**4.2. Lung cancer**

**4. The applications of CTC testing in clinical cancer researches**

types.

160 Tumor Metastasis

**4.1. Breast cancer**

PR status), which are very useful clinically.

As aforementioned, CTC testing are designed to help the diagnosis, early detection and monitoring for response and disease status of cancer patients. Clinical trials to evaluate and validate are inevitable during the developing of any CTC testing. Here, we introduce several important clinical validated studies for the clinical impacts of CTC testing in different cancer

One meta‐analysis reported by Liao et al. [107], 14 studies with 2336 patients were enrolled and found that presence of CTCs in peripheral blood was significantly associated with the size of tumor [OR 0.68, 95% confidence interval (CI) (0.54, 0.87), *P* = 0.002], tumor grade [OR 0.71, 95% CI (0.55, 0.91), *P* = 0.006], estrogen receptor (ER) status [OR 0.72, 95% CI (0.57, 0.91), *P* = 0.007], and progesterone receptor (PR) of tumor status [OR 0.78, 95% CI (0.61, 0.98), *P* = 0.04]. In addition, the presence of CTCs is highly correlated with tumor size, tumor grade, ER, and PR status in patients with breast cancer. Although the analysis did not consider the method of isolation which might be one of the downsides and biases of the analysis, the results suggested a trend of physical (tumor size), functional (tumor grade) and status of drugable targets (ER,

In Zhao et al. [234] performed a meta‐analysis collecting 24 trials with 4013 breast cancer patients and 1333 controls. Poor overall survival was found to be associated with the positive CTC detection (HR = 3.00 [95% CI 2.29–3.94], *P* < 0.0001) and recurrence‐free survival as well (HR = 2.67 [95% CI 2.09–3.42], *P* < 0.0001). CTC‐positive breast cancers were significantly associated with high histological grade (HR = 1.21 [95% CI 1.09–1.35], *P* < 0.0001), tumor size (>2 cm) (HR = 1.12 [95% CI 1.02–1.22], *P* = 0.01), and nodal status (≥1) (HR = 1.10 [95% CI 1.00– 1.21], *P* = 0.037). The studies, different to that of Liao et al. [107], mentioned about prognostic values of CTC testing. However, the two reports did not mention about the isolation methods and might neglect the biases from CTC number is highly correlated to the method of isolation.

For the purpose of technical standardization, Janni et al. [235] conducted a pooled analysis of individual data from 3173 patients with nonmetastatic (stages I–III) breast cancer from five breast cancer institutions. The prevalence and numbers of CTCs were assessed at the time of primary diagnosis with the FDA‐cleared CellSearch System. Results confirmed that ≥1 CTC(s) were detected in 20.2% of the patients and CTC‐positive patients had larger tumors, increased lymph node involvement, and a higher histologic tumor grade than did CTC‐negative patients (all *P* < 0.002). Multivariate Cox regressions confirmed that the presence of CTCs was an independent prognostic factor for disease‐free survival [HR, 1.82; 95% confidence interval (CI), 1.47–2.26], distant disease‐free survival (HR, 1.89; 95% CI, 1.49–2.40), breast cancer‐specific survival (HR, 2.04; 95% CI, 1.52–2.75), and overall survival (HR, 1.97; 95% CI, 1.51–2.59). The study addressed the clinical impacts of CellSearch™ system in breast cancer patients and it

has confirmed the positive results from a large pooled database.

In a meta‐analysis reported in 2013, pooled results from a total of 20 studies, comprising 1576 nonsmall cell lung cancer (NSCLC) patients showed that CTCs were associated with lymph node metastasis (OR = 2.06; 95% CI: 1.18–3.62; *Z* = 2.20; *P* = 0.027) and tumor stage (OR = 1.95; 95% CI: 1.08–3.54; *Z* = 2.53; *P* = 0.011). CTCs were significantly associated with shorter overall survival (relative risk [RR] = 2.19; 95% CI: 1.53–3.12; *Z* = 4.32; *P* < 0.0001) and progression‐free/ disease‐free survival (RR = 2.14; 95% CI: 1.36–3.38; *Z* = 3.28; *P* < 0.0001) [281]. Another study reported the ability to recurrence prediction after curative surgery is positive [282].

For small cell lung cancer, a relatively aggressive subtype with poor prognosis population, a total of seven papers covering 440 SCLC patients were combined in the final analysis. The meta‐analysis revealed that CTCs were significantly associated with shorter overall survival (HR = 1.9; 95% CI: 1.19–3.04; *Z* = 2.67; *P* < 0.0001) and progression‐free survival (HR = 2.6; 95% CI: 1.9–3.54; *Z* = 6.04; *P* < 0.0001) [283].

Interestingly, in a molecular era nowadays, cancer therapy often relies on genetic or molecular information from cancer tissues, CTCs as well. Das et al. [105] checked the status of ERCC1 expression on captured and found that low expression of ERCC1 on CTCs correlates with progression‐free survival (PFS) in patients with metastatic NSCLC receiving platinum‐based therapy. ERCC1 expression was conventionally checked on NSCLC cancer tissue to predict the response to platinum therapy, which has been the first line standard chemotherapy in patients without active EGFR mutation responding to tyrosine kinase inhibitors (TKIs). The impacts of the study suggested that analysis of ERCC1 expression on CTCs in lung cancer patients could predict the chemotherapy responses. It is a predictive role could possible direct therapy in the future if the findings were confirmed in another large‐scale phase III clinical trials. In addition. Yanagita et al. [284] evaluated CTCs and cfDNA in EGFR‐mutant NSCLC patients treated with erlotinib until progression. Among the enrolled 60 patients, rebiopsy was performed in 35/44 patients (80%), with paired CTC/cfDNA analysis in 41/44 samples at baseline and 36/44 samples at progression. T790M was identified in 23/35 (66%) of tissue biopsies and 9/39 (23%) of cfDNA samples. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression‐free survival. Therefore, cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first‐line EGFR TKIs. Recently, *ALK* rearrangement on CTCs are successfully performed and compared with cancer tissues [51, 285]. Chromosome instability and *ROS‐1* rearrangement on CTCs were also proved to be successful [51]. Immune cells analysis, tumor‐associated macrophages (TAMs) accompanied with CTCs analysis were also proven to be possible and CTCs are competent to specifically manipulate TAMs to increase cancer invasiveness, angiogenesis, immunosup‐ pression and possibly lipid catabolism in lung cancer patients [286]. These studies pointed to the driven mutation detection and would directly benefit to NSCLC patients under targeted therapies.

#### **4.3. Gastrointestinal tract cancer**

In 2014, a meta‐analysis comprised 26 studies with peripheral blood samples of 1950 cases for final analysis. The pooled results showed that gastric cancer (GC) patients with detectable CTCs (including circulating miRNAs) had a tendency to experience shortened RFS (HR = 2.91, 95% CI [1.84–4.61], I2 = 52.18%). As for patient deaths, we found a similar association of CTC (including circulating miRNAs) presence with worse OS (HR = 1.78, 95% CI [1.49–2.12], I2 = 30.71%, *n* = 30). Additionally, subgroup analyses indicated strong prognostic powers of CTCs, irrespective of geographical, methodological, detection time and sample size differences of the studies [287]. In addition, the role of EMT status on CTCs correlates with poor treatment outcomes was also revealed. CTCs expressing CD44 were also found to be prognostic and indicated to malignant behaviors of gastric cancer [288].

For pancreatic cancer, a prospective study addressing the role of CTCs, CTMs in 63 pancreatic ductal adenocarcinoma (PDAC) patients before treatment using anti‐EpCAM (epithelial cell adhesion molecule)‐conjugated supported lipid bilayer‐coated microfluidic chips. CTM was an independent prognostic factor of overall survival (OS) and progression free survival (PFS). Patients were stratified into unfavorable and favorable CTM groups on the basis of CTM more or less than 30 per 2 ml blood, respectively. Patients with baseline unfavorable CTM, compared with patients with favorable CTM, had shorter PFS (2.7 versus 12.1 months; *P* < 0.0001) and OS (6.4 versus 19.8 months; *P* < 0.0001). Differences persisted if we stratified patients into early and advanced diseases. The number of CTM before treatment was an independent predictor of PFS and OS after adjustment for clinically significant factors. Therefore, in conclusion, the number of CTM, instead of CTCs, before treatment is an independent predictor of PFS and OS in patients with PDAC [272].

In molecular analysis to predict treatment response, Abdallah et al. [152] found that thymi‐ dylate synthase expression in circulating tumor cells can be useful tool as a 5‐FU resistance predictor biomarker in patients with colorectal cancer while other studies elucidate the prognostic and predictive roles of CTCs [198, 289–291]. Recently, *KRAS* and *BRAF* were successfully detected on CTCs by high‐resolution melt (HRM) and allele‐specific PCR (ASPCR) and *KRAS*‐codon 12/13‐ and *BRAF*‐codon 600‐specific assays. Comparing tumor tissues and CTCs mutation status using HRM, Mohamed Suhaimi et al. [292] reported that a 84.1% concordance in KRAS genotype (*P* = 0.000129) and a 90.9% (*P* = 0.174) concordance in BRAF genotype. Another report utilized ISET system plus PCR for KRAS codons 12 and 13 mutation with a 71% concordance between cancer tissue and CTCs from colorectal cancer patients [293]. In gastrointestinal stroma tumor, Li et al. [294] conducted a trial to elucidate the role of CTCs expressing ANO1(DOG1) in GIST. ANO1s were more frequently detected in unresectable patients. Tumor size, mitotic count, and risk level were associated with ANO1 detection in resectable GIST patients. The presence of ANO1 significantly correlated with poor disease‐free survival (15.3 versus 19.6 months, *P* = 0.038). Most patients turned ANO1‐negative after surgery and inversely, all 21 patients with recurrence turned ANO1‐positive with high ANO1 expression levels. Moreover, in the neoadjuvant setting, decline of ANO1 expression level correlated with the response of imatinib. In the near future, these results would possibly promote the genetic analysis on mutation‐driven cancer therapies although they have not become routine screen tools in CRC patients to date.

#### **4.4. Head and neck cancer**

patients without active EGFR mutation responding to tyrosine kinase inhibitors (TKIs). The impacts of the study suggested that analysis of ERCC1 expression on CTCs in lung cancer patients could predict the chemotherapy responses. It is a predictive role could possible direct therapy in the future if the findings were confirmed in another large‐scale phase III clinical trials. In addition. Yanagita et al. [284] evaluated CTCs and cfDNA in EGFR‐mutant NSCLC patients treated with erlotinib until progression. Among the enrolled 60 patients, rebiopsy was performed in 35/44 patients (80%), with paired CTC/cfDNA analysis in 41/44 samples at baseline and 36/44 samples at progression. T790M was identified in 23/35 (66%) of tissue biopsies and 9/39 (23%) of cfDNA samples. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression‐free survival. Therefore, cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first‐line EGFR TKIs. Recently, *ALK* rearrangement on CTCs are successfully performed and compared with cancer tissues [51, 285]. Chromosome instability and *ROS‐1* rearrangement on CTCs were also proved to be successful [51]. Immune cells analysis, tumor‐associated macrophages (TAMs) accompanied with CTCs analysis were also proven to be possible and CTCs are competent to specifically manipulate TAMs to increase cancer invasiveness, angiogenesis, immunosup‐ pression and possibly lipid catabolism in lung cancer patients [286]. These studies pointed to the driven mutation detection and would directly benefit to NSCLC patients under targeted

In 2014, a meta‐analysis comprised 26 studies with peripheral blood samples of 1950 cases for final analysis. The pooled results showed that gastric cancer (GC) patients with detectable CTCs (including circulating miRNAs) had a tendency to experience shortened RFS (HR = 2.91, 95% CI [1.84–4.61], I2 = 52.18%). As for patient deaths, we found a similar association of CTC (including circulating miRNAs) presence with worse OS (HR = 1.78, 95% CI [1.49–2.12], I2 = 30.71%, *n* = 30). Additionally, subgroup analyses indicated strong prognostic powers of CTCs, irrespective of geographical, methodological, detection time and sample size differences of the studies [287]. In addition, the role of EMT status on CTCs correlates with poor treatment outcomes was also revealed. CTCs expressing CD44 were also found to be prognostic and

For pancreatic cancer, a prospective study addressing the role of CTCs, CTMs in 63 pancreatic ductal adenocarcinoma (PDAC) patients before treatment using anti‐EpCAM (epithelial cell adhesion molecule)‐conjugated supported lipid bilayer‐coated microfluidic chips. CTM was an independent prognostic factor of overall survival (OS) and progression free survival (PFS). Patients were stratified into unfavorable and favorable CTM groups on the basis of CTM more or less than 30 per 2 ml blood, respectively. Patients with baseline unfavorable CTM, compared with patients with favorable CTM, had shorter PFS (2.7 versus 12.1 months; *P* < 0.0001) and OS (6.4 versus 19.8 months; *P* < 0.0001). Differences persisted if we stratified patients into early and advanced diseases. The number of CTM before treatment was an independent predictor of PFS and OS after adjustment for clinically significant factors. Therefore, in conclusion, the

therapies.

162 Tumor Metastasis

**4.3. Gastrointestinal tract cancer**

indicated to malignant behaviors of gastric cancer [288].

Grobe et al. [92] used CellSearch™ for CTC isolation in 80 oral cavity cancer patients and found that 12.5% patients harbored CTCs in peripheral blood, whereas in 20.0% patients DTCs in bone marrow could be detected. Significant correlations could be found for CTCs and tumor size (*P* = 0.04), nodal status and DTCs (*P* = 0.02), and distant metastasis with CTCs (*P* = 0.004) and DTCs (*P* = 0.005). Univariate and multivariate analyses revealed that CTCs and DTCs were significant and independent predictors of recurrence‐free survival (*P* < 0.001) as well as in other findings in HNSCC, including the ability of prediction 6‐month death [231]. In 2015, Oliveira‐ Costa et al. reported that immunohistochemistry was performed in cancer tissues and in CTCs by immunofluorescence and Nanostring. Correlation was shown between PD‐L1 and tumor size and lymph node metastasis, HOXB9 and tumor size, BLNK and perineural invasion, and between ZNF813 and perineural invasion. PD‐L1 positivity was an independent prognostic factor in this cohort (*P* = 0.044, HH = 0.426) in OSCC patients [295]. The results could possibly apply to current immune‐oncology studies.

Wu et al. [296] reported a meta‐analysis conducted a computerized retrieval of literatures. Twenty‐two retrieved studies were eligible for systematic review, of which nine conformed for the diagnostic test meta‐analysis and five for the prognostic analysis. Subgroup analysis showed 24.6% pooled sensitivity and 100% pooled specificity of detections by using positive selection strategy, which moreover presented low heterogeneity. The presence of CTC was significantly associated with shorter disease free survival (DFS, HR 4.62, 95% CI 2.51–8.52). The presence of CTC indicates a worse DFS.

### **4.5. Liver cancer**

Early in 2004, Vona et al. have reported that the presence (*P* = 0.01) and number (*P* = 0.02) of CTCs and microemboli (CTMs) were significantly associated with a shorter survival [161]. Fan et al. [297] reported a meta‐analysis consisting of 23 trials and found that CTC positivity was significantly associated with RFS (HR 3.03, 95% CI: [1.89–4.86]; *P* < 0.00001) and overall survival (OS) (HR 2.45, 95% CI: [1.73–3.48]; *P* < 0.00001). CTC positivity were also significantly associ‐ ated with TNM Stage (RR 1.30, 95% CI: [1.02–1.65]; *P* = 0.03), Tumor size (RR 1.36, 95% CI: [1.09–1.69]; *P* = 0.006), Vascular invasion (RR 1.99, 95% CI: [1.43–2.77]; *P* < 0.0001), Portal vein tumor thrombus (RR 1.73, 95% CI: [1.42–2.11]; *P* = 0.0001), Serum alpha‐fetoprotein (AFP) level (RR 2.05; *P* = 0.01) [297]. Sun et al. found that Stem cell‐like phenotypes are observed in EpCAM <sup>+</sup> CTCs, and a preoperative CTCs of ≥2 is a novel predictor for tumor recurrence in hepatocel‐ lular carcinoma (HCC) patients after surgery, especially in patient subgroups with AFP levels of ≤400 ng/ml or low tumor recurrence risk. EpCAM+ CTCs could serve as a real‐time parameter for monitoring treatment response and a therapeutic target in HCC recurrence [137]. The prognostic value of overall survival of CTCs in HCC patients has been also revealed [298].

## **4.6. Genitourinary tract cancer**

Rink et al. (2012) found that using CellSearch™, CTC were detected in 23 of 100 patients (23%) with nonmetastatic urothelial carcinoma of urinary bladder. CTC‐positive patients had significantly higher risks of disease recurrence and cancer‐specific and overall mortality (*P* values ≤ 0.001). After adjusting for effects of standard clinicopathologic features, CTC posi‐ tivity remained an independent predictor for all end points (hazard ratios: 4.6, 5.2, and 3.5, respectively; *P* values ≤ 0.003). HER2 positivity was found in 3 of 22 patients (14%). There was concordance between CTC, primary tumors, and lymph node metastases in all CTC‐positive cases (100%).

### **4.7. Skin cancer and melanoma**

Conventionally, melanoma cells lack of cytokeratin or EpCAM expression and CTCs by definition are very difficult to identify. However, investigators broke through the strait by combination with CTCs plus cfDNA. Salvianti et al. [299] enrolled 84 melanoma patients and 68 healthy controls for CTC and cell‐free DNA (cfDNA) testing to assess the diagnostic performance of a tumor‐related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs. The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (*P* < 0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. When the CTCs plus RASSF1A cfDNA are jointly considered, a higher sensitivity of the detection of positive cases in invasive and metastatic melanomas could be obtained. A similar finding was obtained to suggest combine cfDNA (GNAQ/GNA11 mutations) and CTCs to identify uveal melanoma patients with poor prognosis [300]. In another reports, a phase III trial of adjuvant immunotherapy after complete resection of stage IV melanoma, quantitative real‐time reverse‐transcriptase polymerase chain reaction (qPCR) for expression of CTC‐specific MART‐1, MAGE‐A3, and PAX3 mRNA biomarkers were found to be not associated with known prognostic factors or treatment arm. In multivariate analysis, pretreatment CTC (>0 versus 0 biomarker) status was significantly associated with disease‐free survival (DFS; HR 1.64, *P* = 0.002) and overall survival (OS; HR 1.53, *P* = 0.028). Serial CTC (>0 versus 0 biomarker) status was also significantly associated with DFS (HR 1.91, *P* = 0.02) and OS (HR 2.57, *P* = 0.012) [301]. The report suggested CTCT could be a new risk factor other than any conventional known factors, which might change the staging systems if the evidence gets solid and validated.
