2.1.1. Patients and Clinical Data
Starting in July 2004, 54 patients with histological proven CRC and 19 controls were consecutively recruited for this study. This sample size allowed us to estimate an expected area under the ROC curve of 0.70 with a standard error of 0.065. Ninety per cent of the subjects were included within the first two years. The clinical characteristics of the patients are shown in .
Patient baseline and clinical characteristics.
The mean age was 62.2 years (SEM 1.84; median, 62 years; range, 43 to 74 years) in the control group and 62.7 (SEM 1.30; median, 62.5; range, 31 to 80 years) in the patient group (t test, p = 0.847). The ratio of males to females was similar in the controls (men 63.2%) and the patients (men 61%) (χ2 test, p = 0.875).
PB samples were obtained after R0 or R1 surgery in 16 patients. In 38 patients, blood samples were obtained before neo-adjuvant chemotherapy or in the presence of active metastatic disease, both of which were categorized as R2. In patients with node-negative disease and R0 resection, the mean number of lymph nodes analyzed was 12.8 (SEM 2.7; range 7–21).
Patients with metastatic CRC (n
= 38) were grouped into high- (19.4%), intermediate- (36.1%) and low-risk groups (44.4%) using performance status, number of tumor sites, alkaline phosphatase and white blood cell count, as suggested by Köhne et al
] Median overall survival (OS) and progression-free survival (PFS) were 98 and 39 weeks, 56 and 26 weeks, and 59 and 14 weeks for the low-, intermediate- and high-risk groups, respectively. The median OS tended to be higher (log-rank p
= 0.061) in the low-risk group (98 weeks; 95% CI, 43.1 to 152.9) compared to the combined intermediate/high-risk group (56 weeks; 95% CI, 47.2 to 64.8).
All patients were followed up until death or the end of the study. Disease progression events occurred in 39 patients (72.2%). There were three relapses among stage I–III patients and 36 progressions of metastatic disease. The median PFS was 44 weeks (95% CI, 24.8 to 63.2 weeks). The median OS was 132 weeks (95% CI, 84.4–179.6 weeks), and 34 patients (63%) died of advanced disease. The mean (SEM) follow-up time for the patients still alive at the time of the analysis was 232 (17.8) weeks (median, 232.5 weeks; range, 67 to 335 weeks).
2.1.2. Expression of AGR2 and LGR5 mRNA Transcripts in Blood Samples
AGR2 mRNA was quantified in 62 blood samples (84.9%), including 43 samples obtained from patients with CRC and 19 from controls. The LGR5 mRNA level was quantified in 67 blood samples (90.5%), 48 from CRC patients and 19 from controls. mRNA was insufficient or its quality was inadequate for qRT-PCR in 11 (15.1%) and 6 (8.2%) patients’ samples for AGR2 and LGR5 respectively.
The mean relative AGR2 mRNA expression was 29.1 (SEM 28.2; median 0.77; range, 0.21 to 536.7) in controls and 418.57 (SEM 84.4; median 191.2; range, 0.05 to 1989.5) in cancer patients (t test, p < 0.001). Likewise, the AGR2 level was significantly increased (ANOVA, p = 0.007) in patients with stage IV CRC (mean 492.6; SEM 114) compared with stage I to III patients (mean 305.4; SEM 122.5) and non-cancer controls (mean 29.1; SEM 28.2).
The mean LGR5 mRNA level was 0.21 (SEM 0.03; median 0.18; range, 0 to 0.4) in controls and 11.6 (SEM 4.9; median 0.08; range, 0.01 to 146.9) in patients (t test, p = 0.026). The LGR5 level was significantly increased (ANOVA, p = 0.038) in patients with stage IV CRC (mean 18.40; SEM 7.70) compared with stage I to III patients (mean 0.20; SEM 0.06) and non-cancer controls (mean 0.21; SEM 0.03). There was no correlation between AGR2 and LGR5 blood levels in the patients group (Pearson correlation coefficient −0.009; p = 0.952).
ROC curves of circulating mRNAs were constructed in order to be able to discriminate different groups ().
AGR2 and LGR5 ROC curves. mRNA relative levels were quantified in blood obtained from patients with colorectal cancer and from controls. Area under the curve (AUC), 95% confidence interval and p-values are shown.
Comparing the relative AGR2 levels in patients and controls, the AUC was 0.722 (95% CI, 0.594–0.849; p = 0.006). According to the ROC curve, a relative level for AGR2 mRNA in the blood of 1.65 was defined as the optimal cutoff value (Youden index) for differentiating patients with CRC from the controls. With this cutoff value for AGR2, the sensitivity and specificity of 62.8% (95% CI, 46.7 to 76.6) and 94.7% (95% CI, 71.9 to 99.7) respectively, were achieved. At this threshold value, AGR2 positivity was associated with CRC diagnostic (p < 0.001).
The ROC curve for LGR5 showed an AUC of 0.376 (95% CI, 0.233–0.520; p = 0.123). A relative blood level of 0.39 was defined as the optimal cutoff point for LGR5. With this cutoff value, the sensitivity and specificity for the LGR5 mRNA assay were 18.8% (95% CI, 9.4 to 33.10) and 100% (95% CI, 79.1 to 99.5) respectively. At this cutoff value, LGR5 positivity tended to associate with CRC diagnostic (p = 0.052).
In CRC patients, relative expression values for AGR2 and/or LGR5 in blood above these cutoff points, defined as the Youden index, were found in 16.7% of stage I–II, in 72.7% of stage III and in 76.9% of stage IV patients (χ2 test; p = 0.016).
markers were analyzed in combination by logistic regression. The predicted probabilities of diagnosis generated a ‘combination marker’ ROC curve. The combination (AGR2
) had an AUC-ROC = 0.767 (95% CI, 0.648–0.886; p
= 0.001) which was slightly improved [24
] compared to AGR2
= 0.25). The sensitivity and specificity of the combination were 67.4% (95% CI, 51.3 to 80.5) and 94.7% (95% CI, 71.9 to 99.7) respectively ().
2.1.3. Clinic Pathological Characteristics and mRNA Markers in Blood
The clinical and pathological characteristics and the AGR2 and LGR5 mRNA expression in blood from cancer patients are shown in .
Distribution of clinical and pathological parameters and levels of AGR2 and LGR5 mRNA in the blood.
A significant higher relative level of AGR2 blood expression was found in pT3-T4 tumors (p = 0.002) and high-grade lesions (p = 0.023). There was a tendency (p = 0.063) to higher AGR2 levels associated with lymph node metastasis. Increased LGR5 expression was found in patients () with stage IV (p = 0.024), R2 resections (p = 0.024) or high-grade tumors (p = 0.024).
Carcinoembryonic antigen (CEA) and carbohydrate antigen 19.9 (CA 19.9) serum levels were increased above the upper limits of normal in 46.3% and 38.9% of the patients, respectively. There were no correlations between AGR2 or LGR5 mRNA levels with CEA or CA 19.9 in serum (Pearson −0.172, −0.155, 0.021 and −0.063 respectively).
To explore the possible influence of recent surgery on the circulation of tumor cells, we analyzed AGR2 and LGR5 levels according to the time interval from operation and blood sampling. The mean time from surgery to blood sampling for mRNA quantification was 52.5 weeks (SEM 8.7 weeks; median, 18 weeks; range, 1 to 202 weeks). The 25th percentile was 6.75 weeks. There was no significant difference in AGR2 and LGR5 levels between time intervals (<6.75 or ≥6.75 weeks) from the last surgery.
In the group of patients with stage IV disease, AGR2
were analyzed according to the prognostic subgroups defined as described previously [19
]. The mean (SEM) relative AGR2
levels were 443.1 (229.6) and 518.8 (129.9) for low- and combined intermediate/high-risk groups, respectively (t
= 0.759). The median (SEM) relative LGR5
levels were 15.5 (11.7) and 20.6 (10.4) for low- and combined intermediate/high-risk groups, respectively (t
2.1.4. Prognostic Significance of AGR2 and LGR5 in Blood
To analyze the relationships between biomarker expression and outcomes (PFS and OS) we estimated the hazard ratios associated with mRNA levels as continuous variables using Cox regression models [25
]. There was a trend for a high risk of disease progression associated with increased AGR2
relative blood expression (HR 1.0; 95% CI, 1.0 to 1.001; p
= 0.093). There was no association with the risk of death (HR 1.0; 95% CI, 0.999 to 1.001; p
= 0.913). However, in stage I to III patients, the risk of disease progression was higher with increasing AGR2
level (HR 1.002; 95% CI, 1 to 1.004; p
Increasing relative blood expression of LGR5 mRNA as a continuous variable was associated with a higher risk of disease progression (HR 1.013; 95% CI, 1.004 to 1.023; p = 0.007). The risk of death was also higher with increasing levels for LGR5 mRNA in the blood (HR 1.01; 95% CI, 1 to 1.020; p = 0.045).
In addition, in order to generate survival curves, we converted continuous mRNAs expression levels measured on qRT-PCR to a dichotomous variable, using the mean levels of expression in the patients group as a threshold. Kaplan-Meier curves for patients categorized according to AGR2 and LGR5 mRNA expression in blood are shown (–).
Figure 2 AGR2 and survival analysis. Kaplan-Meier plots of (a) progression-free survival (PFS) and (b) overall survival (OS) in colorectal cancer patients according to AGR2 mRNA expression in blood. Relative quantification of AGR2 mRNA was calculated by the 2 (more ...)
Figure 4 Combined AGR2/LGR5 and survival analysis. Kaplan-Meier plots of (a) progression-free survival (PFS) and (b) overall survival (OS) in colorectal cancer patients according to combined AGR2/LGR5 mRNA profile in blood. Relative quantification of mRNA was (more ...)
The median PFS for the group with high AGR2 blood expression were 33 weeks (95% CI, 11 to 55) compared with 86 weeks (95% CI, 0 to 305.1) in the group with low AGR2 (log-rank test, p = 0.033). Patients with high AGR2 showed worse OS (median 97 weeks; 95% CI, 0 to 262.9) compared with those with low AGR2 expression (median 192 weeks; 95% CI, 56.6 to 327.4) although this difference was not statistically significant (log-rank test, p = 0.6) ().
Analysis of the patients’ outcome according to LGR5 blood expression revealed that the high LGR5 group exhibited significantly worse PFS (median 22 weeks; 95% CI, 0 to 48.4) compared with patients in the low LGR5 group (median 55 weeks; 95% CI, 5.1 to 104.9) (p = 0.013). Although non-significant, there was a trend (p = 0.061) for a better OS in the group of patients with low LGR5 (median 179 weeks; 95% CI, 74.9 to 283.1) compared with the group with increased LGR5 blood levels (median 61 weeks; 95% CI, 28.6 to 93.4) ().
Figure 3 LGR5 and survival analysis. Kaplan-Meier plots of (a) progression-free survival (PFS) and (b) overall survival (OS) in colorectal cancer patients according to LGR5 mRNA expression in blood. Relative quantification of LGR5 mRNA was calculated by the 2 (more ...)
High mRNA in PB (combined AGR2 and/or LGR5 transcript above the threshold cutoff) was found in 0, 36.4% and 53.8% of stage I–II, III and IV patients, respectively (χ2 test; p = 0.05).
Patients were divided into favorable mRNA profile (both AGR2 and LGR5 below the mean) and unfavorable mRNA profile (AGR2 and/or LGR5 above the mean). At the time of analysis, the mean and the median PFS in the favorable group were 190.8 weeks (95% CI, 131.2 to 250.4 weeks) and not reached in the unfavorable group. The mean and the median PFS were 54.7 weeks (95% CI, 21.2 to 88.1 weeks) and 32 weeks (95% CI, 17.5 to 46.6 weeks) in the unfavorable mRNA profile group (log-rank test p = 0.002) ().
Multivariate Cox regression analyses were performed to determine whether high mRNA in blood were independently statistically predictive of PFS or OS ().
Progression-free survival and overall survival in relation to clinic and pathological characteristics and blood AGR2/LGR5 mRNA: Multivariate Cox proportional hazard analysis.
In testing for the independent prognostic significance of high AGR2/LGR5 expression in a model with pT depth of invasion, lymph node involvement and residual disease (R resection status), the R status (HR of recurrence, 5.8; 95% CI, 1.7 to 19.7; p = 0.005) and the high mRNA blood expression (HR, 2.8; 95% CI, 1.2 to 6.4; p = 0.014) remained associated with PFS (). In this model, the only factor that retained independent prognostic significance for OS was R2-residual disease (HR of death, 7.338; 95% CI, 1.683 to 31.985; p = 0.008).
Highly sensitive detection of CTC and detailed molecular characterization of rare cancer cell subpopulations may not only provide insights into the biology of early metastatic spreading, but these tools can also potentially indicate substantial predictive or prognostic information. PCR amplification of tumor mRNA is a powerful analytical tool for surrogate detection and characterization of CTC. Real-time RT-PCR allows for quantification of the tumor cell load in the PB and, at least theoretically, the determination of cutoff values of mRNA expression of clinical relevance in cancer patients. However, the sensitivity and specificity of this approach both depend on the expression level of candidate biomarkers in tumor cells as well as their background expression in the blood [26
Evidence is rapidly accumulating that cancers are composed of heterogeneous populations of cells. Thus, one would predict that CTC might be enriched in cancer cells that express those biomarkers indicating the greatest invasive and metastatic capacity, including cancer stem cells (CSC) markers. Hence, the selection of appropriate target mRNAs that may be useful for clinical detection of CTC and CSC remains an important outstanding issue.
The current study was intended to assess the diagnostic performance of quantitative RT-PCR detection of AGR2 in the blood as a surrogate marker of CTC. We then hypothesized that a marker indicative of the phenotype of colonic stem cells, such as LGR5, would improve the detection of biologically and clinically relevant CTC.
We found that AGR2 mRNA was significantly elevated in the blood of patients with CRC compared to controls. ROC analysis suggested that at 94.7% specificity, AGR2 achieved 62.8% sensitivity in distinguishing CRC blood samples from the control group. Furthermore, in CRC patients, blood AGR2 mRNA levels correlated with different pathological prognostic factors, including pT3–pT4 depth of invasion and high-grade tumors.
These results are in line with the current evidence indicating that AGR2
can promote cancer growth, cell survival, migration and anchorage-independent growth and cellular transformation [14
]. In the clinical setting, AGR2
protein expression in the primary tumor is an independent prognostic indicator of poor outcome in patients with breast [29
] and prostate adenocarcinomas [30
], and one recent study showed that increased AGR2
protein in plasma is associated with ovarian cancer [31
However, to the best of our knowledge, no comprehensive report has been published about the potential prognostic relevance of AGR2 in colorectal cancer. Our findings indicate for the first time that the quantitative assessment of AGR2 mRNA in blood might indicate a poor patient outcome in CRC. Remarkably, in stage I to III patients, the risk of disease progression was higher with increasing levels of AGR2 in the blood. Likewise, in CRC patients with high AGR2 blood expression, the PFS was significantly reduced, and there was a numerical but non-significant inferior OS.
A recent study [32
] demonstrates that AGR2
induces the expression of the growth-promoting EGFR ligand amphiregulin in human adenocarcinomas. This effect is mechanistically mediated through Yes-associated protein (YAP1) dephosphorylation. Interestingly, YAP1 is also implicated in the regulation of stem cell division through the repression of the Hippo pathway. These data and a previous report [14
] show that proliferating and non-proliferating ISCs, as well as transit-amplifying cells from a secretory lineage express AGR2
and suggest additional mechanisms for oncogenic actions for AGR2
We next explored the expression of the ISC marker LGR5 in the blood of our cohort of controls and CRC patients. We found that LGR5 mRNA was significantly elevated in the blood of patients with colorectal carcinoma compared to controls. However, mean levels of LGR5 mRNA were similar in controls and early stage CRC patients. Nevertheless, there was a significant increase of LGR5 in blood obtained from metastatic CRC patients. When a cutoff point was defined based on the ROC curve, the LGR5 assay achieved only 18.8% sensitivity but 100% specificity in distinguishing CRC and control blood samples. Conversely, LGR5 mRNA in the blood showed a significant correlation with high-grade tumors, metastatic disease and R2 resections. Likewise, LGR5 expression in the blood showed a prognostic value regarding both PFS and OS in CRC patients, as suggested by the Cox regression and Kaplan-Meier analysis. In that sense, our results suggested that LGR5 is expressed only in a rare subset of CTC possibly including cancer stem-like cells. We could speculate that these circulating LGR5-expressing cells might contribute to cancer progression and therapeutic response.
The clinical and biological significances of LGR5
expressing-cells in CRC are poorly understood. A primary tumor profile that encompasses known ISC markers, such as LGR5
, has been strongly associated both with CRC stages and the occurrence of tumor relapse and metastasis [33
protein expression had been associated with a poor PFS in CRC patients [22
]. In contrast, in a recent report [34
] a gene signature defined by methylation silencing of the Wnt-driven ISC marker genes, including LGR5
, in CRC tumors was associated with a poor prognosis.
A number of proposed CSC markers, such as CD44 and CD133, have been explored in CTC detection. Recently, Iinuma H. et al
] demonstrated in patients with Dukes’ stage B and C CRC that the detection of CEA/Cytokeratins (CK) 19/20/CD133 mRNA in blood was useful for determining which patients were at high risk for recurrence and poor prognosis. However, in the CD133 single-marker analysis, no significant differences in OS and PFS were found [7
]. In metastatic CRC, the transcriptional amount of CD133 in blood before resection of hepatic metastases resulted in a high risk of dying of recurrence after apparently curative liver surgery [35
]. Nonetheless, CD133 and other putative markers for CRC stem cells such as CD44 are also expressed in a variety of cells including hematopoietic and/or endothelial cells (reviewed Hundt, S. in [27
]), a factor that could diminish their specificity. The expression patterns of LRG5 and colon differentiation markers such as cytokeratin−20 are mutually exclusive [33
] are of special interest for CTC detection. These facts strengthen the relevance of non-CK mRNA biomarkers for the detection of the most aggressive and specific subpopulations of CTC in CRC patients.
CTC in gastrointestinal cancer patients are increasingly detected when blood is obtained per- or intra-operatively [36
]. However, the postoperative sampling time might reflect the most relevant CTC status [4
]. In our study, blood samples were obtained several weeks after surgery. In order to explore the possible influence of recent surgery on the circulation of tumor cells, AGR2
levels were analyzed according to time intervals between surgery and blood sampling; conversely, no significant differences in biomarker mRNA levels between time intervals were found. From a clinical perspective, assessment of baseline prognostic factors and CTC detection rates may be of interest. In previous studies [38
] including patients with metastatic CRC, the number of CTC detected using the Cell Search System was associated with high LDH level, liver metastasis and poorer performance status. Hence, we performed an exploratory analysis in the subset of stage IV CRC patients, which showed no association between a positive mRNA result and baseline clinical prognostic subgroups categorized according to performance status, white blood cell count, alkaline phosphatase and number of metastatic sites. In addition, levels of AGR2
were not significantly different either.
The combined AGR2
assay resulted in an increased sensitivity (67.4%; AUC-ROC = 0.767; p
= 0.001) to separate cancer patients and controls. Remarkably, and in spite of the limited number of patients, Cox multivariate analysis demonstrated that AGR2
mRNA detection was a significant prognostic factor for PFS (HR, 2.8; 95% CI, 1.2 to 6.4; p
= 0.014). Thus, the transcriptional amount of AGR2
in the PB defined subgroups of CRC patients with significantly different risks of disease progression, improving the so-called biologic specificity [40
] of CTC detection.
Our findings indicate a high sensitivity and specificity for AGR2/LGR5 qRT-PCR for the surrogate detection of CTC in PB samples and it could be useful as a prognostic factor in patients with CRC. However, taking into account the design and sample size of the study, the outcome results could only be considered as generating a hypothesis. Additional possible limitations of this study must be considered. Although the inclusion of patients with different stages and residual tumor status could be considered limitations of the study, we suggest that this pragmatic design accurately reflects the patients attending the oncology clinic every day. Thus, the diagnostic performance of mRNA quantification has been estimated in a cohort of patients truly representative of those found in the clinical setting. However, to adequately assess the prognostic role, if any, of AGR2 and LGR5 mRNA levels in the blood, a larger, more homogeneous cohort of patients is clearly needed. Furthermore, a comparative study with immunofluorescence-based methods such as the Cell Search System is warranted.