Association of ERBB Mutations with Clinical Outcomes of Afatinib- Or

Supplementary Online Content

Goss GD, Felip E, Cobo M, et al. Association of ERBB mutations with clinical outcomes of afatinib- or erlotinib-treated patients with lung squamous cell carcinoma: secondary analysis of the LUX-Lung 8 randomized clinical trial. JAMA Oncol. Published online June 14, 2018. doi:10.1001/jamaoncol.2018.0775

eAppendix. Methods eReferences. eTable 1. Baseline Clinical Characteristics eTable 2. All Genetic Aberrations Detected in the TGA Cohort eTable 3. Relationship Between EGFR Expression Status and PFS and OS eFigure 1. Comparison of Clinical Outcomes in the TGA Cohort and Overall LUX-Lung 8 (Intent-to-Treat) Population eFigure 2. Relationship Between the Most Frequent Tumor Genetic Aberrations and (A) PFS and (B) OS in the LUX-Lung 8 TGA Cohort eFigure 3. (A) Details of ErbB Family Mutations; (B) Location of HER2 Mutations eFigure 4. Standardized Effect Plot for PFS (Left) and OS (Right)

This supplementary material has been provided by the authors to give readers additional information about their work.

Downloaded From: https://jamanetwork.com/ on 09/26/2021 eAppendix. Methods Selection of samples for sequencing Selection of patients was performed in the following order: 1. From the first 176 patients investigated in the LUX-Lung 8 interim analysis (based on independent radiological review [IRR]), samples from patients that were in the upper and lower quartiles of maximum tumor shrinkage or had PFS > 6 months were selected. Upper and lower quartiles of maximum tumor shrinkage were investigated by an independent statistician. 2. From the data available for primary PFS analyses (based on IRR), all patients with a PFS > 2 months (censored or non-censored) were selected. For patients with a PFS ≤ 2 months and with progressive disease as the best overall response, 120 patients , i.e. 60 patients per treatment arm that were non-censored for PFS, were selected randomly.

As the enrichment process may lead to a bias in the statistical analysis, we always carefully interpreted HRs (afatinib vs erlotinib) in any given biomarker subgroup relative to the hazard ratio in the TGA subset and not the HR in the overall LUX-Lung 8 population.. Tumor samples from patients in China were not available for sequencing due to legal requirements, and had to be excluded from this analysis.

Statistical analysis The predictive potential of a binary biomarker variable B (alteration present/absent) was investigated using a Cox’s proportional hazard model. The hazard function of the ith patient is:

∙ ∙ ∙ ∙ ∙,

where t is the time point, is the baseline hazard function stratified by race (Eastern Asian vs Non-eastern Asian), and is the treatment effect. Ties were handled using the Breslow method. The P-value for the biomarker-by- treatment interaction was reported. Further, for each biomarker-defined subgroup, HRs (afatinib vs erlotinib) and their 95% Wald CIs were provided based on the hazard function:

∙ ∙.

Due to the small size of the biomarker-defined subgroups, the subgroup models were not stratified by race.

Kaplan-Meier curves for the two treatments were constructed for the biomarker-defined subgroups. The median of the time-to-event endpoint and its 95% CI were estimated using a log-log transformation.

All biomarker variables were analyzed separately. Multivariate models were not considered. Only patients without missing biomarker information were considered in this analysis.

Unadjusted P -values are reported throughout the main manuscript. Adjustments for multiple comparisons were performed using standardized effect plots (see Supplementary Results). The significance level was defined as α = .05. Analyses were performed using SAS version 9.2 or higher and R version 3.3.2 or higher.

Sample size considerations This was a retrospective and exploratory study, and no formal sample size calculation was performed. In particular, the study was not powered to detect a significant predictive biomarker effect, which typically requires large sample sizes or substantial effects.1

Results Standardized effect plots were produced to investigate if the relative treatment benefit (afatinib vs erlotinib) observed in a subgroup was different to the relative treatment benefit observed in the TGA.2 The standardized effect of a subgroup S was defined as:

log log

Downloaded From: https://jamanetwork.com/ on 09/26/2021 with the hazard ratio in the subgroup, the hazard ratio in the TGA, and the standard deviation of . A standardized effect <0 implied that the relative treatment benefit was more pronounced in the subgroup compared to the relative treatment benefit in the TGA.

P-values for the standardized effects were obtained using a permutation approach (i.e. by randomly permuting the clinical endpoint within each treatment arm R = 2000 times and estimating the standardized effects on these permuted data). In addition, to correct for multiple testing, an adjusted 95% CI and an adjusted P-value for the HER2 mutation-positive subgroup was computed.

Supplementary eFigure 4 displays the results. Unadjusted P-values for the HER2 mutation-positive subgroup were <.05 for PFS and OS. After correction for multiple comparisons, the adjusted P-values for this subgroup were .067 (PFS) and .052 (OS).

eReferences 1. Schmoor C, Sauerbrei W, Schumacher M. Sample size considerations for the evaluation of prognostic factors in survival analysis. Stat Med. 2000;19(4):441-452. 2. Dane, A. Standardised effect plots. Oral presentation presented at: 2016 PSI Conference 'Promoting Statistical Insight and Collaboration in Drug Development'; May 22 – 25, 2016; Berlin.

Downloaded From: https://jamanetwork.com/ on 09/26/2021 eTable 1. Baseline Clinical Characteristics

Characteristic

Overall TGA subset IHC analysis population (n = 245) subset (N = 795) (n = 345) Mean age (standard deviation) 64.1 (8.7) 64.4 (8.4) 63.9 (9.1) Gender (%) Female 16.2 11.8 16.8 Male 83.8 88.2 83.2 Ethnic origin (%) Non-eastern Asian 78.4 80.4 71.3 Eastern Asian 21.6 19.6 28.7 ECOG PS (%) 0 32.7 35.5 31.6 1 66.8 63.7 67.8 2 0.5 0.8 0.6 Smoking history (%) Never smoker 5.5 5.3 6.1 Light ex-smokera 2.9 3.3 1.7 Current or other ex-smoker 91.6 91.4 92.2 Tumor histology (%)b Squamous 96.0 96.7 95.7 Mixed 4.0 3.3 4.3 Best response to first-line Chemotherapy (%) CR/PR 46.7 45.3 47.2 SD 41.3 43.7 40.9 PD 0.9 0.0 1.2 Unknown 11.2 11.0 10.7 Abbreviations: CR, complete response; ECOG PS, Eastern Cooperative Oncology Group performance status; IHC, immunohistochemistry; PD, progressive disease; PR, partial response; SD, stable disease; TGA, tumor genetic analysis. a<15 pack-years and stopped >1 year before diagnosis. b4 patients in the erlotinib group in the overall population and IHC subset had undifferentiated tumor histology but were considered to be squamous by the treating investigator.

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eTable 2. All Genetic Aberrations Detected in the TGA Cohort

Gene At least one SV CNA REAR n % n % n % Total 245 100 245 100 245 100 ABL1 7 2.9 – – – – AKT1 4 1.6 4 1.6 – – AKT2 1 0.4 9 3.7 – – AKT3 7 2.9 2 0.8 – – ALK 24 9.8 – – 2 0.8 ALOX12B 5 2 – – – – APC 14 5.7 1 0.4 1 0.4 APCDD1 10 4.1 2 0.8 – – AR 21 8.6 – – – – ARAF 3 1.2 – – – – ARFRP1 2 0.8 4 1.6 – – ARID1A 27 11 – – 1 0.4 ARID2 13 5.3 2 0.8 – – ASXL1 10 4.1 9 3.7 – – ATM 30 12.2 – – – – ATR 24 9.8 17 6.9 1 0.4 ATRX 9 3.7 – – – – AURKA 5 2 1 0.4 – – AURKB 1 0.4 2 0.8 – – AXL 15 6.1 3 1.2 1 0.4 BACH1 10 4.1 – – 1 0.4 BAP1 5 2 1 0.4 – – BARD1 15 6.1 – – – – BCL2 2 0.8 1 0.4 – – BCL2L2 1 0.4 6 2.4 – – BCL6 10 4.1 75 30.6 – – BCOR 14 5.7 – – – – BCORL1 10 4.1 – – – – BCR – – – – 1 0.4 BLM 13 5.3 – – – – BRAF 7 2.9 – – – – BRCA1 14 5.7 – – – – BRCA2 34 13.9 – – – – BRIP1 24 9.8 1 0.4 – –

Downloaded From: https://jamanetwork.com/ on 09/26/2021 BTK 2 0.8 – – – – C11orf30 9 3.7 – – – – C17orf39 3 1.2 1 0.4 – – CARD11 16 6.5 2 0.8 – – CASP8 6 2.4 4 1.6 – – CBFB – – 1 0.4 – – CBL 13 5.3 – – – – CCND1 2 0.8 32 13.1 – – CCND2 4 1.6 14 5.7 – – CCND3 3 1.2 3 1.2 – – CCNE1 3 1.2 14 5.7 – – CD79A 1 0.4 2 0.8 – – CD79B 3 1.2 1 0.4 – – CDC73 5 2 2 0.8 – – CDH1 9 3.7 – – 1 0.4 CDK12 12 4.9 5 2 1 0.4 CDK4 2 0.8 4 1.6 – – CDK6 1 0.4 9 3.7 – – CDK8 2 0.8 – – – – CDKN1B 4 1.6 9 3.7 – – CDKN2A 70 28.6 36 14.7 2 0.8 CDKN2B 5 2 28 11.4 – – CDKN2C – – 3 1.2 – – CEBPA 1 0.4 21 8.6 – – CHEK1 3 1.2 1 0.4 – – CHEK2 7 2.9 1 0.4 – – CHUK 10 4.1 – – – – CIC 17 6.9 3 1.2 – – CRBN 5 2 – – – – CREBBP 22 9 – – – – CRKL – – 8 3.3 1 0.4 CRLF2 – – 3 1.2 – – CSF1R 16 6.5 1 0.4 – – CTCF 6 2.4 – – – – CTNNA1 9 3.7 – – – – CTNNB1 3 1.2 – – – – CUL4A 2 0.8 7 2.9 1 0.4 CUL4B 6 2.4 – – – – CYP17A1 1 0.4 1 0.4 – –

Downloaded From: https://jamanetwork.com/ on 09/26/2021 DAXX 6 2.4 – – – – DDR2 14 5.7 4 1.6 – – DIS3 6 2.4 1 0.4 – – DNMT3A 12 4.9 – – – – DOT1L 9 3.7 – – – – EGFR 16 6.5 17 6.9 – – EMSY – – 7 2.9 – – EP300 19 7.8 – – – – EPHA3 19 7.8 3 1.2 – – EPHA5 19 7.8 1 0.4 – – EPHB1 18 7.3 8 3.3 – – HER2 12 4.9 9 3.7 – – HER3 15 6.1 – – – – HER4 14 5.7 – – – – ERG 5 2 – – – – ESR1 15 6.1 1 0.4 – – ETV1 – – – – 1 0.4 ETV6 – – – – 1 0.4 EWSR1 – – – – 1 0.4 EZH2 6 2.4 2 0.8 – – FAM123B 7 2.9 – – – – FANCA 17 6.9 – – – – FANCC 5 2 – – – – FANCD2 19 7.8 1 0.4 1 0.4 FANCE 7 2.9 1 0.4 – – FANCF 7 2.9 – – – – FANCG 7 2.9 15 6.1 – – FANCI 6 2.4 – – – – FANCL 2 0.8 4 1.6 – – FANCM 28 11.4 1 0.4 – – FAT3 67 27.3 – – 1 0.4 FBXW7 18 7.3 2 0.8 – – FGF10 6 2.4 18 7.3 – – FGF12 3 1.2 69 28.2 – – FGF14 8 3.3 3 1.2 – – FGF19 2 0.8 31 12.7 – – FGF23 8 3.3 11 4.5 – – FGF3 4 1.6 31 12.7 – – FGF4 1 0.4 26 10.6 – –

Downloaded From: https://jamanetwork.com/ on 09/26/2021 FGF6 6 2.4 11 4.5 – – FGFR1 4 1.6 34 13.9 1 0.4 FGFR2 8 3.3 1 0.4 – – FGFR3 5 2 1 0.4 1 0.4 FGFR4 3 1.2 1 0.4 – – FIP1L1 4 1.6 – – – – FLT1 15 6.1 – – – – FLT3 10 4.1 – – – – FLT4 15 6.1 5 2 – – FOXL2 5 2 – – – – GATA1 5 2 – – – – GATA2 8 3.3 2 0.8 – – GATA3 7 2.9 1 0.4 – – GEN1 1 0.4 – – – – GNA11 3 1.2 – – – – GNA13 1 0.4 6 2.4 – – GNAQ 3 1.2 1 0.4 – – GNAS 18 7.3 1 0.4 2 0.8 GPR124 27 11 24 9.8 1 0.4 GRIN2A 28 11.4 2 0.8 – – GSK3B 5 2 5 2 – – HGF 12 4.9 5 2 – – HRAS 6 2.4 – – – – IDH1 4 1.6 1 0.4 – – IDH2 2 0.8 – – – – IGF1 6 2.4 – – – – IGF1R 12 4.9 2 0.8 – – IGF2 4 1.6 – – – – IGF2R 1 0.4 – – – – IKBKE 13 5.3 3 1.2 – – IKZF1 11 4.5 3 1.2 – – IL7R 9 3.7 24 9.8 – – INHBA 8 3.3 3 1.2 – – INSRR 2 0.8 – – – – IRF4 8 3.3 – – – – IRS2 21 8.6 5 2 – – JAK1 8 3.3 3 1.2 – – JAK2 11 4.5 4 1.6 – – JAK3 11 4.5 4 1.6 – –

Downloaded From: https://jamanetwork.com/ on 09/26/2021 KDM5A 19 7.8 15 6.1 – – KDM5C 6 2.4 – – – – KDM6A 17 6.9 2 0.8 1 0.4 KDR 20 8.2 4 1.6 – – KEAP1 25 10.2 1 0.4 – – KIT 13 5.3 4 1.6 – – KLHL6 5 2 97 39.6 – – KRAS 8 3.3 13 5.3 – – LMO1 2 0.8 – – – – LRP1B 96 39.2 18 7.3 10 4.1 MAP2K1 1 0.4 2 0.8 – – MAP2K2 5 2 – – – – MAP2K4 4 1.6 – – – – MAP3K1 21 8.6 – – – – MAP3K13 14 5.7 79 32.2 – – MCL1 4 1.6 10 4.1 – – MDM2 6 2.4 11 4.5 – – MDM4 5 2 3 1.2 – – MED12 17 6.9 – – – – MEF2B 4 1.6 5 2 – – MEN1 1 0.4 1 0.4 – – MERTK – – 1 0.4 – – MET 12 4.9 2 0.8 1 0.4 MITF 4 1.6 – – – – MLH1 4 1.6 1 0.4 – – MLL 30 12.2 – – – – MLL2 81 33.1 – – 4 1.6 MPL 9 3.7 – – – – MRE11A 14 5.7 – – – – MSH2 6 2.4 – – – – MSH6 10 4.1 1 0.4 – – MST1R 1 0.4 – – – – MTOR 9 3.7 – – – – MUTYH 3 1.2 – – 1 0.4 MYC 3 1.2 26 10.6 – – MYCL1 3 1.2 2 0.8 – – MYCN 6 2.4 2 0.8 – – MYD88 5 2 – – – – MYST3 18 7.3 19 7.8 1 0.4

Downloaded From: https://jamanetwork.com/ on 09/26/2021 NBN 3 1.2 9 3.7 – – NCOR1 20 8.2 2 0.8 1 0.4 NF1 34 13.9 – – – – NF2 7 2.9 2 0.8 – – NFE2L2 43 17.6 8 3.3 1 0.4 NFKBIA 2 0.8 9 3.7 1 0.4 NKX2_1 5 2 9 3.7 – – NOTCH1 42 17.1 – – – – NOTCH2 21 8.6 4 1.6 – – NOTCH3 32 13.1 3 1.2 1 0.4 NOTCH4 22 9 – – – – NPM1 1 0.4 – – – – NRAS 1 0.4 1 0.4 – – NSD1 19 7.8 2 0.8 – – NTRK1 19 7.8 3 1.2 – – NTRK2 5 2 – – – – NTRK3 23 9.4 1 0.4 – – NUP93 6 2.4 1 0.4 – – PAK3 9 3.7 – – – – PAK7 8 3.3 5 2 – – PALB2 10 4.1 – – – – PARP1 7 2.9 3 1.2 – – PARP2 3 1.2 3 1.2 – – PARP3 4 1.6 – – – – PARP4 12 4.9 2 0.8 – – PAX5 7 2.9 6 2.4 – – PBRM1 14 5.7 1 0.4 1 0.4 PDGFRA 13 5.3 3 1.2 – – PDGFRB 12 4.9 1 0.4 – – PDK1 2 0.8 1 0.4 – – PIK3C2G 18 7.3 10 4.1 – – PIK3C3 11 4.5 – – – – PIK3CA 29 11.8 89 36.3 – – PIK3CG 29 11.8 6 2.4 – – PIK3R1 6 2.4 1 0.4 – – PIK3R2 10 4.1 7 2.9 – – PMS2 10 4.1 5 2 – – PNRC1 6 2.4 1 0.4 – – PPP2R1A 5 2 2 0.8 – –

Downloaded From: https://jamanetwork.com/ on 09/26/2021 PRDM1 8 3.3 2 0.8 – – PRKAR1A 3 1.2 5 2 – – PRKDC 40 16.3 10 4.1 – – PRSS8 – – 2 0.8 – – PTCH1 20 8.2 – – – – PTEN 34 13.9 9 3.7 1 0.4 PTPN11 7 2.9 – – – – RAD50 15 6.1 – – – – RAD51 3 1.2 – – – – RAD51B – – 3 1.2 1 0.4 RAD51C 4 1.6 1 0.4 – – RAD51L3 3 1.2 – – – – RAD52 1 0.4 14 5.7 – – RAD54L 4 1.6 – – – – RAF1 3 1.2 – – – – RARA 5 2 2 0.8 – – RB1 19 7.8 6 2.4 2 0.8 REL 3 1.2 7 2.9 – – RET 19 7.8 1 0.4 1 0.4 RICTOR 18 7.3 27 11 – – RNF43 6 2.4 1 0.4 – – ROS1 1 0.4 – – – – RPA1 3 1.2 – – – – RPTOR 15 6.1 1 0.4 – – RUNX1 4 1.6 – – – – RUNX1T1 15 6.1 6 2.4 – – SETD2 22 9 – – – – SF3B1 4 1.6 2 0.8 – – SH2B3 9 3.7 – – – – SMAD2 1 0.4 3 1.2 – – SMAD4 5 2 2 0.8 1 0.4 SMARCA4 17 6.9 1 0.4 2 0.8 SMARCB1 2 0.8 1 0.4 – – SMARCD1 4 1.6 – – – – SMO 11 4.5 – – – – SOCS1 2 0.8 – – – – SOX10 3 1.2 2 0.8 – – SOX2 5 2 105 42.9 – – SPEN 25 10.2 1 0.4 2 0.8

Downloaded From: https://jamanetwork.com/ on 09/26/2021 SPOP 2 0.8 – – – – SRC 2 0.8 4 1.6 – – STAG2 5 2 – – – – STAT4 4 1.6 3 1.2 – – STK11 14 5.7 4 1.6 – – SUFU 4 1.6 2 0.8 – – SYK 4 1.6 – – – – TBX3 15 6.1 – – – – TET2 13 5.3 1 0.4 – – TGFBR2 8 3.3 2 0.8 – – TIPARP 4 1.6 44 18 – – TMPRSS2 1 0.4 TNFAIP3 6 2.4 2 0.8 – – TNFRSF14 1 0.4 1 0.4 – – TOP1 – – 1 0.4 – – TP53 214 87.3 – – 1 0.4 TRRAP 27 11 7 2.9 1 0.4 TSC1 12 4.9 1 0.4 1 0.4 TSC2 25 10.2 – – – – TSHR 5 2 1 0.4 – – VHL 1 0.4 1 0.4 – – WISP3 4 1.6 – – – – WT1 7 2.9 1 0.4 – – XP01 3 1.2 7 2.9 – – XRCC3 2 0.8 5 2.0 – – ZNF217 11 4.5 2 0.8 – – ZNF703 12 4.9 25 10.2 – –

Downloaded From: https://jamanetwork.com/ on 09/26/2021 eTable 3. Relationship Between EGFR Expression Status and PFS and OS

Afatinib (n = 157) Erlotinib (n = 188)

Events/total Median, months (95% CI) Events/total Median, months (95% HR (95% CI) Interaction CI) P-value

PFS

EGFR expression status 81/112 1.94 (1.87–2.66) 97/116 1.87 (1.84–1.87) 0.73 (0.54– .8883 <200 30/45 3.61 (1.87–5.45) 51/72 1.91 (1.87–2.20) 0.98) >200 0.75 (0.48– 1.19) Staining intensity Negative 20/28 1.94 (1.87–5.52) 22/25 1.87 (1.81–2.27) 0.76 (0.41– .9848 Positive 91/129 1.97 (1.87–3.09) 126/163 1.87 (NA–NE) 1.40) 0.74 (0.56– 0.97) OS

EGFR expression status 103/112 7.43 (6.64–8.90) 107/116 6.67 (5.52–8.18) 0.86 (0.66– .2214 <200 37/45 8.38 (5.16–17.64) 67/72 6.41 (3.88–8.51) 1.13) >200 0.66 (0.44– 1.00) Staining intensity Negative 25/28 8.49 (7.10–18.43) 23/25 6.14 (2.89–18.56) 0.81 (0.46– .9072 Positive 115/129 7.43 (6.51–8.90) 151/163 6.64 (5.32–7.95) 1.42) 0.80 (0.63– 1.03)

Abbreviations: CI, confidence interval; EGFR, epidermal growth factor receptor; HR, hazard ratio; NA, not available; NE, not evaluable; OS, overall survival; PFS, progression-free survival.

Downloaded From: https://jamanetwork.com/ on 09/26/2021 eFigure 1. Comparison of Clinical Outcomes in the TGA Cohort and Overall LUX-Lung 8 (Intent-to-Treat) Population

Abbreviations: CI, confidence interval; OS, overall survival; PFS, progression-free survival; TGA, tumor genetic analysis.

Downloaded From: https://jamanetwork.com/ on 09/26/2021 eFigure 2. Relationship Between the Most Frequent Tumor Genetic Aberrations and (A) PFS and (B) OS in the LUX-Lung 8 TGA Cohort

Abbreviations: CI, confidence interval; HR, hazard ratio; LL8, LUX-Lung 8; OS, overall survival; PFS, progression-free survival; TGA, tumor genetic analysis.

Downloaded From: https://jamanetwork.com/ on 09/26/2021 eFigure 3. (A) Details of ErbB Family Mutations; (B) Location of HER2 Mutations

Downloaded From: https://jamanetwork.com/ on 09/26/2021 eFigure 4. Standardized Effect Plot for PFS (Left) and OS (Right)

Abbreviations: EGFR, epidermal growth factor receptor; OS, overall survival; PFS, progression-free survival; TGA, tumor genetic analysis. Displayed P-values are not adjusted for multiple testing. The dashed lines indicate the adjusted 95% confidence interval after correcting for multiple comparisons.

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