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Journal of Gastrointestinal Surgery ISSN 1091-255X J Gastrointest SurgDOI 10.1007/s11605-013-2258-6

Postoperative Detection of CirculatingTumor Cells Predicts Tumor Recurrence inColorectal Cancer Patients

Gennaro Galizia, Marica Gemei, MicheleOrditura, Ciro Romano, Anna Zamboli,Paolo Castellano, Andrea Mabilia,Annamaria Auricchio, et al.

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ORIGINAL ARTICLE

Postoperative Detection of Circulating Tumor Cells PredictsTumor Recurrence in Colorectal Cancer Patients

Gennaro Galizia & Marica Gemei & Michele Orditura & Ciro Romano &

Anna Zamboli & Paolo Castellano & Andrea Mabilia & Annamaria Auricchio &

Ferdinando De Vita & Luigi Del Vecchio & Eva Lieto

Received: 11 May 2013 /Accepted: 11 June 2013# 2013 The Society for Surgery of the Alimentary Tract

AbstractIntroduction Circulating tumor cells are thought to play a crucial role in the development of distant metastases.Their detection in the blood of colorectal cancer patients may be linked to poor outcome, but current evidence iscontroversial.Materials and Methods Pre- and postoperative flow cytometric analysis of blood samples was carried out in 76 colorectalcancer patients undergoing surgical resection. The EpCAM/CD326 epithelial surface antigen was used to identify circulatingtumor cells.Results Fifty-four (71 %) patients showed circulating tumor cells preoperatively, and all metastatic patients showed highlevels of circulating tumor cells. Surgical resection resulted in a significant decrease in the levels of circulating tumor cells.Among 69 patients undergoing radical surgery, 16 had high postoperative levels of circulating tumor cells, and 12 (75 %)experienced tumor recurrence. High postoperative level of circulating tumor cells was the only independent variable related tocancer relapse. In patients without circulating tumor cells, the progression-free survival rate increased from 16 to 86 %, with areduction in the risk of tumor relapse greater than 90 %.Conclusions High postoperative levels of circulating tumor cells accurately predicted tumor recurrence, suggesting thatassessment of circulating tumor cells could optimize tailored management of colorectal cancer patients.

Gennaro Galizia and Marica Gemei contributed equally to this work.

Electronic supplementary material The online version of this article(doi:10.1007/s11605-013-2258-6) contains supplementary material,which is available to authorized users.

G. Galizia (*) :A. Zamboli : P. Castellano :A. Mabilia :A. Auricchio : E. LietoDivision of Surgical Oncology—“F. Magrassi - A. Lanzara”Department of Clinical and Experimental Medicine and Surgery,Second University of Naples School of Medicine, c/o II Policlinico,Edificio 17, Via Pansini 5, 80131 Naples, Italye-mail: gennaro.galizia@unina2.it

M. Gemei : L. Del VecchioCEINGE—Advanced Biotechnologies, Naples, Italy

M. Orditura : F. De VitaDivision of Medical Oncology - “F. Magrassi - A. Lanzara”Department of Clinical and Experimental Medicine and Surgery,Second University of Naples School of Medicine, Naples, Italy

C. RomanoDivision of Internal Medicine, Allergy and Clinical Immunology,Department of Medical and Surgical Sciences,Second University of Naples School of Medicine,Naples, Italy

L. Del VecchioDepartment of Biochemistry and Medical Biotechnologies(DBBM), University of Naples ‘Federico II’,Naples, Italy

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Keywords Colorectal cancers . Circulating tumor cells .

Flow cytometry . Tailored treatments

Introduction

Colorectal cancer (CRC) still represents the second cause ofcancer death inWestern countries.1 Almost 20–25 % of CRCpatients have distant metastases at presentation, and a further30 % will experience distant metastases even after potential-ly curative surgery.2 Despite improvements in surgical tech-niques and chemotherapeutic regimens, distant metastasesremain the leading cause of death.3 Current diagnostic tech-niques, including tumor biomarkers, are often unable toprovide early information about ongoing metastasis and toaccurately predict outcome.4 To improve the survival ofCRC patients, new diagnostic methods able to predict me-tastasis formation and to provide information about real-timeanti-tumor treatment efficacy are eagerly awaited. The met-astatic process can be portrayed as a two-phase system: celltranslocation from primary tumor to distant organs and col-onization, respectively.5 The first phase implies the presenceof cancer cells in the peripheral blood, the so-called circu-lating tumor cells (CTCs);6 their detection in patients withbreast, prostate, lung, and colorectal tumors has recentlybecome a reality.7,8 Evaluation of CTCs is currently a fasci-nating technique which is expected to allow better identifi-cation of high-risk patients and to tailor more effectivetreatments.9 Indeed as shown by both a recent meta-analysis and single series, CTC detection in CRC patientshas been associated with more advanced tumor stage, de-creased survival rate, and chemotherapy failure.10,11 How-ever, due to a considerable degree of heterogeneity amongstudies, the clinical value of CTC detection is still limitedand controversial.10 Pitfalls in CTC evaluation include timeof sampling, cutoff values, paucity of validated markers, andlack of adequate sensitivity for current techniques.12–14 Spe-cifically, very few studies have focused on the relationshipbetween pre- and postoperative CTC counts, with cutoffvalues used to categorize patients ranging widely.15 In addi-tion, some techniques, such as immunohistochemistry (IHC)and reverse transcriptase-polymerase chain reaction (RT-PCR), have been limited by low rates of sensitivity andspecificity.16,17 The CellSearchTM system (Veridex), the onlyFDA-approved method to detect CTCs, has not yet beenapproved by European agencies, and its use requires dedi-cated equipment and kits. On the contrary, engineering prog-ress in flow cytometry automation has now resulted in theavailability of instruments capable to precisely individuateand enrich cells of interest, providing availability of cell-specific monoclonal antibodies. Indeed flow cytometry al-lows detection thresholds as low as one cell in 20 mL of

blood; moreover, cells can also be sorted out and collectedfor further investigations.7,18

EpCAM (epithelial cell adhesion molecule), also knownas CD326 antigen, a 40-kDa transmembrane glycoproteininvolved in cell adhesion, has been proposed for identifica-tion of CTCs.19 The CD326 antigen is widely expressed onthe basolateral surface of epithelial and carcinomatous cells;on the contrary, tumors originating from non-epithelial cellslack CD326 expression. Thus, CD326 can be used as adiagnostic marker for various epithelial cancers.

Interestingly, CD326 is also believed to play a role intumorigenesis and metastasis, thus possibly being a candi-date prognostic marker as well as a potential target forimmunotherapeutic strategies.19,20

In this study, we used a relatively inexpensive and high-throughput technique such as flow cytometry to run aCD326-based easy-to-perform assay, which is potentiallyimplementable in clinical routine. CTC detection was eval-uated pre- and postoperatively in CRC patients to assess theeffects on long-term outcome.

Materials and Methods

Patients

Patients with histologically proven colorectal adenocarcino-ma, referred to our institution from July 2009 to June 2012,were initially eligible for this study. Eighteen patients unfitfor surgery or with unresectable tumor were excluded inorder to avoid inappropriate comparisons with patients whohad resected tumor. Seventy-six consecutive patients (46colon and 30 rectal tumors) entered the study. According topreoperative staging, 12 (40 %) rectal cancer patientsunderwent neo-adjuvant chemoradiotherapy (CRT). Sixty-nine (91 %) patients, including 13 liver-limited metastatictumor cases, underwent potentially curative resection.21 Inthe remaining seven patients, including five patients withdistant metastases, the primary tumor was resected, al-though surgery was judged to be non-curative due to localor distant diffusion.

CTC Detection

Blood samples were collected just before and at 1 monthafter surgery. Peripheral blood samples (7.5 mL) were dilut-ed 1:2 with phosphate buffered saline (PBS) withoutCa++/Mg++. Diluted samples were stratified upon 3 mL ofFicoll-Hystopaque (Sigma-Aldrich, St Louis, MO, USA) ina 15 mL polypropylene tube and centrifuged for 30 min at400g. Mononucleated cells (i.e., lymphocytes and circulat-ing cancer cells) were recovered after centrifugation andwashed two times with 2 mL of PBS at 2,000 rpm for

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3 min. All recovered cells were stained with saturatingamounts of fluorochrome-conjugated monoclonal antibodies(anti-CD326/EpCAM PerCP-Cy5.5 and anti-CD45 APC-Cy7, both from Becton Dickinson, San Jose, CA, USA)and anti-CD326/EpCAMmagnetic beads (Miltenyi Biotech,Auburn, CA, USA). Cells were incubated for 1.5 h on iceand then washed twice with 2 mL of a magnetic separationbuffer composed of PBS plus 0.5 % BSA and 2 mM EDTA.Cells were then resuspended in 1 mL magnetic separationbuffer and loaded upon equilibrated magnetic separation MScolumns (Miltenyi) in the magnetic field of a MACS sepa-rator (Miltenyi). Finally, columns were washed with 2 mL ofmagnetic separation buffer, and following the removal of themagnetic field, CD326/EpCAM+cells were eluted with2 mL of magnetic separation buffer. Cells were immediatelyanalyzed by flow cytometry on a BD FacsARIA III cellsorter (Becton Dickinson). For identification of CTCs, cellswere first gated on the basis of physical parameters (forwardscatter versus side scatter) to exclude debris. Doublet exclu-sion was not performed to avoid cell loss during analysis.Subsequently, in a CD45 versus EpCAM/CD326 dot plot,EpCAM/CD326+/CD45− epithelial circulating cells could beidentified (Fig. 1). Specifically, CTCs stained positive forEpCAM/CD326, but negative for CD45, which is theleukocyte-common antigen and is therefore found only onleukocytes.

Researchers were blinded to the patients’ characteristics,and results were expressed as the number of CTCs per7.5 mL of blood.

Follow-up

All patients were discharged from the hospital andunderwent appropriate postoperative treatment and follow-up. The following clinicopathological parameters wererecorded: age, sex, tumor site, serum carcinoembryonic an-tigen (CEA) levels, performance status, neoadjuvant CRT,postoperative chemotherapy and complication rate, tumorsize, TNM staging system and Dukes’ stage,22 number ofharvested and positive nodes, lymph node ratio,23 histologic

differentiation (well, moderate, or poor), oncologicalradicality, recurrence, and survival rates. No patient was lostto follow-up, and it was complete by December 31, 2012. Allpatients gave their written informed consent, and the studywas approved by the Department of Clinical and Experimen-tal Medicine and Surgery of the Second University ofNaples.

End-points

Primary end-points included accuracy of the cytometric tech-nique, relationship between pre- and postoperative CTClevels, and their correlations with outcome. Progression-freesurvival (PFS) rate, deemed to be a valid surrogate end-pointfor overall survival in CRC, was used to investigate therelationship between CTC levels and tumor recurrence.24,25

Secondary end-points included the relationship between CTClevels and other prognostic factors.

Statistical Analysis

Statistical analysis was carried out using the SPSS statisticalpackage (SPSS Inc., Chicago, IL, USA). In all analyses, thesignificance level was specified as p<0.05. Data wereexpressed as means ± SD (standard deviation), range, andmedian values. The equality of group means and compari-sons between proportions were analyzed by paired Student’st-test and chi-square test, respectively.

To define a prognostic cutoff with respect to the numberof detected CTCs, the internal validation method wasapplied.26 The results obtained in the first 40 patients (train-ing set) were used to select a cutoff level, which was thenvalidated in the next 36 patients (validation set). The receiveroperating characteristic (ROC) curve analysis was used bothto confirm the internal validation and to calculate the accuracyof CTC levels to discriminate the presence of distant metasta-ses, radical operation, and tumor recurrence. Univariate anal-ysis related to PFS was limited to radically resected patientsand determined by log-rank test (Mantel–Cox). Curves wereplotted using the Kaplan–Meier method; p values and hazard

Fig. 1 EpCAM/CD326+ CTCidentification. This picturereports representativecytometric dot plots of EpCAM/CD326 versus CD45 expressionin pre- and postoperativeperipheral blood samples. CTCshave been identified asEpCAM/CD326+/CD45−

(CTCs gate) in pre- (a) andpostoperative (b) sample of thesame patient

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ratio (HR) with 95 % confidence interval (CI) were pro-vided. The independent significance of prognostic vari-ables was determined by multivariate analysis, using theCox’s proportional-hazards model. A stepwise multivariateanalysis was performed in order to estimate the cumulativehazard functions.

Results

Overall, preoperative CTC levels ranged from 0 to 130 per7.5 mL of peripheral blood (mean 16.01±27.3, 95 % CI9.76–22.26, median 8). The optimal prognostic CTC thresh-old was initially determined in the training set. Both HR anddifference in 3-year PFS were greatest when comparinggroups with zero to two CTCs with those with ≥3 CTCsper 7.5 mL of blood [HR=0.25; p=0.06 (Table e1)]. Subse-quently, the CTC threshold was tested in the validation set,which had similar clinicopathological characteristics to thoseof the training set (Table e2). PFS rates were not significantlydifferent between the validation and the training set(p=0.64). The cutoff value from the training set was con-firmed to be the best level to separate two different prognos-tic groups in the validation set. In addition, ROC curveanalysis identified the cutoff level of ≥3 CTCs per 7.5 mLblood as the value with the highest accuracy for predictingPFS (Fig. e1).

Table 1 Clinico-pathological characteristics of the series and relationsbetween preoperative CTC levels and other factors

CTC negative,22 patients

CTC positive,54 patients

p valuea

Ageb

<66 12 26 0.80

>66 10 28

Gender

Male 14 34 0.83

Female 8 20

Tumor site

Right colon 4 9 0.67

Left colon 11 22

Rectum 7 23

Serum CEA levels (ng/mL)c

<3.5 18 27 0.02

>3.5 4 27

Performance status

0 13 25

1 5 24 0.17

2 4 5

Neoadjuvant CRT

No 18 46 0.98

Yes 4 8

Rectum only

No 3 15 0.53

Yes 4 8

Adjuvant chemotherapy

No 5 15 0.86

Yes 17 39

Postoperative complications

No 22 51 0.63

Yes 0 3

Tumor size (cm)b

<4 13 30 0.97

>4 9 24

pT stage

1 5 1

2 0 17 0.001

3 11 21

4 6 15

pN stage

Node negative 14 23 0.15

Node positive 8 31

pM stage

M0 22 36 0.005

M1 0 18

Dukes’ stage

A 5 15

B 9 6 0.001

C 8 15

Table 1 (continued)

CTC negative,22 patients

CTC positive,54 patients

p valuea

D 0 18

Histologic differentiation

Well 11 25 0.15

Moderate 11 21

Poor 0 8

Radical resection

Yes 22 47 0.18

No 0 7

Tumor recurrence

No 20 38 0.16

Yes 2 14

Survival

Yes 22 44 0.07

No 0 10

Performance status according to the ECOG scale

CEA carcinoembryonic antigen, CRT chemoradiotherapya Chi-square testbMedian valuec Normal value in healthy subjects

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Overall, CTCs were not detectable in 14 (18 %) patients,counts were one (three patients) or two (five patients) in eight(11 %) patients, whereas >2 CTCs were recorded in theremaining 54 (71 %) patients. Thus, according to the cutoffvalue, 22 (29 %) patients were CTC-negative, while 54 pa-tients were CTC-positive (Table 1). High preoperative CTClevels tended to be more frequent in undifferentiated,unresectable, and relapsing tumors, while neoadjuvant CRTdid not affect CTC counts. On the contrary, preoperative CTClevels were significantly associated with elevated serum CEAlevels as well as with advanced pT and Dukes’ stage. Of note,all metastatic patients were CTC-positive [test sensitivity100 % (p=0.01)], whereas not all CTC-positive patients haddetectable metastatic disease (only about one third of them,specifically). Thus, CTC-negative patients were conceived tobe not metastatic (Table e3). At 1 month after tumor resection,

CTC levels were found to have dropped significantly [mean1.92±3.06, 95 % CI 1.22–2.62, range 0 to 12, median0 (p<0.001 vs. preoperative levels)]. In no case did postoper-ative CTC levels increase after surgery. Fifty-eight (76 %)patients were recorded as CTC-negative, whereas 18 (24 %)remained CTC-positive. Remarkably, among 69 patients un-dergoing potentially curative surgery, 16 (23 %) still hadelevated postoperative CTC levels, suggesting that postoper-ative CTC levels do not mirror surgical and/or pathologicalradicality (p=0.81), with tumors supposed to have been rad-ically resected still spreading through the bloodstream.

Analysis Related to Outcome

To date, ten (13 %) patients have died of cancer. The 1- to 3-year overall survival rates were 96, 85, and 81%, respectively.

>

>

0

2

4

6

8

10

12

14

16

18

20

0

2

4

6

8

10

12

14

16

18

20

a

b

preOperative postOperative

preOperative postOperative

Fig. 2 Comparison among pre-and postoperative CTC levels in69 colorectal cancer patientsundergoing potentially curativesurgery: a 53 non-recurrentpatients, b 16 recurrent patients

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All patients who died were CTC-positive preoperatively(ROC curve, p=0.09), and all but two patients showed highpostoperative CTC levels (ROC curve, p<0.001). Among 69patients undergoing potentially curative resection, 16 (23 %)

patients suffered from disease recurrence. The 1- to 3-yearPFS rates were 89.6, 76.1, and 68.3 %, respectively. Theperformance of postoperative CTC counts in predicting tumorrecurrence was impressive: test accuracy 88.4 % (Table e3).

Table 2 Univariate analysisrelated to progression-freesurvival in 69 colorectal cancerpatients undergoing potentiallycurative surgery

Performance status according tothe ECOG scale

CEA carcinoembryonic antigen,CRT chemoradiotherapy, LNRthe ratio between node-positiveand resected nodesaMedian valueb Normal value in healthysubjectsc Only node-positive patients andaccording to the cutoff valuedetermined in23

Number ofpatients

42 monthsPFS, %

Hazardratio

95 % CI,hazard ratio

p value

Age ≤66/>66 yearsa 36/33 61/76 2.72 0.93–6.68 0.06

Gender M/F 43/26 67/71 0.96 0.34–2.67 0.94

Tumor site // //

Right colon 13 60

Left colon 27 75 0.68

Rectum 29 76

Serum CEA levels (ng/mL)b

≤3.5 43 83 0.36 0.12–0.94 0.03

>3.5 26 48

Performance status 0/1/2 35/27/7 57/82/100 // // 0.18

Neoadjuvant CRT, no/yes 58/11 74/43 0.51 0.10–1.73 0.23

Adjuvant chemotherapy, no/yes 20/49 100/58 0.00 0.08–0.69 0.008

PO complications, no/yes 66/3 71/0 0.12 0.03–0.21 <0.001

Tumor size ≤4/>4 cma 43/26 67/71 0.92 0.33–2.58 0.88

pT stage

1 6 100

2 17 82 // // 0.30

3 31 56

4 15 68

1–2/3–4 23/46 84/62 0.28 0.13–1.09 0.07

pN stage, negative/positive 37/32 84/53 0.25 0.10–0.73 0.01

Dukes’ stage

A 20 100

B 15 84 // // 0.007

C 21 53

D 13 40

A–B/C–D 35/34 93/48 0.12 0.06–0.49 <0.001

Number of harvested nodesa

≤15 33 60 2.02 0.75–5.45 0.16

>15 36 75

Lymph node ratioc

≤0.1818 12 73 0.43 0.14–1.68 0.25

>0.1818 20 45

Histologic differentiation

Well 32 84

Moderate 31 61 // // 0.41

Poor 6 55

Preoperative CTC levels

Negative 22 89 0.25 0.12–1.00 0.05

Positive 47 58

Postoperative CTC levels

Negative 53 86 0.06 0.00–0.04 <0.001

Positive 16 16

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Indeed among 16 patients showing high postoperative CTClevels, 12 (75 %) relapsed, while only four (7 %) out of 53patients with normal postoperative CTC levels experienceddisease recurrence (Fig. 2). Preoperative CTC positivity wasmarginally related to poor outcome; on the contrary, highpostoperative CTC levels were strongly correlated withtumor relapse (Table 2). The HR for PFS was 0.06 innegative patients, corresponding to an increase in the rateof progression-free survival at 3 years from 16 to 86 %and a reduction of relative risk of tumor relapse greater than90 % (Fig. 3).

On multivariate analysis, high postoperative CTC levelwas the only independent prognostic factor correlated withan increased tumor recurrence rate (Table 3). The strongstatistical power of this variable covered the prognosticsignificance of other factors known to affect long-term out-come, such as Dukes’ stage and presence of distant disease.

Of note, preoperative CTC levels failed to reach independentsignificance. Stepwise multivariate analysis confirmed post-operative CTC level as the only covariate related to long-term outcome. Assuming early Dukes’ stages, absence ofdistant metastases, and normal postoperative CTC levelsas 0 risk of tumor relapse, the cumulative 42-month hazardof cancer recurrence recorded in the complete series wasshown to be significantly lower than that observed in ad-vanced tumor stages and high postoperative CTC levels(Fig. 4).

Discussion

Distant metastases remain the main cause of death in CRCpatients.2 This complex process implies traveling of tumorcells into the bloodstream; thus, their detection may possibly

0 6 12 18 24 30 36 42

100

80

60

40

20

0

months

p < 0.0001

CTCs negative

CTCs positive

Number at riskCTCs negative 53 53 47 35 33 13 9 3

CTCs positive 16 15 9 4 3 2 2 1

Fig. 3 One- to 3-yearprogression-free survival rate in69 colorectal cancer patientsundergoing potentially curativesurgery according topostoperative CTC status CTC-negative vs. CTC-positive:HR=0.06

Table 3 Multivariate analysisrelated to progression-freesurvival in 69 colorectal cancerpatients undergoing potentiallycurative surgery

Multivariate analysis wasperformed including variableswith significant value onunivariate analysis

CI confidence interval

Coefficient Standard error Hazard ratio 95 % CI,hazard ratio

p value

Cox’s proportional-hazards model

Elevated serum CEA levels 1.2113 0.8082 3.35 0.69–16.23 0.13

Adjuvant chemotherapy 1.1055 1.6560 3.02 0.11–76.29 0.50

PO complications 0.0591 0.6617 1.06 0.29–3.85 0.92

Node positive 0.1457 0.9795 1.15 0.17–7.81 0.88

Distant metastases 1.6164 0.9153 5.03 0.84–30.00 0.07

Advanced Dukes’ stage 1.7163 1.0025 5.56 0.78–39.30 0.08

High preoperative CTC levels 0.2314 1.0691 1.26 0.15–10.13 0.82

High postoperative CTC levels 2.3284 1.0413 10.26 1.34–78.18 0.02

Stepwise multivariate analysis

High postoperative CTCs levels 2.8051 0.5853 16.52 5.27–71.55 <0.001

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be useful both to identify cancer patients with misdiagnosedor at high risk of distant metastases and to tailor therapy.12

CTCs were first discovered by Ashwort in 1869 and sub-stantiated by Engell in 1955.27,28 Afterwards, this aspect ofcancer biology remained unaddressed for many years due toconsiderable difficulty in identifying CTCs. However, CTCdetection in the peripheral blood has recently becomepossible,7,8,15 but techniques used to identify CTCs are tooexpensive and time consuming to be used in clinicalroutine.29 Moreover, nucleic acid analysis of cells derivedfrom peripheral blood samples is largely affected by inclu-sion of several contaminants so that current techniques af-ford only a low purity grade.13 On the contrary, flow cytom-etry allows single-cell analysis and permits to include orexclude from the analysis cell populations of doubtful originat any time after sample acquisition.

In this study, we used a cytometric assay able to identifyCTCs in a simple and reliable fashion so as to be possibly usedin cancer management as already described in hemato-oncology.30–32 Anti-CD45 staining allowed ruling out of he-matopoietic cells, whereas detection of the EpCAM/CD326surface antigen unequivocally identified epithelial cells.33–36

Indeed EpCAM/CD326 has been shown to be adequate toidentify CTCs despite the potential pitfall of the so-calledepithelial-to-mesenchymal transition (EMT) cell program.19

EMT has been observed in tumor cells that are prone to enterthe peripheral circulation, with loss of epithelial characteristicsand acquisition of mesenchymal identity.37 In case of EMT,CTCs from colorectal cancers can be supposed to escaperecognition by antibodies directed against epithelial antigens.However, as recently demonstrated, EMT is a dynamicprocess, and most CTCs bear both epithelial and mesen-chymal characteristics, thus maintaining EpCAM/CD326antigen expression.38

A crucial issue is identification of the optimal thresholdfor prognostic evaluation of CTC. In metastatic breast, pros-tate, and non-small-cell lung cancers, a CTC count of ≥5 has

been used to define an unfavorable group,9,39 while in earlybreast cancer this value has been reduced to ≥1.40 Usinginternal validation, a cutoff value of ≥3 CTCs per 7.5 mLof blood was shown to yield the lowest HR and the highestdifference in the 3-year PFS when compared with otherthreshold values. Of note, it perfectly matched with thecutoff value previously identified by others.8,12 In addition,in 25 healthy subjects, we found that only two (8 %) had oneCTC and none had ≥2 CTCs, thus suggesting high specificityfor the technique used in this study.

Previous research, mainly using IHC and RT-PCR tech-niques, reported a rate of preoperative CTC detection rang-ing from 15 to 65 % of the cases.10,41,42 Besides, a recentmeta-analysis, comprising 16 studies and 1,491 metastaticCRC patients, reported a median percentage of cancer cell-positive patients of 35 % (range 10–77 %).15 In our study,CTCs were preoperatively detected in 62 (82 %) CRC pa-tients, and 54 patients (71 %) had ≥3 CTCs per 7.5 mL ofblood. In addition, all patients with distant metastases wereCTC-positive preoperatively, confirming the high sensitivityand reliability of the cytometric technique. Remarkably, nodifferences were seen in the levels of CTCs between colonand rectal cancers, although rectal cancers are believed to farmore likely metastasize via the bloodstream, thus calling forgreater caution in this assumption. Although the small num-ber of patients in each tumor stage prevents robust evidences,it is interesting to note that early CRC stages were associatedwith a non-negligible positivity for preoperative CTCs,suggesting that individual colon cancer cells can acquirethe ability to spread through the bloodstream early.5 Howev-er, only one third of positive patients had distant metastases,confirming the current assumption that the detection oftumor cells in the peripheral blood does not necessarilyimply metastatization at distant sites.5,10,15

Most of previous investigations focused only on preoper-ative CTC assessment and its relationship with pooroutcome.12,15 However, postoperative analysis has been

Fig. 4 Three-year cumulativehazard function of cancerrelapse according to highpostoperative CTC levels,distant metastases, advancedDukes’ stages, and completeseries

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suggested to better reflect the most relevant CTC statusbecause it combines preoperative status, intraoperative bloodspreading, and rapid apoptotic death of shed cells.10 In ourexperience, preoperative CTC levels were demonstrated tobe associated with advanced tumor stage but were onlymarginally related to long-term survival. On the contrary,high postoperative CTC levels were shown to be stronglycorrelated with tumor relapse and to be the only independentprognostic factor of poor outcome. To date, only two studieshave provided separate data on prognosis of CRC patientsbased on pre- and postoperative CTC detection. The resultswere slightly in favor of postoperative sampling, thusneeding to be substantiated by further studies.10,43,44 Consis-tently, here we provide statistical evidence that postoperativeCTC detection is more accurate than preoperative assessmentfor prediction of prognosis. Specifically, 75 % of patients withhigh postoperative CTC levels experienced tumor recurrence;however, four relapsing patients were CTC-negative, thussuggesting that lack of postoperative detection of CTCs cannotcompletely exclude tumor recurrence.

A novel oncologic issue is represented by the prospect tomodulate chemotherapy according to CTC detection. Pre-liminary reports have shown that assessment of CTC levelsduring chemotherapy may help identify patients who couldbenefit from a specific treatment, and this hypothesis iscurrently under evaluation.45–47

In conclusion, flow cytometry was demonstrated to be high-ly accurate in detecting CTCs in CRC patients. PreoperativeCTC-negative patients were certainly non-metastatic; surgeryplayed a critical role in the treatment of CRC patients, althoughpotentially curative resection was not synonymous to CTCnegativity. Postoperative CTC levels strongly predicted tumorrecurrence, albeit normal postoperative CTC levels did notcompletely exclude the chance of tumor relapse. Further studiesare mandatory to refine the identification of CTCs and to assesstheir impact on tailored chemotherapeutic regimens.

References

1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CACancer J Clin 2013;63:11–30.

2. Manfredi S, Lepage C, Hatem C, Coatmeur O, Faivre J, BouvierAM. Epidemiology and management of liver metastases fromcolorectal cancer. Ann Surg 2006;244:254–259.

3. de Haas RJ, Wicherts DA, Andreani P, Pascal G, Saliba F, Ichai P,Adam R, Castaing D, Azoulay D. Impact of expanding criteria forresectability of colorectal metastases on short- and long-term out-comes after hepatic resection. Ann Surg 2011;253:1069–1079.

4. Zlobec I. Novel biomarkers for the prediction of metastasis incolorectal cancer. Expert Opin Med Diagn 2013;7:137–146.

5. Chaffer CL, Weinberg RA. A perspective on cancer cell metas-tasis. Science 2011;331: 1559–1564.

6. Yokobori T, Iinuma H, Shimamura T, Imoto S, Sugimachi K, IshiiH, Iwatsuki M, Ota D, Ohkuma M, Iwaya T, Nishida N, Kogo R,Sudo T, Tanaka F, Shibata K, Toh H, Sato T, Barnard GF, Fukagawa

T, Yamamoto S, Nakanishi H, Sasaki S, Miyano S, Watanabe T,Kuwano H,Mimori K, Pantel K, Mori M. Plastin3 is a novel markerfor circulating tumor cells undergoing the epithelial–mesenchymaltransition and is associated with colorectal cancer prognosis. CancerRes 2013;73:2059–2069.

7. Allard WJ, Matera J, Miller MC, Repollet M, Connelly MC, Rao C,Tibbe AG, Uhr JW, Terstappen LW. Tumour cells circulate in theperipheral blood of all major carcinomas but not in healthy subjectsor patients with nonmalignant diseases. Clin Cancer Res2004;10:6897–6904.

8. Cohen SJ, Punt CJ, Iannotti N, Saidman BH, Sabbath KD, GabrailNY, Picus J, Morse MA, Mitchell E, Miller MC, Doyle GV, TissingH, Terstappen LW, Meropol NJ. Prognostic significance of circu-lating tumor cells in patients with metastatic colorectal cancer. AnnOncol 2009;20:1223–1229.

9. KhanMS, Kirkwood A, Tsigani T, Garcia-Hernandez J, Hartley JA,Caplin ME, Meyer T. Circulating tumor cells as prognostic markersin neuroendocrine tumors. J Clin Oncol 2013; 31:365–372.

10. Rahbari NN, Aigner M, Thorlund K, Mollberg N, Motschall E,Jensen K, Diener MK, Büchler MW, Koch M, Weitz J. Meta-analysis shows that detection of circulating tumor cells indicatespoor prognosis in patients with colorectal cancer. Gastroenterology2010;138:1714–1726.

11. Lu CY, Tsai HL, Uen YH, Hu HM, Chen CW, Cheng TL, Lin SR,Wang JY. Circulating tumor cells as a surrogate marker for deter-mining clinical outcome to mFOLFOX chemotherapy in patientswith stage III colon cancer. Br J Cancer 2013;108:791–797.

12. Negin BP, Cohen SJ. Circulating tumor cells in colorectal cancer:past, present, and future challenges. Curr Treat Options Oncol2010;11:1–13.

13. Sun YF, Yang XR, Zhou J, Qiu SJ, Fan J, Xu Y. Circulating tumorcells: advances in detection, methods, biological issues, and clinicalrelevance. J Cancer Res Clin Oncol 2011;137:1151–1173.

14. Attard G, de Bono JS. Utilizing circulating tumor cells: challengesand pitfall. Curr Opinion in Genetics & Development 2011;21:50–58.

15. Groot KoerkampB,Rahbari NN, Büchler MW, KochM,Weitz J.Circulating tumor cells and prognosis of patients with resectablecolorectal liver metastases or widespread metastatic colorectal can-cer: a meta-analysis. Ann Surg Oncol. 2013 Mar 2, DOI 10.1245/s10434-013-2907-8.

16. Pelkey TJ, Frierson HF Jr, Bruns DE. Molecular and immunolog-ical detection of circulating tumor cells and micrometastases fromsolid tumors. Clin Chem 1996;42:1369–1381.

17. Molnar B, Sipos F, Galamb O, Tulassay Z. Molecular detection ofcirculating cancer cells. Role in diagnosis, prognosis and follow-upof colon cancer patients. Dig Dis 2003;21:320–325.

18. Witzig TE, Bossy B, Kimlinger T, Roche PC, Ingle JN, Grant C,Donohue J, Suman VJ, Harrington D, Torre-Bueno J, Bauer KD.Detection of circulating cytokeratin-positive cells in the blood ofbreast cancer patients using immunomagnetic enrichment and dig-ital microscopy. Clin Cancer Res 2002;8:1085–1091.

19. Patriarca C, Macchi RM, Marschner AK, Mellstedt H. Epithelialcell adhesion molecule expression (CD326) in cancer: a shortreview. Cancer Treat Rev 2012;38:68–75.

20. van der Gun BT, Melchers LJ, Ruiters MH, de Leij LF, McLaughlinPM, Rots MG. EpCAM in carcinogenesis: the good, the bad or theugly. Carcinogenesis 2010;31:1913–1921.

21. Chapuis PH, Dent OF, Bokey EL, Newland RC, Sinclair G. Ad-verse histopathological findings as a guide to patient managementafter curative resection of node-positive colonic cancer. Br J Surg2004;91:349–354.

22. Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A.AJCC cancer staging manual, 7th edn. Chicago: Springer; 2010.

23. Galizia G, Orditura M, Ferraraccio F, Castellano P, Pinto M,Zamboli A, Cecere S, De Vita F, Pignatelli C, Lieto E. The lymph

J Gastrointest Surg

Author's personal copy

node ratio is a powerful prognostic factor of node-positive coloncancers undergoing potentially curative surgery. World J Surg2009;33:2704–2713.

24. Giessen C, Laubender RP, Ankerst DP, Stintzing S, Modest DP,Mansmann U, Heinemann V. Progression-free survival as a surro-gate endpoint for median overall survival in metastatic colorectalcancer: literature-based analysis from 50 randomized first-line tri-als. Clin Cancer Res 2013;19:225–235.

25. Sidhu R, Rong A, Dahlberg S. Evaluation of progression-freesurvival as a surrogate endpoint for survival in chemotherapy andtargeted agent metastatic colorectal cancer trials. Clin Cancer Res2013;19:969–976.

26. Altman DG, Vergouwe Y, Royston P, Moons KG. Prognosis andprognostic research: validating a prognostic model. BMJ 2009;338:1432–1435.

27. Ashworth TR. A case of cancer in which cells similar to those in thetumours were seen in the blood after death. Aus Med J 1869;14:146–149.

28. Engell HC. Cancer cells in the circulating blood; a clinical study onthe occurrence of cancer cells in the peripheral blood and in venousblood draining the tumour area at operation. Acta Chir Scand Suppl1955;201:1–70.

29. Chiappini F. Circulating tumor cells measurements in hepatocellu-lar carcinoma. Int J of Hepatology 2012; 2012:684802, doi:10.1155/2012/684802.

30. Dworzak MN, Gaipa G, Ratei R, Veltroni M, Schumich A,Maglia O, Karawajew L, Benetello A, Pötschger U, Husak Z,Gadner H, Biondi A, Ludwig WD, Basso G. Standardization offlow cytometric minimal residual disease evaluation in acute lym-phoblastic leukemia: multicentric assessment is feasible. Cytome-try B Clin Cytom 2008;74:331–340.

31. Patkar N, Alex AA, Bargavi B, Ahmed R, Abraham A, George B,Vishwabandya A, Srivastava A, Mathews V. Standardizing mini-mal residual disease by flow cytometry for precursor B lineageacute lymphoblastic leukemia in a developing country. CytometryPart B 2012;82:252–258.

32. Loken MR, Alonzo TA, Pardo L, Gerbing RB, Raimondi SC,Hirsch BA, Ho PA, Franklin J, Cooper TM, Gamis AS, MeshinchiS. Residual disease detected by multidimensional flow cytometrysignifies high relapse risk in patients with de novo acute myeloidleukemia: a report from Children’s Oncology Group. Blood2012;120:1581–1588.

33. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M,Peschle C, De Maria R. Identification and expansion of humancolon cancer-initiating cells. Nature 2007;445:111–115.

34. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, Hoey T,GurneyA, Huang EH, SimeoneDM, Shelton AA, Parmiani G, CastelliC, Clarke MF. Phenotypic characterization of human colorectal cancerstem cells. Proc Natl Acad Sci USA 2007;104:10158–10163.

35. Galizia G, Gemei M, Del Vecchio L, Zamboli A, Di Noto R,Mirabelli P, Salvatore F, Castellano P, Orditura M, De Vita F, PintoM, Pignatelli C, Lieto E. Combined CD133/CD44 expression as aprognostic indicator of disease-free survival in patients with colo-rectal cancer. Arch Surg 2012;147:18–24.

36. Gemei M, Mirabelli P, Di Noto R, Corbo C, Iaccarino A, ZamboliA, Troncone G, Galizia G, Lieto E, Del Vecchio L, Salvatore F. CD66cis a novel marker for colorectal cancer stem cell isolation and itssilencing halts tumor growth in vivo. Cancer 2013;119:729–738.

37. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial–mesen-chymal transitions in development and disease. Cell 2009;139:871–890.

38. Armstrong AJ, Marengo MS, Oltean S, Kemeny G, Bitting RL,Turnbull JD, Herold CI, Marcom PK, George DJ, Garcia-BlancoMA. Circulating tumor cells from patients with advanced prostateand breast cancer display both epithelial and mesenchymalmarkers. Mol Cancer Res 2011;9:997–1007.

39. Krebs MG, Hou JM, Sloane R, Lancashire L, Priest L, Nonaka D,Ward TH, Backen A, Clack G, Hughes A, Ranson M, Blackhall FH,Dive C. Analysis of circulating tumor cells in patients with non-smallcell lung cancer using epithelial marker-dependent and -independentapproaches. J Thorac Oncol 2012;7:306–315.

40. Lucci A, Hall CS, Lodhi AK, Bhattacharyya A, Anderson AE, XiaoL, Bedrosian I, Kuerer HM, Krishnamurthy S. Circulating tumourcells in non-metastatic breast cancer: a prospective study. LancetOncol 2012;13:688–695.

41. Nakamori S, Kameyama M, Furukawa H, Takeda O, Sugai S,Imaoka S, Nakamura Y. Genetic detection of colorectal cancer cellsin circulation and lymph nodes. Dis Colon Rectum 1997;40(10suppl):S29–S36.

42. Koch M, Kienle P, Kastrati D, Antolovic D, Schmidt J, Herfarth C,von Knebel Doeberitz M, Weitz J. Prognostic impact of hematog-enous tumor cell dissemination in patients with stage II colorectalcancer. Int J Cancer 2006;118:3072–3077.

43. Hardingham JE, Kotasek D, Sage RE, Eaton MC, Pascoe VH,Dobrovic A. Detection of circulating tumor cells in colorectalcancer by immunobead-PCR is a sensitive prognostic marker forrelapse of disease. Mol Med 1995;1:789–794.

44. Lloyd JM, McIver CM, Stephenson SA, Hewett PJ, Rieger N,Hardingham JE. Identification of early-stage colorectal cancer pa-tients at risk of relapse postresection by immunobead reversetranscription-PCR analysis of peritoneal lavage fluid for malignantcells. Clin Cancer Res 2006;12:417–423.

45. Denlinger CS, Cohen SJ. Progress in the development of prog-nostic and predictive markers for gastrointestinal malignancies.Curr Treat Options Oncol 2007;8:339–351.

46. Tol J, Koopman M, Miller MC, Tibbe A, Cats A, CreemersGJ, Vos AH, Nagtegaal ID, Terstappen LW, Punt CJ. Circulatingtumour cells early predict progression-free and overall survival inadvanced colorectal cancer patients treated with chemotherapy andtargeted agents. Ann Oncol 2010;21:1006–1012.

47. Otsuka K, Imai H, Soeda H, Komine K, Ishioka C, Shibata H.Practical utility of circulating tumour cells as biomarkers in cancerchemotherapy for advanced colorectal cancer. Anticancer Res2013;33:625–629.

Author names in bold designate shared co-first authorship.

J Gastrointest Surg

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1

Supplement eTable1. Table e1. Establishment of Cutoff for CTCs in the training set eTable2. Clinico-pathological characteristics of training and validation sets eTable3. Diagnostic performance of determination of CTC levels

eFigure1. Receiving Operating Characteristics (ROC) curve analyzing different performance of

each CTC value to predict progression-free survival rate. The true positive rate

(Sensitivity) is plotted in function of the false positive rate (100-Specificity) for

different cutoff points of CTCs. Each point on the ROC plot represents a

sensitivity/specificity pair corresponding to a particular decision threshold. The criterion

value corresponding with the highest accuracy (minimal false negative and false

positive results) is indicated with a square sign ( > three circulating cancer cells for 7.5

mL of peripheral blood). Sensitivity 75%; specificity 61%; positive predictive value

36%; negative predictive value 89%, accuracy 75%.

2

Table e1. Establishment of Cutoff for CTCs in the training set Groups (CTC cutoff)

Number in each group

36 Months PFS (%)

Survival Difference

Hazard Ratio

p value

0 > 1

8 32

87 68

19 0.58 0.61

0-1 > 2

10 30

90 65

25 0.39 0.36

0-2 > 3

12 28

92 62

30 0.25 0.06

0-3 > 4

12 28

92 62

30 0.25 0.06

0-4 > 5

16 24

80 67

23 0.40 0.25

0-5 > 6

18 22

82 65

17 0.34 0.16

0-6 > 7

20 20

85 60

25 0.30 0.23

0-7 > 8

20 20

85 60

25 0.30 0.23

0-8 > 9

23 7

80 63

17 0.33 0.10

0-9 > 10

25 15

74 73

1 0.80 0.76

0-10 > 11

30 10

77 65

12 0.51 0.34

0-11 > 12

27 13

74 71

3 0.66 0.57

0-12 > 13

28 12

75 68

7 0.54 0.38

0-13 > 14

28 12

75 68

7 0.54 0.38

0-14 > 15

29 11

76 64

12 0.47 0.28

0-15 > 16

31 9

73 71

2 0.75 0.72

0-16 > 17

31 9

73 71

2 0.75 0.72

0-17 > 18

32 8

73 70

3 0.68 0.64

0-18 > 19

32 8

73 70

3 0.68 0.64

0-19 > 20

34 6

75 55

20 0.39 0.23

0-20 > 20

34 6

75 55

20 0.39 0.23

0-30 > 30

36 4

75 73

2 0.51 0.51

0-54 > 54

37 3

73 67

6 0.40 0.37

CTC indicates circulating cancer cell; PFS indicates progression-free survival

3

Table e2. Clinico-pathological characteristics of training and validation sets

Training Set 40 patients

Validation Set 36 patients

p value a

Age b

< 66 > 66

19 21

19 17

0.81

Gender Male Female

26 14

22 14

0.91

Tumor Site Right Colon Left Colon Rectum

7 17 16

6 16 14

0.98

Serum CEA Levels (ng/mL) c

< 3.5 > 3.5

24 16

21 15

0.93

Performance Status 0 1 2

20 15 5

18 14 4

0.97

NeoAdjuvant CRT No Yes Rectum Only No Yes

33 7 9 7

31 5 9 5

0.90 0.94

Adjuvant Chemotherapy No Yes

10 30

10 26

0.98

PostOperative Complications No Yes

38 2

35 1

0.92

Tumor Size (cm) b

< 4 > 4

23 14

20 16

0.73

pT Stage 1 2 3 4

3 9 17 11

3 8 15 10

0.99

pN Stage Node Negative Node Positive

19 21

18 18

0.99

pM Stage M0 M1

30 10

28 8

0.98

Dukes' Stage A B C D

10 8 12 10

10 7 11 8

0.98

4

Table e2. Clinico-pathological characteristics of training and validation sets

(continued)

Training Set

40 patients

Validation Set

36 patients p value

a

Histologic Differentiation well moderate poor

19 17 4

17 15 4

0.98

Radical Resection Yes No

36 4

33 3

0.88

Tumor Recurrence No Yes

32 8

28 8

0.96

Survival Yes No

35 5

31 5

0.87

Preoperative CTCs Levels Negative Positive

12 28

10 26

0.96

CEA indicates carcinoembryonic antigen; Performance status according to the ECOG scale; CRT chemoradiotherapy a chi-square test

b median value

c normal value in healthy subjects

5

Table e3. Diagnostic performance of determination of CTC levels

Negative CTC

Positive CTC

Sensitivity %

Specificity %

PPV %

NPV %

Accuracy %

p value

a

Distant Metastases b

No Yes

22 0

36 18

100 37.9 33.3 100 52.6 0.012

Operation c

Radical Non Radical

53 5

16 2

28.6 76.8 11.1 91.3 72.3 0.81

Tumor Recurrence c

No Yes

54 4

6 12

75.0 90.0 66.7 93.1 86.8 <0.001

Tumor Recurrence d

No Yes

49 4

4 12

75.0 92.4 75.0 92.5 88.4 <0.001

The analysis was performed by using the Receiver Operating Characteristic (ROC) curve, and estimating the area under the curve with its p value

a

PPV indicates positive predictive value; NPV indicates negative predictive value

b Preoperative blood CTCs levels in 76 patients (22 CTCs negative and 54 CTCs positive)

c Postoperative blood CTCs levels in 76 patients (58 CTCs negative and 18 CTCs positive)

d Postoperative blood CTCs levels in 69 patients undergoing potentially curative resection (53 CTCs negative and 16 CTCs positive)

6

Figure e1

0 20 40 60 80 100

100

80

60

40

20

0

100-Specificity

Sensi

tivit

y