Circulating tumor cells in hepatocellular carcinoma a pilot study of detection enumeration and next

Circulating tumor cells in hepatocellular carcinoma:a pilot study of detection,enumeration,and

next-generation sequencing in cases and controls

Robin K Kelley 1*,Mark Jesus M Magbanua 2,Timothy M Butler 3,Eric A Collisson 2,Jimmy Hwang 2,

Nikoletta Sidiropoulos 4,Kimberley Evason 5,Ryan M McWhirter 2,Bilal Hameed 6,Elizabeth M Wayne 7,Francis Y Yao 8,Alan P Venook 1and John W Park 2

Background

Hepatocellular carcinoma (HCC)is a grim,heteroge-neous disease with limited treatment options despite its enormous global impact as the third leading cause of cancer death worldwide [1].Conventional liver imaging modalities for diagnosis and staging are imprecise and can result in underestimation of the true extent of dis-ease,with microvascular invasion and multifocal tumors

often identified incidentally at resection or transplant and associated with significantly poorer prognosis [2,3].Translational research efforts to better understand the complex tumor biology of HCC,define biomarkers,and identify novel therapeutic targets are further limited by a scarcity of annotated,untreated tumor specimens,owing to the acceptance of radiographic diagnosis without tis-sue confirmation,the prevalence of liver-directed ther-apy before transplantation,and the risks associated with tumor biopsy in this population [4,5].Non-invasive bio-markers for diagnosis and molecular characterization are urgently needed to overcome these pervasive challenges in HCC.

*Correspondence:Katie.kelley@https://www.wendangku.net/doc/1b323929.html, 1

Helen Diller Family Comprehensive Cancer Center and The Liver Center,University of California San Francisco (UCSF),55016th St.,Box 3211,San Francisco,CA 94143,USA

Full list of author information is available at the end of the

article

?2015Kelley et al.;licensee BioMed Central.This is an Open Access article distributed under the terms of the Creative

Commons Attribution License (https://www.wendangku.net/doc/1b323929.html,/licenses/by/4.0),which permits unrestricted use,distribution,and reproduction in any medium,provided the original work is properly credited.The Creative Commons Public Domain Dedication waiver (https://www.wendangku.net/doc/1b323929.html,/publicdomain/zero/1.0/)applies to the data made available in this article,unless otherwise stated.

Kelley et al.BMC Cancer (2015) 15:206 DOI 10.1186/s12885-015-1195-z

Circulating tumor cells(CTCs)in the peripheral blood are a biomarker of poor prognosis in multiple epithelial tumor types[6,7].The CellSearch System(Veridex LLC, Raritan,New Jersey,U.S.A)is an FDA-cleared device for CTC detection using enrichment for cells in the blood expressing the epithelial cell adhesion marker(EpCAM) [6].The absolute numbers of CTCs detected and changes on therapy have been associated with survival and treatment response in breast,colon,and prostate cancers[8-13].Multiple small studies have examined CTCs in patients with HCC using EpCAM-and non-EpCAM-based enrichment methods,with detection rates ranging from approximately30%to over80%de-pending on methodology and population[14-17].As in other epithelial tumor types,the detection of CTCs by CellSearch correlates with poor prognosis in HCC co-horts,including increased recurrence risk after resection and shorter overall survival[14,15].

In order to study CTCs as a biomarker in HCC,how-ever,it is essential to establish that circulating epithelial cells in HCC populations are true tumor cells,rather than benign epithelial cells released into circulation as a consequence of the underlying inflammation or aberrant vasculature associated with liver disease.Though the de-tection of CTCs by CellSearch is extremely rare in healthy volunteers or patients with benign conditions [6,10],there is limited data describing the incidence of circulating EpCAM-positive epithelial cells in the con-text of cirrhosis,viral hepatitis,or other causes of liver injury,conditions present in the majority of patients with HCC[14].

Beyond detection and enumeration,isolation of CTCs in cancer patients holds great promise as a“liquid bi-opsy”,a non-invasive means of accessing real-time tumor tissue in the metastatic state for molecular profil-ing.Array comparative genomic hybridization has dem-onstrated concordance of characteristic copy number aberrations between CTC-derived DNA and archival pri-mary tumor samples in breast,colon,prostate,and lung cancer[12,18-20].Next-generation sequencing technolo-gies now have the ability to sequence very small amounts of input DNA with high accuracy[21,22]. Illumina MiSeq technology can detect characteristic driver mutations in single CTCs derived from patients with metastatic colorectal cancer,concordant with the mutational profile of paired primary tumor specimens [18].To date,the feasibility of efficient CTC isolation and molecular profiling,e.g.next-generation DNA se-quencing,has not been reported in HCC.

We conducted this study to determine the proportion of metastatic HCC patients with detectable circulating EpCAM-positive epithelial cells using the CellSearch System,compared to a relevant control cohort of pa-tients with liver disease,hypothesizing that circulating EpCAM-positive cells are actual tumor cells rather than benign epithelial cells.To characterize their prognostic significance,CTC levels were examined for association with clinical covariates including alpha-fetoprotein(AFP) levels,the presence of vascular invasion,and overall sur-vival.To explore the potential for CTCs to serve as a source of tumor DNA for genomic profiling in HCC, next-generation sequencing using a targeted cancer gene panel was performed using whole genome-amplified DNA derived from pooled purified CTCs,along with DNA from paired archival,paraffin-embedded tumor tissue and per-ipheral blood mononuclear cells when available. Methods

Study design

This pilot study was a non-therapeutic,minimally-invasive biomarker study.The trial was approved by the UCSF Committee on Human Research.All patients pro-vided written informed consent for specimen collection and genetic testing of tumor and germline DNA.The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice.

The primary endpoint was incidence of CTCs detected in metastatic HCC patients compared to a control co-hort with NMLD.Secondary endpoints were enumer-ation of CTCs in each cohort,association with clinical and pathologic characteristics including alpha fetopro-tein(AFP)level,tumor vascular invasion,and etiology of liver disease in the HCC cohort,and association with overall survival in the HCC cohort.An exploratory end-point was to describe performance of and somatic muta-tions identified by next-generation sequencing of CTC whole-genome-amplified DNA along with paired tumor and germline DNA when available.

Patient selection

HCC patients were recruited at the UCSF Helen Diller Family Comprehensive Cancer Center.Principal inclu-sion criteria were:radiographic[4]or histologic diagno-sis of American Joint Committee on Cancer(AJCC) stage IV HCC;≥6weeks post biopsy,surgery,liver-directed interventions,or other invasive procedures;no prior systemic therapy or≥4weeks since last dose of so-rafenib or other systemic therapy for advanced HCC. Non-malignant liver disease(NMLD)control cohort pa-tients were recruited at the UCSF Gastroenterology and Liver Disease Clinic.Principal inclusion criteria were: diagnosis of active hepatitis of any etiology plus clinical or pathologic diagnosis of cirrhosis or hepatic fibrosis (any stage);no evidence liver tumor on ultrasound or cross-sectional imaging within6months;AFP≤20ng/mL within6months;≥6weeks post biopsy,surgery,or other invasive procedures;no prior history of HCC.

Specimen collection

Approximately30mL of whole blood was obtained from study subjects at a single time-point.For HCC patients with available archival tumor tissue from prior biopsy or resection,approximately five10-micron sections of formalin-fixed,paraffin-embedded(FFPE)tumor along with a matching H&E slide were collected from the path-ology files of the University of California,San Francisco. Banked frozen aliquots of peripheral blood mononuclear cell(PBMC)were obtained when available from HCC cohort patients.

Circulating tumor cell enumeration

CTCs were isolated from7.5mL whole blood and enu-merated using the CellSearch System(Veridex LLC, Raritan,NJ)[6-8].Briefly,specific antibodies to EpCAM were used to enrich for epithelial cells.A mixture of fluorescently-labeled monoclonal antibodies to cytokera-tin and the nuclear dye DAPI were used to select for nucleated,keratin-positive cells.CTCs were defined as nucleated,EpCAM-positive cells that stain positive for cytokeratin and negative for leukocyte common antigen, CD45[6].Labeled cells were enumerated using semi-automated fluorescence-based microscopy.Analysis was performed by a trained technician blinded to diagnosis (HCC versus NMLD).

Immunoenrichment and fluorescence-activated cell sorting(IE/FACS)

A novel EpCAM-based immunoenrichment(IE)/fluores-cence-activated cell sorting(FACS)procedure has been developed to isolate purified CTCs without contamin-ation from normal blood cells and has demonstrated correlation with CellSearch System CTC enumeration [12,19,23].For patients found to have>10CTCs in 7.5mL of whole blood by CellSearch System,IE/FACS was then performed to isolate purified CTCs as has been previously described[12,24].Briefly,approximately15–20mL of whole blood was incubated with immunomag-netic particles coated with two different monoclonal antibodies to EpCAM,one conjugated to magnetic parti-cles and the other to a fluorophore.FACS was used to isolate nucleated,EpCAM-positive,CD45-negative cells. Whole genome amplification(WGA)

A ligation-adaptor method of WGA was performed on whole cell lysates from pooled CTCs isolated by IE/ FACS using a GenomePlex whole genome amplification kit(WGA4,Sigma-Aldrich)according to the manufac-turer’s instructions[12,25].DNA was randomly frag-mented and converted to polymerase chain reaction (PCR)-amplifiable library molecules flanked by universal priming sites.PCR amplification of library molecules was performed using universal oligonucleotide primers.DNA extraction from tumor tissue and peripheral blood mononuclear cells(PBMC)

Tumor-containing FFPE sections were identified and marked by a hepatopathologist(KE).DNA was ex-tracted from FFPE sections as well as from banked PBMC using QIAmp kits(Qiagen)according to the manufacturer’s instructions.DNA concentration was quantified using PicoGreen.

Ion semiconductor NGS

Sequencing of DNA extracted from CTCs,FFPE,and PBMC was performed by TMB in the Spellman Laboratory at Oregon Health Sciences University.From each sample, 10ng DNA was PCR-amplified using AmpliSeq Cancer Panel Primer Pools and Library Kit2.0to generate190 multiplexed amplicons(representing46cancer-related genes)[21].Up to11barcoded samples were multiplexed on Ion318chips.Sequencing was performed on a Personal Genome Machine(PGM)sequencer(Ion Torrent)using the Ion PGM200sequencing kit.Torrent Suite software version4.0.1was employed to analyze read counts and quality.Variant Caller software version4.0.1identified vari-ants.Coverage Analysis software version4.0.1determined target coverage.To minimize false positives,variants were required to have sequencing depth of at least20x,an allele frequency of5percent,and not be present in any of the3 PBMC samples sequenced.Variant calls were filtered against the Single Nucleotide Polymorphism Database (dbSNP)version132,using the software ANNOVAR. Protein-altering variants were predicted by Mutation Asses-sor version2(https://www.wendangku.net/doc/1b323929.html,).

Statistical analysis

Based upon the a priori hypothesis that approximately 50%of the HCC cohort and none of the NMLD cohort would have detectable CTCs by CellSearch,the planned sample size for this pilot study was20patients with metastatic HCC and10patients with NMLD,to permit estimation of proportion of detectable CTCs with95% confidence intervals(CI)as(0.30,0.70)in the HCC cohort and(0.01,0.26)in the NMLD cohort.The inci-dence and number of detectable CTCs were analyzed using frequency and proportions with95%CI and com-pared between HCC and NMLD cohorts using the Wilcoxon-Kruskal-Wallis rank test.Cut-points of≥1,≥2,≥3,and≥5CTCs/7.5mL were examined based upon pub-lished literature in HCC and other tumor types [8,10,14,15].Wilcoxon-Kruskal-Wallis rank testing was also used to determine association between the presence of de-tectable CTCs by CellSearch System,AFP elevation using ≥400ng/mL as an established prognostic cut-point[26,27], and the presence of vascular invasion(all binary variables). In the HCC cohort,overall survival was measured in months from date of CTC blood draw to the date of death

with censoring at date of last known vital status if lost to follow-up.Kaplan-Meier methods were used to determine the impact of CTCs at each cut-point and conventional prognostic factors on overall survival.The CTC level,AFP value of400ng/mL,and presence of macrovessel invasion were used to dichotomize for univariate analyses.The Child Pugh score and etiology of liver disease were also examined.A p value of<0.05was considered statistically-significant under log-rank tests.Sequencing coverage depth was compared between sample types using two-tailed t-tests assuming unequal variance.Variant calls were reported descriptively due to small sample size.

Results

Patient characteristics

Twenty patients with a diagnosis of metastatic HCC (HCC cohort)and10patients with underlying non-malignant liver disease without cancer(NMLD cohort) were prospectively enrolled between June2011and April 2012.All HCC patients were followed to date of death. Baseline patient characteristics are shown in Table 1. The median overall survival in the HCC cohort was 9.44months from date of CTC blood draw.One NMLD cohort patient with HCV cirrhosis(Hep25)was found to have a liver mass with adjacent portal vein thrombosis on a surveillance ultrasound after enrollment and was excluded based upon a suspected new diagnosis of HCC, resulting in9eligible patients in the NMLD cohort.The patient was subsequently lost to follow up.Figure1dis-plays the study subject enrollment and samples tested. CTC detection and enumeration by CellSearch

Figure2depicts the number of CTCs detected in each patient.At least1CTC per7.5mL was detected in8of 20(40%,95%CI:17%,64%)HCC patients and1of9 (11%,95%CI:0,37%)eligible NMLD patients(p=0.1, Wilcoxon-Kruskal-Wallis rank test).At least2CTC per 7.5mL were detected in7of20(35%,95%CI:12%,

60%)HCC patients and0of9eligible NMLD patients (p=0.04,Wilcoxon-Kruskal-Wallis rank test).Among the HCC cohort patients,at least1CTC per7.5mL was detected in7of10(70%,95%CI:35%,100%)with AFP≥400ng/mL,versus1of10(10%,95%CI:0,33%)with AFP<400ng/mL(p=0.008).At least1CTC per7.5mL was detected in8of13(62%,95%CI:31%,92%)with vascular invasion versus0of7without(p=0.009) (Wilcoxon-Kruskal-Wallis rank tests).The NMLD control cohort patient Hep25who was removed for ineligibility (due to new liver mass with thrombosis consistent with HCC)was found to have20CTCs per7.5mL peripheral blood.Another NMLD cohort patient with alcoholic cir-rhosis had1CTC detected per7.5mL peripheral blood.It is noteworthy that the single eligible NMLD control pa-tient with detectable CTCs(1in7.5mL)subsequently developed new infiltrative changes in the liver on a sur-veillance ultrasound,raising the possibility of underlying tumor though no formal HCC diagnosis was made before his death of complications of cirrhosis approximately 13months after CTC blood draw.

The median overall survival(OS)in the HCC cohort was9.4months.Among HCC cohort patients with at least1CTC per7.5mL,the median OS was2.8months (95%CI:1.08,15.5),versus11.3months(95%CI:7.49, 12.9)for those without CTCs detected,although the dif-ference was not statistically significant(p=0.62,Log-Rank test)(Figure3).In univariate analysis of CTC levels and conventional prognostic factors(Table2),none showed significant effect on overall survival,though ana-lyses were limited by small sample sizes;no further multivariate analysis was performed.

Table1Patient characteristics

HCC cohort

(n=20)

NMLD control

Cohort(n=10) Median age(range)(years)61.5(50–82)26-91(53.5) Male/female(n)20/09/1

Etiology of liver disease(%)

HBV2520

HCV4560

Co-infection HBV+HCV a100

ETOH510

NAFLD100

PSC010

Unknown50

Race/ethnicity(%)

African-American510

Asian3510 Caucasian5570 Hispanic/Latino530

Non-Hispanic/Latino5040

Native American50

Other/unknown010

Child Pugh score(%)

A/B/C/unknown70/25/5/030/30/30/10 Median AFP(range)(ng/mL)492(3.8-587,134) 5.5(1.7-17.2) BCLC score C(%)b100N/A

Vascular invasion(%)65N/A Extrahepatic spread(%)b100N/A

Median overall survival(months)9.4months Not measured Key:HBV=hepatitis B virus.HCV=hepatitis C virus.ETOH=alcohol.NAFLD= non-alcoholic fatty liver disease.PSC=primary sclerosing cholangitis.BCLC= Barcelona Clinic Liver Cancer.N/A=not applicable.

a Defined as HCV antibody positive plus either HBV surface antigen and/or core antibody positive.

b BCLC C and presence of extrahepati

c sprea

d wer

e required eligibility criteria for HCC cohort.

CTC isolation by IE/FACS

Five patients in the HCC cohort showed greater than10 CTC per7.5mL detected by CellSearch.CTCs were then isolated via IE/FACS performed on the remaining blood samples collected from these patients.IE/FACS was also performed on the specimen from Hep25,the patient removed from the NMLD cohort for the finding of a liver mass with portal vein thrombosis.Absolute CTC counts by CellSearch and IE/FACS for these sam-ples are provided in Additional file1.

CTC,PBMC,and FFPE sequencing performance Sequencing of adequate DNA samples from CTCs,FFPE tumor samples,and banked PBMC from the study co-hort(Figure1,Table3)was performed.Paired FFPE tumor and/or PBMC from patients with adequate CTC DNA for sequencing were available in two cases;two additional cases with paired FFPE tumor and PBMC samples available without adequate CTC DNA also were analyzed from the HCC cohort(Figure1).Sequencing performance according to sample type is displayed in Table3.Sequencing performance data for FFPE tumor samples and banked PBMC(both a source of DNA not re-quiring WGA)were combined due to small sample sizes, for comparison to WGA DNA from CTCs(Table3).The mean amplicon read depth was lower(2258versus2954, p<0.01)and proportion of targeted bases with sequencing coverage of≥100x was significantly lower in CTC samples (43%)than in FFPE tumor plus PBMC samples(87%)(p< 0.025),using two-tailed t-tests.The mean number of vari-ant calls per sample was higher in CTC samples compared to FFPE samples(9vs.2,p<0.04),though the mean fre-quency of individual variant alleles was significantly lower (36%vs.60%,p<0.001)(two-tailed t-tests).Reproducibil-ity of sequencing results was demonstrated by3samples run in duplicate(data not shown).

Sequencing results:variants,SNPs and mutation calls Eighty-six variants overall,58of which were predicted to be protein-altering,were identified from all of the CTC

enrollment and samples tested.a One patient enrolled to NMLD control cohort was removed

with portal vein thrombosis on imaging after enrollment.CTC testing in this patient showed sample each of CTC and FFPE did not yield sufficient DNA for sequencing.c4primary and3metastatic 7of the HCC cohort cases.Paired CTC WGA DNA and FFPE tumor tissue were available in Paired FFPE tumor tissue and PBMC were available from2additional

cases.

Table2Univariate analysis of CTC levels and conventional prognostic factors with overall survival

HCC Cohort(n=20)Mean overall survival

(months)(standard error)Median overall survival

(months)(95%CI)

p value(Log-Rank test)

CTC per7.5mL

<1.0(n=12)10.96(1.95)11.29(2,69,16.06)

1.0(n=8)8.49(3.63)

2.76(0.72,15.54)0.6179

<2.0(n=13)10.37(1.89)10.32(3.25,12.91)0.8021

≥2.0(n=7)9.23(4.11) 2.20(0.72,15.54)0.8510

<3.0(n=14)9.74(1.86)9.45(2.69,12.91)

≥3.0(n=6)10.50(4.62)8.26(0.72,29.14)

Median AFP(ng/mL)

<400(n=10)11.20(2.29)11.32(2.69,16.07)0.4058

≥400(n=10)8.73(2.92) 5.39(0.72,14.32)

Macrovessel invasion

No(n=7)10.12(2.48)10.32(2.69,12.91)0.7493

Yes(n=13)10.45(2.82)8.58(1.58,15.54)

Child Pugh score(%)

A(n=14)10.69(1.87)11.32(2.20,15.54)

B(n=5)9.29(5.39) 3.25(0.72,29.14)0.7181

C(n=1)I

Etiology of liver disease(%)

HBV(n=5)10.28(3.83)8.58(2.20,21.85)

HCV(n=9)10.41(1.96)12.62(1.91,15.54)0.9324

HBV+HCV(n=2)I I

ETOH(n=1)I I

NAFLD(n=2)I I

Unknown(n=1)I I

Kaplan-Meier methods were used to determine the impact of CTC at each cut-point and conventional prognostic factors on overall survival.The CTC level,AFP value of400ng/mL,and presence of macrovessel invasion were used to dichotomize for univariate analyses.The Child Pugh score and etiology of liver disease were also examined.A p value of<0.05was considered statistically-significant under log-rank tests.No factor showed significance in univariate analysis though analyses were limited due to small small sample sizes.Key:CI=confidence interval.ETOH=alcohol.NAFLD=non-alcoholic fatty liver disease.I=sample size insufficient for analysis.

and FFPE tumor samples combined.Approximately54% were low-frequency(occurring in less than10%of the individual sample),among which93%were from CTC-derived DNA.Fifty-eight somatic,non-synonymous vari-ants were called mutations if a matching mutation has been described in liver cancer,if the variant shared the same amino acid residue as a COSMIC mutation in any cancer type,and/or if the variant allele frequency was greater than5%but the variant was not a known SNP and not present in any PBMC sample[28].Frameshift mutations were excluded from analysis due to known limitations of ion semiconductor sequencing to accur-ately detect frameshift mutations.Characteristic muta-tions in HCC(TP53,PTEN)were identified in CTC-derived DNA from two cases.Figure4displays a sum-mary of the somatic,non-synonymous mutations identi-fied in CTC and FFPE tumor samples combined.A listing of all somatic,non-synonymous mutations(ex-cluding frameshift)detected according to sample type is provided in Additional file2.In one HCC case with matched CTC,FFPE tumor,and PBMC DNA,8SNPs were present and concordant in both FFPE tumor and PBMC DNA;5of these(63%)were detected in the CTC DNA.Neither was identified in the paired CTC DNA. Discussion

The ability to detect and characterize malignant cells in circulation holds enormous promise as a biomarker in HCC,a grim cancer challenged by the inability of con-ventional noninvasive diagnostic and staging modalities to encompass its great clinical and biological heterogen-eity,as well as by a scarcity of tumor tissue available for diagnostic or research purposes.In this study,at least one CTC was detected in8/20(40%)of patients with metastatic HCC,compared to1/9(11%)of eligible NMLD patients using the CellSearch System.Though the cut-point of≥1CTC/7.5mL did not achieve signifi-cance between the two groups,a cut-point of≥2CTCs/ 7.5mL was significant,positive in7/20(35%)HCC patients compared with none in the NMLD cohort(p= 0.04),consistent with prior reports[14,15].The one eli-gible NMLD control patient with CTC count of1/ 7.5mL was subsequently found to have ultrasound find-ings suggestive of underlying tumor,although no formal HCC diagnosis was made,and thus he was not removed from the control cohort.Our findings confirm the lim-ited existing data suggesting that circulating EpCAM-positive epithelial cells are rare in patients with non-malignant liver diseases,and that EpCAM-positive cells in HCC patients are generally of tumor origin[14]. Corroborating the prognostic value of EpCAM-positive CTCs in other recent series[14,15],the detec-tion of CTCs in the HCC cohort of this study was sig-nificantly associated with high AFP and the presence of vascular invasion,and there was a non-significant trend toward poorer overall survival in patients with detectable CTCs.These findings support the value of CTCs as a prognostic biomarker in metastatic HCC and suggest fu-ture potential roles for CTCs in treatment decision-making as well as for stratification in clinical research, which historically has been challenged by the great prog-nostic heterogeneity of this disease[29].

The unexpected finding of high CTC levels in a patient initially enrolled to the NMLD cohort,who subsequently was removed for ineligibility due to the finding of a new liver mass with vascular invasion on ultrasound suggestive of HCC,raises the intriguing possibility that CTC detec-tion also may be associated with vascular invasion and poor prognosis in earlier stages of disease.This incidental finding,along with recent results of Schulze et al.and Sun et al.indicating prognostic value of CTC detection in pa-tients with localized HCC[14,15],suggest an important potential role for CTCs as a biomarker of occult vascular invasion,recurrence risk,and overall survival in patients with apparent localized disease undergoing evaluation for surgery or transplantation.

Our finding that EpCAM-positive CTCs are associated with high AFP and the presence of vascular invasion is

Table3Sequencing performance by sample type

Sample type CTC WGA DNA(n=5)FFPE Tumor DNA(n=6)and

PBMC DNA(n=3)(n=9total a)

p value(two-tailed t-test) Mean read length74bp76bp NS

Mean mapped reads per sample653,878bp668,633bp NS

Mean amplicon read depth(std.dev)2258(4389)2954(1379)p<0.01

Proportion with coverage>20x50%97%p<0.0002

Proportion with coverage>100x43%88%p<0.026

Mean non-synonymous variant calls per sample92b p<0.03

Mean variant allele frequency37%61%b p<0.0001

a Data from FFPE and PBMC DNA samples were combined for sequencing performance analyses(but not for genotype analyses)due to small sample size and similar observed coverage.NS=not significant.

b PBMC samples(germline DNA)were excluded from variant analyses,n=3.

in keeping with the results of others[14,15]which indi-cate that EpCAM-positive CTCs have biologic relevance as a diagnostic and prognostic biomarker in HCC. EpCAM expression and an EpCAM-positive gene ex-pression signature are associated with poor differenti-ation,high AFP levels,and activation of Wnt-β-catenin signaling pathways[30-32].EpCAM-positive HCC cells also express markers associated with cancer stem cells and the epithelial to mesenchymal transition,supporting a hypothesis that EpCAM enrichment identifies stem-like cells with potential for metastasis[15,30,31,33].

A key unanswered question is whether EpCAM is the optimal marker for CTC enrichment in HCC.Unlike other epithelial tumor types which demonstrate nearly universal EpCAM expression[34],EpCAM is not expressed on mature hepatocytes and is expressed in only approximately35%to60%of HCC tumors by immunohis-tochemistry or PCR-based methods[30,31,35-37].Thus,it is possible that non-EpCAM-expressing HCC cells exist in circulation and are undetectable by technologies employ-ing EpCAM enrichment,which may account for our in-ability to detect CTCs in some of our HCC patients.Small series of non-EpCAM-based CTC isolation methods,such selection for the expression of asialoglycoprotein receptor or pancytokeratin or by cell size,suggest numerically higher incidence of detectable CTCs in metastatic HCC patients than has been reported with CellSearch,though the data are limited by small sample sizes and are not comparative[16,17,38].Optimal CTC isolation and en-richment in HCC may require combining EpCAM with other markers.

Beyond using CTC detection and enumeration as a prognostic biomarker,however,CTCs offer a dynamic window into the evolution of metastatic disease.The ad-vent of next-generation sequencing has revealed a remark-able degree of heterogeneity within individual tumors and between primary tumors and their metastases[39].With increasingly sensitive and precise technologies for the de-tection and molecular profiling of rare cells,the genomic interrogation of CTCs may offer a powerful new tool to characterize,and someday to target,the dominant tumor subclones responsible for treatment resistance or meta-static progression.Heitzer et al.recently reported the first comprehensive genomic profiling of single CTCs using array comparative genomic hybridization and next-generation sequencing in a study of37patients with meta-static colorectal cancer[18].Among the6patients with adequate(>10)CTCs isolated for genomic profiling,con-cordance on copy number changes and characteristic driver mutations including PIK3CA,APC,and KRAS was shown,along with many additional mutations in the CTCs which were later found to be present at subclonal levels in the primary tumors by deep sequencing.Interestingly,het-erogeneity was observed between CTCs isolated from the same patient at the same time-point.

This pilot study represents the first report of efficient isolation and next-generation sequencing of CTCs in HCC,to our knowledge.In this study,ion semicon-ductor next-generation sequencing showed a signifi-cantly higher proportion of targeted bases with at least 100x coverage depth among FFPE tumor and PBMC samples(87%)compared to CTC-derived DNA

samples

(43%)(p <0.025).The disparate coverage depths accord-ing to sample type may be due in part to the use of an adaptor-ligation PCR WGA method which has been as-sociated with allelic loss;alternate methods of amplifica-tion such as multiple displacement may mitigate this effect [40-42].An alternate or contributory factor lead-ing to the difference in allele frequency between sample types,as well as to the mutational disagreements be-tween FFPE and CTC samples,may be the inherent het-erogeneity of individual CTCs which were pooled for WGA from each patient [43].WGA may also introduce low frequency variants by artifact [40,41].

In our study,86variants were identified from CTC and FFPE tumor samples.One half of the variants were low frequency (<10%)and derived predominantly from the CTC DNA samples.While again this finding could be due to coverage bias or artifact arising from WGA,these results are also consistent with the findings from Heitzer et al.in a colorectal cancer cohort [18],which suggest significant inter-CTC heterogeneity and could explain the prevalence of low-frequency variants arising from pooled DNA derived from multiple CTCs from an individual patient.Characteristic mutations associated with HCC (including TP53and PTEN )were identified in CTC-derived DNA,consistent with tumor origin [44].The overall sequencing accuracy in this study was demon-strated by several cases with available paired PBMC,CTC,and tumor DNA samples showing concordance on SNP calls,along with reproducibility of results in duplicate runs.A significant limitation of the exploratory sequen-cing in this pilot study,however,was its small sample size,along with the limited proportion of cases with paired CTC,FFPE tumor,and PBMC DNA available.

Conclusions

This study strongly supports that circulating epithelial cells are detectable in HCC patients,including via the CellSearch assay;and that these cells are EpCAM-positive tumor cells in circulation,rather than benign epithelial cells released in the setting of liver injury.These findings are based on significant CTC detection in HCC but not in NMLD cohorts,associations between CTC detection and HCC prognostic markers,and the demonstration of characteristic HCC mutations in DNA derived from purified CTCs.The significant association with macrovessel invasion and elevated AFP in this study,along with a trend towards poorer survival,indi-cate the potential value of CTC detection as a prognostic biomarker in metastatic HCC.Prospective analyses of CTCs in earlier stages of disease are warranted to deter-mine surrogacy for vascular invasion in patients undergo-ing evaluation for surgery or liver transplantation.In parallel,we demonstrate that CTCs offer a source of non-invasive tumor DNA for next-generation sequencing and

molecular profiling efforts in HCC.Future studies to de-termine the optimal CTC isolation technology,cut-points by assay and population,and methods for single-cell CTC molecular characterization are essential to develop CTCs as a clinical biomarker as well as a research tool in this grim,complex disease in urgent need of new biomarkers and therapeutic targets.

Additional files

Competing interests

The authors declare that they have no competing interests.

Authors ’contributions

RKK developed study concept,design,and protocol,consented and enrolled patients,managed and analyzed data,and wrote manuscript.MJ-MM performed CTC and WGA assays and contributed to data analysis and writing.TMB performed sequencing and analysis of sequencing data.EAC performed DNA extraction and contributed to study design,analysis,and writing.JH participated in study design and performed statistical analysis.NS performed DNA extraction.KE reviewed and marked pathology specimens for tumor content.RMM assisted in patient consent,blood specimen collection,and study coordination.BH,EMW,and FYY identified and consented control cases.APV participated in study design and data analysis.JWP participated in study design,developed IE/FACS assay,and contributed to data analysis and writing.All authors read and approved the final manuscript.

Acknowledgments

We acknowledge and deeply appreciate the patients who donated their specimens and time to participate in this biomarker study.The study was funded by a grant to RKK from the Mt.Zion Health Fund,University of

California,San Francisco.Support for specimen processing was provided by The Bili Project Foundation,Inc.RKK ’s effort was funded in part by a Young Investigator Award (YIA)from the American Society of Clinical Oncology (ASCO)and by the NHGRI (R01HG007063,PI:Phillips).KE was a Robert Black Fellow of the Damon Runyon Cancer Research Foundation (DRG-109-10)and is supported by the NCI/NIH (1K08CA172288-01A1).We thank Janet Scott,

Eduardo Sosa,and Adam Foye for technical assistance in specimen processing.Author details 1

Helen Diller Family Comprehensive Cancer Center and The Liver Center,University of California San Francisco (UCSF),55016th St.,Box 3211,San Francisco,CA 94143,USA.2Helen Diller Family Comprehensive Cancer

Center,UCSF,San Francisco,CA 94143,USA.3Department of Molecular and Medical Genetics,Oregon Health Sciences University,3181SW Sam Jackson Park Road,Mail Code #L103,Portland,OR 97239,USA.4University of Vermont Medical Center,89Beaumont Ave.,Burlington,VT 05405,USA.5Department of Pathology,UCSF,513Parnassus Ave.,San Francisco,CA 94143,USA.6

Division of Hepatology and Liver Transplant,UCSF,513Parnassus Ave.,S-357,San Francisco,CA 94143,USA.7Department of

Transplantation-Abdominal,UCSF,513Parnassus Ave.,S-357,San Francisco,CA 94143,USA.8Division of Hepatology and Liver Transplant and The Liver Center,UCSF,513Parnassus Ave.,S-357,San Francisco,CA 94143,USA.

Received:27May2014Accepted:16March

2015

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