ORIGINAL RESEARCH

ERCC1 in Advanced Biliary Tract Cancer Patients Treatedwith Chemotherapy: Prognostic and Predictive Roles

Vanessa da Costa Miranda & Maria Ignez Braghiroli & Luiza Dib Batista Bugiato Faria &

Sheila Aparecida Coelho Siqueira & Jorge Sabbaga & Paulo M. Hoff &Rachel P. Riechelmann

Published online: 11 December 2013# Springer Science+Business Media New York 2013

AbstractBackground In oncology, we tend to look for factors thatreflect better prognosis or predict response to treatments inorder to make a selection from which patients will derive thebenefit, avoiding futile therapies and/or toxicities. Definitiveprognostic and predictive factors in advanced biliary cancerremain unknown.Methods We retrospectively analyzed all consecutive patientsin our institution with advanced biliary tract cancer treatedwith palliative cisplatin plus gemcitabine. We evaluated theprognostic and predictive role of the immunohistochemistry(IHC) expression of ERCC1 (excision cross-complementinggene-1) on tumor response and also examined several clinicaland laboratory prognostic factors for overall survival.Results From January 2009 to July 2011, 72 patients wereidentified; their median overall survival was 9.5 months. In-dependent variables associated with shorter survival identifiedby the multivariable Cox regression analysis were ECOG 2-3(HR 8.4; 95 % CI 3.4 to 20.7; p <0.001) and Charlson Co-morbidity Index >1 (HR 9.5; 95 % CI 1.6 to 55.3; p =0.012).

Pathology slides were available from 44 patients: 23 (52 %)stained positive for ERCC1 on IHC (score ≥0.5). In thissubgroup, expression of ERCC-1 was not prognostic andwas not associated with either clinical benefit (partial responseand stable disease) or tumor response (partial response only)to chemotherapy.Conclusions In this cohort of unselected patients with ad-vanced biliary tract cancer treated with first-line gemcitabineplus cisplatin, IHC expression of ERCC1 was not eitherpredictive or prognostic. Patients with ECOG 2-3 and/ormultiple comorbidities had worse survival

Keywords ERCC-1 protein . biliary duct neoplasms .

cisplatin and gemcitabine

Introduction

Biliary tract tumors (BTCs) are rare, representing 3 % of allgastrointestinal tumors, but incidence has progressively in-creased over the past years [1, 2]. Unfortunately, most patientsare diagnosed with advanced stage disease, and only a few areconsidered candidates for curative surgery [1]. In advanceddisease, combination therapy based on 5-fluorouracil orgemcitabine (associated with oxaliplatin or cisplatin) resultedin increased progression-free survival in several phase II trials[1]. The landmark ABC-02 trial established gemcitabine pluscisplatin as the standard first-line treatment for biliary tractcancer. The authors showed an absolute gain of 4 months inoverall survival for those treated with the combination regi-men when compared to gemcitabine alone [3]. Based on thisstudy, most cancer centers have adopted this regimen as thestandard first-line treatment for the majority of metastatic/locally advanced biliary tract cancer.

Taylor of therapeutic management as to avoid futile thera-pies and/or toxicities requires the identification of prognostic

Electronic supplementary material The online version of this article(doi:10.1007/s12029-013-9568-5) contains supplementary material,which is available to authorized users.

V. da Costa Miranda :M. I. Braghiroli : L. D. B. B. Faria :J. Sabbaga : P. M. Hoff : R. P. Riechelmann (*)Disciplina de Radiologia e Oncologia, Instituto do Câncer do Estadode São Paulo, Faculdade de Medicina da Universidade de São Paulo,Avenida Dr. Arnaldo 251,12o andar, São Paulo,SP 01246-000, Brazile-mail: rachel.riechelmann@icesp.org.br

J. Sabbaga : P. M. HoffCentro de Oncologia, Hospital Sírio Libanês, São Paulo, Brazil

S. A. C. SiqueiraDisciplina de Patologia, Instituto do Câncer do Estado de São Paulo,Faculdade de Medicina da Universidade de São Paulo,São Paulo, Brazil

J Gastrointest Canc (2014) 45:80–86DOI 10.1007/s12029-013-9568-5

and/or predictive factors. Some retrospective analyses havesuggested that bilirubin level >10 mg/dL, intrahepaticcholangiocellular carcinoma, poor Eastern Cooperative On-cology Group (ECOG) performance status, and elevated al-kaline phosphatase level are independent predictors of poorprognosis [4, 5]. However, prognostic and predictive factorsin biliary cancer also remain unknown.

DNA repair is an extremely complex process, and many ofthese processes may be altered in various types of tumors. Forplatinum compounds, in vitro data clearly show that nucleo-tide excision repair (NER) pathway is the DNA repair path-way responsible for the repair of cisplatin-DNA damage [6].The mechanism of action of cisplatin is through formation ofbulky intrastrand platinum–DNA adducts; the removal ofthese adducts from genomic DNA is mediated by the NERpathway [6]. High ERCC1 expression has been associatedwith resistance to platinum-containing therapy lung, ovarian,and colorectal cancers [7–9]. However, ERCC1 expressionhas not been investigated in BTCs.

In this study, we have retrospectively evaluated the prognosticand predictive role of immunohistochemistry (IHC) expressionof ERCC1 (excision cross-complementing gene-1) on the re-sponse to chemotherapywith cisplatin plus gemcitabine.We alsohave looked at several clinical and laboratory prognostic factorsthat could influence overall survival in patients with metastaticbiliary tract cancer.

Methods

We performed a retrospective evaluation of all consecutivepatients with histologically confirmed advanced adenocarci-noma of the biliary tract that received palliative chemotherapywith cisplatin plus gemcitabine in our institution (Instituto doCâncer do Estado de São Paulo, Brazil) from January 2009 toJuly 2011. Data from the electronic medical records werecollected, including factors potentially considered to be ofprognostic value: age at initial treatment, sex, comorbidities(defined by the Charlson’s Comorbidity Index) [10], tumorlocation (intra or extrahepatic cholangiocarcinoma or gall-bladder), ECOG performance status, baseline Ca19-9 (within1 month prior to chemotherapy initiation), baseline bilirrubin,billiary tree drainage, number of chemotherapy cycles, treat-ment interruptions, and major toxicities (grade 3 and 4).Images were retrieved for all patients, and response rate wasmeasured by RECIST 1.1 [11].

For the subset of patients who had tumor tissue available(44 out of 72), we performed an evaluation of ERCC-1 proteinexpression by IHC [12, 13]. Expression was evaluated by aninvestigator (SACS) who was blinded to clinical data. Sheindependently evaluated the ERCC1 staining under a lightmicroscope at a magnification of 400× and recorded whethertumor or stromal cells expressed ERCC1. Staining intensity

was graded on a scale of 0 to 3 (with a higher numberindicating a higher intensity). The extension of positive tumornuclei was calculated for each specimen and scored asassigned (0 if 0 %, 0.1 if 1 % to 9 %, 0.5 if 10 % to 49 %,and 1.0 if 50 % or more). This score was multiplied by thestaining intensity of nuclei to obtain a final semiquantitative Hscore. The median value of all H scores was chosen a priori asthe cutoff point for separating ERCC1-positive tumors fromERCC1-negative tumors [12, 13].

We used summary statistics to describe patient characteris-tics. Survival time was defined from the date of first visit to thedate of death, and overall survival was displayed as a Kaplan–Meier curve.

The predictive value of ERCC1 expression and tumorresponse was calculated by the Chi-square test, utilizing themedian as the cut off value. We also evaluated the predictivevalue of ERCC1 as a continuous variable using univariatelogistic regression. For the prognostic evaluation, we per-formed a multivariable COX regression analysis for overallsurvival, using all the clinical and laboratory parameters de-scribed above as well as the ERCC-1 expression as a contin-uous variable. A multivariable analysis for predictive factorsof response was not performed because of the small number ofpatients whose tumor tissue was available (N =44). A two-tailed p value<0.05 was considered significant.

Table 1 Patient characteristics

All – N (%)=72

Median age (range) 60 years (30–80)

Sex

Female 45 (62.5 %)

Male 27 (37.5 %)

Median baseline CA19.9 (ng/ml) 84.7 (0.1–50,000)

Conjugated bilirrubin 0.32 (0.07–25.2)

ECOG

0/1 50 (69.4.3 %)

>1 22 (37.5 %)

Tumor location

Intraheptic 30 (41.7 %)

Extrahepatic 19 (26.4 %)

Gallbladder 23 (31.9 %)

Clinical stage

Locally advanced 25 (34.7 %)

Metastatic 47 (63.3 %)

Charlson index

0/1 65 (90.3 %)

>1 7 (9.7 %)

Drainage before chemotherapy

Yes 32 (44.4 %)

No 40 (55.6 %)

J Gastrointest Canc (2014) 45:80–86 81

Results

Clinical/Laboratorial Data and Survival Analysis

From January 2009 to July 2011, 72 patients were identifiedand their characteristics are summarized in Table 1. Themedian baseline CA 19.9 was 84.7 ng/mL, 42 (62.5 %) werewomen and 69.4 % of patients had an ECOG status of 0 or 1.

Nearly 40 % had an intrahepatic tumor, and two-thirds weremetastatic at diagnosis.

Themedian number of chemotherapy cycles was four (range1–9), and the most common grade 3–4 treatment-related toxic-ity was hematologic (N =28; 37.8 %). During treatment, 30(41.7 %) patients were hospitalized, with 5 (7 %) due tochemotherapy-induced adverse events. Additionally, 18(25 %) patients had chemotherapy discontinued due to toxic-ities (Table 2). Nearly one-third of patients (27.7 %) receivedsecond-line treatment, mostly with fluoropyrimidines.

The median overall survival of our cohort was 9.5 months(range 0.3–35.3 months). Survival estimates according toECOG status showed a median survival of 13.5 monthsamong ECOG 0-1 patients, while the median survival forECOG 2-3 patients was 3.5 months (Fig. 1). MultivariableCox regression analysis for overall survival identified ECOG2-3 and presence of major comorbidities as independent var-iables associated shorter survival (Table 3). Patients with anECOG 2/3 had an 8.4 times higher risk of death (confidenceinterval [CI] 95 % adjusted HR 3.4 to 20.7; p <0.0001) whencompared with patients with ECOG 0 or 1; a ComorbidityCharlson index score of >1 was associated with a 9.5 in-creased risk of death (CI 95 % adjusted HR 1.6 to 55.3; p =0.012) in comparison to patients with lower score. There was atrend for worse survival among patients who did not have a

Table 2 Information on treatment

Median chemotherapy cycles 4 (1–9)

G3-G4 toxicities

Neutropenia 12 (16.6 %)

Thrombocytopenia 9 (11.5 %)

Anemia 7 (9.7 %)

Nausea and vomiting 6 (8.3 %)

Hospitalization during treatment (any cause) 30 (41.7 %)

Hospitalization due to toxicity 5 (7 %)

Treatment discontinuation 18 (25 %)

Second-line treatment 20 (27.7 %)

5 FU+Leucovorin 19

Carboplatin and gemcitabine 1

Fig. 1 Patients overall survivalaccording to ECOG

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biliary drainage before starting chemotherapy (adjusted HR2.3; CI 95 % 1.0 to 5.3; p =0.051).

Response to Treatment and ERCC-1 Hyperexpression

Of the 44 patients with pathology slides available, 27(61 %) had tissue collected from metastatic a lesion, 16(36 %) from primary resection of the tumor, and one(3 %) patient had diagnosis from fine needle biopsy(Table 4). Of the 44 patients, 23 (52 %) patients hadtumors that stained positive for ERCC1 on IHC (themedian score was 0.5). Figures 2 and 3 show positiveand negative IHC expressions of ERCC1, respectively, inpathology slides. ERCC1 positivity (score ≥0.5) was notassociated with either clinical benefit (partial response[PR] or stable disease [SD], p =0.55) or tumor response(complete or partial response only, p =0.57). The meanIHC expression score was 0.8 (median=zero staining) forpatients who had progressive disease (PD) at best responseand 1.1 (median=0.5) for those who achieved clinicalbenefit (p =0.43, OR 1.2 [IC 95 % 0.73–2.05]). Whenthe PD/SD group was combined and compared with thePR group, the mean ERCC1 expression was not statisti-cally different 0.85 (median=0.05) vs 1.36 (median=1.0)(p =0.22, OR 1.4 [IC 95 % 0.80–2.46]).

The ERCC-1 IHC expression score was also not prognosticwhen evaluated as a categorical variable, using the median 0.5as the cut off value, (p =0.51) or as a continuous variable (p =0.82, unadjusted HR 0.96 [IC 95 % 0.71–1.30]) Fig. 4.

Discussion

In this retrospective study, the ERCC1 IHC expression wasnot predictive for response to chemotherapy or prognosticfor overall survival. ECOG 2-3 and multiple comorbiditieswere associated with worse prognosis. Patients with ECOG2-3 had a median overall survival of only 3.5 months com-pared with 13.5 months observed in patients with betterperformance status.

In the ABC 02 trial [3], which led to gemcitabine andcisplatin become the standard treatment for patients withadvanced BTCs, the median overall survival reported was11.7 months. In our study, the median overall survival was9.5 months.We think this difference is due to an out of clinicaltrial context, where patients are selected by strict eligibilitycriteria [3]. In the ABC 02 trial [3], an unplanned subgroupanalysis also found that patients with an ECOG 2 (51/410,12.4 %) had higher risk of death, although the authors do notprovide their median survival. In our study, patients withECOG 2-3 had a median overall survival 10 months shorterthan those with ECOG 0-1 [3]. Similarly, Suzuki et al

Table 3 Cox regression multivariate analysis for survival

Variables p Hazard ratio (CI 95 %)

ECOG 2/3 vs 0/1 p <0.0001 8.4 (3.4–20.7)

Charlson index (>1 vs 0/1) p =0.012 9.5 (1.6–55.3)

Biliary drainage (no vs yes) p =0.051 2.3 (1.0–5.3)

Tumor location(other vs gallbladder)

p =0.078 1.9 (0.9–4.0)

Treatment interruption (no vs yes) p =0.168 0.6 (0.3–1.2)

CA 19.9 (continuous variable) p =0.619 1.0 (1.0–1.1)

Metastatic vs locally advanced p =0.845 1.1 (0.5–2.1)

Table 4 Patients tested for ERCC1

Number ofpatients

Intensity Extension ScoreERCC1

Origin oftumor tissue

1 1 0.10 0.1 Peripancreatic node biopsy

2 1 0.50 0.5 Material obtained fromprimary tumor resection

2 1 0.50 0.5 Liver biopsy

1 2 0.10 0.2 Liver biopsy

3 2 0.50 1 Material obtained fromprimary tumor resection

2 2 0.50 1 Liver biopsy

1 2 0.50 1 Biopsy of omental nodule

3 2 1.00 2 Liver biopsy

1 2 1.00 2 Material obtained fromprimary tumor resection

5 3 1.00 3 Material obtained fromprimary tumor resection

2 3 1.00 3 Liver biopsy

1 3 1.00 3 Bone biopsy

1 3 1.00 3 Peritoneum biopsy

Nineteen patients had an ERCC1 score of zero

Fig. 2 Positive immunehistochemistry expression of ERCC1

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evaluated prognostic factors of biliary tract cancer in Japanesepatients treated with gemcitabine monotherapy and also foundECOG to be a poor prognostic factor for survival [14].

Another independent prognostic variable found in ourstudy was the presence of multiple comorbidities. This isvery important to take into account when deciding first-linetreatment because the outcomes of these patients have notbeen represented in clinical trials. Although it is intuitive toconsider comorbid illnesses prognostic, we are unaware ofother studies that have evaluated this issue in advancedbiliary tract cancer.

In other tumor types, such as lung cancer, the IHChyperexpression of ERCC1 has been associated with platinumresistance [12]. Low expression of ERCC1 has also beenlinked to longer survival among patients with head and neckcancer [13]. In advanced urothelial cancer, ERCC1 expressionpredicted for longer progression-free survival in patients treat-ed with platin-based chemotherapy [15]. Our results showedno association between ERCC-1 expression and either surviv-al or response to treatment. ERCC1 is a key component of theplatinum–DNA repair machinery responsible for NER, andreports have suggested that it is a predictive and prognosticmarker in solid cancers treated with platinum-based chemo-therapy. The cytotoxic effect of platinum-based chemotherapyis related to the formation of bulky platinum–DNA adducts,and cisplatin resistance seems to be associated with the re-moval of these adducts by the NER system, which plays amajor part in cisplatin resistance [7]. ERCC1 messenger RNA(mRNA) has been studied in human ovarian, gastric, cervical,colon, and non-small-cell lung cancer (NSCLC) patients, andhigher tumor levels of ERCC1 were associated with clinicalresistance to cisplatin-based chemotherapy [8, 9, 16–19]. Theabsence of a predictive effect of ERCC1 on tumor response orsurvival in our study could be a result of the small sample size,the method utilized to measure ERCC1 expression (IHC vspolymerase chain reaction), or simply because ERCC1 is notassociated with platinum resistance in biliary cancer.

In our series, 25 % of patients had to discontinue treatmentdue to toxicity. The most common G3-4 adverse events

Fig. 3 Negative immunehistochemistry expression of ERCC1

Fig. 4 Patients overall survivalaccording to ERCC1 score

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observed were nausea/vomiting and myelotoxicity. The ABC-02 trial noted similar adverse events but with higher frequencyof G3-4 fatigue and abnormal liver function. Additionally,patients with advanced biliary cancer often need to be hospi-talized due to infectious complications and biliary obstruction.In our study, 30 (40 %) patients required hospitalization forany cause during treatment with chemotherapy, with the maincauses being cholangitis, febrile neutropenia, and biliary ob-struction, and five patients needed hospitalization due totreatment-related complications. We also noted that biliaryducts drainage prior to starting chemotherapy showed a trendfor better survival. However, the benefit for biliary drainageamong patients with advanced BTC and poor performancestatus is yet to be determined. Crosara et al. [20] retrospec-tively evaluated which cancer patients (solid tumors, mainlyBTCs) could benefit from percutaneous transhepatic biliarydrainage after a malignant obstructive jaundice in our institu-tion. In this retrospective study, authors found that cancerpatients with ECOG >2 and who are not candidates for furtherchemotherapy had a dismal prognosis and should probablynot be offered percutaneous transhepatic biliary drainage,mainly because these patients suffer more hospitalization ratesand complications related to the procedure [20].

In pancreatic tumors, baseline CA19.9 and its decreasealong treatment have shown prognostic importance [21]. Al-though biliary cancers usually express CA 19.9, the prognos-tic role of this tumor marker in biliary cancer is still uncertain.Some studies suggest that pretreatment CA 19-9 levels and itsdecrease after treatment are prognostic; however, other studieshave not shown the same [21, 22]. The largest study toevaluate patients with advanced biliary cancer (ABC-02) didnot asses the possible prognostic role of this marker [23]. Inour population, the baseline CA 19.9 was not an independentvariable for worse survival. Nevertheless, the prognostic in-fluence of Ca19-9 might have been compromised becausesome patients presented cholestasis at the time this markerwas tested.

Because this was a retrospective analysis, the informationon grade 1 or 2 adverse events could have beenunderestimated. The study was conducted in a single centerin Brazil and had a small sample; therefore, the study mayhave been underpowered to detect differences in response rateor survival according to ERCC1 expression. Nevertheless,given the paucity of data about prognostic and predictivefactors in biliary cancer, our study adds relevant informationon how to select patients for palliative chemotherapy.

In conclusion, we found that in this cohort of unselectedpatients with advanced biliary tract cancer who were treatedwith first-line gemcitabine plus cisplatin, IHC expression ofERCC1was neither predictive nor prognostic, while ECOG 2-3 and multiple comorbidities were associated with worseprognosis. Based on our results, we suggest that chemothera-py for biliary cancer should be offered to patients in good

clinical conditions and with minor comorbid illnesses. Wealso suggest that patients with ECOG 0-1 should have theirbiliary tract drained before treatment starts. Prospective col-lection of data, for example, as part of phase III trials, shouldbe conducted in this patient population with the aim toidentify/validate prognostic and predictive markers, as forexample, Ca19-9 and ERCC1, to better tailor the treatmentof patients with metastatic biliary cancer.

Conflict of Interest The authors declare that they have no conflict ofinterest.

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