A 14-gene prognosis signature predicts metastasis risk in node-negative, estrogen receptor-positive,...
1
A 14-gene prognosis signature predicts metastasis risk in node-negative, estrogen receptor-positive, Tamoxifen-treated breast cancer in different ethnogeographic regions H. Iwata 1 , A. Wang 2 , C. Gillett 3 , H. Higashimoto 4 , R. Lagier 2 , C. Rowland 2 , S. Anderson 5 , S. Broder 2 , J. Sninsky 2 , A. Tutt 6,7 1 Department of Breast Oncology, Aichi Cancer Center Hospital, Japan, 2 Celera, LLC, Alameda, CA, USA, 3 Guy’s and St Thomas’ Hospital Breast Research Tissue & Data Bank, London, UK, 4 Research & Development Department, SRL Inc, Japan, 5 Laboratory Corporation of America, Triangle Park, NC, USA, 6 Breakthrough Breast Cancer Research Unit, King’s College, London, UK, 7 Guy’s Hospital, London, UK Hypothesis We previously identified a 14-gene expression signature from non-systemically treated node-negative (N-), estrogen receptor- positive (ER+) breast cancer patients that was prognostic of metastasis (Tutt et al. BMC Cancer 8 ,339 (2008)). The equally- weighted expression signature was not confounded with the presence of treatment response related genes and is independent of routinely used clinical information. Given the modest but important absolute survival gains provided by adjuvant hormonal therapy and reports of significant non-adherence to hormonal therapy in this patient group, we performed the current study to answer two additional questions: 1) Does the gene signature successfully classify outcomes in women receiving adjuvant hormonal therapy; and 2) Is the signature comparably prognostic in a different ethnogeographic population (Asian women). Patients • Two N-, ER+, hormone-treated breast cancer cohorts in community settings at the Aichi Cancer Center (ACC, n=205) in Japan and Guy’s Hospital (GH, n=203) in United Kingdom were selected. RT-PCR Profiling • Five 10 mm sections of FFPE tissues were used for RNA extraction. • A single-step RT-PCR with SYBR Green was used for gene expression profiling. • The assays were performed on the Prism 7900 Real-Time PCR system. • Expression levels were normalized to 3 housekeeping genes and relative changes in gene expression were calculated by Ct method (Livak and Schmittgen Methods 25 , 402 (2001)). Statistical Analyses • Differences between the patient characteristics of the two cohorts were determined by the Wilcoxon rank sum test for continuous variables and Fisher’s exact test for categorical variables. • Kaplan-Meier analysis and Cox proportional hazard models were used to evaluate the metastasis score (MS) on the risk of distant metastasis. The analyses evaluated the MS both as a continuous and as a categorical risk variable. For the categorical analyses, subjects were stratified into high and low risk groups based on a previously established untreated breast cancer cohort median. • The probabilities of distant metastasis for given metastasis scores were calculated from survivor functions as estimated by Cox models including the continuous MS as the explanatory variable. Materials and Methods Table 1. Genes in prognostic signature and its overlap with other signatures 1 Paik et al. NEJM 351 , 2817 (2004), 2 van’t Veer et al. Nature 415 , 530 (2002), 3 Dai et al. Can. Res. 16 , 4059 (2005) Results Table 2. Clinical and pathological characteristics of patients Table 3. Univariate and multivariate analyses of distant- metastasis-free survival (DMFS) using continuous MS AICH ICANCER CENTER H ospitaland R esearch Institute C haracteristics ACC (n=205) n (% ) Guy’s (n=203) n (% ) p-value Age (yrs) < 40 40 – 49 50 – 59 60 – 69 ≥ 70 Median M in.– M ax. 14 (6.8) 63 (30.7) 67 (32.7) 47 (22.9) 14 (6.8) 52 31 – 81 6 (3.0) 27 (13.3) 72 (35.5) 65 (32.0) 33 (16.3) 59 33 – 86 <.0001* Tum ordiameter ≤ 2 cm > 2 cm Median M in.– M ax. 132 (64.4) 73 (35.6) 1.8 0.1 – 5 138 (68.0) 65 (32.0) 2.0 0 # – 3 0.1159* Tum orgrade G rade 1 G rade 2 G rade 3 70 (34.2) 96 (46.8) 39 (19.0) 60 (29.6) 98 (48.3) 45 (22.2) 0.5548** Stage I II 132 (64.4) 73 (35.6) 138 (68.0) 65 (32.02) 0.4650** D istant recurrence Yes No 26 (12.7) 179 (87.3) 16 (7.9) 187 (92.1) 0.1421** D eath ofall cause Yes No 17 (8.3) 188 (91.7) 43 (21.2) 160 (78.8) 0.0002** M edian Follow up 9.0 (0.2-12.4)yrs 9.3 (0.7-16.7)yrs *W ilcoxon rank sum test,**Fisher’s exacttest # tum ors w ere im palpable • MS is significantly associated with risk of distant metastasis when analyzed as a continuous variable • A 1.52, 1.85 and 1.55-fold increase in metastasis risk per 10-unit increase in MS for ACC, Guy’s and combined set, respectively. Fig 1. Kaplan-Meier analysis of DMFS using previously established cut point in Tamoxifen-treated patients Fig 3. Relationship of probability of distant metastasis at 9 years (dash lines as 95% CI) for untreated and Tamoxifen- treated patients Fig 2. Kaplan-Meier estimates of the percent of patients with distant metastasis at 9 years Conclusions • The 14-gene signature, originally derived from non-systemically treated, N-, ER+ breast cancer patients, is significantly associated with risk of distant metastasis when analyzed as a continuous variable in Tamoxifen-treated women with N-, ER+ breast cancer. There is a trend toward significance when a previously established MS cut point for untreated women is used in a categorical analysis. • The prognostic performance of this signature is demonstrated in community settings in widely variant ethnogeographic regions. •There is a trend toward significance in ACC but not Guy’s sample set when a previously established MS cut point for untreated women is used in a categorical analysis. •When previously used cut point for categorical analysis is used in combined analysis, resulting data are significant. •Reduction of risk observed in Tamoxifen-treated relative to untreated women in community settings consistent with previously observed reduction of risk with Tamoxifen treatment in clinical trials. •Dichotomous categorization of differential risk groups results in statistically significant separation in combined sample set. 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 Years Probability of DMFS H L Patients atrisk Low risk 51 46 10 High risk 154 120 37 H R = 2.80 (0.84-9.33) P = 0.093 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 Years Probability of DMFS H L Patients atrisk Low risk 51 46 10 High risk 154 120 37 H R = 2.80 (0.84-9.33) P = 0.093 Patients atrisk Low risk 122 113 50 3 High risk 81 72 28 5 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L H R = 2.00 (0.75-5.38) P = 0.168 Patients atrisk Low risk 122 113 50 3 High risk 81 72 28 5 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L H R = 2.00 (0.75-5.38) P = 0.168 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L Patients atrisk Low risk 173 159 60 3 High risk 235 192 65 5 H R = 2.61 (1.28-5.32) P = 0.008 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L Patients atrisk Low risk 173 159 60 3 High risk 235 192 65 5 H R = 2.61 (1.28-5.32) P = 0.008 ACC Guy’s Com bined 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 Years Probability of DMFS H L Patients atrisk Low risk 51 46 10 High risk 154 120 37 H R = 2.80 (0.84-9.33) P = 0.093 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 Years Probability of DMFS H L Patients atrisk Low risk 51 46 10 High risk 154 120 37 H R = 2.80 (0.84-9.33) P = 0.093 Patients atrisk Low risk 122 113 50 3 High risk 81 72 28 5 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L H R = 2.00 (0.75-5.38) P = 0.168 Patients atrisk Low risk 122 113 50 3 High risk 81 72 28 5 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L H R = 2.00 (0.75-5.38) P = 0.168 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L Patients atrisk Low risk 173 159 60 3 High risk 235 192 65 5 H R = 2.61 (1.28-5.32) P = 0.008 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L Patients atrisk Low risk 173 159 60 3 High risk 235 192 65 5 H R = 2.61 (1.28-5.32) P = 0.008 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 Years Probability of DMFS H L Patients atrisk Low risk 51 46 10 High risk 154 120 37 H R = 2.80 (0.84-9.33) P = 0.093 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 Years Probability of DMFS H L Patients atrisk Low risk 51 46 10 High risk 154 120 37 H R = 2.80 (0.84-9.33) P = 0.093 Patients atrisk Low risk 122 113 50 3 High risk 81 72 28 5 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L H R = 2.00 (0.75-5.38) P = 0.168 Patients atrisk Low risk 122 113 50 3 High risk 81 72 28 5 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L H R = 2.00 (0.75-5.38) P = 0.168 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L Patients atrisk Low risk 173 159 60 3 High risk 235 192 65 5 H R = 2.61 (1.28-5.32) P = 0.008 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0 5 10 15 20 Years Probability of DMFS H L Patients atrisk Low risk 173 159 60 3 High risk 235 192 65 5 H R = 2.61 (1.28-5.32) P = 0.008 ACC Guy’s Com bined Low risk High risk Low risk High risk Low risk High risk Univariate analysis M ultivariate analysis Study Variable H azard ratio (95% C I) p-value H azard ratio (95% C I) p-value 14-gene signature (10 unitscale) 1.43 (1.02-2.00) 0.036 1.52 (1.00-2.30) 0.049 Age (10 yearscale) 1.66 (1.11-2.49) 0.013 1.67 (1.10-2.54) 0.017 Tum orsize 1.59 (1.13-2.23) 0.008 1.69 (1.15-2.50) 0.008 G rade 2 1.19 (0.55-2.56) 0.661 0.96 (0.35-2.66) 0.939 ACC G rade 3 1.21 (0.48-3.01) 0.687 0.60 (0.16-2.30) 0.454 14-gene signature (10 unitscale) 1.49 (1.01-2.21) 0.044 1.85 (1.05-3.25) 0.033 Age (10 yearscale) 1.38 (0.85-2.25) 0.197 1.38 (0.85-2.24) 0.198 Tum orsize 1.20 (0.71-2.01) 0.496 1.08 (0.61-1.90) 0.798 G rade 2 1.81 (0.66-4.98) 0.251 2.47 (0.53-11.61) 0.252 Guy’s G rade 3 1.20 (0.39-3.71) 0.756 0.74 (0.09-5.83) 0.776 14-gene signature (10 unitscale) 1.51 (1.19-1.92) 0.001 1.55 (1.11-2.15) 0.009 Age (10 yearscale) 1.38 (1.03-1.85) 0.033 1.52 (1.10-2.08) 0.010 Tum orsize 1.51 (1.12-2.04) 0.007 1.47 (1.05-2.07) 0.026 G rade 2 1.38 (0.75-2.53) 0.303 1.31 (0.58-2.99) 0.517 Com bined G rade 3 1.18 (0.58-2.39) 0.654 0.66 (0.22-2.02) 0.469 # of events # of rem aining 5 46 51 2 21 23 5 8 13 7 29 36 89 108 197 26 66 92 71 39 110 97 105 202 U ntreated G uy’s set Tam -treated ACC set Tam -treated Com bined set Tam -treated G uy’s set 0 5 10 15 20 25 30 35 AllPatients (n=408) H igh M S (n=235) Low M S (n=173) AllPatients (n=203) H igh M S (n=81) Low M S (n=122) AllPatients (n=205) H igh M S (n=154) Low M S (n=51) AllPatients (n=279) H igh M S (n=172) Low M S (n=107) Percentw ith D M at9 Years # of events # of rem aining 5 46 51 2 21 23 5 8 13 7 29 36 89 108 197 26 66 92 71 39 110 97 105 202 U ntreated G uy’s set Tam -treated ACC set Tam -treated Com bined set Tam -treated G uy’s set 0 5 10 15 20 25 30 35 AllPatients (n=408) H igh M S (n=235) Low M S (n=173) AllPatients (n=203) H igh M S (n=81) Low M S (n=122) AllPatients (n=205) H igh M S (n=154) Low M S (n=51) AllPatients (n=279) H igh M S (n=172) Low M S (n=107) Percentw ith D M at9 Years G ene Paik 1 van'tVeer 2 D ai 3 G ene D escription G ene Function BUB1 x BUB1 budding uninhibited by benzim idazoles 1 hom olog cell cycle,D NA replication CCNB1 x cyclin B1 cell cycle,cell death,D NA replication CENPA x x centrom ere protein A,17kD a cell m orphology,D NA replication D C 13 x D C 13 protein assem bly ofcytochrom e oxidase DIAPH3 x x diaphanous hom olog 3 (D rosophila) actin cytoskeleton organization and biogenesis M ELK x m aternal em bryonic leucine zipperkinase protein am ino acid phosphorylation,ATP binding MYBL2 x v-m yb m yeloblastosis viral oncogene hom olog (avian)-like 2 cell cycle,cell death,cell m orphology ORC6L x x origin recognition com plex,subunit6 hom olog-like (yeast) cell cycle,D NA replication PKMYT1 x m em brane-associated tyrosine-and threonine-specific cdc2-inhibitory kinase cell cycle PRR11 x proline rich 11 cell cycle RACGAP1 x Rac G TPase activating protein 1 cell cycle,cell m orphology RFC4 x replication factorC (activator1)4,37kD a D NA replication TK1 x thym idine kinase 1,soluble D NA replication,cell death UBE2S x ubiquitin-conjugating enzym e E2S ubiquitin cycle 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% -60 -50 -40 -30 -20 -10 0 10 20 M etastasis Score Probability ofD istantM etastasis at9 years U ntreated Tam -treated U ntreated Tam -treated
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Transcript of A 14-gene prognosis signature predicts metastasis risk in node-negative, estrogen receptor-positive,...
A 14-gene prognosis signature predicts metastasis risk in node-negative, estrogen receptor-positive, Tamoxifen-treated breast cancer in different ethnogeographic regionsH. Iwata1, A. Wang2, C. Gillett3, H. Higashimoto4, R. Lagier2, C. Rowland2, S. Anderson5, S. Broder2, J. Sninsky2, A. Tutt6,7
1Department of Breast Oncology, Aichi Cancer Center Hospital, Japan, 2Celera, LLC, Alameda, CA, USA, 3Guy’s and St Thomas’ Hospital Breast Research Tissue & Data Bank, London, UK,4Research & Development Department, SRL Inc, Japan, 5Laboratory Corporation of America, Triangle Park, NC, USA, 6Breakthrough Breast Cancer Research Unit, King’s College, London, UK, 7Guy’s Hospital, London, UK
Hypothesis
We previously identified a 14-gene expression signature from non-systemically treated
node-negative (N-), estrogen receptor-positive (ER+) breast cancer patients that was
prognostic of metastasis (Tutt et al. BMC Cancer 8,339 (2008)). The equally-weighted
expression signature was not confounded with the presence of treatment response
related genes and is independent of routinely used clinical information. Given the
modest but important absolute survival gains provided by adjuvant hormonal therapy
and reports of significant non-adherence to hormonal therapy in this patient group, we
performed the current study to answer two additional questions: 1) Does the gene
signature successfully classify outcomes in women receiving adjuvant hormonal
therapy; and 2) Is the signature comparably prognostic in a different ethnogeographic
population (Asian women).
Patients• Two N-, ER+, hormone-treated breast cancer cohorts in community settings at the
Aichi Cancer Center (ACC, n=205) in Japan and Guy’s Hospital (GH, n=203) in United Kingdom were selected.
RT-PCR Profiling • Five 10 mm sections of FFPE tissues were used for RNA extraction.• A single-step RT-PCR with SYBR Green was used for gene expression profiling.• The assays were performed on the Prism 7900 Real-Time PCR system.• Expression levels were normalized to 3 housekeeping genes and relative changes in
gene expression were calculated by Ct method (Livak and Schmittgen Methods 25, 402 (2001)).
Statistical Analyses• Differences between the patient characteristics of the two cohorts were determined by
the Wilcoxon rank sum test for continuous variables and Fisher’s exact test for categorical variables.
• Kaplan-Meier analysis and Cox proportional hazard models were used to evaluate the metastasis score (MS) on the risk of distant metastasis. The analyses evaluated the MS both as a continuous and as a categorical risk variable. For the categorical analyses, subjects were stratified into high and low risk groups based on a previously established untreated breast cancer cohort median.
• The probabilities of distant metastasis for given metastasis scores were calculated from survivor functions as estimated by Cox models including the continuous MS as the explanatory variable.
Materials and Methods
Table 1. Genes in prognostic signature and its overlap with other signatures
1Paik et al. NEJM 351, 2817 (2004), 2van’t Veer et al. Nature 415, 530 (2002), 3Dai et al. Can. Res. 16, 4059 (2005)
Results
Table 2. Clinical and pathological characteristics of patients
Table 3. Univariate and multivariate analyses of distant-metastasis-free survival (DMFS) using continuous MS
AICHI CANCER CENTERHospital and Research Institute
Characteristics
ACC (n=205) n (%)
Guy’s (n=203) n (%)
p-value
Age (yrs) < 40 40 – 49 50 – 59 60 – 69 ≥ 70 Median Min. – Max.
14 (6.8) 63 (30.7) 67 (32.7) 47 (22.9) 14 (6.8)
52 31 – 81
6 (3.0)
27 (13.3) 72 (35.5) 65 (32.0) 33 (16.3)
59 33 – 86
<.0001*
Tumor diameter ≤ 2 cm > 2 cm Median Min. – Max.
132 (64.4) 73 (35.6)
1.8 0.1 – 5
138 (68.0) 65 (32.0)
2.0 0# – 3
0.1159*
Tumor grade Grade 1 Grade 2 Grade 3
70 (34.2) 96 (46.8) 39 (19.0)
60 (29.6) 98 (48.3) 45 (22.2)
0.5548**
Stage I II
132 (64.4) 73 (35.6)
138 (68.0) 65 (32.02)
0.4650**
Distant recurrence Yes No
26 (12.7)
179 (87.3)
16 (7.9)
187 (92.1)
0.1421**
Death of all cause Yes No
17 (8.3)
188 (91.7)
43 (21.2) 160 (78.8)
0.0002**
Median Follow up 9.0 (0.2-12.4) yrs 9.3 (0.7-16.7) yrs
*Wilcoxon rank sum test, **Fisher’s exact test # tumors were impalpable
• MS is significantly associated with risk of distant metastasis when analyzed as a continuous variable
• A 1.52, 1.85 and 1.55-fold increase in metastasis risk per 10-unit increase in MS for ACC, Guy’s and combined set, respectively.
Fig 1. Kaplan-Meier analysis of DMFS using previously established cut point in Tamoxifen-treated patients
Fig 3. Relationship of probability of distant metastasis at 9 years (dash lines as 95% CI) for untreated and Tamoxifen-treated patients
Fig 2. Kaplan-Meier estimates of the percent of patients with distant metastasis at 9 years
Conclusions
• The 14-gene signature, originally derived from non-systemically treated, N-, ER+ breast cancer patients, is significantly associated with risk of distant metastasis when analyzed as a continuous variable in Tamoxifen-treated women with N-, ER+ breast cancer. There is a trend toward significance when a previously established MS cut point for untreated women is used in a categorical analysis.
• The prognostic performance of this signature is demonstrated in community settings in widely variant ethnogeographic regions.
• The mechanistic basis for the 14-gene signature influence on prognosis is worthy of study.
• There is a trend toward significance in ACC but not Guy’s sample set when a previously established MS cut point for untreated women is used in a categorical analysis.
• When previously used cut point for categorical analysis is used in combined analysis, resulting data are significant.
• Reduction of risk observed in Tamoxifen-treated relative to untreated women in community settings consistent with previously observed reduction of risk with Tamoxifen treatment in clinical trials.
• Dichotomous categorization of differential risk groups results in statistically significant separation in combined sample set.
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
0 5 10 15Years
Pro
babi
lity
of D
MFS
HL
Patients at riskLow risk 51 46 10 High risk 154 120 37
HR = 2.80 (0.84-9.33)P = 0.093
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
0 5 10 15Years
Pro
babi
lity
of D
MFS
HL
Patients at riskLow risk 51 46 10 High risk 154 120 37
HR = 2.80 (0.84-9.33)P = 0.093
Patients at riskLow risk 122 113 50 3High risk 81 72 28 5
0.30
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1.00
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HL
HR = 2.00 (0.75-5.38)P = 0.168
Patients at riskLow risk 122 113 50 3High risk 81 72 28 5
0.30
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HR = 2.00 (0.75-5.38)P = 0.168
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HL
Patients at riskLow risk 173 159 60 3High risk 235 192 65 5
HR = 2.61 (1.28-5.32)P = 0.008
0.30
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Patients at riskLow risk 173 159 60 3High risk 235 192 65 5
HR = 2.61 (1.28-5.32)P = 0.008
ACC Guy’s Combined
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Patients at riskLow risk 51 46 10 High risk 154 120 37
HR = 2.80 (0.84-9.33)P = 0.093
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Patients at riskLow risk 51 46 10 High risk 154 120 37
HR = 2.80 (0.84-9.33)P = 0.093
Patients at riskLow risk 122 113 50 3High risk 81 72 28 5
0.30
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HL
HR = 2.00 (0.75-5.38)P = 0.168
Patients at riskLow risk 122 113 50 3High risk 81 72 28 5
0.30
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HL
HR = 2.00 (0.75-5.38)P = 0.168
0.30
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0.90
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0 5 10 15 20Years
Pro
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MFS
HL
Patients at riskLow risk 173 159 60 3High risk 235 192 65 5
HR = 2.61 (1.28-5.32)P = 0.008
0.30
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0 5 10 15 20Years
Pro
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HL
Patients at riskLow risk 173 159 60 3High risk 235 192 65 5
HR = 2.61 (1.28-5.32)P = 0.008
0.30
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0 5 10 15Years
Pro
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HL
Patients at riskLow risk 51 46 10 High risk 154 120 37
HR = 2.80 (0.84-9.33)P = 0.093
0.30
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0.60
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0.80
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1.00
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MFS
HL
Patients at riskLow risk 51 46 10 High risk 154 120 37
HR = 2.80 (0.84-9.33)P = 0.093
Patients at riskLow risk 122 113 50 3High risk 81 72 28 5
0.30
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0.70
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0 5 10 15 20Years
Pro
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lity
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MFS
HL
HR = 2.00 (0.75-5.38)P = 0.168
Patients at riskLow risk 122 113 50 3High risk 81 72 28 5
0.30
0.40
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0.60
0.70
0.80
0.90
1.00
0 5 10 15 20Years
Pro
babi
lity
of D
MFS
HL
HR = 2.00 (0.75-5.38)P = 0.168
0.30
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0.50
0.60
0.70
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0.90
1.00
0 5 10 15 20Years
Pro
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lity
of D
MFS
HL
Patients at riskLow risk 173 159 60 3High risk 235 192 65 5
HR = 2.61 (1.28-5.32)P = 0.008
0.30
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0.70
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0.90
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0 5 10 15 20Years
Pro
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HL
Patients at riskLow risk 173 159 60 3High risk 235 192 65 5
HR = 2.61 (1.28-5.32)P = 0.008
ACC Guy’s Combined
Low risk
High risk
Low risk
High risk
Low risk
High risk
Univariate analysis Multivariate analysis
Study Variable Hazard ratio
(95% CI) p-value Hazard ratio
(95% CI) p-value 14-gene signature
(10 unit scale) 1.43 (1.02-2.00) 0.036 1.52 (1.00-2.30) 0.049
Age (10 year scale)
1.66 (1.11-2.49) 0.013 1.67 (1.10-2.54) 0.017
Tumor size 1.59 (1.13-2.23) 0.008 1.69 (1.15-2.50) 0.008
Grade 2 1.19 (0.55-2.56) 0.661 0.96 (0.35-2.66) 0.939
ACC
Grade 3 1.21 (0.48-3.01) 0.687 0.60 (0.16-2.30) 0.454
14-gene signature (10 unit scale)
1.49 (1.01-2.21) 0.044 1.85 (1.05-3.25) 0.033
Age (10 year scale)
1.38 (0.85-2.25) 0.197 1.38 (0.85-2.24) 0.198
Tumor size 1.20 (0.71-2.01) 0.496 1.08 (0.61-1.90) 0.798
Grade 2 1.81 (0.66-4.98) 0.251 2.47 (0.53-11.61) 0.252
Guy’s
Grade 3 1.20 (0.39-3.71) 0.756 0.74 (0.09-5.83) 0.776
14-gene signature (10 unit scale)
1.51 (1.19-1.92) 0.001 1.55 (1.11-2.15) 0.009
Age (10 year scale)
1.38 (1.03-1.85) 0.033 1.52 (1.10-2.08) 0.010
Tumor size 1.51 (1.12-2.04) 0.007 1.47 (1.05-2.07) 0.026
Grade 2 1.38 (0.75-2.53) 0.303 1.31 (0.58-2.99) 0.517
Combined
Grade 3 1.18 (0.58-2.39) 0.654 0.66 (0.22-2.02) 0.469
# of events
# of remaining
5
46
51
2
21
23
5
8
13
7
29
36
89
108
197
26
66
92
71
39
110
97
105
202
UntreatedGuy’s set
Tam-treatedACC set
Tam-treatedCombined set
Tam-treatedGuy’s set
0 5 10 15 20 25 30 35
All Patients (n=408)
High MS (n=235)
Low MS (n=173)
All Patients (n=203)
High MS (n=81)
Low MS (n=122)
All Patients (n=205)
High MS (n=154)
Low MS (n=51)
All Patients (n=279)
High MS (n=172)
Low MS (n=107)
Percent with DM at 9 Years
# of events
# of remaining
5
46
51
2
21
23
5
8
13
7
29
36
89
108
197
26
66
92
71
39
110
97
105
202
UntreatedGuy’s set
Tam-treatedACC set
Tam-treatedCombined set
Tam-treatedGuy’s set
0 5 10 15 20 25 30 35
All Patients (n=408)
High MS (n=235)
Low MS (n=173)
All Patients (n=203)
High MS (n=81)
Low MS (n=122)
All Patients (n=205)
High MS (n=154)
Low MS (n=51)
All Patients (n=279)
High MS (n=172)
Low MS (n=107)
Percent with DM at 9 Years
Gene Paik1 van't Veer2 Dai3 Gene Description Gene Function
BUB1 x BUB1 budding uninhibited by benzimidazoles 1 homolog cell cycle, DNA replication
CCNB1 x cyclin B1 cell cycle, cell death, DNA replication
CENPA x x centromere protein A, 17kDa cell morphology, DNA replication
DC13 x DC13 protein assembly of cytochrome oxidase
DIAPH3 x x diaphanous homolog 3 (Drosophila) actin cytoskeleton organization and biogenesis
MELK x maternal embryonic leucine zipper kinase protein amino acid phosphorylation, ATP binding
MYBL2 x v-myb myeloblastosis viral oncogene homolog (avian)-like 2 cell cycle, cell death, cell morphology
ORC6L x x origin recognition complex, subunit 6 homolog-like (yeast) cell cycle, DNA replication
PKMYT1 xmembrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase
cell cycle
PRR11 x proline rich 11 cell cycle
RACGAP1 x Rac GTPase activating protein 1 cell cycle, cell morphology
RFC4 x replication factor C (activator 1) 4, 37kDa DNA replication
TK1 x thymidine kinase 1, soluble DNA replication, cell death
UBE2S x ubiquitin-conjugating enzyme E2S ubiquitin cycle
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
-60 -50 -40 -30 -20 -10 0 10 20
Metastasis Score
Pro
bab
ility
of
Dis
tan
t M
etas
tasi
s at
9 y
ears
UntreatedTam-treated
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
-60 -50 -40 -30 -20 -10 0 10 20
Metastasis Score
Pro
bab
ility
of
Dis
tan
t M
etas
tasi
s at
9 y
ears
UntreatedTam-treatedUntreatedTam-treated
