Kaohsiung Medical University Institutional Repository:Item 310902000/9748

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Persistent Presence of Postoperative Circulating Tumor Cells

is a Poor Prognostic Factor for Patients with Stage I–III

Colorectal Cancer after Curative Resection

Yih-Huei Uen, MD,

1

Chien-Yu Lu, MD,

2

Hsiang-Lin Tsai, MD,

3,4

Fang-Jung Yu, MD,

2

Ming-Yii Huang, MD,

5,6

Tian-Lu Cheng, PhD,

7

Shiu-Ru Lin, PhD,

8

and Jaw-Yuan Wang, MD, PhD

4,6

1

Division of General Surgery, Department of Surgery, Chi Mei Foundation Medical Center, Taipei Medical University, Taipei, Taiwan

2

Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

3

Department of Emergency Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

4

Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 100 Tzyou 1st Road, Kaohsiung 807, Taiwan

5

Department of Radiation Oncology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

6

Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

7

Faculty of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan

8_{Department of Medical Research, Fooyin University Hospital, Kaohsiung, Taiwan}

Aim: To detect pre- and postoperative circulating tumor cells (CTCs) in stage I–III colo-rectal cancer (CRC) patients undergoing curative resection and so identify a subgroup of patients who are at high risk for relapse.

Methods: Four mRNA molecular markers including human telomerase reverse transcrip-tase, cytokeratin-19, cytokeratin-20, and carcinoembryonic antigen mRNA were used to de-tect CTCs in 438 CRC patients underwent curative resection.

Results: Out of 438 patients, 80 CRC patients were classiﬁed to preoperative ()/postop-erative (), 221 patients were preop()/postop-erative (+)/postop()/postop-erative (), while 137 patients were preoperative (+)/postoperative (+). Univariately, postoperative relapse was signiﬁcantly correlated with depth of invasion (P = 0.032), lymph node metastasis (P \ 0.001), vascular invasion (P = 0.001), perineural invasion (P = 0.013), and persistent presence of CTCs (P \ 0.001). Using a multivariate proportional hazards regression analysis, the presence of lymph node metastasis (P = 0.012; HR, 7.652; 95% CI: 4.162–14.827), vascular invasion (P = 0.033; HR, 4.360; 95% CI: 2.793–10.847), and the persistent presence of CTCs (P \ 0.001; HR, 29.486; 95% CI: 10.281–87.792) were demonstrated to be independent pre-dictors for postoperative relapse. Combination of these three independent prepre-dictors showed that patients with any one positive predictor had a hazard ratio of sevenfold to develop postoperative relapse (P \ 0.001; HR, 7.064; 95% CI: 4.354–11.464). Furthermore, the

Published online May 15, 2008.

Shiu-Ru Lin and Jaw-Yuan Wang contributed equally to this paper.

Address correspondence and reprint requests to: Jaw-Yuan Wang, MD, PhD; E-mail: [email protected]

Shiu-Ru Lin, PhD; E-mail: [email protected]

Published by Springer Science+Business Media, LLC 2008 The Society of Surgical Oncology, Inc.

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persistent presence of CTCs was strongly correlated with poorer relapse-free survival rates (all P\ 0.001).

Conclusion: The promising results of this study suggest that persistent presence of post-operative CTCs may be a crucial prognostic factor adjuvant to conventional tumor markers in CRC patients who have undergone curative resection. Identiﬁcation of these high-risk patients of persistent CTCs positivity is important and thus could help to deﬁne patients for adjuvant therapy with this tumor entity.

Key Words: Circulating tumor cells—Molecular markers—Colorectal cancer—Prognosis—

Postoperative surveillance.

Colorectal cancer (CRC) is the third most common cancer and is also the third major cause of cancer-related death in Taiwan, with over 8,000 new cases and 4,000 deaths per year.1In addition, this disease has one of the highest rates of increased incidence in Taiwan, gradually approaching Western figures in recent decades. Even with the recent advances in diagnostic and surgical techniques, the outcome re-mains poor in the cases of advanced disease, and only CRC diagnosed at an early stage is likely to be cured by surgical resection.2–4Pathologic prognostic factors of primary tumor invasion, regional lymph node involvement, and the presence or absence of metas-tasis have been used for many decades as the three major prognostic determinants for CRC patients, and predict the risk of relapse of this disease. Relapses have an important meaning in relation to survival for curative surgical intervention of CRC patients. Unfortunately, despite the curative resection for CRC patients, some patients with apparently local-ized disease at diagnosis will subsequently develop recurrent or metastatic diseases. It is important to note that as many as 25–40% of patients who un-dergo curative resection nevertheless subsequently develop metastatic disease, suggesting that unde-tected micrometastasis exists and may play a key role in relapse.5–7One of the major causes is the presence of disseminated tumor cells shed from the primary carcinoma into circulation prior to, during, or after surgery. The fact that the overall survival rate re-mains poor strongly suggests that the dissemination of these cells occurs early in the disease process and emphasizes the need for finding feasible diagnostic methods with sufficient sensitivity and specificity. Therefore, development of a sensitive, specific and convenient diagnostic method for detecting circulat-ing tumor cells (CTCs) at a very early stage could be used as postoperative surveillance, and ultimately affect future patient prognosis.

The most commonly used technique for the detection of nucleic acid material of disseminated tumor cells is the use of polymerase chain reaction

(PCR), reverse-transcriptase PCR (RT-PCR), or real-time quantitative PCR (Q-PCR) assays, which now permit sensitive detection of CTCs in peripheral blood. Accumulated reports have described the detection of CTCs in the peripheral blood of CRC patients, which has important prognostic and thera-peutic implications.8–12 Due to the heterogeneity of gene marker expression in blood and lymph nodes, a multimarker assay is regarded as more reliable and sensitive than a single-marker assay.13–16Our recently developed membrane array-based multimarker assay can detect CTCs in the peripheral blood of CRC patients; this is found to be a rational approach for the postoperative surveillance of CRC patients.17–20 Though many messenger RNA (mRNA) molecular markers have been evaluated as putative prognostic markers in CRC patients, no information about the multimarker assay [human telomerase reverse trans-criptase (hTERT), cytokeratin-19 (CK-19), cytoker-atin-20 (CK-20), and carcinoembryonic antigen (CEA)] in the detection of CTCs as a prognostic tool for CRC patients undergoing curative resection has been obtained. The aim of this study was to detect both pre- and postoperative CTCs in peripheral blood of stage I–III CRC patients who had under-gone curative resection by a panel of molecular markers using a constructed membrane array meth-od, and evaluate whether persistence of CTCs posi-tivity after primary CRC excision was related to clinical outcome.

PATIENTS AND METHODS

Patients and Sample Collection

Included in this prospective study were Interna-tional Union against Cancer (UICC) stage I–III CRC patients admitted to the Department of Surgery of Kaohsiung Medical University Hospital for elective surgery between January 2002 and December 2005. Patients with other malignant diseases in their medical

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history were excluded. Of a total 461 stage I–III CRC patients, 16 were lost to follow-up, 4 had surgical mortality, and 3 with surgical resection margin posi-tive for tumor invasion were excluded. The remaining 438 stage I–III CRC patients (234 males and 204 fe-males; mean age 65.6 ± 13.1 years) with curative resection for the primary lesion were finally enrolled in the present study. Curative resection was defined as any gross residual tumor that did not remain in the surgical bed and in which the surgical resection margin was pathologically negative for tumor invasion. Pa-tients diagnosed as either high-risk stage II or III re-ceived adjuvant chemotherapy. Patients with risk factors for relapse (tumor poorly differentiated, tumor perforation, number of lymph nodes examined \12 or lymphatic/vascular invasion) were considered as high-risk stage II cases. Patients were administered three to six 8-week cycles of adjuvant chemotherapy. Each cycle consisted of leucovorin (LV) 100 mg/m2 admin-istered as a 2-h infusion and given weekly for three to six cycles, and 5-ﬂuorouracil (5-FU) 500 mg/m2 administered as an intravenous bolus 1 h after the start of LV infusion and repeated weekly for six doses. This cycle was then repeated after a 2-week rest period. CTCs in peripheral blood of these 438 patients were detected using our constructed membrane array method. Postoperative surveillance consisted of med-ical history, physmed-ical examination, and laboratory studies including serum CEA levels every 3 months. Abdominal ultrasonography or computed tomogra-phy was performed every 6 months, and chest radi-ography and total colonoscopy were performed once a year. Patients were followed up at 3-monthly intervals for 2 years and 6-monthly intervals thereafter; median follow-up was 44 months (range 21–66 months). The development of new recurrent or metastatic lesions after operation was defined as a postoperative relapse. The type of postoperative relapse was designated as local recurrence (tumor growth restricted to the anastomosis or the region of primary operation) or distant metastases (distant metastases or diffuse peri-toneal seeding).

A 4-ml sample of peripheral blood was obtained from each CRC patient preoperatively (1 day prior to operation) and postoperatively (1 week after opera-tion) for total RNA isolation. To prevent contami-nation of epithelial cells, peripheral blood samples were obtained through a catheter inserted into a peripheral vessel, and the ﬁrst 5 ml of blood were discarded. Written informed consent was obtained from each subject and/or guardian. Sample acquisi-tion and subsequent use were also approved by the hospital’s institutional review board. Clinical stage

and pathological features of primary tumors were deﬁned according to the criteria of the American Joint Commission on Cancer/International Union Against Cancer (AJCC/UICC).21

Detection of Serum CEA

Additional 3 ml peripheral blood samples from 438 CRC patients were obtained less than 1 week prior to operation (preoperative) and 4 weeks after operation (postoperative). Serum CEA levels were determined by means of an enzyme immunoassay test kit (DPC Diagnostic Product Co., Los Angeles, CA) with the upper limit of 5 ng/ml deﬁned as normal according to the manufacturers of the kits that were used.

mRNA Isolation and First Strand cDNA Synthesis Total RNA was extracted from the fresh whole blood of preoperative/postoperative CRC patients using a QIAmp RNA Blood Mini Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer’s instructions. The RNA concentration was determined spectrophotometrically on the basis of absorbance at 260 nm. First strand cDNA was synthesized from total RNA by using a RT-PCR kit (Promega Corp., Madison, WI).

Membrane Arrays

The procedure of the membrane array method for the detection of CTC-related mRNA molecular markers was performed according to our recent study.17,20,22 Patients overexpressing all four molec-ular markers by membrane-array methods were considered as positive results of CTCs.20In our pre-vious investigation, the sensitivity limit of this tech-nique was established at approximately one tumor cell per 106 white blood cells (5 cells per 1 ml blood).17,22

Statistical Analysis

All data were statistically analyzed using the Sta-tistical Package for the Social Sciences, version 12.0 (SPSS Inc., Chicago, IL). A P value less than 0.05 was considered statistically signiﬁcant. The univariate analysis of clinicopathologic features and expression of preoperative/postoperative molecular markers be-tween the two groups (relapse group versus non-relapse group) was compared using the chi-square test. Expression of molecular markers was also

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ana-lyzed according to types of postoperative relapse, either local recurrence or distant metastasis. Inde-pendent prognostic factors for postoperative relapse were determined using a multivariate Cox propor-tional hazards regression analysis. The combination of lymph node metastasis, vascular invasion, and presence of postoperative CTCs as predictors of postoperative relapse was analyzed using the chi-square test. The relapse-free survival rates of CRC patients were further categorized according to the tumor location. The relapse-free survival rates were calculated by the Kaplan–Meier method, and the differences in survival rates were analyzed by the log-rank test.

RESULTS

Two hundred and thirty-four men (53.4%) and 204 women (46.4%) were recorded. The average age was 65.6 years (range 27–90 years). Two hundred and eighty-two tumors (64.4%) were in the colon and 156 (35.6%) in the rectum. With regard to the histological type of these tumors, 42 (9.6%) were well ated, 342 (78.1%) were moderately well differenti-ated, and 54 (12.3%) were poorly differentiated carcinomas. The clinicopathologic characteristics of these 438 UICC stage I–III CRC patients are listed in Table1; 66 patients were subsequently diagnosed with stage I CRC, 188 with stage II, and 184 with stage III. Overall, 80 of 438 (18.3%) patients were identiﬁed to have no detectable CTCs in neither preoperative nor postoperative peripheral blood; 221 of 438 patients (50.4%) were found to have detectable CTCs in preoperative but not in postoperative peripheral blood; 137 of 438 patients (31.3%) had CTCs in both preoperative and postoperative peripheral blood.

Table2 shows the distribution of preoperative/ postoperative serum carcinoembryonic antigen levels and presence of CTCs according to stage (I–III) of CRC patients. The frequency of abnormal serum preoperative CEA level (‡5 ng/ml) for UICC stage I, II, and III patients was 12.1% (8/66), 46.8% (88/188), and 48.4% (89/184) respectively. Meanwhile, the corresponding values for abnormal serum postoper-ative CEA levels were 4.5% (3/66), 10.6% (20/188), and 14.7% (27/184) respectively. The frequency of preoperative CTCs positivity for UICC stage I, II, and III patients was 33.3% (22/66), 84.6% (159/188), and 96.2% (177/184) respectively. Simultaneously, the corresponding values for frequency of postoper-ative CTCs positivity were 6% (4/66), 30.3% (57/

188), and 41.3% (76/184), respectively. Our results show that only 60 (13.7%) of 185 patients with abnormal serum preoperative CEA level were not found to convert to normal serum CEA level, whereas 137 (38.3%) of 358 patients with preopera-tive presence of CTCs were observed to have persis-tent CTCs positivity despite later curative resection. Distribution of preoperative/postoperative serum CEA levels and presence of CTCs is not related to the tumor location. During the follow-up period, 79 of 282 (28%) colon cancer patients and 51 of 156 (32.7%) rectal cancer patients were identified with postoperative relapse. Of 130 CRC patients with postoperative relapse, 38 patients had local recurrent and 92 patients had distant metastasis diseases.

From the correlation between postoperative re-lapse and clinicopathologic features or the persistent presence of CTCs positivity of 438 CRC patients using univariate analyses, we found the depth of

TABLE 1. Clinicopathologic characteristics of 438 colorec-tal cancer patients undergoing curative resection

Variables Number (%)

Gender

Male/female 234 (53.4)/204 (46.4) Age (years)

\65/‡65 192 (43.8)/246 (56.2)

Maximum tumor size (cm)

\5/‡5 225 (51.4)/213 (48.6)

Tumor location

Colon/rectum 282 (64.4)/156 (35.6) Depth of tumor invasion

T1+T2/T3+T4 96 (21.9)/342 (78.1)

Lymph node metastases

No/yes 254 (58.0)/184 (42.0) UICC stage I/II/III 66 (15.1)/188 (42.9)/ 184 (42.0) Vascular invasion No/yes 309 (70.5)/129 (29.5) Perineural invasion No/yes 333 (76.0)/105 (24.0) Differentiation Well/moderately/poorly 42 (9.6)/342 (78.1)/ 54 (12.3) Type of tumor Adenocarcinoma/mucinous 409 (93.4)/29 (6.6) Presence of CTCs positivity Preoperative ()/postoperative ()/ Preoperative (+)/postoperative ()/ Preoperative (+)/postoperative (+) 80 (18.3)/221 (50.4)/ 137 (31.3) Preoperative colonic obstruction/perforation No/yes 413 (94.3)/25 (5.5) Postoperative relapse No/yes 308 (70.3)/130 (29.7) Patients with postoperative relapse

UICC stage I/II/III 3 (2.3)/54 (41.5)/73 (56.2) CTCs, circulating tumor cells; UICC, International Union against Cancer.

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invasion (P = 0.032), the presence of lymph node metastasis (P \ 0.001), the presence of vascular invasion (P = 0.001), the presence of perineural invasion (P = 0.013), and the persistent presence of CTCs positivity (P \ 0.001; Table3) to show signif-icant correlation. Moreover, no clinicopathologic parameters were signiﬁcantly different between the colon and rectal cancer patients in the groups with or without postoperative relapse. Using a Cox propor-tional hazard regression analysis, the presence of lymph node metastasis (P = 0.012; HR, 7.652; 95% CI: 4.162–14.827), vascular invasion (P = 0.033; HR, 4.360; 95% CI: 2.793–10.847), and the persistent presence of CTCs positivity (P \ 0.001; HR, 29.486; 95% CI: 10.281–87.792) were demonstrated to be independent predictors for postoperative relapse (Table4). Moreover, the combination of presence of lymph node metastasis, vascular invasion, and per-sistent CTCs positivity as high-risk predictors of postoperative relapse is shown in Table5. CRC pa-tients with one high-risk predictor had a relative risk of 7.064 of developing postoperative relapse com-pared to those without any high-risk predictor (P \ 0.001; HR, 7.064; 95% CI: 4.354–11.464). Fur-thermore, statistically significant difference was ob-served in terms of relapse-free survival rate between CRC patients with and those without persistent presence of CTCs positivity using the log-rank test, in all patients with CRC, and in colon cancer or rectal cancer (Fig.1; all P \ 0.001). Patients with a failed conversion of the preoperative detectable CTCs to the postoperative undetectable CTCs showed the worst relapse-free survival rate when compared with the other two groups (P \ 0.001).

DISCUSSION

The recent identification of genes overexpressed in CRC, combined with advances in molecular biology, provides the opportunity to establish more sensitive, specific, and cost-effective ways of identifying meta-static disease. Among the current possibilities, one of the most compelling is the development of a highly sensitive molecular diagnostic procedure that permits the detection of tumor cells in different tissues and biologic fluids, especially peripheral blood. Because metastasis is such a key process, there is an enormous effort to identify markers that can detect dissemi-nated tumor cells in the circulation, which will aid early diagnosis. However, the heterogeneity of the expression of tumor genes and the variable perfor-mance of these assays has posed major problems for the detection of CTCs. Our membrane array assay was able to simultaneously detect a panel of infor-mative molecular markers for the presence of CTCs in CRC patients undergoing curative resection, with advantages in terms of time saving and cost effec-tiveness.20,23 The current investigation has demon-strated that patients identified with persistent presence of CTCs postoperatively using our multi-marker membrane array method exhibit higher inci-dence of postoperative relapse and poorer relapse-free survival rate. Even patients with preoperative CTCs positivity would eventually attain a better prognosis after curative resection when their post-operative CTCs convert to negativity. Because the low frequency (10.4%) of abnormal serum CEA levels could be determined at 4 weeks after operation, the considerably higher frequency (31.3%) of CTCs

TABLE 2. Distribution of preoperative/postoperative serum carcinoembryonic antigen levels and presence of circulating tumor cells according to various stages colon and rectal cancer patients

CEA levels

Pvalue*

Presence of CTCs positivity

Pvalue*

Stage I Stage II Stage III Stage I Stage II Stage III

N(%) N(%) N(%) N(%) N(%) N(%) Preoperative Preoperative \5 ng/ml 58 (87.9) 100 (53.2) 95 (51.6) Negative 44 (66.7) 29 (15.4) 7 (3.8) Colon 42 (24.0) 67 (38.3) 66 (37.7) 0.774 Colon 32 (56.1) 20 (35.1) 5 (8.8) 0.941 Rectum 16 (20.5) 33 (42.3) 29 (37.2) Rectum 12 (52.2) 9 (39.1) 2 (8.7) ‡5 ng/ml 8 (12.1) 88 (46.8) 89 (48.4) Positive 22 (33.3) 159 (84.6) 177 (96.2) Colon 6 (4.8) 59 (47.2) 60 (48.0) 0.889 Colon 15 (6.0) 109 (43.8) 125 (50.2) 0.909 Rectum 2 (3.3) 29 (48.3) 29 (48.3) Rectum 7 (6.4) 50 (45.9) 52 (47.7) Postoperative Postoperative \5 ng/ml 63 (95.5) 168 (89.4) 157 (85.3) Negative 62 (94.0) 131 (69.7) 108 (58.7) Colon 46 (17.0) 115 (42.6) 109 (40.4) 0.797 Colon 44 (19.4) 98 (43.2) 85 (37.4) 0.518 Rectum 17 (14.4) 53 (44.9) 48 (40.7) Rectum 18 (24.3) 33 (44.6) 23 (31.1) ‡5 ng/ml 3 (4.5) 20 (10.6) 27 (14.7) Positive 4 (6.0) 57 (30.3) 76 (41.3) Colon 2 (6.1) 13 (39.4) 18 (54.5) 0.993 Colon 3 (2.9) 43 (41.0) 59 (56.2) 0.954 Rectum 1 (5.9) 7 (41.2) 9 (52.9) Rectum 1 (3.1) 14 (43.8) 17 (53.1)

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positivity at 1 week would play a crucial role in the early detection of micrometastasis and CTCs. Re-cently, our study also revealed that molecular detec-tion of postoperative CTCs is helpful in the early prediction of postoperative relapse in CRC patients with normal perioperative serum CEA levels, with a median lead time of 6 months before the measure-ment of abnormal CEA levels.20Consequently, detecting preoperative/postoperative CTCs seems to be an auxiliary diagnostic tool to conventional serum tumor marker-CEA in the early identiﬁcation of high-risk stage I–III CRC patients having undergone curative resection.

In our current study, CTCs positivity for UICC stage I, II, and III CRC patients was observed with an incidence of 6%, 30.3%, and 41.3% respectively. The detection incidence of our constructed membrane ar-ray methods is almost approaching the incidence of presence of postoperative relapse in stage I, II, and III. The detection incidence of the membrane array in stage I, II, III CRC patients were 6%, 30.3%, and 41.3%. And the incidence of postoperative relapse in stage I, II, and III CRC patients were 2.3%, 28.7%, and 39.7%. Consistent with our findings, Patel et al. have disclosed that 30% of stage I + II and 65% of stage III CRC patients could be identified with CTCs at 1 week after operation, either by CEA or CK-20 mRNA markers.11They pointed out that no signifi-cant reduction in RT-PCR positivity was detected after apparently complete tumor excision among pa-tients with lymph-node-positive tumors (Dukes’ C) in whom there was a high probability of recurrence, compared with the significant reduction detected in node-negative tumors (Dukes’ A or B), where the risk of recurrence was lower. Likewise, multivariate analysis indicated that detection of the simulta-neous presence of CEA/CK-20 mRNA by RT-PCR or Q-PCR in tumor drainage blood is a potent

TABLE 3. Correlation between postoperative relapse and clinicopathologic features of colorectal cancer patients using univariate analysis

Postoperative relapse (+) Postoperative relapse ()

Pvalue** Colon (N = 82) (%) Rectum (N = 48) (%) P value* Colon (N = 200) (%) Rectum (N = 108) (%) P value* Gender Male/female 44/38 22/26 0.389 108/92 60/48 0.794 0.469 Age (years) \65/‡65 27/55 21/27 0.217 92/108 52/56 0.718 0.058 Maximum size (cm) \5/‡5 37/45 25/23 0.443 101/89 52/56 0.470 0.317

Depth of tumor invasion

T1+T2/T3+T4 14/68 6/42 0.486 50/150 26/82 0.857 0.032

Lymph node metastases

No/yes 27/55 14/34 0.656 141/59 72/36 0.487 \0.001 Vascular invasion No/yes 67/15 39/9 0.948 129/71 74/34 0.478 0.001 Perineural invasion No/yes 70/12 40/8 0.757 143/57 81/27 0.510 0.013 Differentiation Well/moderately/poorly 10/58/14 4/37/7 0.740 18/160/22 10/87/11 0.975 0.077 Type of tumor Adenocarcinoma/mucinous 74/8 44/4 0.787 189/11 102/6 0.984 0.154 Presence of CTCs positivity Preoperative ()/postoperative ()/ Preoperative (+)/postoperative ()/ Preoperative (+)/postoperative (+) 4/24/54 2/12/34 0.842 52/115/33 22/70/16 0.443 \0.001

Preoperative colonic obstruction/perforation

No/yes 76/6 54/2 0.355 189/11 104/4 0.485 0.245

* Results of two-sided Pearson v2test between colon and rectal cancer patients; ** results of two-sided Pearson v2test of overall colorectal cancer patients between postoperative relapse (+) and () groups; CTCs, circulating tumor cells.

TABLE 4. Correlation between postoperative relapse and clinicopathologic features of colorectal cancer patients using

multivariate Cox proportional hazard regression analysis

Variables Hazard ratio 95% CI Pvalue Lymph node metastases

(yes/no) 7.652 4.162–14.827 0.012 Vascular invasion (yes/no) 4.360 2.793–10.847 0.033 Presence of CTCs positivity (yes/no) 29.486 10.281–87.792 \0.001

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prognostic factor independent of the traditional path-ologic parameters.10,24,25Furthermore, the disease-free and/or overall survival of patients with CEA/CK-20-positive peripheral blood was signiﬁcantly shorter than

that of marker-gene-negative patients.10,24,25 Similar results were obtained where detection of CTCs in blood samples of patients with stage II CRC identiﬁed patients with poor outcome.23,26,27

Relapse-free survival (years)

6 5 4 3 2 1 0

Pre- and postoperative markers (-) N = 80

Preoperative markers (+) and postoperative markers (-) N = 221

Pre- and postoperative markers (+) N = 137 P< .001 Probab ility 1.0 .8 .6 .4 .2 0.0 Probabi li ty 1.0 A B C .8 .6 .4 .2 0.0 Probabi li ty 1.0 .8 .6 .4 .2 0.0

Pre- and postoperative markers (+) N = 92 P < .001

6 5 4 3 2 1 0

Pre- and postoperative markers (+) N = 45 P < .001

FIG. 1. Relapse-free survival rates of stage I–III colorectal cancer patients analyzed by the Kaplan–Meier method with the differences compared by a log-rank test according to tumor location. (A) All 438 stage I–III colorectal cancer patients with a failed conversion of the preoperative detectable CTCs to the postoperative undetectable were found to have the worst overall survival rate while patients with preoperative/postoperative undetectable CTCs were found to have the best relapse-free survival rate among the three groups (P \ 0.001); (B) All 282 stage I–III colon cancer patients with a failed conversion of the preoperative detectable CTCs to the postoperative undetectable CTCs showed the worst relapse-free survival rate when compared with the other two groups (P \ 0.001); (C) All 156 stage I–III rectal cancer patients with a failed conversion of the preoperative detectable CTCs to the postoperative undetectable CTCs showed the worst relapse-free survival rate when compared with the other two groups (P \ 0.001).

TABLE 5. Combination of the depth, vascular invasion and presence of circulating tumor cells as predictors of postoperative relapse for stage I-III colorectal cancer patients following curative resection

Lymph node (+) or vascular invasion (+) or presence of CTCs positivity (+)

No. of relapse patients (n = 130)

No. of non-relapse patients

(n = 308) Hazard ratio 95% CI Pvalue Any one predictor

Positive 103 108 7.064 4.354–11.464 \0.001

Negative 27 200

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In contrast, some recently published studies report conflicting results regarding the prognostic value of CTCs.28,29A major problem of most of the published studies is that only small, inhomogeneous patient groups with relatively short follow-up periods were evaluated. Moreover, the methods used for CTCs detection also need to be taken into account, as sensitivity and specificity are of major importance and may differ significantly.20,23,30 Our membrane array assay is unlikely to be 100% accurate in pre-dicting postoperative relapse, suggesting that there is room for the improvement of this method. Recently, Khair et al. proposed that the discrepancies in the published results of many PCR studies discussed in this review are most likely because of one or more of the following factors:30 (1) the method of blood sample preparation—some studies have measured CTC by using extracellular markers and others intracellular; (2) time lapse between sample collection and processing, as variations in handling are likely to affect the viability and expression of mRNA in par-ticular; and (3) PCR conditions, i.e., single or multi-ple, number of PCR cycles and whether the PCR is standard, nested or real time. Of three factors, the timing of blood sampling is particularly important in the molecular detection of CTCs. Surgical manipu-lation probably enhances this release of hematogenic tumor cells into the circulation.10 Therefore, the detection of CTCs in blood samples taken during surgery is unstable and unreliable. Patel et al. ob-tained blood samples before and until 3 months after surgery and reported a significant decrease of the CTC-positive patients at 24 h after surgery in Dukes’ A/B CRC patients.11 Analogously, cancer cells experimentally injected into the peripheral vein of mice rapidly decreased with time and were detected 3 days but not 7 days after injection.31 One recent report also suggests the detection of circulating can-cer cells in peripheral blood at 7–10 days after sur-gery was associated with significantly increased risk of recurrence.32 However, Allen-Mersh et al. point out that RT-PCR CTCs-positivity within 24 h of primary CRC resection is a strong predictor of CRC recurrence, and may be useful clinically.33 From the viewpoint of surgical manipulation and clearance of CTCs after operation, it is significant that a direct association was observed between the time when the specimen was obtained and molecular detection of tumor cells.

Combining three independent prognostic markers identiﬁed in our patient cohort, including the pres-ence of lymph node metastasis, the prespres-ence of vas-cular invasion and postoperative persistent presence

of CTCs positivity, stage I–III CRC patients have a sevenfold risk of developing postoperative relapse compared with those without any high-risk predictor. Concomitant molecular diagnosis of CTCs with a multimarker panel is a justifiable supplementary ap-proach to the current pathological staging system, which may help physicians make appropriate judg-ments on clinical management and predictive prog-nosis for stage I–III CRC patients following operation. Additionally, a positive correlation with pathological parameters compatible with more aggressive tumors and CTCs was observed in patients with tumor mRNA present in plasma.12,34 In the future, these criteria might enable the selection of high-risk patients who would beneﬁt from adjuvant treatment. Hence, therapeutic decision-making mod-els are likely to be further redeﬁned by the inclusion of perioperative changes of such mRNA markers.

In conclusion, the constructed membrane array method for the detection of CTCs has been demon-strated to be complementary to conventional serum CEA level for the surveillance of stage I–III CRC patients. We suggest that CRC patients with persis-tent positive CTC blood samples should be evaluated for further adjuvant therapeutic strategies after sur-gery. However, multicenter and long-term clinical follow-up is warranted to address the potential clin-ical significance.

ACKNOWLEDGEMENTS

The authors would like to thank Drs. Jan-Sing Hsieh, Deng-Chyang Wu, Yu-Chung Su, Jeng-Yih Wu, and Che-Jen Huang for contributing materially to the paper. This study was supported by a grant from the Chi Mei Medical Center and Kaohsiung Medical University Research Foundation (97CM-KMU-03).

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