The authors have declared that no competing interests exist.
Conceived and designed the experiments: TSP EvdV MGN RTNM. Performed the experiments: TSP EvdV. Analyzed the data: TSP AH. Contributed reagents/materials/analysis tools: EvdV. Wrote the manuscript: TSP EvdV MGN LABJ JWAS RTNM AH.
Autophagy is a central process in regulation of cell survival, cell death and proliferation and plays an important role in carcinogenesis, including thyroid carcinoma. Genetic variation in autophagy components has been demonstrated to influence the capacity to execute autophagy and is associated with disease susceptibility, progression and outcome. In the present study, we assessed whether genetic variation in autophagy genes contributes to susceptibility to develop thyroid carcinoma, disease progression and/or patient outcome. The results indicate that patients carrying the
Epithelial cell derived non-medullary thyroid cancer (NMTC) is the most common endocrine malignancy with a rising incidence during the last decades of which papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) represent the vast majority of cases
Increasing evidence suggests that autophagy plays an important role in the pathophysiology of the malignant process. Autophagy is a complex process of auto-digestion in conditions of cellular stress, hypoxia or energy deprivation. Upon activation, an autophagosome is formed which engulfs cellular components such as organelles, ribosomes and protein aggregates, which are subsequently degraded by fusion of the autophagosome with a lysosome. These degradation products can be reused for building macromolecules and for cellular energy metabolism
Essential components of the autophagy process are the evolutionary highly conserved ATG proteins, of which more than 30 have currently been identified in yeasts
The study was approved by the Ethical Committee of Radboud University Medical Centre, Nijmegen, The Netherlands. All subjects gave written informed consent. The study has been performed in accordance with the Declaration of Helsinki.
All patients with histologically confirmed non-medullary epithelial cell derived NMTC who visited the outpatient clinic at the Division of Endocrinology of the Department of Internal Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands, were asked to participate in genetic testing. The recruitment of the patients took place between November 2009 and June 2010. Primary treatment of the patients consisted of total or near-total thyroidectomy in all of the patients, and modified radical lymph node dissections in patients with confirmed nodal metastases. This was followed by ablation with radioactive iodine (I131, RAI) of residual thyroid tissue 4–6 weeks after surgery. If necessary, patients were treated multiple times with RAI to reach remission. Initial cure was defined as undetectable Thyroid Stimulating Hormone stimulated thyroglobulin (Tg) in the absence of anti-Tg antibodies and no evidence of loco-regional disease or distant metastasis on whole body iodine scans (WBS) and/or neck ultrasonography examinations at six to nine months after RAI ablation. Tumor recurrence was defined as new evidence of loco-regional disease or distant metastasis after successful primary therapy. Current disease status was defined as “in remission” in case of undetectable Tg in the absence of anti-Tg antibodies and no evidence of loco-regional disease or distant metastases at the last follow-up visit. Persistent disease status was defined as detectable Tg and/or evidence of loco-regional disease or distant metastases.
Demographic and clinical characteristics (tumor histology and TNM staging), treatment (number of RAI therapy sessions, cumulative RAI dose), follow-up time, the number of re-operations and external beam radiation therapy, if applicable, were retrieved from the patient's medical records (
Variable | Total (±SD) | Variable | Total (%) |
Patients (number) | 139 | Cum. RAI dose ≤3.7 GBq | 35 (25.2%) |
Gender (Female/Male) | 104/35 | Cum. RAI dose 3.8–7.4 GBq | 50 (36.0%) |
Age at diagnosis, years (mean ± SD) | 38.9 (±12.8) | Cum. RAI dose >7.4 | 54 (38.8%) |
Tumor histology | Total (±SD) | TNM staging | Total (%) |
Papillary thyroid cancer | 99 | T1 | 41 (29.5%) |
Follicular thyroid cancer | 33 | T2 | 45 (32.3%) |
Both papillary and follicular | 5 | T3 | 23 (16.5%) |
Differentiated thyroid cancer, not further specified | 1 | T4 | 11 (8.0%) |
Poorly differentiated thyroid cancer | 1 | Tx | 19 (13.7%) |
Re-operations | 9 | N0 | 72 (51.8%) |
External beam radiation therapy | 2 | N1 | 46 (33.1%) |
Mean duration follow-up, months (mean ± SD) |
128 (±112) | Nx | 21 (15.1%) |
RAI sessions 0–1 | 82 (59.0%) | M0 | 96 (69.1%) |
RAI sessions ≥2 | 57 (41.0%) | M1 | 3 (2.1%) |
Persistent after ablation | 60 (43.2%) | Mx | 40 (28.8%) |
since diagnosis of NMTC (primary surgery).
Venous blood was drawn from the cubital vein of all participants into 10 ml EDTA tubes (Monoject). DNA was isolated from whole blood by using the isolation kit Puregene (Gentra Sytems, MN, USA), according to the manufacturer's protocol. Coding non-synonymous single nucleotide polymorphisms (SNPs) and a few SNPs in untranslated regions of the analyzed genes were selected based on previously published associations with human diseases and/or known functional effects on protein function or gene expression. A total of 10 SNPs in
Gene | SNP ID | Gene region | Amino acid change |
rs9323945 | Exon 19 | Asn1124Asp | |
rs3759601 | Exon 25 | Gln1383Glu | |
rs2245214 | Intron 6 | - | |
rs3734114 | Exon 1 | Ser62Pro | |
rs1864183 | Exon 4 | Thr212Met | |
rs72553867 | Exon 1 | Thr94Lys | |
rs4958847 | 3′ UTR | - | |
rs9577229 | Exon 3 | Ala204Val | |
rs482912 | Exon 2 | Ile318Val | |
rs883541 | Exon 1 | Thr31Ile |
UTR = untranslated region.
The difference in genotype frequencies between the patients and the control group were analyzed in a dominant, gene dosage and recessive model using logistic regression. The effect of the genotypes on epithelial derived NMTC susceptibility was estimated by calculating odds ratios (ORs) and their 95% confidence intervals (95% CI) using the same statistical methods. We also performed χ2 analysis, and if applicable logistic regression, to determine whether tumor size, cumulative RAI dose, number of RAI treatments, disease status after thyroidectomy plus radio-ablation (if applicable) and current disease status were associated with the genotype of the analyzed autophagy genes. The following parameters were analyzed: 1) the tumor size at time of diagnosis was classified according to the 6th edition of the UICC TNM classification
To test for differences between the three different genotype groups (homozygous wild-type (ancient), heterozygous, homozygous variant (derived)) in mean age at diagnosis, sex distribution or tumor histology (potential confounders), one-way ANOVA and Pearson χ2 analysis were used when appropriate. All statistical analyses were carried out with the SPSS software package (version 20.0). Overall, statistical tests were two-sided and a p-value below 0.05 was considered statistically significant.
From all the patients with NMTC who visited the outpatient clinic of the Radboud University Medical Centre, Nijmegen, The Netherlands between November 2009 and June 2010, 139 patients (104 women; mean age 38.9±12.8 (SD) years at time of blood sampling) agreed to participate in the study. The clinical and demographical characteristics of the NMTC patients are summarized in
Statistical analysis of autophagy genetic variants for NMTC susceptibility revealed a statistically significant assocation with the
Gene | Polymorphism | Allelic distribution | OR (95% CI) |
P-value |
|||
rs9323945 | CC | TC | 0.65 (0.20–2.18) | 0.547 | |||
Asn1124Asp | Patients | 133 (96%) | 6 (4%) | ||||
Controls | 184 (97%) | 5 (3%) | |||||
rs3759601 | CC | GC | GG | 0.70 (0.44–1.11) | 0.125 | ||
Gln1383Glu | Patients | 50 (36%) | 67 (48%) | 22 (16%) | |||
Controls | 54 (29%) | 105 (55%) | 30 (16%) | ||||
rs2245214 | CC | CG | GG | ||||
Intron 6 | Patients | 41 (30%) | 67 (48%) | 31 (22%) | |||
Controls | 66 (35%) | 98 (52%) | 25 (13%) | ||||
rs3734114 | CC | TC | TT | 1.53 (0.98–2.37) | 0.060 | ||
Ser62Pro | Patients | 9 (6%) | 40 (29%) | 90 (65%) | |||
Controls | 12 (6%) | 74 (39%) | 103 (55%) | ||||
rs1864183 | AA | GA | GG | 1.41 (0.85–2.33) | 0.204 | ||
Thr212Met | Patients | 32 (23%) | 68 (49%) | 39 (28%) | |||
Controls | 46 (24%) | 102 (54%) | 41 (22%) | ||||
rs72553867 | CC | CA | 1.59 (0.76–3.33) | 0.256 | |||
Thr94Lys | Patients | 124 (89%) | 15 (11%) | ||||
Controls | 175 (93%) | 14 (7%) | |||||
rs4958847 | AA | GA | GG | 0.88 (0.54–1.43) | 0.620 | ||
3′ UTR | Patients | 1 (1%) | 36 (26%) | 102 (73%) | |||
Controls | 3 (2%) | 44 (23%) | 142 (75%) | ||||
rs9577229 | CC | TC | 0.79 (0.05–12.78) | 1.000 | |||
Ala204Val | Patients | 138 (99%) | 1 (1%) | ||||
Controls | 188 (99%) | 1 (1%) | |||||
rs482912 | AA | GA | GG | 0.78 (0.51–1.20) | 0.276 | ||
Ile318Val | Patients | 11 (8%) | 63 (45%) | 65 (47%) | |||
Controls | 18 (10%) | 70 (37%) | 101 (53%) | ||||
rs883541 | AA | GA | GG | 1.35 (0.88–2.08) | 0.185 | ||
Thr31Ile | Patients | 74 (53%) | 58 (42%) | 7 (5%) | |||
Controls | 115 (61%) | 66 (35%) | 8 (4%) |
* Dominant model.
Within the NMTC patient cohort, associations between genotype and tumor size (T stage), number of I131 treatments, cumulative I131 dose, disease status after ablation and current disease status were assessed using Pearson χ2 analysis. For the
Variable | CC (%) | CG (%) | GG (%) | Total | P-value |
0.962 | |||||
13 (31%) | 18 (27%) | 10 (32%) | 41 | ||
12 (29%) | 22 (33%) | 11 (35%) | 45 | ||
6 (15%) | 13 (19%) | 4 (13%) | 23 | ||
4 (10%) | 5 (8%) | 2 (7%) | 11 | ||
6 (15%) | 9 (13%) | 4 (13%) | 19 | ||
0.176 | |||||
23 (56%) | 33 (49%) | 16 (52%) | 72 | ||
9 (22%) | 27 (40%) | 10 (32%) | 46 | ||
9 (22%) | 7 (11%) | 5 (16%) | 21 | ||
0.633 | |||||
26 (64%) | 49 (73%) | 21 (68%) | 96 | ||
1 (2%) | 2 (3%) | 0 | 3 | ||
14 (34%) | 16 (24%) | 10 (32%) | 40 | ||
0.856 | |||||
24 (59%) | 39 (58%) | 19 (61%) | 82 | ||
17 (41%) | 28 (42%) | 12 (39%) | 57 | ||
0.626 | |||||
8 (20%) | 17 (25%) | 10 (32%) | 35 | ||
17 (41%) | 22 (33%) | 11 (36%) | 50 | ||
16 (39%) | 28 (42%) | 10 (32%) | 54 | ||
0.872 | |||||
25 (61%) | 37 (55%) | 17 (55%) | 79 | ||
16 (39%) | 30 (45%) | 14 (45%) | 60 | ||
0.230 | |||||
32 (78%) | 48 (72%) | 27 (87%) | 107 | ||
7 (17%) | 18 (27%) | 3 (10%) | 28 | ||
2 (5%) | 1 (1%) | 1 (3%) | 4 |
Calculated by Pearson χ2 analysis.
The present study was performed to investigate whether common genetic variants in human autophagy genes are associated with NMTC susceptibility, severity and/or clinical outcome. We found that one of the selected genetic variants, the
All of the investigated proteins are involved in the autophagy machinery, some in the early phase of autophagosome formation (ATG2B, ATG5, ATG10, IRGM and WIPI1), the others in the late phase of autophagosome-lysosome fusion (LAMP1 and LAMP3)
In terms of carcinogenesis, the role of autophagy is complex and depends on the type of cancer and the stage of the disease. Defects in autophagy may mediate carcinogenesis through accumulation of protein aggregates and damaged organelles. On the other hand, in apoptotic-competent cells autophagy is cytoprotective, as these cells depend on autophagy to cover their increased energy expenditure
The present genetic association study revealed that the G allele of the
Genetic variation in
Our previous report of the genetic association of the
Multiple studies have shown the important role of autophagy in NMTC pathogenesis, representing one of the most prominent downstream pathways of the often aberrantly regulated RAS/RAF/MEK/ERK and PI3K/Akt/mTOR pathways in NMTC, leading to inactivation of the autophagy machinery
An important point to be considered is that of correction for multiple testing in this study. It has to be taken into account that, when applying correction for multiple testing, statistical significance of the
In conclusion, we have identified the