CC	18/32	0.32	Reference	NE		21/32	0.96	Reference	NE
CT or TT	56/77		1.30 (0.76–2.21)			54/77		1.01 (0.61–1.67)
CC or CT	53/82	0.21	Reference	NE		56/82	0.48	Reference	NE
TT	21/27		1.38 (0.83–2.29)			19/27		1.20 (0.71–2.03)
CASP3 c.-1191A > G
AA	33/45	0.39	Reference	NE		33/45	0.23	Reference	NE
AG or GG	41/64		0.82 (0.51–1.29)			42/64		0.75 (0.47–1.19)
AA or AG	68/98	0.29	Reference	NE		67/98	0.85	Reference	NE
GG	6/11		0.63 (0.27–1.47)			8/11		0.93 (0.44–1.94)
CASP3 c.-182–247G > T
GG	24/39	0.59	Reference	NE		29/39	0.78	Reference	NE
GT or TT	50/70		1.14 (0.70–1.85)			46/70		0.93 (0.58–1.49)
GG or GT	65/96	0.86	Reference	NE		67/96	0.66	Reference	NE
TT	9/13		0.94 (0.46–1.89)			8/13		0.85 (0.40–1.77)
CASP3 + CASP3
GT	26/36	0.45	1.19 (0.74–1.93)	NE		24/36	0.93	1.01 (0.62–1.65)	NE
Other haplotypes	48/73		Reference			51/73		Reference

N: number, HR: hazard ratio, CI: confidence interval, NE: not evaluated.

P-values ≤ 0.15 are presented in bold letters for univariate Cox analysis.

In multivariate Cox analysis adjusted by gender and tumor stage: ^aP_bootstrap = 0.02; ^bP_bootstrap = 0.02; ^cP_bootstrap = 0.01; ^dP_bootstrap = 0.04. The total number of patients differed from the total quoted in the study (N = 109) because it was not possible to obtain consistent information about histological grade in some cases.

Discussion

We investigated, in this prospective study, the roles of TP53 c.215G > C, FAS c.671A > G and c.1378G > A, FASL c.-844 C > T, CASP3 c.-1191A > G and c.-182-247G > T SNVs, and FAS and CASP3 haplotypes in adverse events, response to therapy, and survival of HNSCC patients treated with RT and concomitant CDDP.

We initially observed that clinicopathological aspects of our patients were in general like those seen in patients from other parts of the world,^29,42–45 which indicate that our sample was representative of HNSCC in medical practice settings. The more consistent difference between our cases and others seemed to occur in HNSCC carcinogenesis. Although tobacco and alcohol consumption and HPV16 infection were significant risk factors for HNSCC worldwide,⁴ HPV was not identified in our patients and was uncommon in those from other regions of the country.^45,46

Second, we found that patients with FAS c.-671GG genotype and FAS c.-671AG or GG genotype had 5.52 and 4.30 more chances of presenting anemia grades 2–4 and lymphopenia grade 3 or 4 with CDDP chemoradiation than those with the remaining genotypes, respectively. Myelotoxicity was not altered by TP53 c.215G > C, FAS c.1378G > A, FASL c.-844 C > T, CASP3 c.-1191A > G and c.-182-247G > T SNVs. Khrunin et al.¹⁷ found association of TP53 c.215CC genotype with severe neutropenia in ovarian cancer patients treated with CDDP-cyclophosphamide. The difference in results obtained in our study and Khrunin’s study may be attributed to distinct types of tumors and treatments. To the best of our knowledge, the role of FAS c.-671A > G SNV in cytopenias induced by CDDP chemoradiation was not previously analyzed.

It is already known that CDDP may induce a deep transient erythropoiesis alteration leading to anemia.^47,48 CDDP inhibits bone marrow cell proliferation by DNA damage,⁴⁹ possibly due to apoptosis induced by FAS activation.⁹ Because the allele G of FAS c.671A > G was previously associated with lower apoptosis of damaged cells,¹³ these results were not expected by us. However, activation of FAS/FASL was described as an important mechanism in erythroid maturation⁵⁰ and regulation of red blood cell homeostasis,⁵¹ and Rehman et al.⁵² observed that G allele of FAS c.-671A > G increased aplastic anemia risk. Moreover, deletion of FAS in lymphocytes caused lymphopenia in mice,⁵³ suggesting that FAS expression in lymphocytes is required for their survival and/or proliferation.⁵⁴ So, we hypothesize that abnormalities in erythroid maturation and lymphocytes survival and/or proliferation may have contributed to the moderate/severe anemia and lymphocitopenia seen in our patients with FAS c.-671GG genotype under CDDP chemoradiation.

We also observed that patients with FASL c.-844CC genotype had 5.77 more chance of presenting ototoxicity after CDDP chemoradiation than those with the remaining genotypes. So far, our knowledge reaches, there are no previous studies focusing on the role of FASL c.-844 C > T in ototoxicity induced by RT and CDDP.

Such association is biologically plausible because FASL c.-844CC genotype was related to higher apoptosis capacity,¹⁴ CDDP induces apoptosis in cells of the cochlea by generating reactive oxygen species (ROS),⁵⁵ and ROS can promote programmed cell death from activation of the FAS/FASL pathway in auditory hair cell.⁵⁶

Third, no association of TP53 c.215G > C, FAS c.-671A > G and c.1378G > A, FASL c.-844 C > T, CASP3 c.-1191A > G and c.-182-247G > T with response to treatment was seen in this study. Shiraishi et al.²⁰ analyzed association of TP53 c.215G > C SNV with response to platinum plus vinorelbine, paclitaxel, docetaxel, or gemcitabine in lung cancer patients, and observed that patients with the CC genotype obtained a better response than others. Again, the distinct types of tumor and treatments may explain differences of results found in both studies.

Fourth, we observed that HNSCC patients with FAS c.-671GG genotype had lower EFS and OS, respectively, than those with the remaining genotypes, but TP53 c.215G > C, FAS c.1378G > A, FASL c.-844 C > T, CASP3 c.-1191A > G and c.-182-247G > T SNVs did not alter patients’ survival. Zhang et al.²² evidenced associations of FAS c.-671AG or GG and FASL c.-844CT or TT genotypes with lower DFS in SCCO patients treated with RT, chemotherapy, and/or surgery, and the associations were particularly seen in patients with HPV16-positive tumors. No significant associations between FAS c.1378G > A SNP and patients’ survival were found in their study. Shiraishi et al.²⁰ found longer DFS and OS in lung cancer patients with CC genotype of TP53 c.215G > C treated with platinum plus vinorelbine, paclitaxel, docetaxel, or gemcitabine, and Azad et al.²¹ observed lower DFS in early-stage HNSCC patients radiation-treated with the CC genotype of TP53 c.215G > C compared with those with the remaining genotypes. FAS c.1378AA genotype was associated with higher OS in chemoradiation-treated HPV16-positive SCCO patients analyzed by Feng et al.²³ Again, the opposed results obtained in our study and previously reported studies may be attributed to the different types of tumors, carcinogenesis, or treatments.

Since the FAS c.-671GG genotype was related to lower apoptosis of cancer cells,¹³ the association of genotype with lower EFS and OS in HNSCC patients is also biologically plausible, and probably results from tumor cells survival and proliferation.

At this time, it is important to comment that although Zhang et al.²² had seen association of FAS c.-671AG or GG genotype with lower DFS particularly in HPV16-positive SCCO patients, we found associations of the GG genotype with DFS and OS of HNSCC, which were strongly related to tobacco smoking and alcohol consumption.

Finally, we postulate that although we have not evaluated combinations of genotypes of distinct genes, we cannot exclude co-participation of TP53 c.215G > C, CASP3 c.-1191A > G, and CASP3 c.-182-247G > T SNVS in associations between FAS and myelotoxicity and patients’ survival and FASL and ototoxicity found in our study.

We are aware that our study has some inherent limitations, such as the relatively small number of patients and the lack of investigation of HPV16 in all tumor samples, which may have limited the obtaining and interpretation of the findings.

In conclusion, our data present, for the first time, associations of FAS c.-671A > G and FASL c.-844 C > T with hematological toxicity, ototoxicity, and survival of tobacco- and alcohol-related HNSCC patients homogeneously treated with CDDP chemoradiation. Nevertheless, large studies with evaluation of HPV-16 in tumor fragments are needed to validate our results and further explore the molecular mechanisms that underlie the observed associations, which may have future utility as clinical prognostic biomarkers.

Supplemental Material

Supplementary_Material – Supplemental material for FAS and FASL variations in outcomes of tobacco- and alcohol-related head and neck squamous cell carcinoma patients

Supplemental material, Supplementary_Material for FAS and FASL variations in outcomes of tobacco- and alcohol-related head and neck squamous cell carcinoma patients by Ericka Francislaine Dias Costa, Tathiane Regine Penna Lima, Leisa Lopes-Aguiar, Guilherme Augusto Silva Nogueira, Marília Berlofa Visacri, Julia Coelho França Quintanilha, Eder Carvalho Pincinato, Luciane Calonga, Fernanda Viviane Mariano, Albina Messias de Almeida Milani Altemani, João Maurício Carrasco Altemani, Patrícia Moriel, Carlos Takahiro Chone, Celso Dario Ramos and Carmen Silvia Passos Lima in Tumor Biology

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by State of São Paulo Research Foundation (FAPESP) (grant numbers 2012/01807-2, 2012/01418-6).

ORCID iD

Carmen Silvia Passos Lima

Supplemental material

Supplemental material for this article is available online.

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