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OPEN A new clinical-genomic model to

predict 10-year recurrence risk in
primary operable breast cancer
patients
Tzu-Ting Huang1, Lei Lei2,3, Ching-Hsuan Andre Chen4, Tzu-Pin Lu4, Chung-Wen Jen5 &
Skye Hung-Chun Cheng5,6*
This study aimed to validate the long-term prognostic value of a new clinical-genomic model, Distant
Genetic Model-Clinical Variable Model 6 (DGM-CM6), developed in Asia as a prognostic panel for all
subtypes of breast cancer. We included 752 operable stage I–III breast cancer patients representing all
subtypes treated from 2005 to 2014 as the validation cohort. The median follow-up was 95.8 months.
The low- and high-risk patients classified by DGM-CM6 (RI-DR) had significant differences in 10-year
distant recurrence-free interval (DRFI) (94.1% vs. 85.0%, P < 0.0001) and relapse-free survival
(RFS) (90.0% vs. 80.5%, P = 0.0003). External validation using EMTAB-365 dataset showed similar
observation (P < 0.0001). DGM-CM6 was an independent prognostic factor by multivariate analysis with
hazard ratios of 3.1 (1.6–6.0) for RFS (P = 0.0009) and 3.8 (1.6–9.0) for DRFI (P = 0.0028). Comparing the
C-index of DGM-CM6 and PAM50-ROR scores, the former performed better than the latter in predicting
long-term DRFI and RFS, especially in N0, ER/PR-positive, and HER2-negative patients.

Breast cancer is the most prevalent and deadly malignant disease among women throughout the world. Despite
recent advances in early breast cancer (EBC) management, recurrent events remain inevitable in high-risk pop-
ulations1. A reliable prognostic algorithm combining clinical and genomic information to help determine treat-
ment strategies for EBC patients is urgently needed. The Oncotype DX (ODx) 21-gene recurrence score (RS) is
known to be a sound prognostic and predictive assay in node-negative hormonal receptor-positive (HR-positive)
and human epidermal growth factor receptor 2 (HER2) negative breast cancer patients. However, its prognos-
tic value in HR-negative patients is unknown. According to the Trial Assigning Individualized Options for
Treatment (TAILORx), the RS cutoff at which chemotherapy in patients 50 years of age or younger can safely be
avoided is under investigated2,3. Women from Asian-Pacific countries experience earlier onset of breast cancer
than women from Western countries, nearly 50% suffering from breast cancer under the age of 504. However,
they usually have a better survival rate. One study using data from Surveillance, Epidemiology and End Results
(SEER) demonstrated that the actuarial risk of death 7 years post-onset for women with stage I breast cancer was
lower among Asian women than that of non-Hispanic white women5. This difference may be related to biological
differences in tumour characteristics between the races. The ODx was mainly developed based on Western popu-
lations and may not be fully applicable to Asian ethnic groups; the TAILORx trial included only 151 Asian women
out of a total of 11,248 (1.3%) patients2. Overestimated prediction for the recurrence risk by ODx in Japanese
populations has been reported, with no recurrence in the intermediate-risk group (cutoff 18–30)6. Considering
the potential impact caused by differences in race and ethnicity, a Korean group developed a multi-gene assay in
2019, which could identify more low-risk patients in the young age group (<50 years) than those identified by
ODx7. In our group, a 34-gene panel was developed in 2006, which could classify the low- and high-risk groups
of local/regional recurrence (LRR) after mastectomy8. This multi-gene panel was further refined and validated

1
Department of Research, Koo Foundation, Sun Yat-Sen Cancer Center, Taipei, Taiwan. 2Department of Breast
Medical Oncology, Cancer Hospital of University of Chinese Academy of Sciences (Zhejiang Cancer Hospital),
ZheJiang, China. 3Visiting Scholar, University of Mississippi Medical Center, Jackson, Mississippi, USA. 4Epidemiology
and Preventive Medicine, Department of Public Health, National Taiwan University, Taipei, Taiwan. 5Department of
Radiation Oncology, Koo Foundation Sun Yat-Sen Cancer Center, Taipei, Taiwan. 6Taitung Cancer Center, Department
of Radiation Oncology, Taitung Christian Hospital, Taitung, Taiwan. *email: skyecheng@gmail.com

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as an 18-gene classifier (18-GC) with more sensitivity, specificity, and accuracy—not only in predicting LRR but
also distant recurrence9,10. This 18-GC utilises the genes BLM, TCF3, PIM1, RCHY1, PTI1, DDX39, BUB1B, STIL,
TPX2, CCNB1, MMP15, CCR1, NFATC2IP, TRPV6, OBSL1, C16ORF7, DTX2, and ENSA, among which 17 were
included in the distant genomic model (DGM).
This clinical model was based on our previous work that identified the most important prognostic factors as
the number of axillary lymph nodes involved, age at diagnosis (≤40vs >40 years), prominent lymphovascular
invasion (LVI), oestrogen receptor (ER) status, tumour grade, and tumour size (>2 cm)11,12. Incorporating both
the genomic and clinical data, DGM-CM6 (recurrence index for distant metastasis [RI-DR]) proved to be the
most predictive13.
In this study, we assessed and validated the prognostic value of DGM-CM6 (RI-DR) in different molecular
subtypes of EBC after surgery based on the independent dataset.

Results
Validation dataset. A total of 752 patients who had undergone Affymetrix microarray testing and had
N0-2 breast cancer were included in the analysis (mastectomy, n = 482; BCS, n = 270). The median follow-up
was 86.9 months for patients without adjuvant chemotherapy and 96 months for patients with chemotherapy.
Patients without adjuvant chemotherapy were significantly older and had favourable pathological features (T1,
HR-positive, HER2-negative, no/focal LVI, and grade I/II) (Supplementary Table S3). The median age of sub-
jects was 49 years (range: 27–88 years), 55.5% (417) were 50 or below and 64.0% (481) were pre-menopausal.
Immunohistochemical analysis revealed that 34.6% (260) of subjects were negative for both ER and progesterone
receptor (PR) and 34.2% (257) were positive for HER2. Prominent LVI tumours were identified in 22.6% (170) of
patients. Adjuvant chemotherapy was used in 89.1% (670) of patients and adjuvant hormone therapy was used in
62.6% (471) of patients. Among mastectomy patients, post-mastectomy radiotherapy was administrated in 65.1%
(314/482). Among HER2-positive patients, 37.7% (97/257) received adjuvant trastuzumab (Table 1).
We examined the relationship between DGM-CM6 and 10-year DRFI and RFS (Fig. 1). The results revealed
that 3.5% (8/232) of the low-risk patients had DR and 6.9% (16/232) experienced any type of relapse or death;
whereas 13.5% (70/520) of the high-risk group patients had DR and 17.3% (90/520) experienced any type of
relapse or death. The estimated 10-year DRFI for low- and high-risk patients by the DGM-CM6 was 94.1% and
85.0% (P < 0.0001); and 10-year RFS was 90.0% and 80.5% (P = 0.0003), respectively (Fig. 1A,B).
Subgroup analyses revealed that DGM-CM6 (RI-DR) and DGM could distinguish the low- and high-risk
patients in luminal, HER2, and triple-negative EBC (Supplementary Table S6). However, DGM score and RI-DR
were not significant factors in patients with HER2-overexpressed and triple-negative breast cancer; the low-risk
group had a trend towards a better outcome than the high-risk group. When we confined the analysis to luminal
N0-N1 patients, DGM and RI-DR could significantly distinguish the low- and high-risk patients (Supplement
Table S6).
For the interaction between DGM-CM6 (RI-DR) and chemotherapy, RI-DR was capable of classifying low-
and high-risk N0-2 patients as 10-year DRFI regardless of chemotherapy administration. The 10-year DRFI for
low- and high-risk patients who did not receive chemotherapy was 97.0% and 82.3% (P = 0.012), respectively.
The corresponding rates in patients receiving chemotherapy were 93.4% and 85.2% (P = 0.0008), respectively
(Fig. 1C). The int eraction between RI-DR and chemotherapy using RFI, DRFS, and RFS as study endpoints was
shown in Supplementary Figs. S1–S3.

Comparison to PAM50 intrinsic subtypes. According to research-based PAM50 intrinsic subtypes, a

heatmap was generated by unsupervised clustering of all 752 patients combining our genomic panel with IHC4
genes (ER, PR, HER2 and MKI67). Our gene panel differentiates each subtype correctly (Fig. 2A); for example,
BUB1B, TPX2, BLM, and DDX39 were clustered together with MKI67; furthermore, the panel was capable of
distinguishing luminal A from luminal B subtypes. TRPV6 and CLCA2 were clustered together with ERBB2 and
the HER2 subtype was differentiated from other subtypes.
The score distributions of DGM were significantly different among PAM50 intrinsic subtypes (Fig. 2B).
Luminal A patients had the lowest DGM scores among all subtypes (P < 0.0001). PAM50 ROR score (ROR-S)
low-risk patients were usually classified as low-risk by DGM; however, DGM further classified some patients
in normal-like and luminal B subtypes as low-risk. DGM divided the ROR-S low-risk group into low-risk and
high-risk groups; the former had a 10-year DRFI of 93.5% (86.3%, 97.0%) and the latter 77.1% (53.1%, 89.9%)
(P = 0.0019) (Table 2). DGM also identified low-risk patients in the ROR-S intermediate-risk group with a
10-year DRFI of 95.7% (87.1%, 98.6%). The gene expression levels of DGM-low and -high patients were signifi-
cantly different (Supplementary Table S4).
Similarly, DGM combined with clinical variables (DGM-CM6 or RI-DR) separated ROR-S low- and
intermediate-risk patients into low- and high-risk groups significantly (Table 2).

Concordance index (C-index). Comparing the performance of DGM, DGM-CM6, and PAM50 ROR
scores from the validation dataset, the C-index by DGM, DGM-CM6, and ROR for RFS in all patients was 0.565,
0.583, and 0.561, respectively (Fig. 2C). The corresponding C-index in N0, ER+/PR+, and HER2- patients was
0.716, 0.748, and 0.651, respectively (Fig. 2D).

Uni- and multivariate analyses of the internal validation cohort. Univariate analysis with the Cox
regression model revealed that RI-DR was a prognostic factor associated with DRFI, RFI, DRFS, and RFS with
hazard ratios of 4.0 (95% CI, 1.9–8.3), 3.8 (1.9–7.6), 2.6 (1.5–4.5) and 2.6 (1.5–4.4), respectively. Tumour stage,
nodal status, and tumour grading were also associated with prognosis (all P < 0.05). Detailed information is illus-
trated in Supplementary Table S5.

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All patients Low-risk* High-risk*

Variables (n = 752) (n = 232) (n = 520) P-value
Median follow-up (Months) 95.8 (0.6–169.3) 95.8 (0.6–169.3) 95.8 (0.9–164.5) 0.2525
Menstruation status <0.0001
Pre-menopausal 481 138 (59.5%) 343 (66.0%)
Post-menopausal 262 91 (39.2%) 171 (32.9%)
Unknown 9 3 (1.3%) 6 (1.2%)
Age <0.0001
<40 138 25 (10.8%) 113 (21.7%)
41–50 279 101 (43.5%) 178 (34.2%)
51–60 223 58 (25.0%) 165 (31.7%)
>60 112 48 (20.7%) 64 (12.3%)
T stage <0.0001
T1 327 152 (65.5%) 175 (33.7%)
T2 408 76 (32.8%) 332 (63.9%)
T3 17 4 (1.7%) 13 (2.5%)
N stage <0.0001
N0 364 152 (65.5%) 212 (40.8%)
N1 282 67 (28.9%) 215 (41.4%)
N2 106 13 (5.6%) 93 (17.9%)
ER and PR status <0.0001
Both Negative 260 14 (6.0%) 246 (47.3%)
ER or PR (+) 492 218 (94.0%) 274 (52.7%)
HER2 overexpression <0.0001*
Negative 492 198 (85.3%) 294 (56.5%)
Positive 257 34 (14.7%) 223 (42.9%)
Indeterminant 3 0 (0.0%) 3 (0.6%)
LVI <0.0001
Absent/focal 582 201 (86.6%) 381 (73.3%)
Prominent 170 31 (13.4%) 139 (26.7%)
Tumour grade <0.0001
Grade I 83 74 (31.9%) 9 (1.7%)
Grade II 238 123 (53.0%) 115 (22.1%)
Grade III 431 35 (15.1%) 396 (76.2%)
PMRT or RNI <0.0001
No 184 78 (33.6%) 106 (20.4%)
Yes 568 154 (66.4%) 414 (79.6%)
Adjuvant C/T <0.0001
No 82 54 (23.3%) 28 (5.4%)
Yes 670 178 (76.7%) 492 (94.6%)
Adjuvant H/T <0.0001
No 281 20 (8.6%) 261 (50.2%)
Yes 471 212 (91.4%) 259 (49.8%)
Adjuvant trastuzumab <0.0001
No 655 220 (94.8%) 435 (83.7%)
Yes 97 12 (5.2%) 85 (16.4%)
IHC subtype <0.0001
ER/PR+ HER2−, Gr 1–2 249 171(73.7%) 78 (15.0%)
ER/PR+ HER2−, Gr 3 89 19 (8.2%) 70 (13.5%)
ER/PR+ HER2+ 152 28 (12.1%) 124 (23.9%)
ER−, PR−, HER2+ 105 6 (2.6%) 99 (19.0%)
ER−, PR−, HER2− 154 8 (3.5%) 146 (28.1%)
PAM50 intrinsic subtypes <0.0001
Luminal A 192 148 (63.8%) 44 (8.5%)
Luminal B 212 43 (18.5%) 169 (32.5%)
HER2 140 1 (0.4%) 139 (26.7%)
Basal 144 2 (0.9%) 142 (27.3%)
Normal 64 38 (16.4%) 26 (5.0%)

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Table 1. Baseline characteristics of subjects in the internal validation dataset. *Defined by DGM-CM6: cutoff
<33 as low-risk, ≥33 as high-risk. BCS, breast-conserving surgery; C/T, chemotherapy; H/T, hormonal therapy;
LVI, lymphovascular invasion; MRM, modified radical mastectomy; PMRT, post-mastectomy radiotherapy;
RNI, regional node irradiation.

Multivariate analysis adjusted for age, T or N stage, ER/PR/HER2 status, tumour grade, and LVI by stepwise
selection revealed that the RI-DR high-risk group and N2 category were poor prognostic factors for DRFI, RFI,
DRFS, and RFS with hazard ratios of 3.8 (1.6–9.0), 3.5 (1.5–8.1), 3.2 (1.6–6.3), and 3.1 (1.6–6.0), respectively
(Table 3).
Multivariate analysis for each subtype revealed that RI-DR was an independent prognostic factor for DRFI,
RFI, DRFS, and RFS in luminal subtype (Supplementary Table S7). RI-DR in HER2 subtype had hazard ratios
of 3.7 (0.4–33.3), 4.6 (0.5–40.3), 1.7 (0.4–7.1), and 2.0 (0.5–7.9) for DRFI, RFI, DRFS, and RFS, respectively
(Supplementary Table S8). Multivariate analysis for triple-negative subtype could not be performed due to none
recurrence being observed in low-risk patients.

Validation in an external dataset. The performance of DGM (clinical data was inadequate to test
DGM-CM6) in predicting the outcomes of N0-2 patients from the EMTAB-365 dataset revealed that the 10-year
DRFS was 62.1% in the high-risk group and 82.3% in the low-risk group (P < 0.0001) (Fig. 3). According to
the PAM50, the ROR-S low-, intermediate- and high-risk patients had 10-year DRFS rates of 80.1%, 67.2% and
57.8%, respectively (Fig. 3).

Discussion
The new clinical-genomic model DGM-CM6 serves as an independent prognostic factor in patients with N0-N2
primary operable breast cancer, especially the luminal subtype; however, its prognostic value in non-luminal
subtype needs to be confirmed with more data. The hazard ratios for DRFI and RFS were 3.8 (1.6–9.0,
P = 0.0028) and 3.1 (1.6–6.0, P = 0.0009), respectively (Table 3). This model also divided PAM50 ROR low- and
intermediate-risk patients into different risk groups (Table 2). The 10-year rates of DRFI in RI-DR low-risk and
ROR low/intermediate-risk groups were excellent, ranging from 94.6% to 98.5%. The data obtained in our study
suggest that our model can identify high-risk patients from the ROR low-risk group and low-risk patients from
the ROR intermediate-risk group (Table 2). As a result, 44/192 (22.9%) PAM50 luminal A patients were identified
as high-risk and 43/212 (20.3%) luminal B patients as low-risk (Table 1).
Although the multi-gene panel was initially developed without considering breast cancer subtypes, the
heatmap and correlation analyses revealed that our panel can differentiate among PAM50 intrinsic subtypes
(Fig. 2A,B). The heatmap showed that the gene expression levels of BUB1B, TPX2, BLM and DDX39 are dif-
ferent between PAM50 luminal A and B subtypes. Other researchers have made similar observations; BUB1B
is associated with poor prognosis in luminal A breast cancer14. TPX2 is the most well-connected gene within a
proliferation network; its knockdown significantly affects metastasis but not tumour proliferation in oestrogen
receptor-positive tumours15. Bloom syndrome helicase (BLM) has key roles in homologous recombination repair;
PAM50 luminal A subtype is more likely to express low levels of BLM mRNA16.
Concordant statistics using the validation dataset revealed DGM-CM6 had higher C-indices than DGM and
PAM50 ROR scores (Fig. 2C). This is understandable as DGM-CM6 incorporates clinical information in the model
that might increase the C-index. Confined to node-negative, ER+/PR+ and HER2-negative patients, the C-indices
of DGM and DGM-CM6 for DRFS and RFS were 0.72–0.75; however, the C-index of ROR-S was 0.65–0.66
(Fig. 2D). This may be related to the fact that our dataset is based on an Asian population with reduced odds of the
basal-like subtype and apparent ethnicity differences17. The C-index of ROR-S for post-menopausal node-negative
luminal women in anastrozole or tamoxifen alone or combined randomised clinical trials was reported as 0.7818.
The main goal of adjuvant chemotherapy is to reduce the risk of distant recurrence. The current study demon-
strated very low-risk DR within 5 years in the DGM-CM6 low-risk group. However, some late recurrences devel-
oped after 5 years (Fig. 1A). Patients in the current study received hormonal therapy for only 5 years; the DR
after 5 years was probably related to the duration of hormonal therapy. The type and risk of recurrence vary
significantly among different molecular subtypes; furthermore, our genomic information is highly correlated
with the PAM50 subtype. Numerous multi-gene panels or clinical-genomic models have been developed to assist
in decision making for adjuvant systemic therapy. However, most of them focus on luminal subtypes and are
rarely shown to play a role in basal-like or HER2 positive subtypes. In our gene panel, TRPV6 and CLCA2 were
clustered together with ERBB2 and could differentiate HER2 from non-HER2 subtypes (Fig. 2A). Both genes are
related to ion channel pathway control19,20 TRPV6 expression leads to reduction in basal calcium influx and cel-
lular proliferation and is significantly elevated in basal-like and HER2 subtypes19. CACL2 is a tumour suppressor,
involved in the p53 tumour suppressor network and has a significant effect on cell migration and invasion21. These
2 genes could be novel targets for HR-negative breast cancer19.
For patients with HER2 positive breast cancer treated with curative surgery, adjuvant trastuzumab for one year
is the standard care. However, identifying patients, who are at a higher risk of recurrence and would, therefore,
benefit more from novel anti-HER2 agents such as pertuzumab and neratinib is paramount. There is an urgent
need for a predictive tool to guide the systemic treatment strategies of these patients. Our clinical-genomic model
can classify breast cancer patients into high recurrence risk and low recurrence risk regardless of molecular sub-
types, which has the potential to help clinicians make more informed decisions about systemic treatments.
A Korean group has developed a clinical-genomic model (GeneWell BCT), which consists of 6 prognos-
tic genes and 2 clinical risk factors and can divide pN0-N1, ER/PR-positive and HER-2 negative patients into

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Figure 1. DGM-CM6 and distant recurrence-free interval (DRFI) and relapse-free survival (RFS). (A,B)
DRFI and RFS of low- and high-risk groups divided by DGM-CM6 (RI-DR); X and Y axes of the Kaplan-Meier
estimate plots show the follow-up interval (months) and estimated probability of events, respectively. (C) The
interaction between DGM-CM6 (RI-DR) and adjuvant chemotherapy (DRFI as an event): (1) Blue line: low
RI-DR and chemotherapy; (2) Red line: low RI-DR and no chemotherapy; (3) Green line: high RI-DR and
chemotherapy; (4) Black line: high RI-DR and no chemotherapy.

low- and high-risk groups22. Comparison of GenesWell BCT score with ODX RS revealed that BCT score clas-
sified more low-risk patients than RS in patients aged 50 years or less (73.0% versus 33.6%)7. Since Asian breast
cancer patients are usually pre-menopausal23, further studies, including our model, are necessary to identify
which test is more accurate in this subpopulation.
There were some limitations to our study. First, the ideal prognostic validation dataset should recruit only
patients who have not received systemic therapies because the risk of recurrence after adjuvant therapy may be
underestimated. We had 82 (10.9%) patients who did not receive chemotherapy, but this number was too small
for further analysis. It is clear that this study cannot provide adequate information for patients to make a decision
about adjuvant chemotherapy. However, the potential prognostic value of our DGM-CM6 model should be noted
for the significant difference between the low- and high-risk breast cancer recurrence in large cohorts. Second,
most patients with HER2-positive breast cancer did not receive anti-HER2 therapy. The utility of this model in
the era of anti-HER2 treatment is unclear. Finally, only a few triple negative breast cancer patients were low-risk
according to our model; further investigation is necessary for this group.
In conclusion, we developed a model combining genomic and clinical information as a prognostic tool for
non-metastatic breast cancer. This multi-gene model can provide not only clinical outcome information before
treatment but also may play a tool to assist in the risk-benefit judgement of systematic adjuvant treatments, espe-
cially in Asian patients.

Materials and Methods

Patient population. Breast cancer patients, who had undergone microarray analysis of their primary
tumour were enrolled in this study. The Consolidated Standards of Reporting Trials (CONSORT) flow diagram
for this study is shown in Fig. 4. Details of the training and testing information for DGM-CM6 has been reported
in our previous publication (Supplementary Tables S1 and S2)13. This study focused on validation using a dataset
obtained from the Affymetrix platform.

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Figure 2. Differentiation of subtypes. (A) Heatmap of DGM, ESR1, PgR, HER2, and MKI67 gene expression
levels in all 752 patients (X-axis for PAM50 subtypes); Unsupervised clustering DGM genes, ESR1, PgR, HER2
and MKI67 (Y-axis); (B) DGM score distribution according to PAM50 subtypes and ROR scores. The X-axis
for ROR score; Y-axis for DGM score. PAM50 subtypes: Orange dots represent the basal-like subtype, grey dots
the HER2 subtype, green dotes luminal A, blue dots luminal B, and pink dots represent the normal type; (C)
C-indices for all subtypes; (D) C-indices for N0 luminal subtype (ER/PR+ and HER2−) patients only.

DGM-low 10-year DRFI DGM-high 10-year DRFI

Log-rank
PAM50 ROR-S Patient # % (95% CI) Patient # % (95% CI) p-value
Low 169 93.5% (86.3%, 97.0%) 26 77.1% (53.1%, 89.9%) 0.0019
Intermediate 83 95.7% (87.1%, 98.6%) 113 88.8% (81.0%, 93.5%) 0.0831
High 21 64.1% (33.6%, 83.4%) 340 85.2% (80.6%, 88.8%) 0.0883
RI-DR low 10-year DRFI RI-DR high 10-year DRFI
Low 151 94.6% (86.7%, 97.8%) 44 80.5% (62.9%, 90.3%) 0.0016
Intermediate 66 98.5% (89.6%, 99.8%) 130 88.5% (81.3%, 93.0%) 0.0263
High 15 77.4% (31.5%, 94.5%) 346 84.2% (79.5%, 87.9%) 0.8150

Table 2. PAM50 ROR score (ROR-S) risk classification and 10-year DRFI by DGM and RI-DR scores.

The internal validation cohort consisted of 752 patients, who had a microarray study performed for their
primary tumours. This study was performed in a prospective way that all alive participants gave written informed
consent to use their frozen tumor tissues from the biobank for the purpose to identify poor or good gene expres-
sion profiling. The inclusion criteria were pathology stage pN0-2 (0–9 axillary lymph nodes were positive) breast
cancer patients after primary surgery with either mastectomy or breast-conserving surgery (BCS). Patients who
had preoperative chemotherapy and pN3, T4, and/or M1 disease were excluded. The protocol and informed con-
sent documents were reviewed and approved by the institutional review board (IRB) of the Koo Foundation Sun
Yat-Sen Cancer Center in Taipei, Taiwan (IRB no. 20131001 A).
The EMTAB-365 dataset was used as the external validation cohort, which is the most extensive dataset using
Affymetrix U133 Plus 2.0 microarray to analyse gene expression profiles of primary tumour tissues24. A total of
426 patients with pN0-N2 regardless of breast subtypes and microarray data were included (http://www.ebi.ac.uk/
arrayexpress).

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DRFI RFI DRFS RFS

HR (95% HR (95% HR (95% HR (95%
Parameter CI) P-value CI) P-value CI) P-value CI) P-value
1.0 1.1 0.8 0.8
<40 0.9113 0.8012 0.4521 0.5555
(0.5–2.3) (0.5–2.4) (0.4–1.5) (0.4–1.6)
Age 0.8 0.9 0.6 0.7
40–60 0.625 0.8634 0.1018 0.1961
(0.4–1.7) (0.5–1.8) (0.4–1.1) (0.4–1.2)
>60 Ref Ref Ref Ref
T1 Ref Ref Ref Ref
1.5 1.6 1.4 1.5
T2 0.1845 0.0986 0.1629 0.0886
T stage (0.8–2.6) (0.9–2.7) (0.9–2.3) (0.9–2.4)
2.5 2.6 2.1 2.2
T3 0.108 0.0987 0.1348 0.1295
(0.8–7.8) (0.8–7.9) (0.8–5.8) (0.8–5.8)
N0 Ref Ref Ref Ref
1.6 1.5 1.7 1.6
N1 0.1237 0.1659 0.0749 0.0989
N stage (0.9–3.0) (0.8–2.8) (1.0–2.9) (0.9–2.7)
4.2 4.1 3.6 3.6
N2 <0.0001 <0.0001 <0.0001 <0.0001
(2.1–8.4) (2.1–7.9) (1.9–6.8) (1.9–6.6)
1.1 1.0 0.7 0.7
Both neg. 0.8453 0.8919 0.2329 0.16
ER/PR status (0.6–1.8) (0.6–1.6) (0.5–1.2) (0.5–1.1)
ER/PR pos. Ref Ref Ref Ref
Neg. Ref Ref Ref Ref
HER2 1.0 1.0 0.9 0.9
Pos. 0.9571 0.8472 0.6851 0.784
(0.6–1.7) (0.6–1.7) (0.6–1.4) (0.6–1.5)
1 Ref Ref Ref Ref
1.8 2.2 2.2 2.5
2 0.3509 0.2149 0.1162 0.0741
Grade (0.5–6.6) (0.6–8.0) (0.8–6.1) (0.9–6.7)
1.1 1.5 1.5 1.8
3 0.9413 0.5862 0.444 0.2597
(0.3–4.1) (0.4–5.6) (0.5–4.4) (0.6–5.4)
Absent/focal Ref Ref Ref Ref
LVI 0.7 0.6 0.7 0.6
Prominent 0.1818 0.1163 0.1331 0.0857
(0.4–1.2) (0.4–1.1) (0.4–1.1) (0.4–1.1)
0.8 0.7 1.0 0.9
MRM 0.337 0.1824 0.9911 0.6674
Surgery (0.4–1.3) (0.4–1.2) (0.6–1.6) (0.6–1.4)
BCT Ref Ref Ref Ref
No Ref Ref Ref Ref
Chemo 0.5 0.4 0.2 0.2
Yes 0.1864 0.0259 <0.0001 <0.0001
(0.2–1.4) (0.1–0.9) (0.1–0.5) (0.1–0.4)
Low Ref Ref Ref Ref
DGM-CM6 3.8 3.5 3.2 3.1
High 0.0028 0.0028 0.0009 0.0009
(1.6–9.0) (1.5–8.1) (1.6–6.3) (1.6–6.0)

Table 3. Multivariate analysis.

Affymetrix microarray and PAM50 subtyping. The mRNA microarray results were reported previ-
ously8,25 RNA was extracted from primary tumour tissue using TRIZOL reagent (Invitrogen/Thermo Fisher
Scientific, Waltham, MA, USA) and purified with an RNEASY Mini Kit (Qiagen, Hilden, Germany); the purity
was evaluated with an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). According to the Affymetrix
protocol, hybridisation targets were prepared from total RNA and hybridised to U133 Plus 2.0 (U133P2) arrays
(Affymetrix, Santa Clara, CA, USA). The details of the study protocol were reported previously25. Each patient
was assigned to an intrinsic molecular subtype of breast cancer (luminal A, luminal B, HER2-enriched, basal-like
and normal-like) using the research-based PAM50 subtyping26,27.
External validation Affymetrix U133P2 dataset was obtained from ArrayExpress (EMTAB-365). Raw CEL
files were pre-processed using the robust multi-array average method in the affy package of R software28,29.
Quantile normalisation was performed to reduce potential systematic biases. The classification of PAM50 sub-
types and calculation of risk of recurrence (ROR) score were performed using genefu R package26,30,31.

Algorithm of DGM and DGM-CM6. The algorithm for the DGM is summarised as follow:
DGM score = β1 × Gene1 + β 2 × Gene2 + … + β N × Gene N, N ≤ 17 (scores rescaled to 1–100)
The RI-DR score was calculated in 2 steps: 1) the genetic score was calculated as described above; and 2) clin-
ical and genetic scores were integrated. The algorithm is summarised as follows:
DGM − CM6 (RI − DR)score = H1 × DGM score + H2 × clinical score(CM6) (scores rescaled to 1 - 100)

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Figure 3. DRFS of patients from the EMTAB-365 dataset. DRFS of low-, intermediate- and high-risk groups
divided by PAM50 (black color); and low- and high-risk groups divided by DGM-CM6 (red color).

Figure 4. Consolidated Standards of Reporting Trials for this study. Using LASSO to select significant
genes. Abbreviation: IHC: immunohistochemistry; DGM: Distant genomic model; CM6: Clinical model 6
(age, lymphovascular invasion, oestrogen receptor, lymph node status, tumor size and grade); ROR: risk of
recurrence; LASSO: Least Absolute Shrinkage and Selection Operator.

Statistical methods. The Kaplan-Meier method was used to estimate the 10-year relapse-free survival
(RFS), DR-free survival (DRFS), and DR-free interval (DRFI); the log-rank test was used to examine whether
the difference in survival curves was significant. All statistical analyses were performed using R v.3.4.1 (http://
www.R-project.org/) and SAS v.9.4 (SAS Institute). P < 0.05 was considered significant.
Patients with DGM score cut-offs < 41 and ≥41 were considered low- and high-risk, respectively. Patients
with DGM-CM6 (RI-DR) scores ≥ 33 and <33 were defined as having a high and low-risk of distant metastasis,
respectively13. Using these predefined cut-offs, we examined the performance of CM6, DGM and RI-DR in train-
ing, testing and validation datasets (Supplementary Table S9).

Protocol approval. The Bio-bank Ethics Committee and the IRB of the Koo Foundation Sun Yat-Sen Cancer
Center approved this study (approval numbers 20131001A and 20150327A). The committee confirmed that all
research was performed in accordance with relevant guidelines/regulations.

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www.nature.com/scientificreports/ www.nature.com/scientificreports

Received: 13 October 2019; Accepted: 28 February 2020;

Published: xx xx xxxx

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Acknowledgements
Ministry of Science and Technology (MOST), Taipei, Taiwan—MOST 105-2314-B-368-001; National Science
Council—NSC 102-3114-Y-368-001.

Author contributions
T.T.H. acquired the data, carried out statistical analyses, and prepared the data presented in this paper. L.L. carried
out statistical analyses, interpreted the data and drafted the manuscript, C.W.J., C.A.C. and T.P.L. participated
in the molecular genetic analysis and data interpretation and edited the manuscript. S.H.C. applied the grants,
designed the research, acquired the data, carried out statistical analyses, prepared the data presented in this paper
and edited the script.

Competing interests
SHC owns a patent related to the content of this manuscript. SHC reports grants from the Ministry of Science
and Technology, Taiwan, and National Science Council, during the conduct of the study. There is no competing
interest for other authors (T.T.H., L.L., C.A.C., T.P.L. and C.W.J.).

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Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-61535-9.
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