MicroRNA MIR21 and T Cells in Colorectal Cancer

Kosuke Mima; Reiko Nishihara; Jonathan A. Nowak; Sun A. Kim; Mingyang Song; Kentaro Inamura; Yasutaka Sukawa; Atsuhiro Masuda; Juhong Yang; Ruoxu Dou; Katsuhiko Nosho; Hideo Baba; Edward L. Giovannucci; Michaela Bowden; Massimo Loda; Marios Giannakis; Adam J. Bass; Glenn Dranoff; Gordon J. Freeman; Andrew T. Chan; Charles S. Fuchs; Zhi Rong Qian; Shuji Ogino

doi:10.1158/2326-6066.CIR-15-0084

DOI: 10.1158/2326-6066.CIR-15-0084 Published January 2016

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Figure 1.
MIR21 expression in colorectal cancer. A, quantitative RT-PCR assays for MIR21 and RNU6-2 using 10-fold dilution series (1:1,000, 1:100, 1:10, and 1:1) from the same specimen. Mean cycle threshold values (± SD) of triplicate runs and the coefficient of determination (r²) in the assays for MIR21 and RNU6-2 are shown. cDNA, complementary DNA. B, MIR21 expression in 54 pairs of colorectal cancer and adjacent nontumor colonic mucosa. A statistical analysis was performed using the two-sided Wilcoxon signed rank test.

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Table 1.

Interassay coefficients of variation in quantitative RT-PCR assays for MIR21 and RNU6-2

	Targets in quantitative RT-PCR assays
	MIR21		RNU6-2
	Mean cycle threshold ± SD	Interassay coefficient of variation (%)	Mean cycle threshold ± SD	Interassay coefficient of variation (%)
Specimen 1	19.5 ± 0.06	0.28	19.9 ± 0.13	0.65
Specimen 2	19.5 ± 0.07	0.36	21.2 ± 0.15	0.73
Specimen 3	19.3 ± 0.09	0.44	21.0 ± 0.18	0.86
Specimen 4	20.0 ± 0.09	0.46	22.1 ± 0.09	0.39
Specimen 5	18.0 ± 0.10	0.55	21.0 ± 0.12	0.59
Mean coefficient of variation (%)		0.42		0.64

NOTE: Interassay coefficient of variation of cycle threshold values from the same specimen were assessed by repeating assays in five different batches with the use of five colorectal cancers.

Table 2.

Clinical, pathologic, and molecular features according to tumor MIR21 expression in 538 colorectal cancer cases

		Tumor MIR21 expression (quartile)
Characteristic^a	Total (N = 538)	Q1 (lowest; n = 135)	Q2 (second; n = 134)	Q3 (third; n = 134)	Q4 (highest; n = 135)	P^b
Mean age ± SD, y	67.6 ± 8.3	66.7 ± 8.2	67.2 ± 8.5	68.8 ± 8.1	67.5 ± 8.3	0.19
Sex						0.044
Men	185 (34%)	50 (37%)	44 (33%)	56 (42%)	35 (26%)
Women	353 (66%)	85 (63%)	90 (67%)	78 (58%)	100 (74%)
Year of diagnosis						0.006
Prior to 1995	201 (38%)	63 (47%)	55 (41%)	42 (32%)	41 (30%)
1996–2000	202 (38%)	43 (32%)	54 (40%)	44 (34%)	61 (45%)
2001–2008	131 (24%)	28 (21%)	25 (19%)	45 (34%)	33 (25%)
Family history of colorectal cancer in a first-degree relative						0.21
Absent	422 (79%)	105 (78%)	111 (83%)	96 (74%)	110 (82%)
Present	109 (21%)	29 (22%)	22 (17%)	34 (26%)	24 (18%)
Tumor location						0.30
Cecum	96 (18%)	20 (15%)	22 (17%)	27 (20%)	27 (20%)
Ascending to transverse colon	173 (32%)	39 (29%)	40 (30%)	43 (32%)	51 (38%)
Splenic flexure to sigmoid	149 (28%)	39 (29%)	44 (33%)	31 (23%)	35 (26%)
Rectosigmoid and rectum	117 (22%)	36 (27%)	27 (20%)	33 (25%)	21 (16%)
Disease stage						0.009
I	110 (21%)	37 (28%)	29 (22%)	23 (18%)	21 (16%)
II	173 (34%)	44 (34%)	45 (35%)	44 (35%)	40 (31%)
III	164 (32%)	31 (24%)	44 (34%)	48 (39%)	41 (32%)
IV	67 (13%)	18 (14%)	11 (8.5%)	10 (8.0%)	28 (21%)
Tumor differentiation						0.24
Well to moderate	486 (91%)	121 (90%)	127 (95%)	119 (89%)	119 (88%)
Poor	51 (9.5%)	13 (9.7%)	7 (5.2%)	15 (11%)	16 (12%)
MSI status						0.26
MSI-low/MSS	440 (84%)	114 (87%)	115 (86%)	107 (82%)	104 (79%)
MSI-high	86 (16%)	17 (13%)	18 (14%)	24 (18%)	27 (21%)
MLH1 hypermethylation						0.15
Absent	461 (87%)	118 (89%)	121 (91%)	109 (83%)	113 (85%)
Present	69 (13%)	14 (11%)	12 (9.0%)	23 (17%)	20 (15%)
CIMP status						0.015
Low/negative	440 (83%)	113 (86%)	119 (89%)	108 (82%)	100 (75%)
High	90 (17%)	19 (14%)	14 (11%)	24 (18%)	33 (25%)
BRAF mutation						0.003
Wild-type	444 (84%)	120 (90%)	116 (88%)	110 (82%)	98 (75%)
Mutant	86 (16%)	13 (9.8%)	16 (12%)	24 (18%)	33 (25%)
KRAS mutation						0.11
Wild-type	311 (59%)	76 (58%)	67 (51%)	88 (66%)	80 (61%)
Mutant	216 (41%)	55 (42%)	64 (49%)	46 (34%)	51 (39%)
PIK3CA mutation						0.86
Wild-type	408 (83%)	99 (84%)	104 (82%)	105 (82%)	100 (85%)
Mutant	82 (17%)	19 (16%)	23 (18%)	23 (18%)	17 (15%)
Mean LINE-1 methylation level (%) ± SD	61.6 ± 9.6	61.6 ± 8.4	59.6 ± 10.4	62.1 ± 10.2	63.0 ± 9.1	0.032

Abbreviations: Q1 to Q4, quartile 1 to quartile 4.

↵^aPercentage indicates the proportion of cases with a specific clinical, pathologic, or molecular feature in colorectal cancer cases with each tumor MIR21 expression. There were cases that had missing values for any of the characteristics except for age and sex.
↵^bTo assess associations between the ordinal categories (first to fourth quartile) of tumor MIR21 expression and categorical data, the χ² test was performed. To compare mean age and mean LINE-1 methylation levels, an ANOVA was performed. We adjusted the two-sided α level to 0.003 (= 0.05/14) by simple Bonferroni correction for multiple hypothesis testing.

Table 3.

Distribution of colorectal cancer cases according to tumor MIR21 expression and the density of T cells

		Tumor MIR21 expression (quartile)
	Total	Q1 (lowest)	Q2 (second)	Q3 (third)	Q4 (highest)	P_trend^a
CD3⁺ cell density (quartile)						0.0004
Q1 (0–115 cells/mm²)	130 (25%)	26 (20%)	29 (22%)	31 (25%)	44 (33%)
Q2 (116–252 cells/mm²)	129 (25%)	26 (20%)	30 (23%)	35 (28%)	38 (29%)
Q3 (253–533 cells/mm²)	130 (25%)	42 (32%)	33 (25%)	30 (24%)	25 (19%)
Q4 (≥534 cells/mm²)	129 (25%)	38 (28%)	38 (30%)	28 (23%)	25 (19%)
CD8⁺ cell density (quartile)						0.27
Q1 (0–66 cells/mm²)	128 (25%)	23 (18%)	31 (24%)	40 (32%)	34 (26%)
Q2 (67–185 cells/mm²)	127 (25%)	42 (33%)	26 (20%)	28 (23%)	31 (24%)
Q3 (186–410 cells/mm²)	128 (25%)	30 (24%)	34 (26%)	28 (23%)	36 (27%)
Q4 (≥411 cells/mm²)	127 (25%)	31 (25%)	39 (30%)	27 (22%)	30 (23%)
CD45RO⁺ cell density (quartile)						0.0002
Q1 (0–183 cells/mm²)	131 (25%)	24 (18%)	30 (23%)	31 (24%)	46 (35%)
Q2 (184–430 cells/mm²)	130 (25%)	32 (25%)	33 (25%)	39 (31%)	26 (20%)
Q3 (431–805 cells/mm²)	131 (25%)	26 (20%)	37 (27%)	30 (24%)	38 (29%)
Q4 (≥806 cells/mm²)	130 (25%)	48 (37%)	33 (25%)	27 (21%)	22 (16%)
FOXP3⁺ cell density (quartile)						0.032
Q1 (0–14 cells/mm²)	124 (25%)	25 (20%)	31 (26%)	32 (27%)	36 (28%)
Q2 (15–25 cells/mm²)	124 (25%)	29 (23%)	24 (20%)	34 (28%)	37 (28%)
Q3 (26–48 cells/mm²)	124 (25%)	38 (31%)	27 (22%)	27 (22%)	32 (25%)
Q4 (≥49 cells/mm²)	123 (25%)	32 (26%)	38 (32%)	28 (23%)	25 (19%)

Abbreviations: Q1 to Q4, quartile 1 to quartile 4.

↵^aP_trend value was calculated by the linear trend test across the ordinal (first to fourth quartile) categories of tumor MIR21 expression as a continuous variable in a univariable ordinal logistic regression model for the density of CD3⁺, CD8⁺, CD45RO⁺, or FOXP3⁺ T cells (an ordinal quartile outcome variable). Because we assessed four primary outcome variables, we adjusted the two-sided α level to 0.012 (= 0.05/4) by simple Bonferroni correction.

Table 4.

The association of tumor MIR21 expression with the density of T cells

		Univariable OR (95% CI)	Multivariable OR (95% CI)^a
Model for CD3⁺ cell density (n = 518, as an outcome variable)
MIR21 expression	Q1 (lowest)	1 (reference)	1 (reference)
	Q2 (second)	0.88 (0.57–1.36)	0.85 (0.55–1.31)
	Q3 (third)	0.67 (0.43–1.04)	0.59 (0.37–0.92)
	Q4 (highest)	0.47 (0.31–0.73)	0.44 (0.28–0.68)
	P_trend^b	0.0004	<0.0001
Model for CD8⁺ cell density (n = 510, as an outcome variable)
MIR21 expression	Q1 (lowest)	1 (reference)	1 (reference)
	Q2 (second)	1.14 (0.74–1.77)	1.25 (0.80–1.96)
	Q3 (third)	0.72 (0.46–1.12)	0.76 (0.48–1.19)
	Q4 (highest)	0.89 (0.58–1.38)	0.99 (0.63–1.54)
	P_trend^b	0.27	0.50
Model for CD45RO⁺ cell density (n = 522, as an outcome variable)
MIR21 expression	Q1 (lowest)	1 (reference)	1 (reference)
	Q2 (second)	0.70 (0.46–1.09)	0.72 (0.46–1.12)
	Q3 (third)	0.57 (0.37–0.89)	0.54 (0.34–0.84)
	Q4 (highest)	0.45 (0.29–0.70)	0.41 (0.26–0.64)
	P_trend^b	0.0002	<0.0001
Model for FOXP3⁺ cell density (n = 495, as an outcome variable)
MIR21 expression	Q1 (lowest)	1 (reference)	1 (reference)
	Q2 (second)	0.98 (0.63–1.54)	0.93 (0.59–1.46)
	Q3 (third)	0.73 (0.47–1.14)	0.61 (0.39–0.96)
	Q4 (highest)	0.66 (0.42–1.02)	0.55 (0.35–0.86)
	P_trend^b	0.032	0.003

Abbreviations: Q1 to Q4, quartile 1 to quartile 4.

↵^aThe multivariable ordinal logistic regression analysis model initially included age; sex; year of diagnosis; family history of colorectal cancer in parent or sibling; tumor location; tumor differentiation; MSI; CpG island methylator phenotype; KRAS, BRAF, and PIK3CA mutations; and LINE-1 methylation level. A backward stepwise elimination with a threshold of P = 0.05 was used to select variables in the final models. Variables remaining in the final multivariable ordinal logistic regression models are shown in Supplementary Table S2.
↵^bP_trend value was calculated by the linear trend across the ordinal (first to fourth quartile) categories of MIR21 expression as a continuous variable in the ordinal logistic regression model for the density of CD3⁺, CD8⁺, CD45RO⁺, or FOXP3⁺ T cells (an ordinal quartile outcome variable). Because we assessed four primary outcome variables, we adjusted the two-sided α level to 0.012 (= 0.05/4) by simple Bonferroni correction.