Qian et al. Radiation Oncology (2015) 10:125 DOI 10.1186/s13014-015-0428-2

RESEARCH Open Access

Microinvasion of liver metastases fromcolorectal cancer: predictive factors andapplication for determining clinical targetvolume

Yang Qian1, Zhao-Chong Zeng1*, Yuan Ji2 and Yin-Ping Xiao2

Abstract

Objectives: This study evaluates the microscopic characteristics of liver metastases from colorectal cancer (LMCRC)invasion and provides a reference for expansion from gross tumor volume (GTV) to clinical targeting volume (CTV).

Methods: Data from 129 LMCRC patients treated by surgical resection at our hospital between January 2008 andSeptember 2009 were collected for study. Tissue sections used for pathology and clinical data were reviewed.Patient information used for the study included gender, age, original tumor site, number of tumors, tumor size,levels of carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199), synchronous or metachronousliver metastases, and whether patients received chemotherapy. The distance of liver microinvasion from the tumorboundary was measured microscopically by two senior pathologists.

Results: Of 129 patients evaluated, 81 (62.8 %) presented microinvasion distances from the tumor boundary rangingbetween 1.0− 7.0 mm. A GTV-to-CTV expansion of 5, 6.7, or 7.0 mm was required to provide a 95, 99, or 100 %probability, respectively, of obtaining clear resection margins by microscopic observation. The extent of invasion wasnot related to gender, age, synchronous or metachronous liver metastases, tumor size, CA199 level, or chemotherapy.The extent of invasion was related to original tumor site, CEA level, and number of tumors. A scoring system wasestablished based on the latter three positive predictors. Using this system, an invasion distance less than 3 mm wasmeasured in 93.4 % of patients with a score of ≤1 point, but in only 85.7 % of patients with a score of ≤2 points.

Conclusions: The extent of tumor invasion in our LMCRC patient cohort correlated with original tumor site, CEA level,and number of tumors. These positive predictors may potentially be used as a scoring system for determiningGTV-to-CTV expansion.

Keywords: Liver metastases, Colorectal cancer, Radiotherapy, Pathological characteristics, Tumor invasion

SummaryData from 129 patients with liver metastases from colo-rectal cancer (LMCRC) were used to analyze factors as-sociated with hepatic metastatic microextensions. Theextent of invasion was positively correlated with theoriginal primary tumor site, CEA levels, and number oftumors. These predictors may potentially be used as ascoring system for determining gross tumor volume(GTV) to clinical tumor volume (CTV) expansion.

* Correspondence: zeng.zhaochong@zs-hospital.sh.cn1Department of Radiation Oncology, Zhongshan Hospital, Fudan University,136 Yi Xue Yuan Road, Shanghai 200032, ChinaFull list of author information is available at the end of the article

© 2015 Qian et al. This is an Open Access artLicense (http://creativecommons.org/licenseany medium, provided the original work is p(http://creativecommons.org/publicdomain/zero

IntroductionColorectal cancer is one of the most common gastro-intestinal carcinomas, and worldwide incidence andmortality rates are continually increasing. The five-yearsurvival rate after radical surgery is 40−56 %. The majorfailure pattern involves liver metastases. During follow-up observation, 50−70 % of patients diagnosed withcolorectal cancer exhibit liver involvement, which pre-sents as a solitary nodule in one-half of these patients[1]. In approximately 25 % of cases diagnosed with colo-rectal cancer, liver metastases are found at the time ofdiagnosis. Among those without liver metastases at the

icle distributed under the terms of the Creative Commons Attributions/by/4.0), which permits unrestricted use, distribution, and reproduction inroperly credited. The Creative Commons Public Domain Dedication waiver/1.0/) applies to the data made available in this article, unless otherwise stated.

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time of diagnosis, 80−90 % will have liver metastaseswithin 2–3 years after surgery [2].In 2010, colorectal cancer guidelines proposed that

carefully selected patients should be considered for con-formal radiation therapy and included in some clinicaltrials. Studies in the literature show that stereotacticbody radiation therapy (SBRT) for liver metastases fromcolorectal cancer (LMCRC) is an effective ablation therapywith a favorable toxicity profile [3, 4]. Conformal radio-therapy accurately shapes the high-dose radiotherapybeam closely around the outline of the visible tumors andsubclinical lesions. Precise outlining of the gross tumorvolume (GTV) and the clinical targeting volume (CTV)can focus the radiation dose to the target tumor and re-duce exposure and toxicity to normal tissue.GTV is the volume of the visible tumor. CTV includes

GTV and is defined as the volume of tissue that has asignificant probability of containing microscopic tumorextensions (microextensions), which represents subclin-ical diseases. Few studies have analyzed microextensioncharacteristics in liver metastasis. This knowledge deficitlimits the application of conformal radiotherapy forLMCRC. The objectives of this study were to performhistological quantification of microextension at livermetastatic lesions, and to correlate pathological macro-scopic tumor dimensions with MRI measurements todefine CTV as precisely as possible.

MethodsEthics approval and patients in the studyThis study was approved by the ethical review board ofZhongshan Hospital, Fudan University and in compli-ance with the Helsinki Declaration of 1975, as revised in2000. Between January 2008 and September 2009, 129patients with liver metastasis from colorectal cancerunderwent radical surgical resection at ZhongshanHospital. Patient selection was based on the followingcriteria. Patients must have undergone a radical resectionfor liver lesions at our hospital, and must have been patho-logically diagnosed with metastatic adenocarcinoma. Toavoid potential influences on the presence of microexten-sions, patients may not have received recent local treat-ment for liver metastatic lesions. Complete clinical datamust have been available for patients, including tumorinformation and laboratory values. Normal liver tissuesurrounding tumors must have had at least a 1-cmmargin extending beyond the tumor boundary, so thatall microextensions could be observed properly. Manylesions are excluded from radiotherapy due to possibleradiation damage of normal liver [5, 6]. Patients wereexcluded from the current study if more than four lesionswere detected by preoperative radiography or laparotomy.If more than one lesion was present, we selected thelargest one to evaluate.

Pathological analysisAfter radical resection, surgical specimens were analyzedby surgeons to determine the pathological boundary typeand maximum diameter. Then, some specimens werefixed in phosphate-buffered saline (PBS) containing 10 %(v/v) formalin; in this study, these are designated aspathology specimens. Unfixed specimens are designatedas surgical specimens. All specimens were sectioned into1.5 × 1.0 × 0.4-cm slices, dehydrated, and then embeddedin paraffin. Tissue blocks were prepared for routinehistological examination, and 5-μm sections on slideswere stained with hematoxylin and eosin (H&E) for lightmicroscopy. Three hundred slides from these specimenswere reviewed. To avoid variations between observa-tions, a single pathologist assessed all specimens to iden-tify microscopic evidence of microextension.

Analysis and definitions of microextensionsOn each histological slide, tumor margins were assessedon the cut surface by gross findings and then outlined bymarker pen with indelible ink. A microextension was de-fined as an extension of the tumor through the markedmargin, and was analyzed by light microscopy using anOlympus BX 40 (Olympus; Tokyo, Japan) at low-powermagnification (×40) to identify the boundary between thetumor and normal liver tissues. These observations alsowere confirmed by examination at a higher magnification(×100 or × 400). On each slide, the maximum distance tonormal liver tissue around the tumor periphery was deter-mined using an eyepiece with micrometer. If two or moremicroinvasions were observed (Fig. 1f), the longest inva-sive distance was recorded. If the primary tumor was com-pletely or partially surrounded by a fibrous pseudocapsule(Fig. 1b), the microextension was considered to be themaximum distance from the outer border of the pseudo-capsule to the outer boundary of visible nests of tumorcells [7]. If a fibrous pseudocapsule surrounding theprimary tumor was absent (Fig. 1c), the tumor mass wasdefined as the area that lacked (or essentially lacked) liverparenchyma interspersed between tumor cells as deter-mined by microscopic examination. If the tumor had acapsule and did not exhibit microinvasion (Fig. 1d), themicroextension was considered to be the maximum dis-tance from the outer border of the pseudocapsule to theouter boundary of visible nests of tumor cells. The tumorborder was defined as the line along whose length liver tis-sue and nests of tumor cells became interchanged; in thiscase, the microextension was defined as the distance fromthe primary tumor border to the outer boundary of visiblenests of tumor cells (Fig. 1) [8].

Magnetic resonance imaging and computed tomographyWe collected data from magnetic resonance imaging(MRI) and computed tomography (CT) images to compare

A

D

B C

FE

B

F

Fig. 1 Survey of observations of liver microinvasion from colorectal tumor metastases. a Tumor with a capsule (black cross) and showing adistinct boundary. b Tumor and microinvasions (black arrows) with capsules. c Tumor and microinvasions without capsules. d Tumor with acapsule that does not exhibit microinvasions. e Irregularly protruding microinvasions. f Multiple microinvasions (black arrows) with different sizesand shapes that show obscure boundaries

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with the pathological measurements. To allow as little timeas possible for additional tumor development before per-forming surgery, the scans had to be performed within3 days before resection. Of the two imaging modalities,MRI was preferred to CT for its superior assessment ofmalignant focal liver lesions [9, 10].Of the 129 patients in this study, only 49 underwent

MRI at our hospital. An MRI scan of the liver was per-formed with a 1.5 T MR scanner (SignaHDxt; GeneralElectric, WI, USA). This preoperative MRI included aT2-weighted (w) fast-spin echo (FSE) single-shot (SS)sequence, a T1-w gradient-echo (GE) sequence, and aT1-w dynamic multiphase gadolinium-enhanced (DMGE)sequence (Fig. 2a–d). All MRI sequences were performedwhile the patient held his breath (after exhalation). TheT2-w FSE FS sequence also was performed with the sys-tem triggered to expiration. The slice thickness for T1-wGE and T2-w FSE SS sequences was 8 mm, and for theT1-w DMGE sequence it was 5 mm. Liver metastases vary

in their T1 and T2 signal intensities but are usually pro-longed, which results in hypointensity to isointensity onT1-w images and isointensity to hyperintensity on T2-wimages. Some metastases may show a hyperintense halo ofviable tumor surrounding a central region of hypointensitydue to necrosis. The pathologically determined GTV cor-related best with unenhanced MRI compared with venousenhanced MRI. The tumor contours revealed by T1-w GEwere reviewed by an experienced liver radiologist.

Comparison of preoperative MRI with macroscopicpathologyTo define the GTV by radiography, we calculated thatthe coefficient of specimen contraction during fixationfor a pathology slide was 90.1 %, as reported by Bi et al.[11] from their analysis of 66 primary liver carcinomaspecimens. The pathology specimens, which includedboth tumor and normal liver tissues, were cut into aregular rectangle of 2 × 1.5 cm. The width and length of

A

DC

B

Fig. 2 Liver metastasis (white arrow) of preoperative magnetic resonance images. a T1-w GE. b T1-w arterial enhanced. c T1-w venous enhanced.d T2-w

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the cut tissue were measured. Two to four pieces of thespecimens were placed into an automatic tissue hydro-extractor and subjected to a dehydration cycle. H&E-stained slides were prepared using routine methods, andthe resulting tissue sections were measured. Finally, thespecimen contraction coefficient was calculated basedon the two measured specimen sizes.

Definitions of clinical factorsThe preoperative evaluation included a medical history,physical examination, complete blood-cell count, liverfunction tests, measurements of carcinoembryonic anti-gen (CEA) and carbohydrate antigen 19–9 (CA199)levels, chest X-ray, abdominal ultrasonography, and en-hanced CT or MRI (or both). Levels of CEA and CA199tumor markers were measured using an electrochemilu-minescence immunoassay system (Roche ModularE170). Liver functions were tested by an automatedmethod and analyzed statistically (Hitachi 7600–120).All laboratory tests were performed less than 1 week

before surgery at the Clinical Laboratory Department ofZhongshan Hospital. The presence of multiple lesions isdefined as more than one lesion but less than four le-sions. Chemotherapy is defined as having undergone atleast one chemotherapeutic regimen before live surgery.Liver metastases are defined as synchronous when theyoccur within 6 months after the original tumor surgeryand as metachronous when they occur after 6 months.The surgeons specified the original tumor site as rectumwhen it was located within <12 cm from the perianalskin border, and as colon when it was located in cecum,ascending colon, transverse colon, descending colon, orsigmoid colon. All patients had hepatic lesions that wereconsistent with metastases from a histologically docu-mented colorectal carcinoma.

Statistical analysis of dataStatistical analysis was performed using SPSS version19.0 (SPSS Inc., Chicago, IL). The association of char-acteristics with microinvasion distance was analyzed by

Table 1 Factors associated with the presence of microextension

Clinicopathologicalfactor

n Microextension, n (%) x2 P-value*

Absent Present

Sex 0.790 0.598

Male 75 28 (37.3 %) 47 (62.7 %)

Female 54 20 (37.0 %) 34 (63.0 %)

Age 0.794 0.624

<65 years 93 34 (36.6 %) 59 (63.4 %)

≥65 years 36 14 (38.9 %) 22 (61.1 %)

Synchronous ormetachronous

0.982 0.965

Synchronous 70 25 (35.7 %) 45 (64.3 %)

Metachronous 59 23 (39 %) 36 (61 %)

Original site 3.185 0.009

Rectum 48 25 (52.1 %) 23 (47.9 %)

Colon 81 23 (28.4 %) 58 (71.6 %)

Number of tumors 2.537 0.045

1 85 38 (44.7 %) 47 (55.3 %)

>1 44 10 (22.7 %) 34 (77.3 %)

Max tumor diameter 1.345 0.52

<3 cm 39 17 (43.6 %) 22 (56.4 %)

≥3 cm 90 31 (34.4 %) 59 (65.6 %)

CEA (ng/ml) 4.805 0.001

≤5 35 20 (57.1 %) 15 (42.9 %)

>5 94 28 (29.8 %) 66 (70.2 %)

CA199 (U/ml) 0.478 0.114

≤37 68 24 (35.3 %) 44 (64.7 %)

>37 61 24 (39.3 %) 37 (60.7 %)

Chemotherapy 0.906 0.817

Yes 55 21 (38.2 %) 34 (61.8 %)

No 74 27 (36.5 %) 47 (63.5 %)

CEA Carcinoembryonic antigen, CA199 Carbohydrate antigen 19–9*Determined by binary logistic regression

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chi-square test for categorical variables, and by binary lo-gistic regression for predictor variables of the significantparameters. P values ≤0.05 were considered significant.

ResultsDemographic dataOne hundred and twenty-nine LMCRC patients who ful-filled the specified criteria were recruited, including 75men and 54 women. The patient ages ranged from 32 to80 years, with a mean of 59 years.Microextension was noted in 62.8 % (81/129) of the

patients. Microinvasion was assessed from H&E-stainedsections. Microscopic analysis of tissue specimens fromthese patients revealed two microinvasion patterns inLMCRC patients. In one, the region of microextensionand tumor foci was separated by normal liver tissue(Fig. 1b). The second pattern was irregular protrusionextending beyond the tumor (Fig. 1e). The microexten-sion distances ranged from 1–7 mm, with an averagedistance of 1.50 ± 1.51 mm. The microextension distancewas 1–2 mm in 48 (37.2 %) patients, 2.1 − 4.0 mm in 24(18.6 %) patients, and 4.1 − 7.0 mm in 9 (7 %) patients.This distance had to be expanded to 5 mm during mi-croscopy to include at least 98 % of all microextensions.

Factors associated with the presence of microextensionsClinical factors associated with the presence of microex-tension are listed in Table 1. The data indicated that thepresence of microextensions was associated with highserum levels of CEA (P = 0.002), primary tumor site incolon (P = 0.008), and multiple lesions (P = 0.045).

Scoring system to estimate risk of microextensions withvarying distances for all patientsPlots showing the cumulative distribution of microinva-sion according to the clinicopathologic factors are pre-sented in Fig. 3. We found that among patients withmicroinvasion distance of ≤2.4 mm, 94.3 % had CEAlevels of ≤5 U/ml. When the microinvasion distancewas ≤4.3 mm, sensitivity was approximately 95 % forpatients in which the primary tumor site was rectum. Amicroinvasion distance of ≤4 mm was associated with asensitivity of 95 % for patients with a single liver meta-static lesion (unilesional). The best single predictor formicroinvasion was the CEA level.To improve the success of prediction for microinva-

sion, we developed a scoring system based on a com-bination of the associated factors that were identified.Patients were assigned a score of 0 for each of the fol-lowing: CEA level ≤5; original site of primary tumor inrectum; and unilesional liver metastasis. Otherwise, pa-tients received a score of 1 for factors not meetingthose criteria. To account for ≥95 % probability ofmicroinvasion, one must select an expansion of 2 mm

for a score of ≤1, and an expansion of 5 mm for a scoreof ≥2 (Fig. 3d).

Tumor sizes determined from radiographs, surgicalspecimens, and pathology specimensIn samples from the 49 patients undergoing a preopera-tive MRI scan at our hospital, only 49/129 tumors(38 %) received the same boundary classifications basedon gross analyses of surgical specimens and radiographs.The mean tumor diameter measured 3.3 cm (range, 0.3–9.0 cm) in radiographs, 3.5 cm (range, 0.8 − 9.5 cm) insurgical specimens, and 3.2 cm (range, 0.8–9.5 cm) inpathology specimens. Regression analysis of tumor sizein radiographs, surgical specimens, and pathology speci-mens revealed that the three measurements were signifi-cantly correlated (P < 0.001). This was especially true of

Fig. 3 Cumulative distributions of LMCRC microextensions according to serum CEA level. a Serum CEA levels. b Original primary tumor site.c Intrahepatic tumor number. d A score derived from a combination of these factors

Fig. 4 Correlation between tumor sizes measured using magneticresonance images and pathology specimen slides

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measurements taken from radiographs and surgicalspecimens. Measurements from pathology specimenswere slightly smaller than those taken from radiographs(r = 0.92, P < 0.001). A comparison of radiographic mea-surements with those obtained from pathology specimensshowed that radiographic measurements were larger in34.7 % (17/49) of tumors, smaller in 40.8 % (20/49) of tu-mors, and identical in 24.5 % (12/49) of tumors. Overall,measurements taken from pathology specimens weresmaller than those obtained from radiographs (r = 0.92,P < 0.001; see Fig. 4), with pathology specimen measure-ments reduced by 8 % relative to those from radiographs.The radiograph-slide contraction coefficient was 82.9 %

(0.92 × 0.90), and a distance of 5 mm was required to in-clude 98 % of microextensions. By extrapolation, theequivalent distance on radiographic specimens had to be6.1 mm (5.0/0.829). For a score of ≤1, an expansion dis-tance of 2.41 mm (2.0/0.829) on radiographic images canbe accepted, which accounts for ≥95 % probability ofmicroextension. For a score of ≥2, an expansion distance

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of 6.1 mm on radiographic images must be selected. AGTV-to-CTV expansion of 8.5 mm in radiographicimages of LMCRC is required to encompass the grosstumor and all subclinical microscopic disease with100 % accuracy.

DiscussionThe liver is the target of many metastatic cancers, par-ticularly those of colorectal cancer. Major advances inthe diagnosis and treatment of metastatic liver cancershave occurred during the last two decades. Surgery isstill considered the gold-standard therapy for patients di-agnosed with LMCRC because it confers the best chanceof long-term survival. However, only 25 % of patientswith liver metastases are candidates for curative resec-tion [2]. The majority are not eligible for surgery due toextensive disease, multiplicity of tumor, concomitantmajor systemic disease, or poor hepatic reserve. Duringthe past 20 years, SBRT has evolved as another localtreatment option for primary and metastatic liver tu-mors. Local control rates have been improved by doseescalation protocols, whereas acceptable levels of toxicityhave been maintained [6, 7, 12, 13].To further optimize SBRT treatment, the definition of

the target volume needs to be improved. Determiningthe degree of GTV expansion to include the CTV posesa considerable challenge to radiologists. To preciselydefine the limits of the target volume, the correlationbetween macroscopic tumor dimensions that are visiblein radiological images and those that are visible in path-ology specimens should be evaluated. To our knowledge,reports in the literature on this topic are scarce [14, 15].The current study was designed as a prospective pilotstudy to establish all the necessary procedures to obtain agood clinicopathologic correlation between the method-ologies and reproducible microextension measurements.We found that expansions of 5.0 or 6.7 mm in micros-

copy specimens were required to encompass the grosstumor and any microscopic disease with 95 % or 99 %accuracy, respectively. A more important issue was tomeasure the tissue contraction coefficient that occursafter formalin fixation of specimens and syneresis in theautomatic tissue hydroextractor. Our analysis of 66 livercarcinoma pathology specimens on slides revealed thatthe prepared specimen sizes were 91.4 % of the originalspecimens. The radiograph-slide contraction coefficientwas 82.9 % (0.92 × 0.90). These calculations indicate thatan expansion of at least 6.1 mm on radiographic imagesshould be considered during radiological treatment ofLMCRC.The microextension frequency observed in the present

study is in agreement with other published reports.Prospective and retrospective surgical reports describethe presence of microextensions with a large variation

(2–58 %) in occurrence [16, 17]. Our results were inagreement with those of Gandhi et al., who reportedmeasurable microscopic disease (mean, 1.25 mm) in 7of 24 (29.2 %) tumors analyzed [18]. However, thedepths of microextension infiltration (0.15–38.00 mm)described in the literature are not consistent with ourresults, which ranged from 1–7 mm. One possible rea-son for the discrepancy could be that the criteria forpatient selection differed in the two studies. Further-more, it is impossible to entirely exclude whether someof our tumor nests (observed in a two-dimensionalmicroscopic field) were not in reality attached to theprimary tumor at another level, because some of thecolorectal metastases had very irregular borders. Wetried to correct for this weakness as much as possibleby thoroughly inspecting the slices located just aboveand beneath the one in which we observed the micro-extension. In general, all the metastases included in thepresent study showed rather irregular shapes, and thismade it difficult to establish a relationship between theirregular tumor shape and more frequent or deepermicroextensions.We found that microextension was related to the ori-

ginal primary tumor site, CEA levels, and multiple le-sions. Reports on prognosis in colon and rectal cancersare limited. In our study, the median microextensionlevels in liver were significantly different depending onwhether the origin of metastasis was from the colon orrectum. A possible reason for this may be the differencesin their anatomical locations, which could account for dif-ferent pathways in lymph node metastases and specificbiological behaviors. However, further study is needed tounderstand the phenomenon.There are some reports that serum CEA level is an inde-

pendent risk factor for LMCRC. Patients with elevatedCEA levels are more likely to develop recurrent diseaseand to have poorer overall survival than patients withnormal CEA levels [19, 20]. Our results are in agreementwith this assessment. The presence of multiple lesions isanother factor that correlates with microextension,although the P value was only 0.045. The presence ofmultiple lesions suggests that the cancer cells aremore highly prone to invasion.It is unclear whether pretreatment with chemotherapy

might influence metastatic microextensions. A limitationin our study is that the impact of tumor regression maynot be the same after treatment with 5-fluorouracil (5-FU)or tritherapy with or without targeted agents such as theanti-VEGF monoclonal antibody bevacizumab. In ourstudy, analysis of patients with or without chemotherapydid not identify any significant differences in microexten-sion (P = 0.906). We used a specimen-slide contractioncoefficient that was derived only from primary livercarcinoma specimens. We do not currently know if the

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LMCRC specimens would have the same coefficient.Additional research is needed to answer this question.Only 49 patients had a preoperative MRI scan in ourstudy, which may lead to biases in the results. Most ofthe 129 patients included in the study had a preopera-tive ultrasonic sound examination. Only patients withan obscure mass or a small tumor were referred foradditional examination using CT or MRI. This referralwas dependent on the surgeon’s decision.Analysis of the cumulative distribution of microinvasion

in LMCRT patients with different tumor characteristics(Fig. 3) revealed that the CEA level was the best predictorof microextension. However, the pathologic stage cannotbe accurately determined without surgery. Therefore, webelieve that the scoring system developed in this paper,which is based on factors that can be accurately deter-mined preoperatively (i.e., CEA level, original primarytumor site, and number of liver metastases), will holdgreater promise for prognosis.The average tumor size in this patient cohort was

2.99 cm. Considering that a tumor is approximated as asphere, a reduction in the GTV-to-CTV expansion from6.1 mm (score ≥ 2) to 2.41 mm (score ≤ 1) would reducethe CTV diameter from 4.21 to 3.47 cm. Accordingly, theCTV volumes would be reduced from 39.05 to 21.87 cm3

(i.e., a 50 % reduction). This suggests that the delineationof CTV could vary depending on differences in patient-specific clinical characteristics. Another important point isthat the average tumor size in our cohort was very small(2.99 cm). Further studies based on our analyses will facili-tate potential clinical applications of this promising meth-odology for conformal radiotherapy of LMCRT patients.

ConclusionsThis study showed that an expansion of 2.41 mm onradiographic images with a score of ≤1 can be accepted;however, to account for 98 % probability of microexten-sion, an expansion of 6.1 mm on radiographic images witha score of ≥2 must be selected. A GTV-to-CTV expansionof 8.5 mm in radiographs of LMCRT patients is requiredto encompass the gross tumor and all microscopic diseasewith 100 % accuracy. Microextension distance is corre-lated with original primary tumor site, serum CEA levels,and multiple metastatic lesions in liver. Our multifactorscoring system, which takes CEA level into consideration,was a good predictor of microextension in our dataset.

AbbreviationsLMCRC: Liver metastases from colorectal cancer; GTV: Gross tumor volume;CTV: Clinical tumor volume; CEA: Carcinoembryonic antigen;CA199: Carbohydrate antigen 199; SBRT: Stereotactic body radiation therapy;PBS: Phosphate-buffered saline; MRI: Magnetic resonance imaging;CT: Computed tomography; 5-FU: 5-fluorouracil.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsYQ: implemented a scoring system for determining GTV to CTV expansion,was involved in the collection of data, the design of study drafting of themanuscript, and the critical revision of the manuscript for scientific content.ZCZ: conceived of the study, and participated in its design and coordinationand helped to draft the manuscript. YJ: assessed all specimens to identifymicroextension and interpretation of data. YPX: assessed all specimens toidentify microextension and the critical revision of the manuscript forscientific content. All authors read and approved the final manuscript.

AcknowledgementThe authors would like to thank colleagues of pathological department fortheir technical supports.

Author details1Department of Radiation Oncology, Zhongshan Hospital, Fudan University,136 Yi Xue Yuan Road, Shanghai 200032, China. 2Department of Pathology,Zhongshan Hospital, Fudan University, Shanghai, China.

Received: 11 October 2014 Accepted: 25 May 2015

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