Area under curves (AUC) as a measure of predictive strength for risk-prediction models based on different indicators^a.

a<p>The environmental model is based on consumption of salted fish and preserved vegetables, and cumulative amount of smoking. The family history of NPC model includes family history of NPC only. The epidemiological model combines both environmental and family history of NPC predictors. The genetic risk score model includes a score derived from seven SNPs identified in the Cantonese GWAS. The inclusive model integrates all data on epidemiological and genetic predictors.</p>b<p>χ² statistic and <i>P</i> value was calculated from the Hosmer–Lemeshow Goodness-of-Fit test, a model with χ² statistic <20 (<i>P</i>>0.01) is considered as a good calibration.</p>c<p>AUC of the models were compared with a nonparametric approach, and <i>P</i> value was obtained from the comparison of the inclusive model with the other models.</p

Loss of KLHDC4 reduces NPC cell migration and invasion.

<p>(A) Wound-healing assays were performed at 0 and 20 hours with control and KLHDC4 KO cells. Left panels: representative images; Scale bars: 50 μm. Right panels: quantification of the wound closure area calculated by measuring the decreaseinthe wound bed surface overtime. **<i>P</i> <0.01. (B) KLHDC4 KO significantly reduced the invasive ability of CNE2 cells. Left panels: representative images; Scale bars: 50 μm. Right panels: quantification of average number of cells per field. ***<i>P</i> <0.001.</p

KLHDC4 is overexpressed in NPC tissue and cell lines.

<p>(A) Immunohistochemical image of three NPC samples containing both normal adjacent epithelial and tumor tissues stained with an anti-KLHDC4 antibody. N: normal adjacent epithelial tissues; T: tumor tissues. (B) Western blot of KLHDC4 expression in the above cell lines with GAPDH as the loading control. (C) Increased levels of KLHDC4 mRNA are commonly detected in various types of human cancers, as revealed by analyzing Gene Expression Omnibus microarray datasets. The line inside the boxes represents the median value. The box length indicates the interquartile range. The solid round dots have values > 1.5 interquartile ranges but < 3 interquartile ranges from the end of the box. N: normal tissues; T: tumor tissues; M: metastatic tissues. The numbers under the tissue type indicate the total cases of each cancer type. The <i>P</i> values separated with slashes indicate comparisons of normal/tumor, tumor/metastatic and normal/metastatic tissues.</p

Loss of KLHDC4 induces apoptosis in NPC cells.

<p>(A) Control and KLHDC4 KO cells were stained with DAPI and photographed by fluorescence microscopy under fluorescence and light fields. Left panel: representative images; the arrows indicated the condensed or fragmented nuclear and multiblebbing cells. Right panels: quantification of cells with condensed nuclear per field. **<i>P</i> <0.01. (B) Representative flow cytometric analysis of apoptotic CNE2 control and KLHDC4 KO cells stained for annexin V-FITC/PI under both non-treated and cis-platin treated conditions. The results are summarized in the right panel. Bars denote the S.E.M. (C) Western blot analysis of the expression of cleaved caspase-3, cleaved PARP and GAPDH, as the loading control, under both non-treated and cis-platin treated conditions. (D) Immunohistochemical analysis of KLHDC4, cleaved caspase-3, cleaved PARP, and Ki-67 expression in xenografts generated from CNE2 control and KLHDC4 KO cells. Left panels: representative images; Scale bars: 100 μm. Right panels: quantification of average percentage of cells with positive staining per field; CC3: cleaved caspase-3; CP: cleaved PARP. ***<i>P</i> <0.001.</p

Reclassification of data for use in epidemiological and inclusive models^a.

a<p>NRI: net reclassification improvement; IDI: integrated discrimination index; Reclassification was calculated for strata of predicted risk of <0.2, 0.2 to 0.3, and ≥0.3.</p

Distribution of risk for NPC by genetic risk score (in quintiles).

<p>Risk of NPC (expressed as OR ±95% CI) was adjusted for age, sex, education level, dialect, residential area, family history of NPC, pack-years smoked, salted fish and preserved vegetables consumption. The boundaries for each genetic risk score quintile are shown on the <i>x</i>-axis.</p

Knockout of KLHDC4 reduces NPC cell growth.

<p>(A) A T7E1 assay showed that mutation at the KLHDC4 locus was induced by transgenic Cas9 and sgRNA#2 but not sgRNA#1. (B) Western blotting confirmed that KLHDC4 was absent in KO cells. (C) Indel mutations induced by transgenic Cas9 and sgRNA#2 at the KLHDC4 locus. Representative DNA sequencing results for PCR products from KLHDC4 KO cells showing indel mutations at the targeted locus. Red dashes, deleted bases; Red bases, insertions; Asterisks, consensus bases; Red triangle, putative excision site. (D) KLHDC4 KO reduced the growth rate of CNE2 cells <i>in vitro</i>. **<i>P</i> <0.01. (E) KLHDC4 KO dramatically reduced colony formation in soft agar. Scale bars: 200 μm. Bars denote S.E.M. ***<i>P</i> <0.001. (F) All tumors isolated from mice are shown. Note none of the 8 mice inoculated with the control cells showed visible tumor growth, whereas only 3 of the 8 mice inoculated with KLHDC4 KO cells did. (G-H) Growth curves (G) and tumor weights (H) of xenograft tumors from experiments with nude mice. Changes in tumor volumes measured on the indicated days are shown. Bars denote S.E.M. *<i>P</i> <0.05, **<i>P</i> <0.01.</p

Distribution of genetic risk score.

<p>Distribution of the seven SNPs-based genetic risk score in 1,387 NPC cases (black bars) and 1,459 controls (grey bars). Individual risk for NPC was calculated by weighting each risk allele with its corresponding risk coefficient, which was derived from logistic regression.</p

Elevated KLHDC4 expression correlates with poor prognosis in NPC patients.

<p>(A) Criteria for scoring of KLHDC4 expression intensity. Representative images are shown. All images were obtained and processed under identical conditions. Scale bars: 100 μm. (B-C) Kaplan-Meier analysis of KLHDC4 expression and overall survival (B) and metastasis-free survival rate (C) of NPC patients.</p

Associations between genetic variants, epidemiological risk factors and risk of nasopharyngeal carcinoma.

a<p>OR = odds ratio; CI = confidence interval. OR and 95% CI were derived from logistic regression, with adjustment for age, sex, education level, dialect, household type (rural/urban).</p>b<p>OR and 95% CI were derived using logistic regression adjusted for age, sex, education level, dialect, rural or urban household type, and all other variables listed in the table.</p>c<p><i>P</i> values for trend (two-sided) were derived from Cochran- Armitage trend tests.</p