Long Non-Coding RNAs ASB16-AS1 and AFAP1-AS1: Diagnostic, Prognostic Impact and Survival Analysis in Colorectal Cancer

Figures & data

Table 1 Comparison Between the Three Studied Groups According to Demographic and Laboratory Parameters

	Group I (n = 47)	Group II (n = 49)	Group III (n = 50)	Test of Sig.	p	Sig. Bet. Grps
						I vs II	I vs III	II vs III
Sex
Male	19 (40.4%)	17 (34.7%)	25 (50%)	χ^2=2.436	0.296	>0.05	>0.05	>0.05
Female	28 (59.6%)	32 (65.3%)	25 (50%)
Age (years)
Mean ± SD.	52.79 ± 14.59	51.10 ± 143	52.78 ± 13.63	F= 0.232	0.793	>0.05	>0.05	>0.05
Median (Min. – Max.)	55 (26–85)	54 (25–82)	53.50 (25–85)
BMI
Mean ± SD.	27.29 ± 53	26.4 ± 4.48	22.26 ± 2.92	F= 17.965	<0.001*	0.331	<0.001*	<0.001*
Median (Min. – Max.)	26.63 (18.26–41.62)	26.93 (18.6–382)	21.76 (18.65–31.22)
Hb% (gm/dl)
Mean ± SD.	8.65 ± 1.87	9.39 ± 2.42	13.45 ± 0.78	F = 99.512*	<0.001*	0.117	<0.001*	<0.001*
Median (Min. – Max.)	8.9 (4.9–11.6)	9.40 (4.5–13.9)	13.5 (12.3–15.0)
ALT (IU/L)
Mean ± SD.	24.64 ± 5.87	22.59 ± 8.72	23.32 ± 9.50	H= 11.830*	0.003*	0.002*	0.004*	0.855
Median (Min. – Max.)	22.0 (18–46)	20.0 (15. – 54)	20.0 (16–54)
AST (IU/L)
Mean ± SD.	25.66 ± 4.41	22.78 ± 8.33	22.64 ± 8.63	H = 23.016*	<0.001*	<0.001*	<0.001*	0.735
Median (Min. – Max.)	25.0 (19–33.0)	21.0 (15–63)	20.50 (14–63)
CEA (ng/mL)
Mean ± SD.	24.40 ± 11.28	4.01 ± 2.93	3.56 ± 3.96	H= 90.441*	<0.001*	<0.001*	<0.001*	0.148
Median (Min. – Max.)	25.0 (9–43)	3.10 (1.1–15)	2.50 (0.80–22)
CA19-9 (U/mL)
Mean ± SD.	32.06 ± 23.59	15.16 ± 8.52	13.88 ± 6.45	H= 28.376*	<0.001*	<0.001*	<0.001*	0.475
Median (Min. – Max.)	30.0 (10–122)	16.0 (1–37)	13.50 (2–29)
Tissue.LncRNA.ASB16-AS1
Mean ± SD.	31.4 ± 17.4	5.6 ± 2.1	2.1 ± 1.6	H= 112.238*	<0.001*	<0.001*	<0.001*	<0.001*
Median (Min. – Max.)	28.6 (4.3–69.1)	5.7 (1.2–10.2)	1.7 (0.3–6.9)
Plasma.LncRNA.ASB16-AS1
Mean ± SD.	13.0 ± 9.6	1.6 ± 0.8	0.8 ± 0.5	H= 95.278*	<0.001*	<0.001*	<0.001*	0.020*
Median (Min. – Max.)	10.3 (0.9–35.2)	1.7 (0.3–3.6)	0.9 (0.1–2.0)
Tissue.LncRNA.AFAP1-AS1
Mean ± SD.	20.6 ± 14.1	3.0 ± 1.6	1.9 ± 0.9	H= 92.016*	<0.001*	<0.001*	<0.001*	0.001*
Median (Min. – Max.)	19.3 (2.3–52.4)	2.7 (0.5–9.0)	1.7 (0.1–3.9)
Plasma.LncRNA.AFAP1-AS1
Mean ± SD.	7.9 ± 6.5	1.3 ± 1.0	0.9 ± 0.7	H= 76.670*	<0.001*	<0.001*	<0.001*	0.178
Median (Min. – Max.)	5.5 (1.0–29.6)	1.1 (0.2–4.2)	0.8 (0.0–3.1)

Notes: F, p, F and p values for ANOVA test, Sig. bet. grps was done using Post Hoc Test (Tukey). H: H for Kruskal Wallis test, Pairwise comparison bet. each 2 groups was done using Post Hoc Test (Dunn’s for multiple comparisons test) p: p value for comparing between the three groups, *Statistically significant at p < 0.05. Group I: CRC, Group II: Benign colonic lesion, Group III: Healthy control.

Abbreviations: BMI, body mass index; Hb, hemoglobin concentration; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CEA, carcinoembryonic antigen; CA19-9, carbohydrate antigen 19–9.

Figure 1 Comparison between groups regarding lncRNA ASB16-AS1 and lncRNA AFAP1-AS1 expression levels and ROC curves for their diagnostic performance. (A) Comparison between early-stage CRC patients and benign group according to lncRNA ASB16-AS1 and lncRNA AFAP1-AS1 expression levels. *Significant between early CRC and Group II. (B) Comparison between early-stage CRC patients and controls according to the lncRNA ASB16-AS1 and lncRNA AFAP1-AS1 expression levels. *Significant between early CRC and Group III. (C) ROC curve for lncRNA ASB16-AS1, lncRNA AFAP1-AS1, CEA, and CA19-9 to discriminate CRC group from benign group. (D) ROC curve for lncRNA ASB16-AS1 and lncRNA AFAP1-AS1 to distinguish CRC group from the control group. (E) ROC curve for lncRNA ASB16-AS1 and lncRNA AFAP1-AS1to discriminate early stages CRC patients from benign group. (F) ROC curve lncRNA ASB16-AS1 and lncRNA AFAP1-AS1to discriminate early stages CRC patients and controls.

Table 2 Distribution of the Studied CRC Cases According to Pathological Finding, Staging and Survival (n = 47)

CRC Group (n = 47)	No. (%)
Pathology
Mucinous	9 (19.1%)
Signet ring	4 (8.5%)
Adenocarcinoma	34 (72.3%)
Performance state
0	18 (38.3%)
1	20 (42.6%)
2	9 (19.1%)
Grade
I	4 (8.5%)
II	28 (59.6%)
III	15 (31.9%)
Stage
II	17 (36.2%)
III	22 (46.8%)
IV	8 (17%)
Early (II)	17 (36.2%)
Late (III&IV)	30 (63.8%)
Presence of Metastasis	8 (17%)
Site of metastasis (n = 8)
Liver	4 (50%)
Liver and lung	4 (50%)
Progression	28 (59.6%)
Fate
Alive	26 (55.3%)
Dead	21 (44.7%)
Overall survival
Mean ± SD.	28.32 ± 9.34
Median (Min. – Max.)	36.0 (7.0–36.0)

Table 3 Validity (AUC, Sensitivity, Specificity) of the lncRNAs to Discriminate Between Different Groups

	AUC	p	95% C.I	Cut Off	Sensitivity	Specificity	PPV	NPV
CRC group (n= 47) from benign group (n= 49)
Tissue.LncRNA.ASB16-AS1	0.967	<0.001*	0.928–1.0	>8.24	93.62	89.80	89.80	93.62
Plasma.LncRNA.ASB16-AS1	0.934	<0.001*	0.874–0.993	>2.93	89.36	85.71	85.71	89.36
Tissue.LncRNA.AFAP1-AS1	0.944	<0.001*	0.900–0.988	>5.06	91.49	83.67	84.31	91.11
Plasma.LncRNA.AFAP1-AS1	0.923	<0.001*	0.871–0.975	>1.99	85.11	79.59	80.0	84.78
CEA (ng/mL)	0.888	<0.001*	0.825–0.950	>12	78.72	75.51	75.5	78.7
CA19-9 (U/mL)	0.754	<0.001*	0.657–0.851	>18	63.83	65.31	63.8	65.3
CRC group (n= 47) from control (n= 50)
Tissue.LncRNA.ASB16-AS1	0.996	<0.001*	0.989–1.0	>5.57	95.74	92.0	91.84	95.83
Plasma.LncRNA.ASB16-AS1	0.974	<0.001*	0.945–1.0	>1.37	91.49	88.0	87.76	89.69
Tissue.LncRNA.AFAP1-AS1	0.984	<0.001*	0.966–1.0	>3.03	91.49	90.0	89.58	91.84
Plasma.LncRNA.AFAP1-AS1	0.965	<0.001*	0.935–0.994	>1.52	87.23	84.0	83.67	87.50
Early-stage (n= 17) from benign group (n= 49)
Tissue.LncRNA.ASB16-AS1	0.909	<0.001*	0.807–1.0	>7.36	88.24	81.63	62.50	95.24
Plasma.LncRNA.ASB16-AS1	0.888	<0.001*	0.760–1.0	>2.99	82.35	91.84	77.8	93.7
Tissue.LncRNA.AFAP1-AS1	0.900	<0.001*	0.819–0.980	>5.08	76.47	85.71	65.0	91.3
Plasma.LncRNA.AFAP1-AS1	0.842	<0.001*	0.729–0.954	>2	70.59	81.63	57.1	88.9
Early-stage (n= 17) from control (n= 50)
Tissue.LncRNA.ASB16-AS1	0.988	<0.001*	0.969–1.0	>4.81	94.12	92.0	80.0	97.9
Plasma.LncRNA.ASB16-AS1	0.949	<0.001*	0.879–1.0	>0.95	88.24	62.0	44.1	93.9
Tissue.LncRNA.AFAP1-AS1	0.973	<0.001*	0.938–1.0	>3	82.35	90.0	73.7	93.7
Plasma.LncRNA.AFAP1-AS1	0.925	<0.001*	0.861–0.989	>1.52	70.59	84.0	60.0	89.4

Note: *Statistically significant at p ≤ 0.05.

Abbreviations: AUC, area under a curve; p value, probability value; CI, confidence intervals; NPV, negative predictive value; PPV, positive predictive value.

Table 4 Relation Between Studied Markers with Different Parameters in CRC Patients (n = 47)

	N	Tissue LncRNA ASB16-AS1	Plasma LncRNA ASB16-AS1	Tissue LncRNA AFAP1-AS1	Plasma LncRNA AFAP1-AS1
		Median (Min. – Max.)	Median (Min. – Max.)	Median (Min. – Max.)	Median (Min. – Max.)
Pathology
Mucinous	9	32.11 (4.29–62.01)	13.70 (0.87–35.24)	20.21 (2.30–51.09)	5.45 (1.01–27.18)
Signet ring	4	38.33 (30.07–59.20)	21.96 (10.71–29.66)	28.10 (20.06–47.72)	8.94 (5.20–18.21)
Adenocarcinoma	34	24.17 (5.15–69.07)	9.60 (0.95–32.59)	18.51 (2.90–52.40)	5.46 (1.01–29.58)
H(p)		2.815 (0.245)	4.237 (0.115)	3.425 (0.0180)	1.858 (0.395)
Grade
I	4	11.26 (7.51–23.22)	4.09 (3.09–9.40)	5.62 (5.08–19.09)	3.80 (1.39–8.20)
II	28	22.53 (4.29–59.13)	9.60 (0.87–27.21)	17.02 (2.30–42.13)	5.78 (1.01–18.21)
III	15	42.98 (19.31–69.07)	18.27 (6.72–35.24)	29.10 (17.98–52.40)	7.21 (2.79–29.58)
H(p)		13.346* (0.001*)	13.938* (0.001*)	15.422* (<0.001*)	2.910 (0.233)
Stage
II	17	17.90 (4.29–38.80)	4.92 (0.87–13.98)	6.80 (2.80–21.03)	4.48 (1.01–13.20)
III	22	31.49 (10.14–62.01)	12.35 (1.23–35.24)	20.45 (2.30–51.09)	8.51 (1.22–29.58)
IV	8	55.31 (29.19–69.07)	26.74 (8.48–32.59)	31.86 (18.32–52.40)	7.85 (4.53–17.91)
H(p)		19.426* (<0.001*)	16.610* (<0.001*)	16.877* (<0.001*)	7.324* (0.026*)
Metastasis
No	39	23.22 (4.29–62.01)	9.69 (0.87–35.24)	17.98 (2.30–51.09)	5.41 (1.01–29.58)
Yes	8	55.31 (29.19–69.07)	26.74 (8.48–32.59)	31.86 (18.32–52.40)	7.85 (4.53–17.91)
U(p)		38.50* (<0.001*)	52.0* (0.002*)	52.0* (0.002*)	111.0 (0.212)
Site of lesion
Right	25	30.86 (12.68–62.01)	12 (3.85–15.31)	21.03 (2.30–51.9)	8.49 (1.97–27.18)
Left	18	20.91 (4.29–69.07)	8.34 (0.87–32.59)	6.41 (2.80–52.40)	5.35 (1.01–29.58)
Rectum	4	17.22 (9.83–18.86)	4.52 (1.32–6.65)	7.39 (5.80–8.89)	3.13 (1.22–6.52)
H(p)		7.911* (0.019*)	9.024 (0.011*)	8.782* (0.012*)	5.679 (0.058)

Note: *Statistically significant at p ≤ 0.05 shown in bold. p: p value for comparing between the studied groups.

Abbreviations: H, H for Kruskal Wallis test; U, Mann Whitney test.

Figure 2 Correlation between studied lnRNA and CEA & CA19-9. (A) Correlation between tissue lncRNA ASB16-AS1 with CEA in CRC group. (B) Correlation between plasma lncRNA ASB16-AS1 with CEA in CRC group. (C) Correlation between tissue lncRNA ASB16-AS1 with CA19-9 in CRC group. (D) Correlation between plasma lncRNA ASB16-AS1 with CA19-9 in CRC group. (E) Correlation between tissue lncRNA AFAP1-AS1 with CA19-9 in CRC group.

Figure 3 Kaplan-Meier survival curve for overall survival and progression-free survival with studied lncRNA. (A) Kaplan-Meier survival curve for overall survival with tissue lncRNA ASB16-AS1. (B) Kaplan-Meier survival curve for overall survival with plasma lncRNA ASB16-AS1. (C) Kaplan-Meier survival curve for overall survival with tissue lncRNA AFAP1-AS1. (D) Kaplan-Meier survival curve for overall survival with plasma lncRNA AFAP1- AS1. (E) Kaplan-Meier survival curve for progression-free survival with tissue lncRNA ASB16-AS1. (F) Kaplan-Meier survival curve for progression-free survival with plasma lncRNA ASB16-AS1. (G) Kaplan-Meier survival curve for progression-free survival with tissue lncRNA AFAP1-AS1. (H) Kaplan-Meier survival curve for progression-free survival with plasma lncRNA AFAP1-AS1.

Table 5 Univariate and Multivariate Cox Regression Analysis for the Parameters Affecting Patients’ Survival

	Univariate		^#Multivariate
	p	HR (95%C.I)	p	HR (95%C.I)
Age	0.344	1.015 (0.985–1.045)
Sex (male)	0.801	1.118 (0.471–2.654)
BMI	0.344	1.015 (0.985–1.045)
CEA	0.030*	1.044 (1.004–1.085)	0.740	1.012 (0.943–1.086)
CA19-9	0.046*	1.015 (1.0–1.031)	0.813	1.004 (0.974–1.034)
Stage (late)	0.038*	3.174 (1.065–9.457)	0.972	1.025 (0.259–4.053)
Grade (III)	0.036*	2.505 (1.060–5.923)	1.000	1.000 (0.236–4.234)
Metastasis	<0.001*	16.773 (5.731–49.092)	0.016*	7.757 (1.475–40.788)
Pathology (Adenocarcinoma)	0.996	1.003 (0.388–2.588)
Tissue LncRNA ASB16-AS1	<0.001*	1.067 (1.038–1.098)	1.000	1.000 (0.913–1.095)
Plasma LncRNA ASB16-AS1	<0.001*	1.099 (1.053–1.147)	0.882	0.990 (0.866–1.131)
Tissue LncRNA AFAP1-AS1	<0.001*	1.072 (1.040–1.105)	0.180	1.057 (0.974–1.148)
Plasma LncRNA AFAP1-AS1	0.012*	1.072 (1.015–1.131)	0.740	1.017 (0.920–1.124)

Notes: #: All variables with p < 0.05 was included in the multivariate. *Statistically significant at p ≤ 0.05.

Abbreviations: HR, hazard ratio; C.I, confidence interval; LL, lower limit; UL, upper limit.