Tumor-associated Macrophages as Prognostic and Predictive … · adjuvant chemotherapy for patients in traditional high-risk group of recurrence (signiﬁcant interaction for DFS)

Precision Medicine and Imaging

Tumor-associated Macrophages as Prognosticand Predictive Biomarkers for PostoperativeAdjuvant Chemotherapy in Patients with Stage IIColon CancerQingyang Feng1,2,Wenju Chang1,2, Yihao Mao1,2, Guodong He1,2, Peng Zheng1,2,Wentao Tang1,2, Ye Wei1,2, Li Ren1,2, Dexiang Zhu1,2, Meiling Ji1,2, Yongjiu Tu3, Xinyu Qin1,2,and Jianmin Xu1,2

Abstract

Purpose: For stage II colon cancer, the efficacy of postop-erative adjuvant chemotherapy remains controversial. It iswellknown that tumor-associatedmacrophages (TAMs) are impor-tant in tumor progression. In this study, TAMs were investi-gated as prognostic and predictive biomarkers for the efficacyof adjuvant chemotherapy for stage II colon cancer after radicalresection.

ExperimentalDesign: This study enrolled two independentcohorts of consecutive patients from one medical center withpathologic stage II colon cancer after radical resections.Macro-phages were detected using IHC staining of CD68 and CD206.Infiltration densities of CD68þ TAMs, CD206þ TAMs, andratio of CD206þ TAMs/CD68þ TAMs (CD206/CD68 ratio)were calculated as prognostic and predictive biomarkers.

Results: The primary and validation cohorts consisted of521 and 314 patients, respectively. In both cohorts, high

CD206/CD68 ratio was significantly associated with poordisease-free survival (DFS) and overall survival (OS). As anindependent risk factor, CD206/CD68 ratio also had signif-icantly better prognostic efficacy than CD68þ TAM density,CD206þ TAM density, and traditional clinicopathologic high-risk factors. Moreover, adjuvant chemotherapy significantlyimproved DFS and OS for patients with high CD206/CD68ratio but not for those with low CD206/CD68 ratio. Theinteraction analyses were also significant for DFS. In subgroupanalysis, CD206/CD68 ratiowas still a significant predictor foradjuvant chemotherapy for patients in traditional high-riskgroup of recurrence (significant interaction for DFS).

Conclusions: For stage II colon cancer, CD206/CD68 ratiois a better prognostic and predictive biomarker for postoper-ative adjuvant chemotherapy. Together with clinicopathologichigh-risk factors, it will aid in precision treatment.

IntroductionColon cancer is common around the world. Approximately

one-quarter of patients with colon cancer present with stage IIdisease (1). Approximately 15%–25% of these patients sufferfrom systemic recurrence (including local recurrence and distantmetastasis) and die even after surgery with curative intent (2, 3).Fluorouracil (FU)-based adjuvant chemotherapy after surgery has

been widely used to reduce colon cancer recurrence and improvesurvival (4, 5). However, its efficacy for stage II disease remainscontroversial (5–7), with an improved survival rate of no morethan 5% on average (8–10). Considering the adverse events, cost,and inconvenience, it is important to identify patients with a highrisk of recurrence who can benefit from adjuvant chemotherapy.Thus, accurate prognostic factors, such as traditional clinicopath-ologic high-risk factors (T4 stage, poorly differentiated histology,lymphatic/vascular invasion, perineural invasion, bowel obstruc-tion, localized perforation, positive margins, and <12 lymphnodes examined) andmicrosatellite instability (MSI) status, werealways expected to be predictive for the efficacy of adjuvantchemotherapy. According to these widely recognized prognosticfactors, patients with stage II colon cancer after surgery are dividedinto three groups according to recurrence risk by the NationalComprehensiveCancerNetwork (NCCN) guidelines (8): low-riskgroup as T3 (MSI-H) with no high-risk factors; mid-risk group asT3 (MSS/MSI-L) with no high-risk factors; and high-risk group asT3 with high-risk factors or T4. It has been shown that patients inthe low-risk group have a good prognosis and do not benefit fromadjuvant chemotherapy (11, 12). However, for the remaining85%–90% of patients in the mid-risk and high-risk groups,clinicopathologic high-risk factors combined with MSI statuscannot identify patients who benefit from adjuvant chemother-apy (5–7). There is still a need for better prognostic and predictivebiomarkers.

1General Surgery Department, Zhongshan Hospital, Fudan University, Shanghai,China. 2Shanghai Engineering Research Center of Colorectal Cancer MinimallyInvasive, Shanghai, China. 3The 174th Hospital of PLA, Xiamen, Fujian Province,China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Q. Feng, W. Chang, Y. Mao, and G. He contributed equally to this article.

CorrespondingAuthors: Jianmin Xu, ZhongshanHospital, FudanUniversity, 180Feng-Lin Road, Shanghai 200032, P.R. China. Phone: 8621-6404-1990; Fax:8621-6403-8038; E-mail: [email protected], [email protected] Qin, Zhongshan Hospital, Fudan University, 180 Feng-Lin Road, Shanghai200032, P.R. China. Phone: 8621-6404-1990; Fax: 8621-6403-8038; E-mail:[email protected].

Clin Cancer Res 2019;25:3896–907

doi: 10.1158/1078-0432.CCR-18-2076

�2019 American Association for Cancer Research.

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Clin Cancer Res; 25(13) July 1, 20193896

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It is well known that the immune microenvironment signifi-cantly affects tumor development. Among the various immunecells recruited to the tumor site, tumor-associated macrophages(TAM) are particularly abundant at all stages of tumor progressionand play an important role. TAMs are macrophages that infiltratein tumor tissues. Generally, macrophages are divided into twophenotypes (13–15): the classically activated (M1) type, withiNOS, CD86, CD169, etc. as markers, and the alternatively acti-vated (M2) type,withCD163,CD206,CD204, etc. asmarkers.M1TAMs in tumor tissues stimulate tumor immunity and suppresstumor progression. In contrast, M2 TAMs enhance tumor cellinvasion, motility, and intravasation, stimulate angiogenesis,suppress the immune response, and prevent tumor cell attack bynatural killer andT cells (14–16). There are also studies of TAMs asprognostic factors for colorectal cancer (17–19). However, noclinical evidence for TAMs predicting the efficacy of postoperativeadjuvant chemotherapy has been reported.

In this study, we detected TAMs andM2 subtype that infiltratedin the tumor tissues of patients with stage II colon cancer. Weassessed the prognostic and predictive accuracy of TAMs as bio-markers for postoperative adjuvant chemotherapy. We also com-pared their prognostic and predictive efficacy with traditionalclinicopathologic high-risk factors.

Materials and MethodsPatient eligibility

This study retrospectively enrolled two independent cohortsof consecutive patients at different time periods from theGeneral Surgery Department, Zhongshan Hospital, FudanUniversity (Shanghai, China). The primary cohort was com-prised of patients admitted from July 2009 to June 2012 todefine the cut-off value of TAMs and to determine theirprognostic and predictive efficacy. The validation cohort wascomprised of patients admitted from July 2012 to December2013 to verify the cut-off value and to confirm the prognosticand predictive efficacy of TAMs. All hypotheses were developedon the primary cohort and then tested on the validationcohort.

In both cohorts, the inclusion criteria were the same, as follows:ages between 18 and 80 years; pathologically confirmed colonadenocarcinoma, mucinous adenocarcinoma, or signet-ring cellcarcinoma with stage II disease (T3-4, N0, M0) according to theAJCC/UICC TNM staging system 8th edition; and radical (R0)resectionof theprimary tumor. The exclusion criteriawere also thesame in both cohorts, as follows: emergency surgery because of anacute intestinal obstruction, bleeding or perforation; evidenceof distant metastases; preoperative cancer therapy; hereditarycolorectal cancer, such as familial adenomatous polyposis,Lynch syndrome, and MYH-associated polyposis; multiple pri-mary tumors; a history of other malignancies (except foradequately treated basocellular carcinoma of the skin or in situcarcinoma of the cervix uteri); tissue specimen unavailable; andfollow-up data unavailable. Colon cancer was defined as atumor localized �15 cm away from the anal verge. The appli-cation of postoperative adjuvant chemotherapy was performedaccording to high-risk factors for systemic recurrence (8), phys-ical status, treatment tolerance, and patient preference. Notargeted agents were used in postoperative adjuvant chemo-therapy. All clinicopathologic and follow-up data wereobtained from a prospective database. This study was approvedby the institutional review board of Zhongshan Hospital,Fudan University (Shanghai, China), and was carried out inaccordance with the Declaration of Helsinki. All patients pro-vided written and oral informed consent.

Follow-up principles and sample size estimationFollow-up principles were based on the Chinese guidelines for

colorectal cancer. Patients were asked to come to the hospital fordocumented visits and the following examinations at the outpa-tient department: medical history; physical examination; serumcarcino-embryonic antigen (CEA) level; abdominal ultrasoundevery 3 months for 2 years, then every 6 months for 5 years, thenevery year after 5 years; chest/abdominal/pelvic CT scan every 6months for 2 years, then every year after 2 years; and colonoscopyat 6 months after primary tumor resection, then every year for 5years, then every 2 years after 5 years. For patients who did notreport on time, phone calls and letters were used.

In this study, a retrospective chart review was performed.Recurrence included both local recurrence and distantmetastases.Once recurrence was suspected, detailed examinations, includingcontrast CT/MRI, PET-CT, colonoscopy, and biopsy (if necessary),were conducted for confirmation. The primary endpoint, disease-free survival (DFS), was defined as the time between surgery andthe first recurrence, a newoccurrence of colorectal cancer, or deathfrom any cause. The overall survival (OS) was defined as the timebetween surgery and death. The estimated sample size for theprimary cohort was 495 patients. Details are shown in Supple-mentary Information S1. According to the actual sample size andstudy results, post hoc power analysis was also conducted asdescribed in Supplementary Information S2. The actual powerwas in line with expectations.

IHCIn the primary cohort, a tissue microarray (TMA) of tumor and

normal mucosa tissue was constructed using formalin-fixed par-affin-embedded (FFPE) surgical specimens as described previous-ly (20, 21). For each patient, two cores of tumor tissues were takenfrom two paraffin blocks of different areas of one tumor. In thevalidation cohort, a TMA of only tumor tissue was constructed in

Translational Relevance

For stage II colon cancer, postoperative adjuvant chemo-therapy generally has no significant effect, with an improvedsurvival rate of less than5%at 5 years after surgery. There is stillno effective biomarker for prediction. According to this article,for patients with stage II colon cancer after radical resection,the CD206/CD68 ratio of tumor-associated macrophages(TAMs) can identify those with a poor prognosis and a highrisk of recurrence who can benefit from adjuvant chemother-apy. For the other patients identified as having a good prog-nosis and a low risk of recurrence, adjuvant chemotherapy hasno benefit and will not be used. Therefore, patients will betreated with adjuvant chemotherapy more accurately, thusimproving efficacy, reducing adverse events, and saving finan-cial costs. Moreover, this test of TAMs is cheap, not timeconsuming, and easily conducted in the laboratories of mosthospitals usingmethods involving IHC staining and commonantibodies.

TAMs Predict Adjuvant Chemotherapy for Stage II Colon Cancer

www.aacrjournals.org Clin Cancer Res; 25(13) July 1, 2019 3897




the same method as the primary cohort for each patient. Nonormal mucosa tissue was used in the validation cohort. Forfurther clinical application, in the validation cohort, two normalFFPE sections of tumor tissue were taken from the two paraffinblocks used for tumor cores. Details are shown in SupplementaryInformation S3.

IHC was subsequently conducted on the TMA for both theprimary and validation cohorts and on normal FFPE sections forthe validation cohort. Primary antibodies against human CD68(Clone KP1, 1:200; Abcam) and CD206 (Clone 5C11, 1:200;Abcam) were used to detect macrophages and M2 subtypes,respectively. The IHC protocols were performed as describedpreviously (20, 21). Details are shown in Supplementary Infor-mation S3.

In most solid tumors as well as in colorectal cancer, tumor areais composed of tumor nest and stroma (22, 23). TAMs mainlyinfiltrate in the tumor stroma (24). In this study, only TAMs thatinfiltrated in the tumor stroma were counted and correctedaccording to the stroma area. TAMs that infiltrated in the tumornest were not analyzed because of very low density. Details areshown in Supplementary Information S3.

The infiltration density of macrophages per field was evaluatedby two independent pathologists who were blinded to thepatients' clinical data using Image-Pro Plus 6.0 (Media Cybernet-ics Inc.) for assistance. For each tissue core or normal section, threerandomized fields of positive-stained cells were counted under ahigh-power field (HPF) of 200�. The density ofmacrophages wascalculated as the mean number of all fields from cores or normalsections.Details are shown inSupplementary Information S3. Thestability and repeatability of the detectionmethod for TAMs wereevaluated, and showed good performance (details are shown inSupplementary Information S4). The infiltration densities ofCD68þ TAMs, CD206þ TAMs, and the ratio of CD206þ TAMs/CD68þ TAMs (CD206/CD68 ratio) were calculated as prognosticand predictive biomarkers.

A previous study (25) demonstrated high concordancebetween the IHC-based mismatch repair (MMR) test and thePCR-based MSI test. In this study, MSI status was based on theIHC testing of MMR with four markers (25, 26): MLH1 (CloneG168-15, 1:50; BD Pharmingen), MSH2 (Clone FE11, 1:200;Invitrogen),MSH6 (Clone44, 1:100; BDPharmingen), andPMS2(CloneA16-4, 1:100; BDPharmingen). Patientswith tumor tissuethat exhibited positive staining for all these markers were con-sidered MSS or MSI-L. Patients with negative staining for at leastone marker were considered MSI-H.

Statistical analysisPatient baseline characteristics and disease factors were sum-

marized using descriptive statistics. Categorical variables werecompared using the two-sided Pearson x2 test or Fisher exact testas appropriate. Continuous variables were compared using a t test(normal distribution) or the Wilcoxon rank test (abnormal dis-tribution) as appropriate. The correlation analysis was conductedusing Spearman rank correlation test. Summary statistics on time-to-event variables, such as DFS andOS, were calculated accordingto the Kaplan–Meier method and compared by the log-rank test.Cox regression was used for univariate and multivariate analyseswithHRs and 95%confidence intervals (CI).Only factors with P <0.1 in the univariate analysis were included in the multivariateanalysis. Interaction analysis was also conducted using Coxregression. To compare the prognostic efficacy of different bio-

markers,Harrell concordance index (C-index, calculated using theHmisc package, Soft R version 2.11.1) was used (27). The higherthe C-index, the more effective the biomarker is. Comparisonsbetween different prognostic factors were performed with thercorrp.cens function in the Hmisc package. To evaluate the sta-bility and repeatability of the detection method for TAMs, inter-rater agreement (kappa) was used for categorical parameters, andintraclass correlation coefficient (ICC) was used for continuousvariables. All P values were two-sided and considered significantwhen <0.05.

The cut-off values of TAMs as prognostic biomarkers weredefined according to the DFS data of the primary cohort andthen applied in the validation cohort. To obtain the best prog-nostic efficacy, X-Tile Software (YaleUniversity, version 3.6.1)wasused as described previously (28).

ResultsPatient characteristics

In this study, the primary cohort consecutively enrolled 521eligible patients, and the validation cohort consecutively enrolled314 eligible patients. The flow diagram of cohort selection isshown in Fig. 1. By June 2016, the median follow-up time in theprimary cohort was 69.0 months (range, 13.0–84.0; interquartilerange, IQR, 54.0–76.0). By June 2017, themedian follow-up timein the validation cohort was 55.0months (range, 15.0–60.0; IQR,50.0–58.0). In the primary cohort, 103 (19.8%) patients sufferedfrom recurrence (local recurrence or distant metastases) and 70(13.4%) patients died, including 6 (1.2%) deaths without evi-dence of recurrence. In the validation cohort, 59 (18.8%) patientssuffered from recurrence and 38 (12.1%) patients died, including2 (0.6%) without recurrence. The basic demographics and clin-icopathologic characteristics of both cohorts are shown in Table 1.No significant difference was observed. In both cohorts, postop-erative adjuvant chemotherapy was not balanced. Significantlymore patients with clinicopathologic high-risk factors receivedadjuvant chemotherapy. Details are shown in SupplementaryTable S1.

IHC findingsPositive IHCstaining ofCD68þ andCD206þmacrophages that

infiltrated inprimary tumor tissues andnormal tissues is shown inSupplementary Fig. S1. In the primary cohort, CD68þ macro-phage density, CD206þ macrophage density, and the ratio ofCD206þ/CD68þmacrophages were significantly higher in tumortissues than in normal tissues. Details are shown in Supplemen-tary Table S2.

Definition of cut-off valuesFor CD68þ TAM density, �118/HPF was defined as high and

<118/HPF was defined as low. For CD206þ TAM density, �74/HPFwas defined as high and <74/HPFwas defined as low. For theCD206/CD68 ratio, �0.77 was defined as high and <0.77 wasdefined as low. Details are shown in Supplementary InformationS5, S6, and S7. The C-index analysis of different cut-off values forTAMs is shown in Supplementary Fig. S2.

According to the cut-off value, 148 (28.4%) patients in theprimary cohort and 75 (23.9%) patients in the validation cohortwere defined as having high CD68þ TAM density; 102 (19.6%)patients in the primary cohort and 73 (23.2%) patients in thevalidation cohort were defined as having high CD206þ TAM

Feng et al.

Clin Cancer Res; 25(13) July 1, 2019 Clinical Cancer Research3898




density; and 86 (16.5%) patients in the primary cohort and 65(20.7%) patients in the validation cohort were defined as having ahigh CD206/CD68 ratio.

Correlations between TAMs and clinicopathologic featuresIn both the primary and validation cohorts, low CD68þ TAM

density was significantly associated with perineural invasion(primary cohort P ¼ 0.040, validation cohort P ¼ 0.042). HighCD206þ TAM density was significantly associated with poordifferentiation (P ¼ 0.035, P ¼ 0.032). A high CD206/CD68ratio was significantly associated with poor differentiation (P ¼0.001, P ¼ 0.003), pathologic T4 stage (P ¼ 0.002, P ¼ 0.004),lymphatic/vascular invasion (P ¼ 0.002, P ¼ 0.003), and peri-neural invasion (P ¼ 0.002, P ¼ 0.003). Significantly morepatients in the high-risk group had a higher CD206/CD68 ratiothan those in the mid-risk and low-risk groups (P ¼ 0.002, P ¼0.007). In addition,more patientswith a highCD206/CD68 ratio

received postoperative adjuvant chemotherapy in the primarycohort (P ¼ 0.014), but not in the validation cohort (P ¼0.451).Details of the correlationanalysis, chemotherapy regimen,andDFS events are shown inSupplementary Tables S3, S4, andS5.

TAMs as prognostic biomarkersIn theprimary cohort, CD68þTAMdensitywas not a significant

prognostic biomarker for DFS (P ¼ 0.135) or OS (P ¼ 0.739).However, patients with high CD206þ TAM density had signifi-cantly worse DFS (P < 0.001) and OS (P < 0.001) than those withlow density. Patients with a high CD206/CD68 ratio also hadsignificantly worseDFS (P < 0.001) andOS (P < 0.001) than thosewith a low CD206/CD68 ratio. Details are shown in Fig. 2. In thevalidation cohort, the results were similar. CD68þ TAM densitywas still not a significant prognostic biomarker for DFS (P ¼0.624) or OS (P ¼ 0.355). Patients with high CD206þ TAMdensity had significantly worse DFS (P ¼ 0.005) and OS (P ¼

Primary cohortGeneral Surgery Department

Zhongshan Hospital, Fudan UniversityFrom 2009-07 to 2012-06

Pathologically confirmed colorectal cancer(adenocarcinoma/mucinous

adenocarcinoma/signet-ring cell carcinoma)

N = 2923

Colon cancer(tumor localized ≥ 15 cm from anal verge)

N = 1686

Pathologic stage II disease(T3-4, N0, M0)

N = 594

• > 80 years old: 33• Emergency surgery: 8• P 9• Hereditary colorectal cancer: 2• M 7• History of other malignancies: 5• Specimen unavailable: 4• Follow-up data unavailable: 11

Finally enrolled in primary cohortN = 521

years old and follow-up data unavailable;

emergency surgery; 1 ent had

primary tumors.

General Surgery DepartmentZhongshan Hospital, Fudan University

From 2012-07 to 2013-12Pathologically confirmed colorectal cancer

(adenocarcinoma/mucinous adenocarcinoma/signet-ring cell carcinoma)

N = 1944

Colon cancer(tumor localized ≥ 15 cm from anal verge)

N = 1197

Pathologic stage II disease(T3-4, N0, M0)

N = 361

s#

• > 80 years old: 23• Emergency surgery: 5• P 6• Hereditary colorectal cancer: 1• M 5• History of other malignancies: 4• Specimen unavailable: 2• Follow-up data unavailable: 5

N = 314

: 2years old and follow-up data unavailable;

emergency surgery; 1 old and had history of other malignancies.

Figure 1.

Flow diagram of cohort selection. The inclusion and exclusion process of the primary cohort and the validation cohort. The pathologic stage was according to theAJCC/UICC TNM staging system 8th edition.






0.009). Patients with a high CD206/CD68 ratio also had signif-icantly worse DFS (P < 0.001) and OS (P < 0.001). Details areshown in Supplementary Fig. S3.

CD206/CD68 ratio as a better prognostic biomarkerThe efficacy of prognostic biomarkers (CD68þ TAM density,

CD206þ TAMdensity, andCD206/CD68 ratio)was compared. Inthe primary cohort, the efficacy of the CD206/CD68 ratio as aprognostic biomarker forDFS (C-index¼ 0.632)was significantlybetter than CD68þ TAMdensity (C-index¼ 0.524; P < 0.001) andCD206þ TAM density (C-index ¼ 0.605; P ¼ 0.001). For OS, theCD206/CD68 ratio (C-index ¼ 0.654) was also significantlybetter than CD68þ TAM density (C-index ¼ 0.512; P < 0.001)and CD206þ TAM density (C-index ¼ 0.633; P ¼ 0.009). Detailsare shown in Supplementary Table S6. In the validation cohort,the CD206/CD68 ratio was also significantly better than CD68þ

TAM density and CD206þ TAM density for both DFS and OS.Details are shown in Supplementary Table S7.

The CD206/CD68 ratio was also compared with clinicopath-ologic high-risk prognostic factors. In the primary cohort, for bothDFS and OS, the efficacy of the CD206/CD68 ratio was signifi-cantly better than primary tumor differentiation combined withMSI status, pathologic T stage, lymph nodes examined, lymphat-ic/vascular invasion, perineural invasion, and MSI status. Detailsare shown in Supplementary Table S6. In the validation cohort,the CD206/CD68 ratio was also significantly better than theseclinicopathologic high-risk factors for both DFS and OS. Detailsare shown in Supplementary Table S7.

CD206/CD68 ratio as an independent prognostic factor forDFSIn both the primary and validation cohorts, univariate analysis

showed that DFS was associated with tumor differentiation com-bined with MSI status (primary cohort HR ¼ 1.744, P ¼ 0.006;validation cohort HR¼ 2.131, P¼ 0.004), pathologic T stage (HR¼ 3.375, P < 0.001; HR ¼ 2.995, P < 0.001), number of lymphnodes examined (HR¼ 1.903,P¼ 0.011;HR¼ 1.841,P¼ 0.091),

lymphatic/vascular invasion (HR¼ 2.658, P < 0.001;HR¼ 2.465,P ¼ 0.002), perineural invasion (HR ¼ 2.679, P < 0.001; HR ¼2.459, P ¼ 0.005), MSI status (HR ¼ 0.427, P ¼ 0.021; HR ¼0.359, P ¼ 0.047), and CD206/CD68 ratio (HR ¼ 4.158, P <0.001; HR ¼ 3.730, P < 0.001). For these factors included in themultivariate analysis, only pathologic T stage (HR ¼ 2.613, P <0.001; HR ¼ 2.093, P ¼ 0.012) and CD206/CD68 ratio (HR ¼3.346, P < 0.001; HR ¼ 2.867, P < 0.001) were identified asindependent prognostic factors in both cohorts. Lymphatic/vas-cular invasion was considered an independent factor in theprimary cohort (HR ¼ 1.621; P ¼ 0.044) but not the validationcohort (HR ¼ 1.356; P ¼ 0.355). Details are shown in Supple-mentary Tables S8 and S9.

CD206/CD68 ratio as a predictive biomarker for the efficacy ofpostoperative adjuvant chemotherapy

In the primary cohort, postoperative adjuvant chemotherapyhad no significant benefit on DFS (P ¼ 0.512) or OS (P ¼ 0.806)for all patients. For patients with a low CD206/CD68 ratio,adjuvant chemotherapy had no benefit on DFS (P ¼ 0.986) orOS (P¼ 0.706). However, for patients with a high CD206/CD68ratio, adjuvant chemotherapy significantly improved theDFS ratefrom 38.9% to 68.0% at 3 years and from 33.1% to 66.0% at 5years (P¼ 0.003) and the OS rate from 75.0% to 82.0% at 3 yearsand from 47.8% to 75.8% at 5 years (P ¼ 0.029). The interactionanalysis between the CD206/CD68 ratio and adjuvant chemo-therapy was significant for DFS (P ¼ 0.023 for interaction) andpotentially significant forOS (P¼0.063 for interaction), revealingthat the benefit of adjuvant chemotherapy in patients with a highCD206/CD68 ratio was superior to that in patients with a lowCD206/CD68 ratio. Details are shown in Fig. 3.

In the validation cohort, the results were similar. For allpatients, postoperative adjuvant chemotherapy hadno significantbenefit onDFS (P¼ 0.216) orOS (P¼ 0.293). Patients with a lowCD206/CD68 ratio still did not benefit from postoperative adju-vant chemotherapy regarding DFS (P¼ 0.868) orOS (P¼ 0.556).

Table 1. Demographics and clinicopathologic characteristics of patients

Primary cohort Validation cohortCharacteristics, n (%) n ¼ 521 n ¼ 314 P

Sex: male/female 288 (55.3)/233 (44.7) 191 (60.8)/123 (39.2) 0.116Age (year): �60/>60 249 (47.8)/272 (52.2) 141 (44.9)/173 (55.1) 0.418Preoperative CEA (ng/mL): <5/�5 320 (61.4)/201 (38.6) 189 (60.2)/125 (39.8) 0.724Tumor site: right-sided/left-sided 295 (56.6)/226 (43.4) 163 (51.9)/151 (48.1) 0.185Tumor size (cm): �4/>4 276 (53.0)/245 (47.0) 181 (57.6)/133 (42.4) 0.189Histologic type: nonmucinous/mucinous 433 (83.1)/88 (16.9) 259 (82.5)/55 (17.5) 0.816Differentiation: 0.303Well to moderate 371 (71.2) 213 (67.8)Poor to undifferentiated 150 (28.8) 101 (32.2)

Differentiation combined with MSI status: 0.927Well to moderate, and poor to undifferentiated with MSI-H 398 (76.4) 239 (76.1)Poor to undifferentiated without MSI-H 123 (23.6) 75 (23.9)

Pathologic T stage: T3/T4 404 (77.5)/117 (22.5) 244 (77.7)/70 (22.3) 0.956Lymph nodes examined: �12/<12 466 (89.4)/55 (10.6) 285 (90.8)/29 (9.2) 0.539Lymphatic/vascular invasion: no/yes 453 (86.9)/68 (13.1) 268 (85.4)/46 (14.6) 0.515Perineural invasion: no/yes 458 (87.9)/63 (12.1) 282 (89.8)/32 (10.2) 0.402MSI status: MSS or MSI-L/MSI-H 444 (85.2)/77 (14.8) 265 (84.4)/49 (15.6) 0.747Risk group for recurrence: 0.487Low-risk group: T3 (MSI-H) with no high-risk factors 35 (6.7) 26 (8.3)Mid-risk group: T3 (MSS/MSI-L) with no high-risk factors 241 (46.3) 152 (48.4)High-risk group: T3 with high-risk factors or T4 245 (47.0) 136 (43.3)

Postoperative adjuvant chemotherapy: no/yes 281 (53.9)/240 (46.1) 158 (50.3)/156 (49.7) 0.311

Abbreviations: CEA, carcino-embryonic antigen; MSI-L/H, microsatellite instability-low/high; MSS, microsatellite stable; high-risk factors, including poorlydifferentiated histology (exclusive of those tumors with MSI-H), lymphatic/vascular invasion, <12 lymph nodes examined, and perineural invasion.

Feng et al.





Low, event = 84 High, event = 25

CD68+ TAM densityLog rank P = 0.135HR (95%CI), 0.713 (0.456–1.115)

Low, event = 51High, event = 19

CD68+ TAM densityLog rank P = 0.739HR (95%CI), 0.914 (0.540–1.548)

373 367 320 300 272 206 124 0

148 147 137 129 119 93 69 0

Low

High

No. at risk

373 373 363 346 301 227 130 0

148 148 145 140 126 98 72 0

Low

High

No. at risk


CD206+ TAM densityLog rank P < 0.001HR (95%CI), 2.927 (1.985–4.314)


CD206+ TAM densityLog rank P < 0.001HR (95%CI), 3.687 (2.300–5.911)

419 415 381 363 331 258 168 0

102 99 76 66 60 41 25 0

Low

High

No. at risk

419 419 413 399 357 277 175 0

102 102 95 87 70 48 27 0

Low

High

No. at risk


CD206/CD68 ratioLog rank P < 0.001HR (95%CI), 4.158 (2.824–6.124)


CD206/CD68 ratioLog rank P < 0.001HR (95%CI), 4.843 (3.020–7.768)

435 430 401 381 347 272 180 0

86 84 56 48 44 27 13 0

Low

High

No. at risk

435 435 429 418 372 290 187 0

86 86 79 68 55 35 15 0

Low

High

No. at risk

All, event = 109All patients in primary cohort

3-year DFS rate: 82.3%5-year DFS rate: 79.4%

All, event = 70All patients in primary cohort

3-year OS rate: 93.3%5-year OS rate: 86.6%

521 514 457 429 391 299 193 0All

No. at risk

521 521 508 486 427 325 202 0All

No. at risk

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B

Figure 2.

TAMs as prognostic factors in the primary cohort. A, The DFS curve of all patients in the primary cohort. B, The OS curve of all patients in the primary cohort. C,Comparing the DFS curves of patients with low and high CD68þ TAM density. D, Comparing the OS curves of patients with low and high CD68þ TAM density. E,Comparing the DFS curves of patients with low and high CDCD206þ TAM density. F, Comparing the OS curves of patients with low and high CDCD206þ TAMdensity. G, Comparing the DFS curves of patients with low and high CD206/CD68 ratio. H, Comparing the OS curves of patients with low and high CD206/CD68ratio.






DC

Yes, event = 29No, event = 38

Adjuvant chemotherapyLog rank P = 0.986HR (95%CI), 0.996 (0.614–1.615)



190 187 174 165 147 116 77 0

245 243 227 216 200 156 103 0

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50 50 38 34 33 22 09

36 34 18 14 11 5 4 0

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Interaction analysis HR = 0.404 (0.185–0.882), P = 0.023

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190 190 188 182 155 121 78 0

245 245 241 236 217 169 109 0

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A



240 237 212 199 180 138 8 06

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240 240 234 223 192 146 88 0

281 281 274 263 235 179 11 04

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Ove

rall

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Figure 3.

TAMs as predictive factors for the efficacy of postoperative adjuvant chemotherapy in the primary cohort. A, In the primary cohort, comparing the DFS curves ofpatients receiving adjuvant chemotherapy or not. B, In the primary cohort, comparing the OS curves of patients receiving adjuvant chemotherapy or not. C, Inpatients with low CD206/CD68 ratio, comparing the DFS curves of receiving adjuvant chemotherapy or not.D, In patients with high CD206/CD68 ratio,comparing the DFS curves of receiving adjuvant chemotherapy or not. E, In patients with low CD206/CD68 ratio, comparing the OS curves of receiving adjuvantchemotherapy or not. F, In patients with high CD206/CD68 ratio, comparing the OS curves of receiving adjuvant chemotherapy or not. The interaction analysiswas conducted between low (C) and high (D) CD206/CD68 ratio on DFS, and between low (E) and high (F) CD206/CD68 ratio on OS.

Feng et al.





For patients with a high CD206/CD68 ratio, the benefit ofadjuvant chemotherapy was significant on DFS (P ¼ 0.007) andOS (P ¼ 0.011). The interaction analysis was significant for bothDFS (P ¼ 0.030 for interaction) and OS (P ¼ 0.024 for interac-tion). Details are shown in Supplementary Fig. S4.

However, in both the primary and validation cohorts,neither CD68þ TAM density nor CD206þ TAM density

could significantly identify patients benefiting from adju-vant chemotherapy. No significant interaction was observedbetween CD68þ TAM density, CD206þ TAM density, andadjuvant chemotherapy. Details for the primary cohort areshown in Supplementary Figs. S5 and S6. Details for thevalidation cohort are shown in Supplementary Figs. S7and S8.

High-risk factor CT, n (%)

No CT, n(%)

CT / No CT HR (95% CI)

Overall 240 (46.1) 281 (53.9) 0.881 (0.603–1.288)Differentiation combined with MSI status, interaction analysis HR = 0.773 (0.330–1.809), P = 0.552

Well to moderate, and poor toundifferentiated with MSI-H

147 (36.9) 251 (63.1) 0.765 (0.467–1.254)

Poor to undifferentiated withoutMSI-H

93 (75.6) 30 (24.4) 0.597 (0.299–1.194)

Pathologic T stage, interaction analysis HR = 0.962 (0.435–2.127), P = 0.923 0.632 (0.364–1.098))1.06(342)9.93(1613T0.613 (0.347–1.084))5.23(83)5.76(974T

Lymph nodes examined, interaction analysis HR = 0.801 (0.294–2.186), P = 0.665 ≥ 0.850 (0.558–1.296))2.65(262)8.34(40221

0.673 (0.270–1.675))5.43(91)5.56(6321<Lymphatic/vascular invasion, interaction analysis HR = 0.800 (0.325–1.969), P = 0.627

0.768 (0.490–1.204))6.75(162)4.24(291oN0.631 (0.289–1.378))4.92(02)6.07(84seY

Perineural invasion, interaction analysis HR = 0.726 (0.295–1.788), P = 0.4860.775 (0.495–1.215))6.75(462)4.24(491oN0.548 (0.251–1.199))0.72(71)0.37(64seY

MSI status, interaction analysis HR = 2.131 (0.504–9.005), P = 0.3030.804 (0.542–1.192))5.25(332)5.74(112L-ISM/SSM1.689 (0.422–6.760))3.26(84)7.73(92H-ISM

Risk group, interaction analysis HR = 0.940 (0.387–2.280), P = 0.891Low-risk group: T3 (MSI-H) withno high-risk factors

10 (28.6) 25 (71.4)Not evaluable with

no DFS events Mid-risk group: T3 (MSS/MSI-L) with no high-risk factors

61 (25.3) 180 (74.7) 0.690 (0.333–1.429)

High-risk group: T3 withhigh-risk factors or T4

169 (69.0) 76 (31.0) 0.651 (0.393–1.079)

CD68+ TAM density, interaction analysis HR = 1.033 (0.420–2.542), P = 0.943 0.875 (0.568–1.348))9.35(102)1.64(271woL0.907 (0.412–1.998))1.45(08)9.54(86hgiH

CD206+ TAM density, interaction analysis HR = 1.061 (0.486–2.318), P = 0.881 0.834 (0.515–1.352))4.45(822)6.54(191woL0.894 (0.484–1.651))0.25(35)0.84(94hgiH

CD206/CD68 ratio, interaction analysis HR = 0.404 (0.185–0.882), P = 0.0230.996 (0.614–1.615))3.65(542)7.34(091woL0.417 (0.225–0.771))9.14(63)1.85(05hgiH

1.00.50.250.125 2.0 4.0 8.0

CT Better No CT Better

Figure 4.

Subgroup analysis of the efficacy of postoperative adjuvant chemotherapy on each high-risk factor in the primary cohort. The subgroups were divided accordingto the traditional clinicopathologic high-risk factors and TAMs. The efficacy of adjuvant chemotherapy was compared in each subgroup. Interaction analysis wasalso conducted in each subgroup to verify the predictive efficacy of each factor. CT, chemotherapy; MSI-L/H, microsatellite instability - low/high; MSS,microsatellite stable; high-risk factors, including poorly differentiated histology (exclusive of those tumors with MSI-H), lymphatic/vascular invasion, <12 lymphnodes examined, and perineural invasion.






Clinicopathologic high-risk factors as predictive biomarkersfor the efficacy of postoperative adjuvant chemotherapy

Interaction analyses were conducted to evaluate clinicopatho-logic high-risk factors in predicting the efficacy of postoperativeadjuvant chemotherapy (as shown in Fig. 4 and SupplementaryTable S10). In both the primary and validation cohorts, nosignificant interaction was observed between adjuvant chemo-therapy and traditional clinicopathologic high-risk factors. Thetraditionalmid-/high- risk groups could not identify patients whowould benefit from adjuvant chemotherapy (more details areshown in Supplementary Figs. S9 and S10). Only the CD206/CD68 ratio was identified as a significant predictive factor.

Subgroup analysis of the CD206/CD68 ratio as a predictivebiomarker for the efficacy of postoperative adjuvantchemotherapy

In subgroup analysis, patients were divided into three sub-groups according to the NCCN guidelines (8): low-risk group asT3 (MSI-H) with no high-risk factors, mid-risk group as T3 (MSS/MSI-L) with no high-risk factors, and high-risk group as T3 withhigh-risk factors or T4. Interaction analysis was conducted in eachsubgroup.

In theprimary cohort, for patients in the low-risk group (n¼35,6.7%), no recurrence or death occurred. The interaction analysiswas unavailable. In the mid-risk group (n ¼ 241, 46.3%), nosignificant benefit of adjuvant chemotherapy for DFS or OS wasobserved in patients with low or high CD206/CD68 ratios. Therewas no significant interaction for DFS (P¼ 0.412 for interaction)or OS (P ¼ 0.547 for interaction). In the high-risk group, asignificant benefit of adjuvant chemotherapy was observed forboth DFS (P¼ 0.001) and OS (P¼ 0.039) in patients with a highCD206/CD68 ratio but not in patients with a low CD206/CD68ratio. The interaction analysis was significant for DFS (P ¼ 0.024for interaction) but not for OS (P¼ 0.136 for interaction). Detailsare shown in Fig. 5.

In the validation cohort, the results were similar. In the low-riskgroup (n¼26, 8.3%), no recurrence or death occurred. In themid-risk group (n ¼ 152, 48.4%), no significant benefit of adjuvantchemotherapy for DFS or OSwas observed. In the high-risk group(n ¼ 136, 43.3%), the benefit of adjuvant chemotherapy wassignificant for DFS (P ¼ 0.010) and potentially significant for OS(P¼ 0.072) in patients with a high CD206/CD68 ratio but not inpatients with a low CD206/CD68 ratio. The interaction analysiswas significant for DFS (P¼ 0.046 for interaction) but not for OS(P ¼ 0.091 for interaction). Details are shown in SupplementaryFig. S11.

Comparison between normal sections and TMA for IHCstaining

In validation cohort, normal sections were also used for IHCstaining, and were compared with TMA. Results of high ICC andkappa showed that normal sectionswere in good consistencywithTMA (as shown in Supplementary Information S8). The CD206/CD68 ratio detected using normal sections was also confirmedeffective prognostic and predictive biomarker. Details are shownin Supplementary Figs. S12, S13, and S14.

DiscussionFor stage II colon cancer, most patients can be cured by radical

resection alone (29). Only approximately 15%–25% of patients

suffering from recurrence may benefit from postoperative adju-vant chemotherapy. Thus, prognostic factors are expected toidentify patients with a high risk of recurrence, which can bepredictive of the efficacy of postoperative adjuvant chemotherapy.To identify these patients, traditional clinicopathologic high-riskfactors are too broad, classifying more than half of patients ashaving a high risk of recurrence needing adjuvant chemothera-py (6, 7). Thus, the benefit of adjuvant chemotherapy is easilyconcealed and still not significant (5, 7). There has been someprogress in molecular biomarkers, such as CDX-2 (30) and themulti-miRNA model (1), but more reliable evidence is stillneeded. MSI status has proven accurate and effective enough inidentifying approximately 10%–15% of patients who do notbenefit from FU-based adjuvant chemotherapy (11, 12). How-ever, for the remainder of patients, there is still a need for betterpredictive biomarkers for adjuvant chemotherapy.

For stage II colon cancer, few patients suffer from local recur-rence, which means that surgery is always effective. However, forpatients suffering from distant metastases, tiny metastatic lesionscould form at early time before primary tumor resection. AndM2TAMs play an important role in releasing circulating tumor cells(CTC). Studies have proved that M2 TAMs promote tumor cellvessel directional migration and invasion by the paracrine loop oftumor-derivedCSF-1 and TAM-derived EGF/EGF-like ligands (31,32) and secrete osteonectin (33), cathepsin (34), TGFb (35), etc.M2 TAMs also promote tumor angiogenesis by releasingVEGF (36). Through these pathways, CTCs are significantlyincreased, leading to distant metastases. Thus, tiny metastaticlesions could be effectively predicted by M2 TAMs as biomarkersand eliminated by postoperative adjuvant chemotherapy.

CD206þ M2 TAMs identified by the IHC method have beenconfirmed as significant prognostic biomarkers for pancreaticadenocarcinoma (37, 38), renal cell carcinoma (39), gastriccancer (40), hepatocellular carcinoma (41, 42), and some othermalignant tumors. In this study, we also confirmed CD206þ TAMdensity as a significant prognostic biomarker for stage II coloncancer. However, CD206þ TAM density could still not signifi-cantly predict adjuvant chemotherapy for stage II colon cancer. Itshould be noted that TAMs have two aspects (14–16): immunestimulation and immune inhibition. CD206þ TAMdensity couldonly reflect the inhibitory factors as the M2 subtype, but not thestimulatory factors as the M1 subtype. Thus, the prognosticefficacywas not accurate enough. As an improvement,we detectedthe CD206/CD68 ratio as the proportion of M2 TAMs in totalTAMs, reflecting both immune stimulatory and inhibitory factors.The results showed that the CD206/CD68 ratio was a betterprognostic factor than CD206þ TAM density and traditionalclinicopathologic high-risk factors. More importantly, theCD206/CD68 ratio could successfully identify patients who reallybenefited from postoperative adjuvant chemotherapy. Theseresults were consistent in two independent cohorts of the samemedical center.

However, there were still limitations to this study. First, toconduct a real-world study, we enrolled consecutive patients tocomprise the study cohort. Thus, there were imbalances at base-line, especially in the application of adjuvant chemotherapy andin the variation in regimens. Significantly more patients withhigh-risk factors received adjuvant chemotherapy. This imbalancecould have interfered with the results. However, predictive bio-markers must face it in clinical applications. Second, the twocohorts of patients in this study came from the same medical

Feng et al.





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52 51 48 47 45 35 27 0

161 160 148 140 132 101 63 0

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Adjuvant chemotherapyLog rank P = 0.001HR (95%CI), 0.285(0.132–0.616)

129 127 117 109 95 75 46 0

62 61 57 54 47 38 31 0

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14 12 000235

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129 129 128 123 101 79 47 0

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%)

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Figure 5.

The CD206/CD68 ratio as a predictivebiomarker for the efficacy ofpostoperative adjuvant chemotherapy inthe different clinicopathologic risk groupsin the primary cohort. In each figure,survival curves were compared forpatients receiving adjuvant chemotherapyor not. In the mid-risk group: comparingthe DFS curves in patients with lowCD206/CD68 ratio (A); comparing theDFS curves in patients with high CD206/CD68 ratio (B); comparing the OS curvesin patients with low CD206/CD68 ratio(C); comparing the OS curves in patientswith high CD206/CD68 ratio (D); theinteraction analysis was conductedbetween low (A) and high (B) CD206/CD68 ratio on DFS, and between low (C)and high (D) CD206/CD68 ratio on OS. Inthe high-risk group: comparing the DFScurves in patients with low CD206/CD68ratio (E); comparing the DFS curves inpatients with high CD206/CD68 ratio (F);comparing the OS curves in patients withlow CD206/CD68 ratio (G); comparingthe OS curves in patients with highCD206/CD68 ratio (H); the interactionanalysis was conducted between low (E)and high (F) CD206/CD68 ratio on DFS,and between low (G) and high (H)CD206/CD68 ratio on OS. Mid-risk group,T3 (MSS/MSI-L) with no high-risk factors;high-risk group, T3 with high-riskfactors or T4.






center, lacking external validation. Thus, the extrapolation ofresults was limited. Third, our detection method of TAMs wasnot automated as it still needed the involvement of pathologists.Bias in manual detection could affect the results. Our detectionmethod has been evaluated, and showed good stability andrepeatability. However, the best way to solve this problem is todevelop an automated detection method. And this would alsosignificantly enhance the clinical applicability. Fourth, the pre-dictive efficacy was significant for DFS in the interaction analysisbut not significant forOS. For colon cancer, livermetastases play amajor part inDFS events (67.0% in the primary cohort and 72.9%in the validation cohort in this study). However, some livermetastases can be cured by radical surgery, interfering withlong-term survival.

In summary, our study showed that the CD206/CD68 ratio ofTAMs could effectively classify patients with stage II colon cancerinto groups with a low and high risk of tumor recurrence.Moreover, the CD206/CD68 ratio could identify patients whobenefited from postoperative adjuvant chemotherapy. Therefore,patients will be treated with adjuvant chemotherapy more accu-rately, thus improving efficacy, reducing adverse events, andsaving financial costs. At the same time, TAM detection usingIHC methods is cheap, not time consuming, and can be easilyconducted in the laboratories ofmost hospitals.Most patients canafford TAM detection for individual medical management. Withfurther large-scale clinical verification, theCD206/CD68 ratiowillaid in precision treatment for patients with stage II colon cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Q. Feng, W. Chang, G. He, P. Zheng, Y. Wei, Y. Tu,X. Qin, J. XuDevelopment ofmethodology:Q. Feng, Y.Mao,G.He, P. Zheng, Y.Wei, L. Ren,D. Zhu, Y. Tu, J. XuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Q. Feng, Y. Mao, G. He, P. Zheng, W. Tang, Y. Wei,L. Ren, D. Zhu, Y. Tu, J. XuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Q. Feng, Y. Mao, G. He, P. Zheng, Y. Wei, D. Zhu,Y. Tu, J. XuWriting, review, and/or revision of the manuscript: Q. Feng, Y. Mao, G. He,Y. Wei, L. Ren, Y. Tu, J. XuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):Q. Feng,W.Chang, Y.Mao, Y.Wei, D. Zhu,M. Ji,J. XuStudy supervision: Q. Feng, G. He, Y. Wei, Y. Tu, J. Xu

AcknowledgmentsThis work was supported by National Natural Science Foundation of China

(grant No. 81602040, to Q. Feng), National Natural Science Foundation ofChina (grant No. 81602035, to W. Chang), National Natural Science Founda-tion of China (grant No. 81472228, to J. Xu), and Science and TechnologyProject of Xiamen (grant No. 3502Z20154040, to Y. Tu).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 30, 2018; revised November 6, 2018; accepted April 9, 2019;published first April 15, 2019.

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2019;25:3896-3907. Published OnlineFirst April 15, 2019.Clin Cancer Res Qingyang Feng, Wenju Chang, Yihao Mao, et al. with Stage II Colon CancerBiomarkers for Postoperative Adjuvant Chemotherapy in Patients Tumor-associated Macrophages as Prognostic and Predictive

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