
==== Front
ESMO Open
ESMO Open
ESMO Open
2059-7029
Elsevier

S2059-7029(24)01417-0
10.1016/j.esmoop.2024.103648
103648
Original Research
Treatments and clinical outcomes in stage II colon cancer patients with 12-gene Oncotype DX Colon Recurrence Score® assay-guided therapy: real-world data
Brenner B. brennerb@clalit.org.il
12∗
Shulman K. 34
Hubert A. 5
Man S. 6
Geva R. 7
Ben-Aharon I. 8
Fennig S. 9
Mishaeli M. 10
Yarom N. 11
Bar-Sela G. 412
Brenner R. 13
Shai A. 414†
Baehner F.L. 15
Russell C. 15
Soussan-Gutman L. 16
Voet H. 17
Bareket-Samish A. 18
Liebermann N. 19
1 Davidoff Cancer Center, Rabin Medical Center, Petah Tikva
2 Faculty of Medicine, Tel Aviv University, Tel Aviv
3 Oncology Department, Lin Medical Center, Haifa
4 Rappaport Faculty of Medicine, Technion Israeli Institute of Technology, Haifa
5 Sharett Institute of Oncology, Hadassah-Hebrew University Hospital, Jerusalem
6 Department of Clinical Oncology and Radiology, Soroka University Medical Center, Beer Sheva
7 Division of Oncology, Sourasky Medical Center, Tel Aviv
8 Department of Oncology, Rambam Health Care Campus, Haifa
9 Institute of Oncology, Kaplan Medical Center, Rehovot
10 Oncology Department, Meir Medical Center, Kfar Saba
11 Oncology Department, Shamir Medical Center, Be’er Ya’akov
12 Oncology Department, Emek Medical Center, Afula
13 Oncology Department, Wolfson Medical Center, Holon
14 Oncology Department, Galilee Medical Center, Nahariya, Israel
15 Medical Department, Exact Sciences, Redwood City, USA
16 Oncotest, Rhenium, Modi’in
17 Environmental Economics and Management, Hebrew University of Jerusalem, Rehovot
18 BioInsight, Ltd., Binyamina
19 Community Division, Clalit Health Services, Tel Aviv, Israel
∗ Correspondence to: Prof. Baruch Brenner, Institute of Oncology, Davidoff Cancer Center, Rabin Medical Center, 39 Jabotinski St., Petah Tikva, 49100, Israel; Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Tel: +972-3-9377990; Fax: +972-3-9377902 brennerb@clalit.org.il
† Present address: Department of Oncology, Rambam Healthcare Campus, Haifa, Israel.

12 8 2024
8 2024
12 8 2024
9 8 103648© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Background

The 12-gene Oncotype DX Colon Recurrence Score® result quantifies the recurrence risk in stage II/III colon cancer (CC). This real-world study investigated stage II CC patients whose treatment decisions incorporated the Recurrence Score® (RS) result.

Materials and methods

This retrospective analysis of a prospectively designed cohort included all stage II, mismatch repair-proficient CC patients who underwent 12-gene testing through Clalit between January 2011 and December 2016 and had available data with a minimum 3-year follow-up.

Results

The analysis included 938 patients {median age 68 [interquartile range (IQR) 60-76] years; 96% T3 tumors}. The median RS was 26 (IQR 19-33) and the three RS categories (0-29, 30-40, 41-100) included 65%, 24%, and 11% of patients, respectively. Chemotherapy (CT) use differed significantly between the three RS categories (14%, 36%, and 60%, respectively; P < 0.001). The CT and observation-only groups were imbalanced with worse clinicopathologic characteristics in the former. Among observation-only patients, Kaplan–Meier (KM) estimates for recurrence-free interval (RFI) and CC-specific survival (CCSS) differed significantly between the three RS categories (P < 0.001). Clinical outcomes by treatment (CT versus observation) within each RS category revealed no differences in RFI and CCSS in the RS 0-29 and 30-40 categories. In contrast, in the RS 41-100 category, the difference in RFI trended toward significance (P = 0.066), and for CCSS, a statistically significant difference was observed, with better outcomes among CT-treated patients (P = 0.035).

Conclusions

RS results are prognostic in stage II CC. Among RS 41-100 patients, outcomes were better in CT-treated versus observation-only patients despite worse clinicopathologic characteristics, suggesting that CT confers clinical benefit in high-risk patients.

Highlights

• The 12-gene Oncotype DX Colon Recurrence Score® assay quantifies recurrence risk in mismatch repair-proficient stage II/III CC.

• This real-world clinical practice study found that this assay provides independent prognostic information in these patients.

• The study found that CT seems to confer clinical benefit in patients with high risk according to the assay.

• Our results further validate the assay in such patients and support its use for guiding adjuvant treatment decisions.

Key words

12-gene colon cancer assay
adjuvant chemotherapy
clinical outcomes
colon cancer
Recurrence Score®
==== Body
pmcIntroduction

Colon cancer (CC) is the fifth most common cancer worldwide with ∼1 150 000 new cases and 580 000 deaths estimated in 2020, representing 6.0% of all newly diagnosed cancers (excluding basal cell carcinoma) and 5.8% of all cancer deaths.1 Approximately a quarter-to-third of these patients present with stage II disease, for which surgical resection alone is potentially curative.2 The role of adjuvant chemotherapy (CT) in stage II disease is debated, and adjuvant treatment decisions are often based on risk assessment using traditional clinicopathologic characteristics.3

The 12-gene Oncotype DX Colon Recurrence Score® test (Genomic Health, Inc., Redwood City, CA, a wholly owned subsidiary of Exact Sciences) is a validated multigene assay for quantifying the recurrence risk in patients with stage II, mismatch repair-proficient (MMR-P), and stage III A/B CCs. The assay evaluates the expression of seven cancer-related and five reference genes using reverse transcription–polymerase chain reaction on formalin-fixed paraffin-embedded CC tumor samples and provides a Recurrence Score® (RS) result, which ranges from 0 to 100, and provides an estimate of recurrence risk beyond traditional clinicopathologic parameters.4,5 All validation studies reported thus far employed a prospective–retrospective approach using archival specimens from four clinical trials (the QUASAR, CALGB 9581, NSABP C-07, and SUNRISE trials).6, 7, 8, 9 Outcome data from CC patients for whom treatment decisions incorporated the RS results in real-world clinical practice are lacking.

The 12-gene assay became commercially available in 2010. In Israel, the assay has been reimbursed by Clalit Health Services (CHS), the largest Israeli health maintenance organization with over 4 million members, from 2011 to 2022. The reimbursement was in the context of CHS-initiated observational real-world clinical study, in which a prospectively designed database was generated and analyzed. An earlier analysis of this database involved 269 stage II CC patients and compared treatment recommendations before knowing the RS results to the actual adjuvant treatments received after the RS results became available. The study showed a treatment change in 38% of the patients in a direction that was aligned with the RS results (i.e. increased treatment intensity in high RS patients and decreased intensity in low RS patients).10

The objective of the current analysis of the larger and maturing CHS database was to assess, in contemporary real-world clinical practice, the prognostic and predictive utility of the 12-gene assay by evaluating treatment decisions and clinical outcomes in MMR-P stage II CC patients for whom treatment decisions incorporated the RS results.

Materials and methods

Study design and patient population

This was a retrospective analysis of a prospectively designed cohort, with a pre-defined statistical analysis plan. The cohort was derived from a database that includes all CC patients who underwent the 12-gene assay through CHS.

The analysis included all patients with stage II, MMR-P CC who underwent definitive surgery, were tested through CHS between January 2011 and December 2016 (to allow for ≥5 years of follow-up), received treatment in the participating centers, and had available therapy and clinical outcome data. Patients with MMR deficiency, those who experienced recurrence within 3 months of the initial diagnosis (presumably indicating the presence of recurrence already at the time of testing), and patients who were lost to follow-up within 3 years of the initial diagnosis were excluded. Patients for whom two RS results were available were included once, with the higher RS result.

The study was approved by the institutional review board (IRB) of CHS as well as the IRBs of each participating institution. Informed consent was waived due to the retrospective design.

Data source

Patient/tumor characteristics and clinical information regarding adjuvant treatments received, recurrences, and death were obtained from patients’ medical records.

Statistical considerations

Descriptive statistics were used to summarize clinicopathologic characteristics. The chi-square or Fisher’s exact test was used to compare categorical parameters and the Mann–Whitney test was used to compare continuous parameters between CT-treated and observation-only patients.

The RS categories used in the main analyses were those used in the validation studies: RS 0-29 (low), RS 30-40 (intermediate), and RS 41-100 (high).6, 7, 8, 9 In addition, an exploratory analysis evaluated patients with RS 0-15 (the lower half of the low RS range, defined hereafter as ‘very low risk’). CT use across the three main RS categories (0-29, 30-40, 41-100) was compared using the chi-square test.

Multivariate logistic regression was used to estimate odds ratios and 95% confidence intervals (CI) for receiving CT for all patients and in each RS category separately. The variables used were age and RS as continuous parameters, and grade (2 versus 1, 3 versus 1, 3 versus 2), histological type (adenocarcinoma versus all others), number of nodes examined (≤12 versus >12), and having at least one of the following (lymphovascular, venous, or perineural invasion, obstruction, or perforation) as categorical parameters. Tests and CIs were Wald-based.

Kaplan–Meier (KM) estimates and log-rank tests were used to compare recurrence-free interval (RFI), an endpoint chosen for alignment with the validation studies,6, 7, 8, 9 and CC-specific survival (CCSS), among observation-only patients across RS categories, and between CT-treated and observation-only patients within each RS category. RFI and CCSS were determined from the date of definitive surgery. Patients who died with recurrence were considered to have had CC-related deaths. For RFI analysis, patients without recurrence were censored at the time of last follow-up, date of medical records review, or time of death (of any cause). For CCSS, patients without CC death were censored at the time of last follow-up or date of medical records review, or time of non-CC death (recurrences were ignored for this endpoint). Five-year rates for RFI and CCSS and 95% CI were calculated.

Multivariate Cox proportional hazards regression models for recurrence and CC death were estimated using variables identified as statistically significant on univariate analysis. Hazard ratios (HRs) and 95% CIs were calculated. The C-index was calculated to assess the fit of the multivariate model. Additional multivariate models were fitted including the RS category, treatment, and an interaction in the model.

JMP® Version 16 (SAS Institute Inc., Cary, NC) was used for the analysis. All tests were two-sided. P ≤ 0.05 was considered statistically significant.

Results

Patient population

Between January 2011 and December 2016, 1141 patients with stage II CC who were treated in the participating medical centers were referred to 12-gene testing through CHS, of whom 15 had MMR-deficient tumors and did not undergo the test. Of all the other 1126 patients with MMR-P tumors who were tested and had RS results, 3 were tested twice, and for each of these patients, the higher RS result was included in the analysis. Overall, 151 patients were excluded from the analysis due to missing data on adjuvant treatments received (n = 25), clinical outcomes (n = 27), or both (n = 99), and 37 patients were excluded for other reasons (20 had follow-up of <3 years, 15 had metastatic disease at or within 3 months of diagnosis, and 2 had rectal cancer) (Supplementary Figure S1, available at https://doi.org/10.1016/j.esmoop.2024.103648). The final analysis included 938 patients who met all eligibility criteria and for whom treatment and clinical outcome data were available with a minimum follow-up of 3 years.

Of the study patients, 228 (24.3%) received adjuvant CT, and 710 (75.7%) were only observed. Patient and tumor baseline characteristics overall and by treatment (CT versus observation only) are summarized in Table 1. Overall, nearly half (44.4%) of the patients were 70 years or older when tested, with an almost equal gender distribution. Most patients had T3 disease (96.4%), and the most common histology was adenocarcinoma (89.3%). Examination of at least 12 nodes, as recommended in current guidelines,11 was conducted in the vast majority of patients (88.9%). The CT-treated and observation-only groups were imbalanced in several characteristics; the CT-treated patients were younger, had a higher rate of T4 tumors, and had tumors presenting with obstruction/perforation or lymphovascular/venous invasion (LVI/VVI) (Table 1).Table 1 Baseline patient and tumor characteristics

	CT treated n = 228	Observation n = 710	All
N = 938	P valuea	
Female, n (%)	115 (50.4)	347 (48.9)	462 (49.3)	0.68	
Age at testing					
 Median (IQR) age, years	63 (56-69)	70 (63-77)	68 (60-76)	<0.001	
Age category, n (%)					
 <50 years	32 (14.0)	36 (5.1)	68 (7.3)	<0.001	
 50-69 years	140 (61.4)	314 (44.2)	454 (48.4)	
 ≥70 years	56 (24.6)	360 (50.7)	416 (44.4)	
T category, n (%)				0.030	
 1	0 (0)	1 (0.1)	1 (0.1)	
 2	0 (0)	7 (1.0)	7 (0.8)	
 3	216 (94.7)	688 (96.9)	904 (96.4)	
 4	11 (4.8)	13 (1.8)	24 (2.6)	
 Not available	1 (0.4)	1 (0.1)	2 (0.2)	
Tumor grade category, n (%)				0.14	
 Grade 1	59 (25.9)	146 (20.6)	205 (21.9)	
 Grade 2	144 (63.2)	500 (70.4)	644 (68.7)	
 Grade 3	20 (8.8)	53 (7.5)	73 (7.8)	
 Not available	5 (2.2)	11 (1.6)	16 (1.7)	
Histology, n (%)				0.13	
 Adenocarcinoma	197 (86.4)	641 (90.3)	838 (89.3)	
 Mucinous adenocarcinoma	27 (11.8)	56 (7.9)	83 (8.9)	
 Signet cell carcinoma	1 (0.4)	3 (0.4)	4 (0.4)	
 Other/not available	3 (1.3)	10 (1.4)	13 (1.4)	
Tumor location,bn (%)					
 Left	125 (54.8)	355 (50.0)	480 (51.2)	0.082	
 Right	83 (36.4)	322 (45.4)	405 (43.2)	
 Left plus right	0 (0)	4 (0.6)	4 (0.4)	
 Not available	20 (8.8)	29 (4.1)	49 (5.2)	
Number of nodes examined					
 Median (IQR)	17 (13-24)	17 (13-23)	17 (13-23)	0.75	
 ≥12, n (%)	197 (86.4)	637 (89.7)	834 (88.9)	0.17	
Obstruction or perforation, n (%)					
 Yes	53 (23.3)	74 (10.4)	127 (13.5)	<0.001	
 No	164 (71.9)	602 (84.8)	766 (81.7)	
 Not available	11 (4.8)	34 (4.8)	45 (4.8)	
Lymphovascular invasion, n (%)					
 Yes	19 (8.3)	30 (4.2)	49 (5.2)	0.018	
 No	206 (90.4)	661 (93.1)	867 (92.4)	
 Not available	3 (1.2)	19 (2.7)	22 (2.4)	
Venous invasion, n (%)					
 Yes	23 (10.1)	26 (3.7)	49 (5.2)	<0.001	
 No	189 (82.9)	655 (92.3)	844 (90.0)	
 Not available	16 (7.0)	29 (4.1)	45 (4.8)	
Perineural invasion, n (%)					
 Yes	10 (4.4)	18 (2.5)	28 (3.0)	0.13	
 No	202 (88.6)	655 (92.3)	857 (91.4)	
 Not available	16 (7.0)	37 (5.2)	53 (5.7)	
Median (range) CEA,c ng/ml					
 Pre-surgery	3.1 (0.5-124)	2.6 (0.2-131)	2.8 (0.2-131)	0.18	
 Post-surgery	1.8 (0.2-16.3)	1.8 (0.2-98)	1.8 (0.2-98)	0.54	
CEA pre-surgery >5 ng/ml, n (%)					
 Yes	41 (18.0)	107 (15.1)	148 (15.8)	0.64	
 No	97 (42.5)	280 (39.4)	377 (40.2)	
 Not available	90 (39.5)	323 (45.5)	413 (44.0)	
CEA post-surgery >5 ng/ml, n (%)					
 Yes	11 (4.8)	30 (4.2)	41 (4.4)	0.73	
 No	135 (59.2)	417 (58.7)	552 (58.9)	
 Not available	82 (36.0)	263 (37.0)	345 (36.8)	
Recurrence Score®					
 Median (range)	34 (6-70)	24 (0-71)	26 (0-71)	<0.001	
Recurrence Score® distribution, n (%)					
 RS 0-15 (very low)	15 (6.6)	121 (17.0)	136 (14.5)		
 RS 0-29 (low)	82 (36.0)	524 (73.8)	606 (64.6)	<0.001d	
 RS 30-40 (intermediate)	82 (36.0)	143 (20.1)	225 (24.0)		
 RS 41-100 (high)	64 (28.1)	43 (6.1)	107 (11.4)		
CT, chemotherapy; IQR, interquartile range; RS, Recurrence Score®.

a For all categorical variables, distribution in CT-treated patients was compared to that in the observation-only patients without the missing values using chi-square test, except for T where Fisher’s exact test was used. For continuous parameters, the Mann–Whitney test was used.

b Right-sided tumor colon was defined as the cecum, ascending colon, hepatic flexure, and transverse colon (transverse colon was defined as the hepatic flexure, splenic colon, and the splenic flexure).

c Pre-surgery data were available for 138 CT-treated and 387 untreated patients; post-surgery, data were available for 146 CT-treated and 447 untreated patients.

d Chi-square test for comparing the three main RS categories (low, intermediate, high).

For the entire study cohort, the median RS result was 26 [range 0-71, interquartile range (IQR) 19-33], with 64.6%, 24.0%, and 11.4% having RS 0-29 (low), RS 30-40 (intermediate), and RS 41-100 (high) results, respectively. RS 0-15 (very low) results were reported for 14.5% of patients (Table 1). Distribution of the RS results by 5-unit intervals was approximately bell-shaped. The vast majority of patients (87.5%) had RS results ≤39 (Supplementary Figure S2, available at https://doi.org/10.1016/j.esmoop.2024.103648). The CT-treated and observation-only groups were also imbalanced with respect to the RS results and distribution, with CT-treated patients characterized by higher RS results compared to observation-only patients [median (range) RS result of 34 (6-70) versus 24 (0-71), P < 0.001] and a larger proportion of patients with RS 41-100 (28.1% versus 6.1%, P < 0.001) (Table 1).

Patient and tumor characteristics within each RS category were also analyzed and are summarized in Supplementary Table S1, available at https://doi.org/10.1016/j.esmoop.2024.103648. Within each of the RS categories, the age between CT-treated and observation-only patients differed significantly (CT-treated patients were younger), whereas the imbalance in T distribution that was observed in the cohort as a whole was no longer noted in any of the RS categories. Within the RS 0-29 category, CT-treated and observation-only patients were also imbalanced in the rate of obstruction/perforation and VVI (higher among CT-treated patients), as well as in the distribution of tumor location (more left-sided tumors among CT-treated patients). Within the RS 30-40 category, CT-treated and observation-only patients were balanced in the obstruction/perforation rates but imbalanced in both LVI/VVI rates (higher among CT-treated patients). Notably, in the RS 41-100 category, the CT-treated and observation-only patients were balanced in all evaluated disease characteristics (Supplementary Table S1, available at https://doi.org/10.1016/j.esmoop.2024.103648).

Treatments received by RS category

Overall, 228 patients (24.3%) were treated with adjuvant CT. The proportions of patients receiving adjuvant CT varied significantly between the RS categories and were aligned with the RS results (Figure 1). Adjuvant treatment with 5-fluorouracil (5-FU) or capecitabine alone, or in combination with oxaliplatin, was received by 13.5% of the RS 0-29 patients, 36.4% of the RS 30-40 patients, and 59.8% of the RS 41-100 patients (P <0.001). Of the patients with RS 0-15 (very low category), only 11.0% received adjuvant treatment. The majority of CT-treated patients received 5-FU/capecitabine alone. Only 1.5%, 1.8%, and 3.7% of RS 0-29, RS 30-40, and RS 41-100 patients, respectively, received 5-FU/capecitabine plus oxaliplatin.Figure 1 Adjuvant systemic treatment by RS category. 5-FU, 5-fluoraouracil; FOLFOX, leucovorin calcium (folinic acid), 5-FU, and oxaliplatin; RS, Recurrence Score®; XELOX, capecitabine and oxaliplatin. aDue to rounding, the sum of percentages does not equal the total.

Four multivariate logistic regression analyses were carried out modeling the probability of receiving CT for the entire cohort, as well as in each of the three main RS categories separately, as a function of age (as a continuous variable), gender, grade, number of nodes examined (<12 versus ≥12), having invasion (LVI/VVI/perineural), perforation, or obstruction present, and the RS result (as a continuous variable for the entire cohort and within each category). The analysis of the entire cohort demonstrated that higher RS results, younger age at testing, <12 nodes examined, and tumors with invasion/perforation/obstruction were all significantly associated with increased odds of receiving CT (Supplementary Table S2, available at https://doi.org/10.1016/j.esmoop.2024.103648). Similarly, in all three RS categories, younger age and having tumors with invasion/perforation/obstruction were significantly associated with increased odds of receiving CT. Notably, only in the RS 0-29 category, the number of nodes examined was also a statistically significant variable (having <12 nodes examined increased the odds of receiving CT), and only in the RS 30-40 category, the RS result (within this RS range) was a statistically significant variable, with higher RS results associated with increased odds of receiving CT. None of the other variables were found to be statistically significant, including gender, tumor grade, and histology (Supplementary Table S2, available at https://doi.org/10.1016/j.esmoop.2024.103648).

Clinical outcomes overall and by RS category for observation-only patients

In a KM analysis for the entire cohort (CT-treated and observation-only patients), the 5-year RFI was 84.3% (95% CI 81.8% to 86.5%) and the 5-year CCSS was 93.9% (95% CI 92.1% to 95.3%).

With a median follow-up of 6.7 years (IQR 5.4-8.5 years), KM estimates for RFI and CCSS in patients who did not receive active treatment differed significantly between the RS 0-29, 30-40, and 41-100 categories (P < 0.001, both outcomes for comparing these three categories) (Figure 2). The exploratory analysis in RS 0-15 patients (very low risk) demonstrated that these patients had excellent clinical outcomes with observation only [5-year RFI: 93.8% (95% CI 87.6% to 97.0%); 5-year CCSS: 96.6% (95% CI 91.2% to 98.7%)] (Figure 2). Patients with low and intermediate risk (RS 0-29 and 30-40, respectively) also had very good outcomes (overlapping CIs with the RS 0-15 category). In contrast, patients with high risk according to the RS (RS 41-100) had worse clinical outcomes [5-year RFI: 69.4% (95% CI 53.4% to 81.8%); 5-year CCSS: 81.0% (95% CI 65.1% to 90.7%)] (Figure 2).Figure 2 KM analyses for observation-only patients. KM curves for RFI (A) and CCSS (B) by RS risk categories in observation-only patients. The box under each graph presents the number of patients at risk at each time point. Two-degree of freedom log-rank P values were calculated from all the data for the three-group comparison (RS 0-29, 30-40, 41-100). CCSS, colon cancer-specific survival; KM, Kaplan–Meier; RFI, recurrence-free interval; RS, Recurrence Score®.

Clinical outcomes in CT-treated versus observation-only patients

With a median follow-up of 6.9 years (IQR 5.5-8.6 years), clinical outcomes by treatment (CT versus observation) within each RS category revealed no statistically significant differences in the estimated RFI and CCSS in the RS 0-15, 0-29, and 30-40 categories, trending even for a more favorable outcome in the observation group in the low-risk (RS 0-15 and even RS 0-29) patients (Figure 3A-C).Figure 3 KM analyses by RS category and treatment. KM curves for RFI and CCSS by treatment (CT versus observation) in the RS 0-15 (A), RS 0-29 (B), RS 30-40 (C), and RS 41-100 (D) categories. The box under each graph presents the number of patients at risk at each time point. One-degree of freedom log-rank P values were calculated from all the data. CCSS, colon cancer-specific survival; CT, chemotherapy; KM, Kaplan–Meier; RFI, recurrence-free interval; RS, Recurrence Score®.

In contrast, in the RS 41-100 category, the difference in KM estimates for RFI trended toward statistical significance (P = 0.066) and for CCSS, a statistically significant difference was observed between CT-treated and observation-only patients, with better outcomes among CT-treated patients (P = 0.035) (Figure 3D). In a multivariate model where the RS category, treatment, and an interaction were fitted in the model, the P value for the interaction was nonsignificant (Pinteraction = 0.12).

Clinical outcomes: univariate and multivariate analyses

The univariate analysis for risk of recurrence demonstrated that higher RS results, having invasion/obstruction/perforation present, and male gender were all significantly associated with increased risk, whereas age at testing, T category (pT4 versus pT1-3), histology (adenocarcinoma versus others), grade (3 versus 1 + 2), tumor location, number of lymph nodes examined (≥12 versus <12), preoperation carcinoembryonic antigen (>5 ng/ml versus ≤5 ng/ml), and CT treatment were nonsignificant variables. A similar analysis conducted for the risk of CC death revealed similar results, except that male gender was no longer a prognostic factor, whereas age at diagnosis was significant (Table 2). The identified significant covariates remained significant prognosticators in a multivariate model (Table 2).Table 2 Univariate and multivariate analysis: association of clinicopathologic characteristics, RS results, and treatments received with risk of recurrence/colon cancer death

	Recurrence risk	Colon cancer death risk	
Variable	HR (95% CI)	P value	HR (95% CI)	P value	
Univariate analysis					
 Gender (male versus female)	1.63 (1.20-2.23)	0.0021	1.02 (0.67-1.56)	0.91	
 Age (per year)	1.01 (0.99-1.02)	0.43	1.02 (1.004-1.045)	0.022	
 pT4 versus pT1-3	1.77 (0.80-3.37)	0.12	1.97 (0.69-4.38)	0.14	
 Histology (adenocarcinoma versus other)	1.00 (0.62-1.71)	0.998	1.15 (0.59-2.58)	0.71	
 Grade (3 versus 1 + 2)	1.14 (0.63-1.90)	0.65	1.40 (0.65-2.64)	0.34	
 Invasion/perforation/obstruction (present versus not)a	1.64 (1.15-2.28)	0.0047	2.51 (1.60-3.92)	<0.001	
 Tumor location (left versus right)	1.15 (0.83-1.60)	0.40	0.98 (0.63-1.54)	0.93	
 Number of lymph nodes examined (≥12 versus <12)	1.15 (0.70-1.90)	0.59	1.27 (0.62-2.63)	0.52	
 Preoperation CEA (>5 ng/ml versus ≤5 ng/ml)	1.31 (0.84-2.0)	0.21	1.28 (0.70-2.25)	0.41	
 RS result (per unit)	1.03 (1.02-1.04)	<0.001	1.03 (1.02-1.05)	<0.001	
 Adjuvant CT (yes versus no)	1.24 (0.88-1.72)	0.20	0.95 (0.57-1.51)	0.83	
Multivariate analysis			
	C-index (95% CI), 0.593 (0.591-0.595)	C-index (95% CI), 0.600 (0.597-0.602)	
 Gender (male versus female)	1.53 (1.10-2.14)	0.012	0.85 (0.54-1.33)	0.47	
 Age (per year)	1.00 (0.99-1.02)	0.63	1.02 (1.003-1.05)	0.029	
 Invasion/perforation/obstruction (present versus not)a	1.49 (1.05-2.10)	0.022	2.35 (1.49-3.68)	<0.001	
 RS (per unit)	1.03 (1.02-1.04)	<0.001	1.03 (1.01-1.05)	0.0035	
CEA, carcinoembryonic antigen; CI, confidence interval; CT, chemotherapy; HR, hazard ratio; RS, Recurrence Score®.

a Lymphovascular, venous, or perineural invasion, perforation, or obstruction present.

Discussion

This is the first study investigating the impact of using the 12-gene assay on adjuvant treatment decisions and clinical outcomes in MMR-P stage II CC patients in real-world contemporary clinical practice. The various clinical characteristics of the patient population were overall aligned with those of stage II CC patients in a real-life clinical setting, suggesting that the study population does reflect a typical contemporary stage II CC patient population.12, 13, 14, 15 The study showed that clinicians treated patients according to the RS result while considering other clinicopathologic factors, that the RS was highly prognostic, and that CT seemed to have provided clinical benefit in patients with RS 41-100.

The findings that treatment decisions were aligned with the RS results are consistent with the first analysis of this CHS database, which included a subset of the current cohort, focused solely on the decision impact of the 12-gene assay, and showed treatment decision changes in 38% of patients.10 Notably, decision impact studies in other countries, including Japan and the United States, also demonstrated that knowing the RS result changed treatment decisions in 29%-45% of patients in a direction aligned with the RS result.16, 17, 18, 19 Moreover, as shown by our earlier study and others, the RS-based treatment decisions tend to decrease the use of adjuvant CT, from a range of 45%-57% to 28%-51%.10,16,18 In accordance with this, only 24% of the patients in the current study received adjuvant treatment, proving once again the ability of the test to support oncological decisions and reduce medical resources. Of interest, the fact that the extent of adjuvant CT administration in the current study is at the lower range of earlier reports may reflect a growing confidence of clinicians, facing the accumulating data, in the RS results.

Unlike prior studies, which focused on assessing treatment recommendation changes by RS category,10,16, 17, 18, 19 the current study also examined the clinicopathologic factors that impacted treatment decisions within each RS category. The results demonstrated that within each RS category, clinicians considered age and the presence of invasion/perforation/obstruction in their treatment decisions. Only in the RS 0-29 category, patients with an inadequate number of lymph nodes examined (<12) were more likely to receive CT, probably due to a concern over under-staging and the current evidence suggesting poorer outcomes in such patients.20 Notably, only within the RS 30-40 category, clinicians also considered the RS result as a continuous parameter in their decision making.

Our finding that the RS result provided independent prognostic information is consistent with the validation studies6, 7, 8, 9; however, this is the first study to demonstrate it in real-world clinical practice. Similar to the NSABP C-07 and the SUNRISE validation studies, our analysis also demonstrated that the main difference in outcomes was between the patients in the low/intermediate RS categories and those in the high-risk RS category.8,9 Also, we attempted to define a new very-low RS category in order to identify patients with ultralow risk, for whom adjuvant systemic therapy can be safely omitted. Our results do suggest that RS 0-15 patients have excellent clinical outcomes and further study is required to potentially establish an ‘ultralow’ risk RS category.

Interestingly, the recurrence rates observed in the current study in the three RS categories are somewhat higher compared to the corresponding recurrence rates for stage II patients in the validation studies (for observation-only and CT-treated patients), although the CIs do overlap.6, 7, 8, 9 The seemingly higher recurrence rates in the current study likely reflect differences in the study design in this observational study versus randomized clinical trial, and in the patient population (this is the first 12-gene clinical outcome study in an Israeli population). Nevertheless, the clinical outcomes in the current study are slightly better than those reported for stage II CC patients in other real-life studies.12, 13, 14, 15 This finding may suggest that the higher recurrence rates in our study compared with the validation studies mainly result from the different populations in daily practice studies versus prospective clinical trials. These differences further emphasize the need for real-world evidence when applying novel technologies evaluated in prospective studies into routine daily practice.

The role and the actual impact of adjuvant CT in stage II CC is a subject of much debate, as for the majority of patients, the disease is curable with surgery alone.3 Thus, the main challenge is identifying the patients with high risk of recurrence, and ideally, those who are likely to respond to adjuvant CT. Traditional clinicopathologic characteristics are imperfect prognosticators, as many high-risk patients according to these characteristics do not experience recurrence. Additional prognostic tools have been developed in the last 15 years including several multigene assays (the 12-gene assay, ColDX, and the ColoPrint assays), the Immunoscore, and post-surgical circulating tumor DNA-based assays. The National Comprehensive Cancer Network® (NCCN) guidelines do not recommend using these assays to estimate recurrence risk or to guide adjuvant treatment, due to insufficient data and lack of evidence for their role as predictors of CT benefit.11 The European Society for Medical Oncology (ESMO) guidelines do not recommend the routine use of multigene assays, due to the lack of predictive value and the small prognostic differentiation margins between the risk categories, but they do suggest that they may be used to complement clinicopathologic information in intermediate-risk stage II CC.21

The results of the current study provide preliminary evidence in support of the potential role of the RS result as a predictor of CT benefit, although the Pinteraction was nonsignificant. In patients with RS results of 41-100, CT-treated patients had better clinical outcomes than observation-only patients, despite having worse clinicopathologic characteristics, suggesting that CT conferred a clinical benefit in these high-risk patients. In contrast, in patients with RS 30-40, CT-treated and observation-only patients had similar clinical outcomes. Moreover, in low-risk patients (RS 0-29), although not statistically significant, the results suggest that CT may have been associated with even slightly worse outcomes than observation alone. Importantly, the potential benefit of CT in these RS categories cannot be ruled out, as the CT-treated patients had worse clinicopathologic characteristics that could have balanced out and masked the CT benefit. Our study is the first to support a potential predictive role for the RS result, as such a role was not identified in the only validation study that could have been used to investigate this question (the QUASAR validation study).6 The discrepancy with the QUASAR validation study could stem from differences in the patient populations (higher-risk patients in QUASAR), and/or because QUASAR finalized enrollment over 20 years ago, and does not represent contemporary clinical practice. Regardless, given the lack of additional available cohorts of relevant patients randomly assigned to adjuvant CT versus no CT, our findings provide an important insight into the potential predictive role of the RS result. Nonetheless, additional study on this matter is clearly warranted.

The main strengths of the study include having a large cohort of stage II patients with a long follow-up, and its representation of real-world clinical practice on a national level, since no exclusion criteria were applied with respect to gender, age, location, socioeconomic status, comorbidities, prior malignancies, family history, etc. The study is limited by its nonrandomized design, which led to imbalanced treatment groups, whereby patients with clinicopathologic characteristics associated with worse clinical outcomes (e.g. younger age at testing, having invasion/obstruction/perforation) were more likely to receive CT.

Conclusions

Our findings support the use of the 12-gene assay as a tool to guide treatment decisions in stage II CC. Further studies are needed to validate the suggested role of the RS result as a predictor of CT benefit.

Supplementary data

Supplementary data

Funding

The study received financial support from Genomic Health, a wholly owned subsidiary of 10.13039/100030841 Exact Sciences , Ltd (GHI grant for IIS protocol 05-062). FLB and CR who contributed to data interpretation and writing/editing, are employed by 10.13039/100030841 Exact Sciences Ltd.

Disclosure

BB: consultant for Astellas Pharma and MSD; speaker’s bureau for 10.13039/100002491 Bristol-Myers Squibb and MSD; research funds from Bristol-Myers Squibb and Merck Serono; and travel expenses from Bristol-Myers Squibb, MSD, and Rhenium/Oncotest. RG: leadership role at Pyxis; stock/other ownership interests in BOL Pharma and Pyxis; honoraria from BMS, Janssen, Medison, Merck, MSD, Pfizer, and Roche; a consultant for AstraZeneca, Bayer, BOL Pharma, JNJ, MSD, Rhenium Medical, and Roche; and travel expenses from Medison and Takeda. NY: consultant for MSD Oncology. GBS: honoraria from Abbott, Bristol-Myers Squibb, and MSD; and travel expenses from MSD. RB: consultant for Orgenesis. AS: honoraria from AstraZeneca, Novartis, and Pfizer; a consultant for Novartis; and travel expenses from Gilead Sciences. FLB: employment by, a leadership role at, and owning stock of Exact Sciences. CR: employment by and owning stock of 10.13039/100030841 Exact Sciences . LSG: employment by Oncotest Rhenium. HV: consultant for BioInsight Ltd. ABS: consultant for Oncotest, Exact Sciences, Can-Fite, and Pfizer. All other authors have declared no conflicts of interests.

Data sharing

Data are available from the corresponding author upon reasonable request.
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