Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Bredel M, Scholtens DM, Yadav AK, et al. NFKBIA deletion in glioblastomas. N Engl J Med 2;364:627-37. DOI:.56/NEJMoa632.
TUMOR SAMPLES AND PATIENTS
CELL LINES AND CELL CULTURE
GENOMIC DNA PREPARATION GENOMIC DATA PRE-PROCESSING COPY NUMBER VARIATION ANALYSIS
MUTATIONAL ANALYSES
REAL-TIME POLYMERASE CHAIN REACTION RETROVIRAL INFECTION AND EGF STIMULATION μ μ μ
PROTEIN EXTRACTION AND IMMUNOBLOTTING CELL VIABILITY ASSAY μ μ CELL CYCLE ANALYSIS CELLULAR SENESCENCE ASSAY
WOUND HEALING ASSAY SOFT AGAR ASSAY APOPTOSIS ASSAYS μ μ
STATISTICS
75 Glioblastomas of Study Set NFKBIA gene expression (log2r/g) 2 3 p =.9 NFKBIA wt/egfr wt NFKBIA del only EGFR + only NFKBIA del/egfr + Linear regression LOWESS smooth fit 9 4 5 6 7 8 9 2 EGFR gene expression (log2r/g) Suppl. Figure. Relationship between NFKBIA and EGFR Expression in Glioblastoma. Linear regression of EGFR gene expression on NFKBIA gene expression in 75 glioblastomas of Study Set indicates a statistically significant relationship between both transcripts in 75 glioblastomas of Study Set : tumors with diminished NFKIBA expression due to gene deletion (del) have comparatively low EGFR expression, and tumors with high EGFR expression due to gene amplification (+) have intact NFKBIA expression. Gene expression values are expressed as log2r/g ratios estimated by the RMA pre-processing algorithm. Locally weighted least squares (LOWESS) smooth fit confirmed the appropriateness of a linear regression analysis.
U8 U87 LN229 ctrl ctrl NFKBIA+ ctrl NFKBIA+ NFKBIA+ ectopic endog. NFKBIA Flag α-tubulin NFKBIA protein (%) 25 2 5 5 U8 U87 LN229 ctrl NFKBIA+ Suppl. Figure 2. Retroviral Expression of NFKBIA in Human Glioblastoma Cells. Endogenous (ctrl) and ectopic (FLAG-tagged) NFKBIA protein expression assessed by immunoblotting in three glioblastoma cell lines (U8, U87, and LN229) with heterozygous NFKBIA deletions retrovirally transduced with NFKBIA (NFKBIA+).
A B p =.4 p =.35 C U87-ctrl Debris Dip G: 39.% Dip G2: 3.9% Dip S: 47.% U87-NFKBIA+ Debris Dip G: 62.9% Dip G2: 9.6% Dip S: 27.6% D Cell viability (normal. to øctrl).9.8.7.6.5.4.3.2. Cell viability (normal. to øwt) U8-ctrl U8-NFKBIA+.9.8.7.6.5.4.3.2. U87-ctrl U87-NFKBIA+ (5%) LN229-ctrl LN229-NFKBIA+ U87-ctrl U87-NFKBIA+ Debris Dip G: 54.5% Dip G2: 26.% Dip S: 9.5% Debris Dip G: 62.% Dip G2: 22.2% Dip S: 5.8% (5%) E U87-ctrl U87-NFKBIA+ F U87-ctrl U8-ctrl U87-NFKBIA+ U8-NFKBIA+ % soft agar colony 2 p =. 8 6 4 2 2 LN229-ctrl U8-ctrl LN229-NFKBIA+ U8-NFKBIA+ U8-ctrl LN229-ctrl U8-NFKBIA+ LN229-NFKBIA+ % soft agar colony % soft agar colony 8 6 4 2 2 8 6 4 2 p =.2 p =. Suppl. Figure 3. NFKBIA Functions as a Tumor Suppressor in Glioblastomas. (A) Retroviral re-expression of NFKBIA (NFKBIA+) in glioblastoma cells decreases cell proliferation and foci formation in culture and induces a vacuolated, senescence-like phenotype, compared to control (ctrl) cells. (B) NFKBIA re-expression decreases glioblastoma cell viability (mean ± SE) as measured spectrophotometrically via bioreduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye by dehydrogenase enzymes of metabolically active cells. p values according to unpaired t-test. (C) NFKBIA reexpression induces accumulation of cells in G/G and reduction of the transition from the G2 to the S phase of the cell cycle. (D) NFKBIA re-expression induces cellular senescence, as indicated by expression of senescence-associated β-galactosidase (SA-β-gal) (green); percentage of positively staining cells in a broader view indicated in parentheses. (E) NFKBIA re-rexpression reduces the migratory potential of glioblastoma cells, as assessed by a wound healing assay. (F) NFKBIA re-expression constrains the tumorigenic potential of glioblastoma cells, as measured by colony formation in soft agar (mean ± SE). p values according to unpaired t-test.
A C Cell viability (normal. to øwt) 4.9.8.7.6.5.4.3.2. U87-ctrl p =. U87-ctrl (TMZ) p =.7 U87-NFKBIA+ (TMZ) B U8-ctrl U8 (TMZ) U8-NFKBIA+ (TMZ) U87-ctrl (TMZ) U87-NFKBIA+ (TMZ) LN229-ctrl (TMZ) LN229-NFKBIA+ (TMZ) viable cells late stage 3.% 4 viable cells 9.44% 4 viable cells.95% 4 viable cells 5.85% all cells all cells all cells all cells 3 3 3 3 FL3 Log 2 FL3 Log 2 FL3 Log 2 FL3 Log 2 2 3 4 FL Log early stage 5.% 2 3 4 FL Log.4% 2 3 4 FL Log 2.55% 2 3 4 FL Log 9.35% Suppl. Figure 4. NFKBIA Sensitizes Glioblastoma Cells to Temozolomide. (A) Cell viability (mean ± SE) in mock (plain media)-treated U87 control (ctrl) cells, TMZ-treated U87 cells, and TMZ-treated U87 cells re-expressing NFKBIA (NFKBIA+), based on bioreduction of MTT dye by dehydrogenase enzymes of metabolically active cells. p values according to unpaired t-test. (B) Fluorescence microscopy and Hoechst staining of mock- and TMZ-treated U8-ctrl and U8-NFKBIA+ cells. Arrows: Nuclear chromatin condensation (pyknosis) indicates early apoptosis. (C) Flow cytometry analysis for early and late stages of apoptosis based on annexin V and propidium iodide staining in TMZ-treated U87-ctrl vs. U87-NFKBIA+ cells and LN229-ctrl vs. LN229-NFKBIA+ cells.
A 47 Glioblastomas of Study Set 8 Probability of Survival..8.6.4.2 NFKBIA high NFKBIA low Log-rank p =.5 B 9 Glioblastomas of Study Set 9 Probability of Survival..8.6.4.2 Log-rank p =. NFKBIA high NFKBIA low. 25 5 75 25 5 75 No. at Risk Time (weeks) 24 2 4 8 6 3 23 4 7 2 2 2 2. No. at Risk 95 96 5 5 2 25 3 Time (weeks) 62 33 22 5 9 49 3 6 5 2 35 8 2 4 45 5 Suppl. Figure 5. NFKBIA Expression Associated with Glioblastoma Outcome. Kaplan-Meier estimated survival functions for 47 glioblastoma patients of Study Set 8 (A) and for 9 patients of Study Set 9 (B). In both sets, patients were stratified according to median NFKBIA expression. Patients with tumors having high vs. low NFKBIA expression had estimated median survival durations of 8 vs. 32 weeks in Study Set 8 and estimated median survival durations of 77 vs. 5 weeks in Study Set 9 (see main text for corresponding Cox model statistics). Small vertical lines across the curves indicate patients who were alive at last follow-up. The numbers at risk refer to the numbers of patients at risk entering each indicated time interval. p values according to log-rank test.
22 Malignant gliomas of Study Set 7 NFKBIA expression (R/G x^3) 6 2 8 4 p =.2 p >.5 p >.5 Probability of Survival..8.6.4.2 NFKBIA Pri-high NFKBIA Pri-low Log-rank p =.2 Pri Rec (NFKBIA high) Pri Rec (NFKBIA low). No. at Risk 5 8 9 5 2 Time (weeks) 5 3 25 3 35 Suppl. Figure 6. NFKBIA Expression in Initial and Recurrent Tumor. Expression of NFKBIA in 22 matched malignant glioma pairs from primary (Pri) and recurrent (Rec) disease in Study Set 7 and corresponding estimates of overall survival for the patients with high vs. low ( vs. ) NFKBIA abundance at initial disease (left panel) indicate the survival significance of NFKBIA status at initial diagnosis in these patients. Gene expression values indicate the ratio of red (R, Cy5) to green (G, Cy3) fluorescence dye intensity values. Box plots depict the smallest and largest observations (upper and lower whiskers, respectively), interquartile range (IQR, box), and median (white line). Data observations more than.5 IQR lower than the first quartile or.5 IQR higher than the third quartile were considered as outliers. Subgroups in the left panel according to median NFKBIA abundance (Pri-high vs. Pri-low) in primary disease. Significant downregulation of NFKBIA in the recurrent disease of tumors with initially high expression (p =.2, Wilcoxon signed-rank test) implies that loss of NFKBIA could be a molecular feature associated with disease progression. Small vertical lines across the curves in the right panel indicate patients who were alive at last follow-up. The numbers at risk refer to the numbers of patients at risk entering each indicated time interval. p values according to log-rank test.
A 29 Glioblastomas of Study Set 7 B. MGMT high-risk/nfkbia high MGMT high-risk/nfkbia low.8 28 Glioblastomas of Study Set 8..8 MGMT high-risk/nfkbia high MGMT high-risk/nfkbia low Probability of Survival.6.4 Log-rank p =.3 Probability of Survival.6.4 Log-rank p =.37.2.2. No. at Risk 4 5 5 5 2 25 3 Time (weeks) 3 9 5 3 2 2 9 35. No. at Risk 4 4 25 5 7 8 75 Time (weeks) 4 3 25 3 5 75 C 4 Glioblastomas of Study Set 9 D. MGMT high-risk/nfkbia high MGMT high-risk/nfkbia low.8 2 Glioblastomas of Study Set treated with TMZ/RT..8 MGMT unmethyl./nfkbia high MGMT unmethyl./nfkbia low Probability of Survival.6.4 Log-rank p =.8 Probability of Survival.6.4 Log-rank p =..2.2. No. at Risk 57 57 5 5 2 25 3 Time (weeks) 34 6 9 5 4 25 5 3 3 35 3 4 45 5. No. at Risk 25 5 7 8 2 75 Time (weeks) 25 Suppl. Figure 7. Survival Association for NFKBIA in MGMT High-Risk Glioblastomas. Kaplan-Meier estimates of overall survival in glioblastoma patients of Study Sets 7, 8, 9, and. Small vertical lines across the curves indicate patients who were alive at last follow-up. The numbers at risk refer to the number of patients at risk entering each indicated time interval. p values according to log-rank test. (A) Estimated survival function for the instance of likely unmethylated MGMT gene promoter status (6% of tumors with highest MGMT expression) with high-risk for unfavorable disease in 29 glioblastomas of Study Set 7, separated into two groups according to median NFKBIA expression (see main text for corresponding Cox model statistic). (B) Survival estimates for a similar model in 28 glioblastomas of Study Set 8 (Cox model HR:.47, 95%-CI:.6-.98 for high vs. low NFKBIA expression). Estimated median durations of survival for high vs. low NFKBIA expression were 53 vs. 29.5 weeks. (C) Survival estimates for a similar model in 4 glioblastomas of Study Set 9 (Cox model HR:.63, 95%-CI:.43-.93 for high vs. low NFKBIA expression). Estimated median durations of survival for high vs. low NFKBIA expression were 59 vs. 44 weeks. (D) Survival estimates for 2 newly diagnosed glioblastoma patients of Study Set with unmethylated MGMT gene promoter status and treated with radiotherapy (RT) plus concomitant and adjuvant temozolomide (TMZ), stratified according to median NFKBIA expression (see main text for corresponding Cox model statistic).
A 9 Glioblastomas of Study Set 9 B 74 Glioblastomas of Study Set..8 NFKBIA/MGMT low-risk NFKBIA/MGMT intermediate-risk NFKBIA/MGMT high-risk..8 NFKBIA/MGMT low-risk NFKBIA/MGMT intermediate-risk NFKBIA/MGMT high-risk Probability of Survival.6.4 Log-rank p =.6 Probability of Survival.6.4 Log-rank p =.7.2.2. 5 5 2 25 3 35 4 45 5. 5 5 2 25 3 35 No. at Risk Time (weeks) No. at Risk Time (weeks) 49 35 2 6 6 6 92 53 2 9 6 4 3 5 23 4 3 3 24 2 9 4 3 3 9 8 3 3 2 2 8 Suppl. Figure 8. Combined NFKBIA/MGMT Predictor Model in Glioblastoma. Combined NFKBIA/MGMT risk group models in 9 glioblastomas of Study Set 9 (Panel A) and in 74 glioblastoma patients of Study Set (Panel B). Assignment of patients to low, moderate, and high-risk groups is based on NFKBIA expression (dichotomized at the median) and MGMT status (MGMT expression dichotomized at the median or based on MGMT gene promoter methylation). Higher-than-median NFKBIA expression and lower-thanmedian MGMT expression denotes a low-risk group in Panel A, as does higher-than-median NFKBIA expression and methylated MGMT promoter status in Panel B; lower-than-median NFKBIA expression and higher-than-median MGMT expression denotes a high-risk group in Panel A, as does lower-than-median NFKBIA expression and unmethylated MGMT promoter status in panel B; all other cases were assigned to an intermediate risk group. Small vertical lines across the curves indicate patients who were alive at last follow-up. The numbers at risk refer to the numbers of patients at risk entering each indicated time interval. p values according to log-rank test.