The Genomic Grade Assay Compared with Ki67 to Determine Risk Of

Supplementary Online Content

Ignatiadis M, Azim HA Jr, Desmedt C, et al. The Genomic Grade Assay Compared With Ki67 Prevalence to Determine Risk of Distant Breast Cancer Recurrence. JAMA Oncol. Published online December 3, 2015. doi:10.1001/jamaoncol.2015.4377.

eMethods. Summary of development and validation of the Genomic Grade (GG) qRT-PCR based assay.

eFigure. Comparison in Distant recurrence-free interval (DRFI) between BIG 1-98 patients included and not included in the genomic grade sub-study.

eTable 1. Centrally reviewed histological grade (HG) versus genomic grade (GG) in the BIG 1- 98 sub-study.

eTable 2. Cox model showing the prognostic value of the different parameters included in the CP model, in terms of distant recurrence-free interval (DRFI), disease-free survival (DFS) and overall survival (OS)

eTable 3. Cox model showing the added value of genomic grade (GG) or centrally reviewed Ki67; both tested as continuous variables on Adjuvant!online (AOL) in predicting distant recurrence-free interval (DRFI), disease-free survival (DFS) and overall survival (OS).

eTable 4. Cox model showing the added value of genomic grade (GG) and centrally reviewed Ki67; both tested as continuous variables on clinicopathological (CP) model in predicting disease-free (DFS) and overall survival (OS).

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

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SUPPLEMENT LEGENDS

eMethods: Summary of development and validation of the Genomic Grade (GG) qRT-

PCR based assay.

eFigure

eFigure: Comparison in Distant recurrence-free interval (DRFI) between BIG 1-98

patients included and not included in the genomic grade sub-study.

eTables

eTable 1: Centrally reviewed histological grade (HG) versus genomic grade (GG) in the

BIG 1-98 sub-study.

eTable 2: Cox model showing the prognostic value of the different parameters included

in the CP model, in terms of distant recurrence-free interval (DRFI), disease-free

survival (DFS) and overall survival (OS)

eTable 3: Cox model showing the added value of genomic grade (GG) or centrally

reviewed Ki67; both tested as continuous variables on Adjuvant!online (AOL) in

predicting distant recurrence-free interval (DRFI), disease-free survival (DFS) and

overall survival (OS).

eTable 4: Cox model showing the added value of genomic grade (GG) and centrally

reviewed Ki67; both tested as continuous variables on clinicopathological (CP) model in

predicting disease-free (DFS) and overall survival (OS).

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eMethods: Summary of development and validation of the Genomic Grade (GG) qRT-

PCR based assay.

Selection of genes for the GG qRT-PCR

The GG qRT-PCR assay was developed from the original microarray based 97-gene

Genomic Grade Index (GGI) (1), in order to improve its clinical application by testing on

formalin fixed paraffin embedded tissue (FFPE).

We started by selecting a subset of genes from the original GGI signature based on

their performance in assessing histological grade (HG) and prognosis using 15

independent publicly available microarray datasets including 1,192 patients.

Accordingly, we selected six reporter genes that were mostly overexpressed in grade 3

tumors and 3 reference genes (Table A).

Table A: GG qRT-PCR composition (9 genes, 6 reporter genes and 3 control genes)

Reporter genes Role in cell cycle Upregulation

HG1 HG3

MCM10 Minichromosome Maintenance DNA replication √

Complex Component 10 factor, regulation of

DNA synthesis

CCNB2 Cyclin B2 Phosphorylation of √

proteins involved in

G2/M transition

ASPM ASP (abnormal spindle) homolog, Spindle √

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microcephaly associated organization,

(Drosophilia) spindle positioning

and cytokinesis

PTTG1 Pituitary Tumor Transorfming 1 Control of sister √

chromatids

segregation.

FRY Furry Homolog (Drosophilia) Microtubule binding √

fidelity of mitotic

chromosomes

alignment

CX3CR1 Chemokine receptor 1 √

Control Genes

GUS Glucoronidase, beta

TBP TATA box Binding Protein

RPLP0 Ribosomal Protein, Large, P0

We tested the analytical performance of the reduced gene signature in 4 samples (5

RNA extractions and 3 qRT-PCR reactions per sample) in order to define a 95%

Confidence Interval (CI) around the GG low/high (GG1/GG3) cut-off. We considered

cases with a qRT-PCR GG falling into the 95% CI as equivocal (GGEq).

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Concordance between GG and the original microarray-based GGI

We then assessed the concordance between GG and the original 97-gene signature in

44 paired Frozen and FFPE samples identified at the Department of Pathology, in

Institut Jules Bordet, Brussels, Belgium. The frozen samples were assessed by

microarray (Affymetrix U133 Plus2.0) and results were compared to GG assessed on

paired FFPE samples. We found that the concordance between GG and HG was 91%.

We also found a 95% overall concordance and a high correlation (r=0.87) between the

frozen and FFPE samples (Figure A, Table B).

Figure A

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Table B: Concordance between GG and the original microarray-based GGI

Microarray GGI (N=44)

GGI 1 GGI 3

GG1 19 1

GG equivocal 0 0

GG3 1 23

Validation of the GG in a hospital-based series

Table C: Patient characteristics of samples included in the validation set

N = 388

Age Median, range 61 (31 – 91)

Histology Ductal 326 (84%)

Lobular 62 (16%)

Nodal status N0 387 (99%)

Size Mean, range 16 mm (15 – 80)

≤ 2cm 336 (85.5%)

> 2cm 46 (13%)

Not available 6 (1.5%)

ER / PgR status ER + / PgR + 291 (75%)

ER + / PgR - 97 (25%)

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Histological grade (HG) HG1 105 (27%)

HG2 217 (56%)

HG3 66 (17%)

HER2 status Negative 388 (100%)

60 samples (15%) were excluded due to low tumor cellularity (<30% invasive tumor

cells) leaving 336 samples evaluable for this analysis. RNA was extracted from two

deparaffinized 10μm FFPE slides using RNAeasy FFPE (Qiagen). rTT-PCR assays

were run on 384 well plates on an ABI 7900 HT instrument (Applied Biosystem) with set

quality controls. GG qRT-PCR was successful in 96% of samples (n=336) (Figure B).

Figure B

Table 3 summarizes the concordance between genomic grade and HG. We observed

an overall concordance of 90% between GG and HG. Concordance was similar

according to histology, 91% for ductal and 89% for lobular cancers. 82% of HG2 tumors

reclassified into 74% GG1 and 26% GG3. Overall, there was 52 (16%) of 322 evaluable

samples that were considered equivocal.

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Table D: Concordance between GG and HG in the validation set

HG1 HG2 HG3 Total

GG1 71 107 6 184 (57%)

GG equivocal 9 31 12 52 (16%)

GG3 6 38 42 86 (27%)

Total 86 (27%) 176 (55%) 60 (18%) 322

References

1. Sotiriou C, Wirapati P, Loi S, Harris A, Fox S, Smeds J, et al. Gene expression

profiling in breast cancer: understanding the molecular basis of histologic grade to

improve prognosis. J Natl Cancer Inst 2006;98(4):262-72.

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eFigure 1: Comparison in distant recurrence free interval (DRFI) between BIG 1-98 patients included and not included in the genomic grade (GG) sub-study.

GG Sub-study 5 Year DRFI (ITT) 95% Confidence Interval

No 0.90 0.89 0.91

Yes 0.94 0.93 0.96 GG Sub-study 8 Year DRFI (ITT) 95% Confidence Interval

No 0.86 0.85 0.88

Yes 0.90 0.88 0.92

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eTable 1: Centrally reviewed histological grade (HG) versus genomic grade (GG) in the BIG 1-98 sub-study.

HG1 HG2 HG3 Total

GG1 92 (68%) 202(40%) 23 (10%) 317(36%)

GGEq 38 (28%) 220 (44%) 94 (39%) 352 (40%)

GG3 5 (4%) 80 (16%) 124 (51%) 209 (24%)

Total 135 (100%) 502 (100%) 241 (100%) 878

HG1: histological grade 1; HG2: histological grade 2; HG3: histological grade 3; GG1: genomic grade 1; GG3: genomic grade 3; GGEq: genomic grade equivocal

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eTable 2: Cox model showing the prognostic value of the different parameters of the clinicopathologiacl (CP) model, in terms of distant recurrence-free interval (DRFI), disease-free survival (DFS) and overall survival (OS).

a) Distant recurrence-free survival (DRFI, n=848)

Hazard ratio 95% CI p-value Age (1-unit increase) 1.03 1.00 – 1.06 0.08 Nodal status - 0 1 - 1-3 2.01 1.17 – 3.45 0.01 - > 3 2.37 1.21 – 4.64 0.01 Log2 Tumor size 1.45 1.07 – 1.96 0.02 Histological grade - 1 1 - 2 2.26 0.80 – 6.38 0.012 - 3 3.37 1.16 – 9.81 0.03 ER (1-unit increase) 0.99 1.00 – 1.00 0.18 HER2 - Negative 1 - Positive 0.84 0.39 – 1.80 0.66 Log2 PgR (1-unit increase) 0.92 0.83 – 1.01 0.08

b) Disease-free survival (DFS, n=848)

Hazard ratio 95% CI p-value Age (1-unit increase) 1.06 1.04 – 1.09 <0.0001 Nodal status - 0 1 - 1-3 1.45 1.04 – 2.00 0.03 - > 3 1.77 1.14 – 2.75 0.01 Log2 Tumor size 1.27 1.03 – 2.75 0.02 Histological grade - 1 1 - 2 1.29 0.80 – 2.07 0.29 - 3 1.74 1.04 – 2.91 0.03 ER (1-unit increase) 1.00 0.99 – 1.00 0.99 HER2 - Negative 1 - Positive 1.21 0.74 – 1.99 0.44 Log2 PgR (1-unit increase) 0.99 0.92 – 1.06 0.70

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c) Overall survival (OS, n=848)

Hazard ratio 95% CI p-value Age (1-unit increase) 1.11 1.08 – 1.14 <0.0001 Nodal status - 0 1 - 1-3 1.38 0.89 – 2.12 0.15 - > 3 1.33 0.75 – 2.38 0.32 Log2 Tumor size 1.64 1.27 – 2.13 0.0002 Histological grade - 1 1 - 2 1.10 0.58 – 2.10 0.78 - 3 1.71 0.87 – 3.37 0.12 ER (1-unit increase) 0.99 0.99 – 1.00 0.20 HER2 - Negative 1 - Positive 0.95 0.49 – 1.87 0.89 Log2 PgR (1-unit increase) 1.03 0.94 – 1.13 0.53

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eTable 3: Cox model showing the added value of genomic grade (GG) or centrally-reviewed Ki67; both tested as continuous variables on Adjuvant!online (AOL) in predicting distant recurrence-free interval (DRFI), disease-free survival (DFS) and overall survival (OS).

N. Patients N. Events (%) Likelihood p-value ratio (X2) A) DRFI

AOL + GG vs. AOL 864 82 (9.4%) 12.02 0.0005

AOL + ki67 vs. AOL 844 77 (9.1%) 7.34 0.006

B) DFS

AOL + GG vs. AOL 864 214 (24.7%) 0.52 0.47

AOL + ki67 vs. AOL 844 205 (24.2%) 0.22 0.63

C) OS

AOL + GG vs. AOL 864 122 (14.1%) 3.25 0.07

AOL + ki67 vs. AOL 844 118 (13.9%) 1.03 0.31

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eTable 4: Cox model showing the added value of genomic grade (GG) and centrally reviewed Ki67; both tested as continuous variables on clinicopathological (CP) * model in predicting disease-free (DFS) and overall survival (OS).

N. Patients N. Events (%) Likelihood p-value ratio (X2) A) DFS

GG alone 883 220 7.9 <0.0001

Ki67 alone 862 210 7.29 <0.0001

GG + ki67 vs. GG 862 210 1.3 0.25

GG + ki67 vs. ki67 862 220 3.6 0.057

CP + GG vs. CP 848 207 0.16 0.68

CP + ki67 vs. CP 833 201 0.42 0.51

CP + GG + ki67 vs. CP + GG 833 201 0.17 0.68

CP + ki67 + GG vs. CP + ki67 833 201 0.11 0.74

B) OS

GG alone 883 126 13.81 0.0002

Ki67 alone 862 121 7.78 0.005

GG + ki67 vs. GG 862 121 0.71 0.39

GG + ki67 vs. ki67 862 121 6.01 0.01

CP + GG vs. CP 848 117 2.83 0.09

CP + ki67 vs. CP 833 114 2.32 0.12

CP + GG + ki67 vs. CP + GG 833 114 0.75 0.38

CP + ki67 + GG vs. CP + ki67 833 114 0.75 0.38

* Model includes age, tumor size, nodal status, histological grade, ER, PgR, HER2

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