Purpose: MicroRNAs (miRNA) have potential prognostic value for colorectal cancers; however, their value based on patient race/ethnicity and pathologic stage has not been determined. The goal was to ascertain the prognostic value of 5 miRNAs with increased expression in colorectal cancers of African American (black) and non-Hispanic Caucasian (white) patients.

Experimental Design: TaqMan quantitative real-time PCR was used to quantify expression of miR-20a, miR-21, miR-106a, miR-181b, and miR-203 in paired normal and tumor colorectal cancer archival tissues collected from 106 black and 239 white patients. The results were correlated with overall survival based on patient race/ethnicity and pathologic stage. Because decisions about adjuvant therapy are important for stage III colorectal cancers, and because miR-181b seemed to have prognostic value only for stage III black patients, we assessed its prognostic value in a separate cohort of 36 stage III colorectal cancers of blacks.

Results: All 5 miRNAs had higher expression in colorectal cancers (>1.0-fold) than in corresponding normal tissues. High expression of miR-203 was associated with poor survival of whites with stage IV colorectal cancers (HR = 3.00; 95% CI, 1.29–7.53), but in blacks it was an indicator of poor survival of patients with stages I and II colorectal cancers (HR = 5.63; 95% CI, 1.03–30.64). Increased miR-21 expression correlated with poor prognosis for white stage IV patients (HR = 2.50; 95% CI, 1.07–5.83). In both test and validation cohorts, high miR-181b expression correlated with poor survival of only black patients with stage III colorectal cancers (HR = 1.94; 95% CI, 1.03–3.67).

Conclusion: These preliminary findings suggest that the prognostic value of miRNAs in colorectal cancers varies with patient race/ethnicity and stage of disease. Clin Cancer Res; 19(14); 3955–65. ©2013 AACR.

Translational Relevance

The clinical utility of microRNAs (miRNA), especially for assessing patient prognoses and predicting the efficacy of therapy, is promising. Because most prior studies were conducted in white patient populations, the value of miRNAs in colorectal cancers based on race/ethnicity has not been assessed. We have showed that miRNAs have distinct prognostic value. Increased expression of miR-21 and miR-203 were associated with poor survival of whites with stage IV colorectal cancers, whereas miR-203 was an independent indicator for blacks with early stage colorectal cancers. In contrast, miR-181b was an independent prognostic marker only for stage III colorectal cancers of blacks. These findings suggest that, for evaluation of the clinical utility of miRNAs in relation to colorectal cancers, patient race/ethnicity and tumor stage should be considered. Furthermore, these findings have clinical implications in identifying aggressive phenotypes of colorectal cancers and in identifying high-risk patients, thus maximizing the benefits of adjuvant therapy.

In the United States, colorectal cancer is the third most common malignancy and the second leading cause of cancer-related deaths among men and women (1). Colorectal cancer is a heterogeneous disease, especially with respect to patient race/ethnicity, tumor stage, and genetic alterations that contribute to its progression. Identification of molecular determinants involved in the progression of colorectal cancers will aid in evaluating patient prognosis and/or provide targets for cancer prevention and/or therapy. MicroRNAs (miRNA) are implicated in the progression of and prognosis for colorectal cancers (2–5).

miRNAs are a family of small (20–24 nucleotides) single-stranded, noncoding RNAs that regulate gene expression at the posttranscriptional level (6). They are thought regulate protein production by binding to a complementary site in mRNA, preventing it from being translated or targeting it for destruction, or by transcriptional gene silencing at the chromatin level (7–9). The number of known miRNAs in humans now exceeds 1,500 (10). Each miRNA may regulate hundreds of mRNAs (11).

About half of the human miRNAs are located at cancer-associated regions of the genome, suggesting that they are involved in tumorigenesis (12). Based on cancer-associated alterations in their expression, miRNAs may act as tumor suppressors or oncogenes (13) and are implicated in tumor progression and metastasis (14). Many are dysregulated in human tumors (9). Microarray analyses have revealed specific downregulation of let-7 expression in lung tumors, but not in breast or colon cancers (15), suggesting that the effects of miRNAs are organ specific.

Because of deletion of the p53 tumor suppressor gene in colon cancer cell lines, several miRNAs are characterized as abnormal. Among these, miR-15b, miR-181b, miR-191, and miR-200c are overexpressed in colorectal cancers compared to normal colorectal tissues, and survival analyses indicate that patients with higher miR-200c expression have shorter survival times compared to patients with lower expression (2). Accumulation of miR-143 and miR-145 is downregulated in cells derived from colorectal cancers (16). MiR-21 is expressed at high levels in colonic carcinoma cells, and high miR-21 expression is associated with poor survival and decreased therapeutic response (3). In colorectal cancers, miR-133b and miR-145 are downregulated and miR-31, miR-96, miR-135b, and miR-183 are upregulated (17).

Although previous studies have highlighted the potential value of miRNA expression profiles in the prognosis for colorectal cancer patients (2–5), their value with respect to race/ethnicity and tumor stage is not known. In the present investigation, we determined the expression levels of 5 miRNAs (miR-20a, miR-21, miR-106a, miR-181b, and miR-203), which were the most highly overexpressed miRNAs among a panel of 389 miRNAs assayed in colorectal cancers (3). Furthermore, their prognostic value was analyzed in a consecutive, retrospective colorectal cancer patient cohort of 142 African Americans (blacks) and 239 non-Hispanic Caucasians (whites) with respect to race/ethnicity and tumor stage.

### Patients and tissue sample collection

Included were 142 black and 239 white colorectal cancer patients who had undergone surgical resection for “first primary” sporadic colorectal cancer between 1985 and 2004 at the University of Alabama at Birmingham (UAB). Thus, this was an “unselected” patient population. Patients with multiple primaries within the colorectum, with multiple malignancies, or with a family or personal history of cancer (due to distinctive molecular pathways) were excluded. We excluded from the study population those patients who died within a week of their surgery; those with surgical margin involvement, unspecified tumor location, multiple primaries within the colorectum, or multiple malignancies; and those patients with a family history of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis (FAP), or personal histories of colorectal cancer. Because the information from the patient charts may not be reliable in identifying the familial versus sporadic nature of colorectal cancers, our cohort can be described as a “consecutive” patient population. The intent of using patients from this time period was to maximize postsurgery follow-up. Formalin-fixed, paraffin-embedded (FFPE) tissue blocks of colorectal cancers and their corresponding normal (benign colonic epithelial) surgical specimens were collected from UAB. Of 142 blacks, 106 were included as an initial test cohort and 36 as a separate validation cohort. The sample size and power were estimated based on a previous study (3). Demographic, clinical, and pathologic information was collected from medical records, physician charts, and pathology and radiology reports. These data included age, race, gender, tumor location, tumor-node-metastasis (TNM) stage, tumor size, survival times, status, and relapse. Hematoxylin and eosin–stained slides were reviewed by 2 pathologists (C.K. Shanmugam and W.E. Grizzle) together. Pathologic staging was done according to the criteria of the American Joint Commission on Cancer (18). Histologic grade was assessed according to World Health Organization criteria. Well and moderately differentiated tumors were pooled into a low-grade group and poor and undifferentiated tumors into a high-grade group (19). The Institutional Review Board of UAB approved this study.

### Patient race/ethnicity and follow-up information

Information on patient race/ethnicity was obtained from their charts, and assignment was self-described or self-identified. We recognize, however, that there is some diversity in identification within any race/ethnic group.

Follow-up information was retrieved from the UAB Tumor Registry. Patients were followed either by the patient's physician or by the UAB Tumor Registry until their death or the date of the last documented contact within the study time frame. The Tumor Registry ascertained outcome (mortality) information directly from patients (or living relatives) and from the physicians of the patients through telephone and mail contacts. This information was further validated against the state Death Registry. Demographic data, including patient age at diagnosis, gender, race/ethnicity, date of surgery, date of the last follow-up (if alive), date of recurrence (if any), and date of death, were collected. The Tumor Registry updated follow-up information every 6 months, and follow-up of our retrospective cohorts ended in September 2012. The median follow-up periods for blacks and whites were 19 years (range 7–29 years) and 15 years (range 7–30 years), respectively.

### RNA isolation and qRT-PCR

Total RNA was extracted from macrodissected tumor and corresponding normal FFPE samples using TRIzol Reagent (Invitrogen). The quality of RNA was determined with a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific). Quantitative real-time PCR (qRT-PCR) of miRNAs was conducted using TaqMan miRNA assays (Applied Biosystems) and was conducted by two-step RT-PCR according to the manufacturer's protocol. Briefly, cDNA synthesis was first accomplished using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). The template consisted of 10 ng of total RNA, and miRNA-specific primers were used (provided in TaqMan MicroRNA kits). This PCR reaction was in a 15-μL-reaction mixture and was done on the iQ 5 Real-Time PCR Detection System (Bio-Rad) for 30 minutes at 16°C, 30 minutes at 42°C, and 5 minutes at 85°C. The synthesized target cDNA was amplified using sequence-specific primers from the TaqMan kits. This PCR reaction was accomplished in a 20-μL mixture (1.33 μL of template cDNA) and was conducted for 10 minutes at 95°C, 15 seconds at 95°C, and 1 minute at 60°C for 40 cycles. Signals were collected at the end of each cycle.

Relative expression values were calculated using the comparative CT method and by normalizing with miRNA RNU6B as an endogenous control. Fold change = |$2^{-\Delta\Delta C_{\rm T}}$|⁠, where ΔΔCT = ΔCT(CTmiRNACTRNU6B)tumor − ΔCT(CTmiRNACT RNU6B)normal. The experiments were done in triplicate, and the investigators were blinded to clinical data and survival outcome information until completion of all assays.

### Statistical analyses and validation

#### Sample size and power calculations.

The sample size and power analysis were estimated based on a prior study of miRNAs in colorectal cancers (3). In that analysis, increased expression of miR-21 was a poor prognostic indicator of survival, and 26 of 72 patients (36%) were positive for its overexpression. The HR was 2.5 (P = 0.01). Thus, we proposed to evaluate 96 samples. A minimum of 96 samples in each racial group will provide enough power to detect an HR ≥ 2.2. Therefore, the sample size (n = 96) was sufficient to identify a statistically significant prognostic value for miRNA expression.

#### Statistical analysis.

Chi-square tests were used to assess the univariate associations of baseline characteristics with miRNA expression for black and white patients, separately. The baseline characteristics included demographic variables (age and gender) and pathologic variables (tumor location, size, grade, nodal status, distant metastasis, and stage). Cluster analyses, conducted to define miRNA expression cutoff points, were based on Euclidean distances, which are geometric distances in multidimensional space. This method was chosen to establish cut-off points based on numerical technique. The cutoff of each miRNA was calculated for blacks, whites, and combined patients separately, but remained constant within each race throughout the study. Descriptive statistics were used to describe the basic features of the fold change of each miRNA (Table 2). The type I error rate of each test was controlled at <0.05. All analyses were conducted (by B. Zhang and S. Bae) with SAS application version 9.2 (SAS Institute Inc.).

Deaths due to colorectal cancer were the outcomes (events) of interest. Survival analysis was used to model the relationship between the time to death due to colorectal cancer and miRNA expression. Those patients who died of any cause other than colorectal cancer and those who were alive at the end of the study were considered to be censored. A log-rank test and Kaplan–Meier survival curves were used to compare patient survival in the high and low expression groups for each miRNA. Multivariate Cox regression survival models were built by including all 5 miRNAs and the known confounding covariates (age, sex, tumor location, tumor stage, tumor size, and tumor grade). To detect the relationship for patients with different tumor stages, survival analyses were also conducted for patient groups with each tumor stage (I + II, III, and IV) separately. The final Cox model was obtained based on stepwise selection criteria. All the above analyses were conducted for black, white, and combined patient populations separately. The final Cox model was obtained based on stepwise selection criteria. All the above analyses were conducted for black, white, and combined patient populations separately, and Benjamini–Hochberg corrections were used to adjust for multiple comparisons testing errors (20). This method is an accepted approach for adjusting for multiple comparisons, and similar corrections have been used in other studies of cancer biomarkers (21, 22).

### miRNA expression profiles of colorectal cancers

The characteristics of black (test and validation) and white patient cohorts are given in Table 1. Expression levels of 5 miRNAs in the combined population, blacks, and whites are given separately in Table 2. We recently reported that all 5 miRNAs are stable in FFPE tissues stored over long periods of time (23). For the combined population, all miRNAs had higher expression in colorectal cancers compared to normal tissues (>1.0-fold; range 1.33–2.10). All 5 had higher expression in colorectal cancers from blacks (range 3.97–5.98) as compared to colorectal cancers from whites (1.06–2.03; Table 2).

Table 1.

Clinicopathological and molecular features of colorectal cancer patients in study

VariableBlacksWhites
Test cohortStage III validation cohort
N = 106 (%)N = 36 (%)N = 239 (%)
Age group (years)
<65 52 (49) 13 (36) 117 (49)
≥65 54 (51) 23 (64) 122 (51)
Gender
Female 71 (67) 18 (50) 104 (44)
Male 35 (33) 18 (50) 135 (56)
Tumor location
Proximal colon 28 (26) 19 (53) 114 (48)
Distal colon 69 (65) 12 (33) 69 (29)
Rectum 9 (9) 5 (14) 56 (23)
Depth of tumor invasion
pT0 0 (0) 0 (0) 0 (0)
pT1 4 (4) 0 (0) 8 (3)
pT2 11(10) 2 (6) 29 (12)
pT3 73 (69) 27 (75) 165 (69)
pT4 18 (17) 7 (19) 37 (15)
Nodal statusa
N0 57 (54) 0 (0) 132 (55)
N1-3 49 (46) 36 (100) 97 (41)
Distant metastasisb
M0 89 (85) 36 (100) 199 (83)
M1 16 (15) 0 (0) 40 (17)
Tumor stage
I 9 (9)  29 (12)
II 46 (43)  100 (42)
III 35 (33) 36 (100) 71 (30)
IV 16 (15)  39 (16)
Low 86 (81) 31 (86) 185 (78)
High 20 (19) 5 (14) 53 (22)
Tumor size (cm)c
≤5 66 (62) 18 (50) 152 (64)
>5 40 (38) 16 (44) 87 (36)
VariableBlacksWhites
Test cohortStage III validation cohort
N = 106 (%)N = 36 (%)N = 239 (%)
Age group (years)
<65 52 (49) 13 (36) 117 (49)
≥65 54 (51) 23 (64) 122 (51)
Gender
Female 71 (67) 18 (50) 104 (44)
Male 35 (33) 18 (50) 135 (56)
Tumor location
Proximal colon 28 (26) 19 (53) 114 (48)
Distal colon 69 (65) 12 (33) 69 (29)
Rectum 9 (9) 5 (14) 56 (23)
Depth of tumor invasion
pT0 0 (0) 0 (0) 0 (0)
pT1 4 (4) 0 (0) 8 (3)
pT2 11(10) 2 (6) 29 (12)
pT3 73 (69) 27 (75) 165 (69)
pT4 18 (17) 7 (19) 37 (15)
Nodal statusa
N0 57 (54) 0 (0) 132 (55)
N1-3 49 (46) 36 (100) 97 (41)
Distant metastasisb
M0 89 (85) 36 (100) 199 (83)
M1 16 (15) 0 (0) 40 (17)
Tumor stage
I 9 (9)  29 (12)
II 46 (43)  100 (42)
III 35 (33) 36 (100) 71 (30)
IV 16 (15)  39 (16)
Low 86 (81) 31 (86) 185 (78)
High 20 (19) 5 (14) 53 (22)
Tumor size (cm)c
≤5 66 (62) 18 (50) 152 (64)
>5 40 (38) 16 (44) 87 (36)

NOTE: N, total number of cases; %, percentage of N.

aInformation missing for 10 white cases.

bInformation missing for 1 black case (test).

cInformation missing for 2 black cases (validation).

Table 2.

Expression of miRNAs in colorectal cancers

miRNACombined (N = 345)Blacks (N = 106)Whites (N = 239)
FCa,b (Q1–Q3)cFCa,b (Q1–Q3)cFCa,b (Q1–Q3)c
miR-20a 2.05 (0.72–7.31) 4.14 (1.51–27.3) 1.60 (0.61–4.78)
miR-21 2.10 (0.92–5.38) 3.79 (0.85–11.2) 2.03 (1.00–4.10)
miR-106a 2.00 (0.61–8.14) 4.33 (1.06–47.5) 1.58 (0.58–4.58)
miR-181b 1.33 (0.59–3.97) 3.97 (0.68–22.8) 1.06 (0.58–2.34)
miR-203 2.10 (0.40–9.06) 5.98 (0.69–71.5) 1.49 (0.38–5.70)
miRNACombined (N = 345)Blacks (N = 106)Whites (N = 239)
FCa,b (Q1–Q3)cFCa,b (Q1–Q3)cFCa,b (Q1–Q3)c
miR-20a 2.05 (0.72–7.31) 4.14 (1.51–27.3) 1.60 (0.61–4.78)
miR-21 2.10 (0.92–5.38) 3.79 (0.85–11.2) 2.03 (1.00–4.10)
miR-106a 2.00 (0.61–8.14) 4.33 (1.06–47.5) 1.58 (0.58–4.58)
miR-181b 1.33 (0.59–3.97) 3.97 (0.68–22.8) 1.06 (0.58–2.34)
miR-203 2.10 (0.40–9.06) 5.98 (0.69–71.5) 1.49 (0.38–5.70)

Abbreviations: N, total number of cases; FC, fold change.

aThreshold cycle (CT) is the unit of measurement in qRT-PCR to measure relative gene expression. Difference in CT (ΔΔCT) = tumor change in Ct minus paired nontumor change in CT.

bFold change calculated from ΔΔCt, where fold change = 2−(median ΔΔCT). Median fold change values are represented.

cQ1 and Q3 for interquartile range of fold change values.

### Survival analyses

Data on both univariate Kaplan–Meier and multivariate Cox regression survival analyses of the combined patient population as well as patient groups categorized according to race/ethnicity, tumor stage, and miRNA status are provided in Tables 3 and 4. Univariate survival curve figures are provided only for test and validation data on miR-181b to show its independent prognostic value in blacks with stage III colorectal cancers.

Table 3.

Univariate survival analyses based on miRNA status

RacemiRNALog rank P-valuea,bTotal (N)Low (N)High (N)
Combined
All stages miR-20a 0.020 344 233 111
miR-21 0.223 344 232 112
miR-106a 0.005 344 231 113
miR-181b 0.004 344 234 110
miR-203 0.002 344 233 111
Stage I + II miR-20a 0.236 184 136 48
miR-21 0.396 184 125 59
miR-106a 0.236 184 136 48
miR-181b 0.303 184 138 46
miR-203 0.360 184 136 48
Stage III miR-20a 0.877 105 68 37
miR-21 0.567 105 69 36
miR-106a 0.158 105 63 42
miR-181b 0.125 105 64 41
miR-203 0.045 105 63 42
Stage IV miR-20a 0.965 55 29 26
miR-21 0.155 55 38 17
miR-106a 0.585 55 32 23
miR-181b 0.722 55 32 23
miR-203 0.920 55 34 21
Blacks
All stages miR-20a 0.065 106 72 34
miR-21 0.149 106 72 34
miR-106a 0.049 106 72 34
miR-181b 0.003 106 72 34
miR-203 0.005 106 71 35
Stage I + II miR-20a 0.368 55 36 19
miR-21 0.287 55 38 17
miR-106a 0.471 55 38 17
miR-181b 0.189 55 38 17
miR-203 0.021 55 39 16
Stage III miR-20a 0.017 35 26
miR-21 0.331 35 24 11
miR-106a 0.039 35 25 10
miR-181b 0.008 35 24 11
miR-203 0.071 35 22 13
Stage IV miR-20a 0.879 16 10
miR-21 0.891 16 10
miR-106a 0.766 16
miR-181b 0.891 16 10
miR-203 0.879 16 10
Whites
miR-20a 0.978 238 161 77
miR-21 0.680 238 161 77
miR-106a 0.415 238 160 78
miR-181b 0.123 238 162 76
miR-203 0.696 238 163 75
Stage I + II miR-20a 0.873 129 94 35
miR-21 0.399 129 93 36
miR-106a 0.486 129 96 33
miR-181b 0.371 129 99 30
miR-203 0.408 129 97 32
Stage III miR-20a 0.513 70 42 28
miR-21 0.089 70 42 28
miR-106a 0.919 70 38 32
miR-181b 0.509 70 42 28
miR-203 0.811 70 39 31
Stage IV miR-20a 0.482 39 25 14
miR-21 0.023 39 26 13
miR-106a 0.184 39 26 13
miR-181b 0.752 39 21 18
miR-203 0.029 39 27 12
RacemiRNALog rank P-valuea,bTotal (N)Low (N)High (N)
Combined
All stages miR-20a 0.020 344 233 111
miR-21 0.223 344 232 112
miR-106a 0.005 344 231 113
miR-181b 0.004 344 234 110
miR-203 0.002 344 233 111
Stage I + II miR-20a 0.236 184 136 48
miR-21 0.396 184 125 59
miR-106a 0.236 184 136 48
miR-181b 0.303 184 138 46
miR-203 0.360 184 136 48
Stage III miR-20a 0.877 105 68 37
miR-21 0.567 105 69 36
miR-106a 0.158 105 63 42
miR-181b 0.125 105 64 41
miR-203 0.045 105 63 42
Stage IV miR-20a 0.965 55 29 26
miR-21 0.155 55 38 17
miR-106a 0.585 55 32 23
miR-181b 0.722 55 32 23
miR-203 0.920 55 34 21
Blacks
All stages miR-20a 0.065 106 72 34
miR-21 0.149 106 72 34
miR-106a 0.049 106 72 34
miR-181b 0.003 106 72 34
miR-203 0.005 106 71 35
Stage I + II miR-20a 0.368 55 36 19
miR-21 0.287 55 38 17
miR-106a 0.471 55 38 17
miR-181b 0.189 55 38 17
miR-203 0.021 55 39 16
Stage III miR-20a 0.017 35 26
miR-21 0.331 35 24 11
miR-106a 0.039 35 25 10
miR-181b 0.008 35 24 11
miR-203 0.071 35 22 13
Stage IV miR-20a 0.879 16 10
miR-21 0.891 16 10
miR-106a 0.766 16
miR-181b 0.891 16 10
miR-203 0.879 16 10
Whites
miR-20a 0.978 238 161 77
miR-21 0.680 238 161 77
miR-106a 0.415 238 160 78
miR-181b 0.123 238 162 76
miR-203 0.696 238 163 75
Stage I + II miR-20a 0.873 129 94 35
miR-21 0.399 129 93 36
miR-106a 0.486 129 96 33
miR-181b 0.371 129 99 30
miR-203 0.408 129 97 32
Stage III miR-20a 0.513 70 42 28
miR-21 0.089 70 42 28
miR-106a 0.919 70 38 32
miR-181b 0.509 70 42 28
miR-203 0.811 70 39 31
Stage IV miR-20a 0.482 39 25 14
miR-21 0.023 39 26 13
miR-106a 0.184 39 26 13
miR-181b 0.752 39 21 18
miR-203 0.029 39 27 12

NOTE: Combined population includes all stages and all races/ethnic groups.

aLog-rank, P-value estimated from Kaplan–Meier univariate analysis.

bAdjustment for multiple comparisons has been made for the P values using the Benjamini–Hochberg method.

Table 4.

Cox multivariate regression analysis to evaluate the independent prognostic value of miRNAs in colorectal cancers

CombinedBlacksWhites
Prognostic variablesIndicator of poor prognosisHRa (95% CI)P valuecPrognostic variablesIndicator of poor prognosisHRa (95% CI)P valuecPrognostic variablesIndicator of poor prognosisHRa (95% CI)P valuec
All stages (N = 344) All stages (N = 106) All stages (N = 238)
miRNA    miRNA    Tumor stage
miR-203 High 1.49 (1.03–2.17) 0.036 miR-181b High 3.55 (1.50–8.41) 0.004  II vs. I II 3.55 (1.08–11.63) 0.038
Tumor stage    Tumor stage     III vs. I III 6.22 (1.90–20.37) 0.002
II vs. I II 1.60 (0.75–3.40) 0.224  II vs. I II 0.80 (0.27–2.40) 0.701  IV vs. I IV 26.43 (7.91–88.32) <0.0001
III vs. I III 2.97 (1.41–6.28) 0.004  III vs. I III 2.38 (0.83–6.79) 0.107
IV vs. I IV 10.11 (4.66–21.93) <0.0001  IV vs. I IV 4.19 (1.31–13.41) 0.017 Tumor grade
Tumor grade    Tumor location     High vs. low High 1.54 (0.99–2.38) 0.057
High vs. low High 1.60 (1.13–2.28) 0.009 Proximal colon vs. Rectum Proximal colon 1.60 (1.13–2.28) 0.061
Distal colon vs. Rectum Distal colon 1.43 (0.33–6.21) 0.644
High vs. low High 2.21 (1.14–4.29) 0.019
Stage I + II (N = 184) Stage I + II (N = 55) Stage I + II (N = 129)
High vs. low High 1.68 (0.86–3.28) 0.132 miR-203 High 5.63 (1.03–30.64) 0.046  High vs. low High 1.52 (0.60–3.83) 0.382
High vs. low High 3.67 (1.30–10.37) 0.015
Stage III (N = 105) Stage III (N = 35) Stage III (N = 70)
miR-203 High 1.90 (1.05–3.42) 0.034 miR-181b High 7.94 (1.60–39.30) 0.012  High vs. low High 1.40 (0.63–3.12) 0.416
High vs. low High 1.27 (0.66–2.42) 0.480  High vs. low High 0.28 (0.03–2.42) 0.249
Stage IV (N = 55) Stage IVb (N = 16) Stage IV (N = 39)
miRNA        miRNA
miR-21 High 3.25 (1.37–7.72) 0.008     miR-21 High 2.50 (1.07–5.83) 0.034
miR-203 High 2.19 (1.10–4.33) 0.026     miR-203 High 3.01 (1.30–6.98) 0.011
High vs. low High 3.76 (1.85–7.63) 0.0003      High vs. low High 3.12 (1.29–7.53) 0.012
CombinedBlacksWhites
Prognostic variablesIndicator of poor prognosisHRa (95% CI)P valuecPrognostic variablesIndicator of poor prognosisHRa (95% CI)P valuecPrognostic variablesIndicator of poor prognosisHRa (95% CI)P valuec
All stages (N = 344) All stages (N = 106) All stages (N = 238)
miRNA    miRNA    Tumor stage
miR-203 High 1.49 (1.03–2.17) 0.036 miR-181b High 3.55 (1.50–8.41) 0.004  II vs. I II 3.55 (1.08–11.63) 0.038
Tumor stage    Tumor stage     III vs. I III 6.22 (1.90–20.37) 0.002
II vs. I II 1.60 (0.75–3.40) 0.224  II vs. I II 0.80 (0.27–2.40) 0.701  IV vs. I IV 26.43 (7.91–88.32) <0.0001
III vs. I III 2.97 (1.41–6.28) 0.004  III vs. I III 2.38 (0.83–6.79) 0.107
IV vs. I IV 10.11 (4.66–21.93) <0.0001  IV vs. I IV 4.19 (1.31–13.41) 0.017 Tumor grade
Tumor grade    Tumor location     High vs. low High 1.54 (0.99–2.38) 0.057
High vs. low High 1.60 (1.13–2.28) 0.009 Proximal colon vs. Rectum Proximal colon 1.60 (1.13–2.28) 0.061
Distal colon vs. Rectum Distal colon 1.43 (0.33–6.21) 0.644
High vs. low High 2.21 (1.14–4.29) 0.019
Stage I + II (N = 184) Stage I + II (N = 55) Stage I + II (N = 129)
High vs. low High 1.68 (0.86–3.28) 0.132 miR-203 High 5.63 (1.03–30.64) 0.046  High vs. low High 1.52 (0.60–3.83) 0.382
High vs. low High 3.67 (1.30–10.37) 0.015
Stage III (N = 105) Stage III (N = 35) Stage III (N = 70)
miR-203 High 1.90 (1.05–3.42) 0.034 miR-181b High 7.94 (1.60–39.30) 0.012  High vs. low High 1.40 (0.63–3.12) 0.416
High vs. low High 1.27 (0.66–2.42) 0.480  High vs. low High 0.28 (0.03–2.42) 0.249
Stage IV (N = 55) Stage IVb (N = 16) Stage IV (N = 39)
miRNA        miRNA
miR-21 High 3.25 (1.37–7.72) 0.008     miR-21 High 2.50 (1.07–5.83) 0.034
miR-203 High 2.19 (1.10–4.33) 0.026     miR-203 High 3.01 (1.30–6.98) 0.011
High vs. low High 3.76 (1.85–7.63) 0.0003      High vs. low High 3.12 (1.29–7.53) 0.012

aAdjusted for miRNA expression levels, age, gender, TNM tumor stage, tumor location within the colorectum, tumor grade, tumor size, and race (for analyses of the Combined patient population); CI, confidence interval.

bBecause the small number of available cases, multivariate analyses was not completed for stage IV black colorectal cancer patients.

cAdjustment for multiple comparisons has been made for the P values using the Benjamini–Hochberg method.

#### Prognostic significance of miR-20a and miR-106a.

Univariate survival analyses showed that high expression of miR-20a was associated with shorter overall survival (OS) in the combined patient population when all stages were considered (log-rank, P = 0.02) and especially for stage III black colorectal cancer patients (log-rank, P = 0.017; Table 3). Similarly, high expression of miR-106a was associated with shorter OS for the combined population; for blacks when all stages were considered; and for blacks with stage III colorectal cancers (log-rank, P = 0.005, P = 0.049, and P = 0.039, respectively; Table 3). In our multivariate analyses, however, these 2 miRNAs were not established as independent markers (Table 4).

#### Prognostic significance of miR-21.

Increased expression of miR-21 correlated with shorter OS of only stage IV patients (HR = 3.25; 95% CI, 1.37–7.72; P = 0.008; Table 4), especially for white patients with stage IV colorectal cancers (log-rank, P = 0.023; Table 3 and HR = 2.50; 95% CI, 1.07–5.83; P = 0.034; Table 4).

#### Prognostic significance of miR-181b.

Although high expression of miR-181b was associated with shorter OS of the combined patient population (log-rank, P = 0.004; Table 3), it was an independent prognostic marker only for black patients (log-rank, P = 0.003; Table 3 and HR = 3.55; 95% CI, 1.50–8.41; Table 4), especially for those with stage III colorectal cancers (test-set, log-rank, P = 0.008; Table 3 and Fig. 1A; HR = 7.94; 95% CI, 1.60–39.30; Table 4).

Figure 1.

Kaplan–Meier univariate survival analysis based on miR-181b expression levels in the test (A) and validation (B) cohorts of black stage III colorectal cancer patients.

Figure 1.

Kaplan–Meier univariate survival analysis based on miR-181b expression levels in the test (A) and validation (B) cohorts of black stage III colorectal cancer patients.

Close modal

Because miR-181b seemed to have a prognostic value for stage III, especially for black patients, both in univariate and multivariate analyses, and studies showing that markers are useful in determining prognosis of minority populations are rare, we validated the prognostic value of miR-181b in a separate cohort of 36 stage III colorectal cancers of blacks. Moreover, traditional pathologic features coupled with novel molecular markers could identify aggressive phenotypes within stage III tumors and aid in identifying high-risk patients to maximize the benefits of adjuvant therapy. The demographic and tumor characteristics for this validation cohort are given in Table 1. Because of nonavailability of follow-up information, one case in the validation cohort was excluded from survival analyses.

Similar to the findings of the test cohort of stage III black patients, univariate analysis showed that high expression of miR-181b was associated with short patient survival in the validation cohort (log-rank, P = 0.005; Fig. 1B). Because the findings of multivariate Cox regression analysis of the validation cohort were similar to findings for the test cohort (HR = 2.75; 95% CI, 1.17–6.48; data not shown), and to increase our sample size to increase statistical power, the test and validation cohorts were pooled for multivariate analysis. Multivariate analyses showed that blacks with stage III colorectal cancers with high expression of miR-181b were 1.94 times more likely to die of colorectal cancer than patients in the combined population who had low miR-181b expression (HR = 1.94; 95% CI, 1.03–3.67; Table 5).

Table 5.

Cox multivariate regression analysis to evaluate the independent prognostic value of miR-181b in the combined test and validation cohorts of black stage III colorectal cancers (N = 70)

Prognostic variablesIndicator of poor prognosisHR (95% CI)aP valueb
miRNA
miR-181b High expression 1.94 (1.03–3.67) 0.043
High vs. low High 1.89 (0.91–3.91) 0.092
Prognostic variablesIndicator of poor prognosisHR (95% CI)aP valueb
miRNA
miR-181b High expression 1.94 (1.03–3.67) 0.043
High vs. low High 1.89 (0.91–3.91) 0.092

aAdjusted for miRNA expression levels, age, gender, TNM stage, tumor location within the colorectum, tumor grade, and tumor size; CI, confidence interval.

bAdjustment for multiple comparisons has been made for the P values using the Benjamini–Hochberg method.

#### Prognostic significance of miR-203.

High expression of miR-203 in colorectal cancers correlated with short survival in different groups of patients (Tables 3 and 4). Increased miR-203 expression had an independent prognostic value for all colorectal cancer patients (combined population, log-rank, P = 0.002; HR = 1.49; 95% CI, 1.03–2.17), but especially for blacks with early stage (I + II) colorectal cancers (log-rank, P = 0.021; HR = 5.63; 95% CI, 1.03–30.64) and for whites with stage IV disease (log-rank, P = 0.029; HR = 3.01; 95% CI, 1.30–6.98).

A panel of miRNAs that are overexpressed in colorectal cancers (3) was analyzed to determine their prognostic value for black and white colorectal cancer patients. The results show an increase in the expression of all the miRNAs in colorectal cancers, which is consistent with previous reports (2, 3, 24, 25). Although the prognostic value of these miRNAs has been investigated in various cancers, including colorectal cancers (2, 3, 24, 25), none of the earlier studies dealt with race/ethnicity and tumor stage. The current investigation shows that the prognostic value of different miRNAs varies with tumor stage and patient race/ethnicity. The results show a greater fold increase for all 5 miRNAs in colorectal cancers of blacks than for their white counterparts.

In normal prostate tissues, miR-301, miR-219, miR-26a, miR-1b-1, and miR-30c-1 are expressed three times higher in blacks than in whites (26). Similarly, prostate cancer cell lines generated from blacks have higher expression of miR-26a compared to lines derived from whites of similar stage and pathologic grade (27). These results suggest that racial differences exist in the expression levels of some miRNAs. Plausible mechanisms for aberrant expression of miRNAs include the alteration of miRNA copy numbers, epigenetic modification of miRNAs and/or miRNA processing proteins, and single-nucleotide polymorphisms (SNP) in miRNA genes (28–31). Because SNPs are specific to race/ethnicity (32), associations between SNPs in miRNAs or in their target genes may contribute to distinct phenotypic features in different race/ethnic groups. Also, dysregulations of specific miRNAs are associated with stage of the disease and survival in several malignancies (3, 33–36). Furthermore, miRNA expression profiles can reflect specific stages in tumor progression (37); and several miRNAs, referred to as “metastamirs,” are involved in tumor metastasis, even though some of these may not have obvious roles in tumorigenesis (38). These reports support our findings related to race- and stage-specific miRNAs in the prognosis of colorectal cancer patients.

In this investigation, there was no substantial correlation between increased miR-21 levels and poor patient prognosis when all stages were considered, as previously reported (3, 39, 40). However, once the population was stratified by patient race/ethnicity and tumor stage, high expression of miR-21 was associated with poor prognosis of white patients with stage IV colorectal cancers. In this study, miRNAs were isolated from macrodissected tumor tissues, whereas prior studies evaluated miRNAs from whole colorectal cancer tissues (3, 27, 39). Highly expressed in the stroma of colorectal cancers, miR-21 is associated with shorter disease-free survival (41). Moreover, there is a differential pattern of expression of miRNAs during colorectal cancer progression (42). These factors may have contributed to the inconsistency between our findings and those of previous studies. Similar to our findings, the high expression of miR-21 in advanced stages of colorectal cancers correlates with distant metastases and shorter patient survival (39, 40, 43). Furthermore, highly expressed miR-21 is more common in stage IV colorectal cancers than in stages II and III colorectal cancers (44). These reports support our finding of miR-21 as an independent prognostic indicator of patients with stage IV colorectal cancers.

Increased miR-181b was identified and validated as an independent marker of poor prognosis for black patients with stage III colorectal cancers. There are high levels of miR-181b in colorectal cancers (2) and in sessile serrated adenomas, know aggressive lesions, relative to hyperplastic polyps (45). Even though there is high miR-181b expression in colorectal cancers, it was considered to have no prognostic value (46). Our findings, however, suggest that, for stage III black patients, miR-181b is a prognostic marker that could aid in identifying high-risk patients who would benefit from adjuvant therapy. MiR-181b acts as an epigenetic switch to inhibit the tumor suppressor CYLD, thereby increasing NF-κB activity and maintaining the transformed state of tumor cells (47). In silico analyses have predicted several target genes of miR-181b that are involved in cell-cycle regulation, cell signaling, and chemosensitivity (2). SNPs in the miR-181b binding sites of the targets may be a reason for increased expression of miR-181b in colorectal cancers. For example, in one of the proposed targets of miR-181b, GATA6, which has an oncogenic function in gastrointestinal cancers (48), 3 SNPs have been identified (49). Because SNPs are race/ethnic specific and have prognostic value in colorectal cancers (50), there may be an association between miR-181b and SNPs in its targets that contributes to colorectal cancer carcinogenesis in black patients. However, the underlying mechanisms for the prognostic value of miR-181b in black patients with stage III colorectal cancers need to be explored.

Our study has found, for the combined population, that high expression of miR-203 is an independent marker of poor prognosis of colorectal cancers. Furthermore, miR-203 is an independent marker of poor prognosis for blacks with stage I or II disease, but, in whites, it is a marker for stage IV disease. In colorectal cancer patients <40 years of age, there is increased expression of miR-203 that correlates with aggressive tumor behavior (25). Elevated expression of miR-203 is a predictor of poor prognosis for pancreatic adenocarcinoma (51, 52) and for breast cancer (53). In contrast, there is decreased expression of miR-203 in lung cancer cells, and it inhibits proliferation and invasion (54). These findings suggest that the role of miR-203 varies with the type and stage of cancer and patient race.

Although this study did not find an independent prognostic value for miR-20a or miR-106a, in univariate analyses, their increased expression correlated with poor survival in the combined population. A meta-analysis of 20 studies on miRNA expression levels in colorectal cancers found upregulation of miR-20a in more than one study (55). High levels of miR-20a in gastric and gastrointestinal cancers correlate with reduced survival (56, 57). miR-106a highly is expressed in metastatic colorectal cancer cell lines (58), colorectal cancers tissues (3, 59), and stool samples of colorectal cancer patients (60). With respect to colorectal cancer prognosis, conversely to what we found, low expression of miR-106a was associated with decreased survival in a Spanish colorectal cancer population (61).

We acknowledge that there are limitations of our study. To begin, this study was conducted with a sample set collected at a single medical center, thus, it is not a population-based study. The samples used were remnants of diagnostic tissues that were processed and archived for more than two decades. Thus, there may be biases related to tissue collection and processing (62). For the discovery and validation of biomarkers, reference sample sets that are collected following standardized protocols are ideal. Furthermore, the validation studies should ideally be conducted on a sample cohort collected from a different institution. However, because limited resources and the exploratory nature of this study, both our test and validation cohorts were collected from the same institution. Nevertheless, our future studies will focus on validating these findings in reference colorectal cancer samples collected from blacks and whites of different geographical regions of the United States. Finally, although we used self-identified race to categorize patients into blacks and whites, we recognize that there is some diversity in identification within any race or ethnic group.

The present investigation is the first to evaluate the prognostic value of miR-20a, miR-21, miR-106a, miR-181b, and miR-203 in colorectal cancers based on patient race/ethnicity and tumor stage. These miRNAs were expressed greater than twofold higher in primary colorectal cancers of black patients than in their white counterparts. Furthermore, miR-21 is an independent prognostic marker for stage IV colorectal cancers of whites; miR-181b is an independent prognostic marker for stage III colorectal cancers of blacks; and miR-203 is an independent prognostic marker of blacks with early-stage colorectal cancers and for whites with late-stage colorectal cancers. Although these findings need to be validated in prospective studies, the results warrant that race/ethnicity of patients and stage of the disease should be considered in assessing the clinical utility of miRNAs in colorectal cancers.

No potential conflicts of interest were disclosed.

Conception and design: L.C. Bovell, C. Shanmugam, S. Bae, W.E. Grizzle, U. Manne

Development of methodology: L.C. Bovell, B.D.K. Putcha, B. Zhang, S. Bae, U. Manne

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L.C. Bovell, C. Shanmugam, U. Manne

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L.C. Bovell, C. Shanmugam, V.R. Katkoori, B. Zhang, S. Bae, K.P. Singh, W.E. Grizzle, U. Manne

Writing, review, and/or revision of the manuscript: L.C. Bovell, C. Shanmugam, B. Zhang, S. Bae, K.P. Singh, W.E. Grizzle, U. Manne

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L.C. Bovell, W.E. Grizzle, U. Manne

Study supervision: W.E. Grizzle, U. Manne

The authors thank D.L. Hill, Ph.D., Division of Preventive Medicine, University of Alabama at Birmingham, for his critical review of this article.

This work was supported by grants from the National Institute of Health/National Cancer Institute (NCI; 2U54-CA118948 and CA098932) to U. Manne, and NCI Cancer Training Grant (5R25 CA47888) and UAB Breast SPORE minority supplement (P50 CA089019) to L.C. Bovell.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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