a phase i pharmacologic study of ercc2 mutations correlate with necitumumab (imc … · 1140 |...

1140 | CANCER DISCOVERY�OCTOBER 2014 www.aacrjournals.org

RESEARCH ARTICLE

A Phase I Pharmacologic Study of Necitumumab (IMC-11F8), a Fully Human IgG1 Monoclonal AntibodyAndreas G. Bader1, David Brown1, and Matthew Winkler1,2

ABSTRACT The Mammary Prevention 3 (MAP.3) placebo-controlled randomized trial in 4,560

high-risk postmenopausal women showed a 65% reduction in invasive breast can-

cer with the use of exemestane at 35 months of median follow-up. Few differences in adverse events

were observed between the arms, suggesting a promising risk:benefi t balance with exemestane for

use in chemoprevention. Yet, the MAP.3 design and implementation raise concerns about limited data

maturity and not prospectively including key bone-related and other toxicities as study endpoints.

Exemestane for prevention is juxtaposed against selective estrogen receptor modulators and the other

aromatase inhibitors. Additional issues for prevention, including the infl uence of obesity, alternative

dosing, and biomarker use in phase III trials, are addressed.

SIGNIFICANCE: The recently completed MAP.3 trial of exemestane for breast cancer prevention offers

a potential new standard for pharmaceutical risk reduction in high-risk postmenopausal women. In

addition to describing key fi ndings from the publication of MAP.3 and related trials, our review under-

takes a detailed analysis of the strengths and weaknesses of MAP.3 as well as the implications for

future prevention research. Cancer Discov; 2(X); XXX–XXX. ©2012 AACR.

ABSTRACT Cisplatin-based chemotherapy is the standard of care for patients with muscle-inva-

sive urothelial carcinoma. Pathologic downstaging to pT0/pTis after neoadjuvant

cisplatin-based chemotherapy is associated with improved survival, although molecular determinants

of cisplatin response are incompletely understood. We performed whole-exome sequencing on pre-

treatment tumor and germline DNA from 50 patients with muscle-invasive urothelial carcinoma who

received neoadjuvant cisplatin-based chemotherapy followed by cystectomy (25 pT0/pTis “respond-

ers,” 25 pT2+ “nonresponders”) to identify somatic mutations that occurred preferentially in respond-

ers. ERCC2 , a nucleotide excision repair gene, was the only signifi cantly mutated gene enriched in the

cisplatin responders compared with nonresponders ( q < 0.01). Expression of representative ERCC2

mutants in an ERCC2 -defi cient cell line failed to rescue cisplatin and UV sensitivity compared with

wild-type ERCC2. The lack of normal ERCC2 function may contribute to cisplatin sensitivity in urothe-

lial cancer, and somatic ERCC2 mutation status may inform cisplatin-containing regimen usage in

muscle-invasive urothelial carcinoma.

SIGNIFICANCE: Somatic ERCC2 mutations correlate with complete response to cisplatin-based chemo-

sensitivity in muscle-invasive urothelial carcinoma, and clinically identifi ed mutations lead to cisplatin

sensitivity in vitro . Nucleotide excision repair pathway defects may drive exceptional response to

conventional chemotherapy. Cancer Discov; 4(10); 1140–53. ©2014 AACR.

See related commentary by Turchi et al., p. 1118.

RESEARCH ARTICLE

Somatic ERCC2 Mutations Correlate with Cisplatin Sensitivity in Muscle-Invasive Urothelial Carcinoma Eliezer M. Van Allen 1,2 , Kent W. Mouw 3,4 , Philip Kim 5 , Gopa Iyer 6,7 , Nikhil Wagle 1,2 , Hikmat Al-Ahmadie 6,8 , Cong Zhu 2 , Irina Ostrovnaya 9 , Gregory V. Kryukov 2 , Kevin W. O’Connor 3 , John Sfakianos 5 , Ilana Garcia-Grossman 7 , Jaegil Kim 2 , Elizabeth A. Guancial 10 , Richard Bambury 7 , Samira Bahl 2 , Namrata Gupta 2 , Deborah Farlow 2 , Angela Qu 1 , Sabina Signoretti 11 , Justine A. Barletta 11 , Victor Reuter 6,8 , Jesse Boehm 2 , Michael Lawrence 2 , Gad Getz 2,12 , Philip Kantoff 1 , Bernard H. Bochner 5,6 , Toni K. Choueiri 1 , Dean F. Bajorin 6,7 , David B. Solit 6,7,13 , Stacey Gabriel 1 , Alan D’Andrea 3,4 , Levi A. Garraway 1,2 ,and Jonathan E. Rosenberg 6,7

1 Department of Medical Oncology, Dana-Farber Cancer Institute, Har-vard Medical School, Boston, Massachusetts. 2 Broad Institute of MIT and Harvard, Cambridge, Massachusetts. 3 Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massa-chusetts. 4 Harvard Radiation Oncology Program, Boston, Massachusetts. 5 Urology Service, Department of Surgery, Memorial Sloan Kettering Can-cer Center, New York, New York. 6 Weill Cornell Medical College, Cornell University, New York, New York. 7 Genitourinary Oncology Service, Depart-ment of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York. 8 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York. 9 Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New York. 10 Division of Hematology/Oncology, Department of Medicine, University of Roches-ter Medical Center School of Medicine and Dentistry, Rochester, New York. 11 Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. 12 Massachusetts General Hospi-tal Cancer Center and Department of Pathology, Boston, Massachusetts.

13 Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York.

Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

E.M. Van Allen and K.W. Mouw contributed equally to this article.

L.A. Garraway and J.E. Rosenberg contributed equally to this article.

Corresponding Authors: Jonathan E. Rosenberg, Genitourinary Oncology Service, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065. Phone: 646-422-4461; Fax: 646-227-2417; E-mail: [email protected] ; and Levi A. Garraway, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, D1542, Boston, MA 02115. Phone: 617-632-6689; Fax: 617-582-7880; E-mail: [email protected]

doi: 10.1158/2159-8290.CD-14-0623

©2014 American Association for Cancer Research.

Research. on September 1, 2021. © 2014 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst August 5, 2014; DOI: 10.1158/2159-8290.CD-14-0623

http://cancerdiscovery.aacrjournals.org/

OCTOBER 2014�CANCER DISCOVERY | 1141

INTRODUCTION

Platinum-based chemotherapy has been the standard of

care for patients with muscle-invasive and metastatic urothe-

lial carcinoma for more than 20 years ( 1–3 ), and neoadjuvant

cisplatin-based chemotherapy leads to a 14% to 25% relative

risk reduction for death from muscle-invasive urothelial car-

cinoma (cT2-T4aN0M0; refs. 3–5 ). Pathologic downstaging

to complete response (pT0) or carcinoma in situ (pTis) at

cystectomy occurs in 26% to 38% of patients treated with

neoadjuvant chemotherapy compared with 12.3% to 15%

for patients undergoing cystectomy alone ( 3, 4 , 6 ), and the

5-year survival for pT0/pTis patients is 85% after neoadju-

vant chemotherapy ( 3 ) compared with 43% for patients with

persistent muscle-invasive disease (≥pT2; ref. 7 ). Therefore,

the benefi t of neoadjuvant chemotherapy seems to be most

dramatic in patients who are found to have pathologic com-

plete responses at the time of surgical resection. However, the

inability to predict which patients will derive clinical benefi t

has limited the use of this toxic approach in the urologic

community ( 8–10 ).

Cisplatin causes accumulation of DNA cross-links, which

interfere with DNA replication and gene transcription, and

eventually promotes cell death. Repair of cisplatin-induced

DNA damage occurs primarily through DNA repair pathways

such as nucleotide excision repair (NER; ref. 11 ) and homolo-

gous recombination (which includes BRCA1 and BRCA2 ;

ref. 12 ). The NER pathway involves multiple genes, includ-

ing ERCC1–5 , CDK7 , DDB1–2 , XPA , and XPC ( 13 ). Germline

alterations in NER genes result in multiple recessive inherited

disorders, including xeroderma pigmentosum (XP; ref. 13 ).

Because of a defi ciency in NER, patients with XP have sig-

nifi cantly increased risk of developing skin cancers and other

malignancies ( 14 ).

Germline or expression-based changes in several NER

genes have been suggested to modulate clinical response to

cisplatin-based chemotherapy ( 15, 16 ). However, prospec-

tive studies have not confi rmed these observations ( 17, 18 ).

ERCC2 (an NER helicase) loss-of-function correlates with cis-

platin sensitivity in preclinical models ( 19 ), whereas ERCC2

overexpression leads to cisplatin resistance ( 20 ). These data

suggest that tumors with loss of NER function may exhibit

increased cisplatin sensitivity, and recent data have identi-

fi ed somatic ERCC2 mutations in 7% to 12% of urothelial

carcinomas ( 21, 22 ). We hypothesized that somatic muta-

tions in the NER pathway may correlate with response

to cisplatin-based neoadjuvant chemotherapy in patients

with urothelial carcinoma. To test this, and more gener-

ally to defi ne genomic correlates of chemotherapy response,

we performed whole-exome sequencing (WES) of urothelial





Van Allen et al.RESEARCH ARTICLE

carcinoma tumors from patients with extreme responses to

neoadjuvant cisplatin-based combination chemotherapy.

RESULTS Somatic Genetic Alterations in Muscle-Invasive Urothelial Carcinoma

We sequenced pretreatment tumor and germline DNA

from 50 patients treated with neoadjuvant cisplatin-based

chemotherapy for muscle-invasive urothelial carcinoma; 25

“responders” had no residual invasive disease (pT0/pTis)

on pathologic examination following cystectomy, and 25

“nonresponders” had residual muscle-invasive (≥pT2) disease

( Fig. 1A ; Methods). Although multiple chemotherapeutic reg-

imens were used, the only common agent among all patients

was cisplatin ( Table 1 and Supplementary Table S1). No sig-

nifi cant differences in clinical characteristics were identifi ed

between responders and nonresponders at baseline ( P > 0.05;

Mann–Whitney test).

The mean target coverage from WES was 121× for tumors

and 130× for paired germline samples (Supplementary

Table S1). The median mutation rate was 9.7 mutations per

megabase (mutations/Mb) for responders and 4.4 mutations/

Mb for nonresponders ( P = 0.0003; Mann–Whitney test; Fig. 1B ;

Supplementary Fig. S1A and S1B; Supplementary Table S1),

raising the possibility of reduced DNA repair fi delity among

cisplatin responders. All observed somatic mutations and short

insertion/deletions are reported in Supplementary Table S2.

A statistical assessment ( 23 ) of the base mutations and

short insertion/deletions across both responders and

Figure 1. Study design, mutation rates, and aggregate signifi cant somatic mutations. A, patients with muscle-invasive urothelial carcinoma cancer were split into cases and controls based on their pathologic response to cisplatin-based neoadjuvant chemotherapy. TURBT, transurethral resection of bladder tumor. Nine cases could not complete sequencing due to technical reasons (failed sequencing or elevated contamination). B, data are arranged so that each column represents a tumor and each row represents a gene. The center panel is divided into responders (left and black) and nonresponders (right and yellow). The mutation rates of responders are elevated compared with nonresponders (top). The alteration landscape (center) of the aggregate cohort ( n = 50 patients) demonstrates a set of statistically signifi cant genes that are altered in urothelial carcinoma ( TP53 , RB1 , KDM6A , and A RID1A ). The negative log of the q values for the signifi cance level of mutated genes is shown (for all genes with q < 0.1) on the right. ERCC2 mutation status is also shown below the other genes, although ERCC2 was not signifi cantly mutated across the combined cohort. Additional data about allelic fraction ranges for each case (bottom), mutation rates (top), and mutational frequency (left) are also summarized.

Syn.

Nonsyn.

30

# M

uta

tions/

Mb

2520151050

56% TP53

RB1

KDM6A

ARID1A

ERCC2 0

0204060

Alle

licfr

act

ion (

%)

80100

1 2–log10(q-value)

3 4

24%

24%

26%

18%

30 20 10# Cases

with mutations

MS

KC

C–0296

MS

KC

C–0500

MS

KC

C–0100

MS

KC

C–0389

MS

KC

C–0411

MS

KC

C–0741

DF

CI–

32

MS

KC

C–0677

DF

CI–

35

MS

KC

C–0343

DF

CI–

04

MS

KC

C–0446

MS

KC

C–0425

DF

Cl–

05

MS

KC

C–0464

DF

CI–

26

MS

KC

C–0821

MS

KC

C–0231

DF

CI–

07

MS

KC

C–0620

MS

KC

C–0547

DF

CI–

29

DF

CI–

37

DF

CI–

06

MS

KC

C–0445

DF

CI–

33

MS

KC

C–0255

DF

Cl–

34

DF

Cl–

21

DF

Cl–

31

DF

Cl–

38

MS

KC

C–0745

MS

KC

C–0101

MS

KC

C–0007

MS

KC

C–0567

MS

KC

C–0651

MS

KC

C–0730

MS

KC

C–0373

MS

KC

C–0301

MS

KC

C–0754

MS

KC

C–0439

MS

KC

C–0279

MS

KC

C–0760

MS

KC

C–0820

MS

KC

C–0331

MS

KC

C–0243

MS

KC

C–0234

MS

KC

C–0450

DF

CI–

43

DF

CI–

09

0

Responder Nonresponder

Missense Frame shiftIn-frame indelOther nonsyn.

Splice siteNonsense

A

B

TURBT:Muscle-invasiveurothelialcarcinoma

Whole-exomeSequencing

(n = 59)

Technicalfailure (n = 9)

Nominategenomic correlates

ResponderspT0/pTis(n = 25)

Nonresponders≥pT2

(n = 25)

Surgery and pathologicevaluation

Neoadjuvantcisplatin-basedchemotherapy

GermlineDNA





ERCC2 and Cisplatin Sensitivity in Urothelial Carcinoma RESEARCH ARTICLE

Table 1. Patient characteristics

Total ( N = 50) Responders ( n = 25) Nonresponders ( n = 25)

Age at TUR, y 62.5 ± 8.9 61 ± 10.1 64 ± 7.3 P > 0.05

Female sex, n (%) 13 (26) 6 (24) 7 (28)

Ethnicity, n (%)

�Hispanic/Latino 1 (2) 0 (0) 1 (4)

� Non-Hispanic/

non-Latino

48 (96) 24 (96) 24 (96)

�Unknown 1 (2) 1 (4) 0 (0)

Race, n (%)

�Caucasian 49 (98) 25 (100) 24 (96)

�African American 1 (2) 0 (0) 1 (4)

Smoking status, n (%)

�Never 13 (26) 8 (32) 5 (20)

�Former 26 (52) 13 (52) 13 (52)

�Current 11 (22) 4 (16) 7 (28)

Clinical staging, n (%)

�T2 37 (74) 19 (76) 18 (72)

�T3 10 (20) 5 (20) 5 (20)

�T4 3 (6) 1 (4) 2 (8)

�N0 40 (80) 18 (72) 22 (88)

�Node positive 10 (20) 7 (28) 3 (12)

TUR histology, n (%)

�TCC 32 (64) 19 (76) 13 (52)

�Mixed TCC 18 (36) 6 (24) 12 (48)

Neoadjuvant chemotherapy regimen, n (%)

�GC 31 (62) 14 (56) 17 (68)

�ddMVAC 16 (32) 9 (36) 7 (28)

�GC-sunitinib 2 (4) 2 (8) 0 (0)

�ddGC 1 (2) 0 (0) 1 (4)

Median interval

from chemotherapy

to cystectomy

(days ± SD)

47 ± 29.9 46 ± 26.0 47 ± 33.8 P > 0.05

Median length of

follow-up (days ±

SD) a

351 ± 363.2 372.5 ± 416.2 329.5 ± 287.1 P > 0.05

Patients with

recurrence, n (%)

13 (26) 2 (8) 11 (44)

NOTE: Clinical characteristics of the total patient cohort, as well as data stratifi ed by responder or nonresponder status. Plus–minus values are medians ± SD. Abbreviations: ddGC, dose-dense gemcitabine and cisplatin; ddMVAC, dose-dense methotrexate, vinblastine, doxorubicin, and cisplatin; GC, gemcitabine and cisplatin; TUR, transuretheral resection. a For only patients alive at the time of this study. P < 0.05 is considered signifi cant (Mann–Whitney test).

nonresponders nominated four signifi cantly altered genes

previously implicated in urothelial carcinoma ( 21, 22 , 24 ):

TP53 , RB1 , KDM6A , and ARID1A ( Fig. 1B and Supplemen-

tary Tables S3 and S4). In addition, nine nonsynonymous

somatic mutations were observed in ERCC2 ( Fig. 1B ; Sup-

plementary Fig. S2; Supplementary Table S2). Although

ERCC2 did not reach cohort-wide statistical signifi cance, its

known role in DNA repair and report of being recurrently

mutated in bladder cancer ( 21, 22 ) raised the possibility that

ERCC2 mutations might associate with cisplatin response.

Somatic ERCC2 Mutations in Cisplatin-Based Chemotherapy Responders

We performed an enrichment analysis to identify genes that

were selectively mutated in the responders compared with

nonresponders (Supplementary Methods). Among 3,277 genes

with at least one possibly damaging somatic alteration (Sup-

plementary Methods), ERCC2 was the only gene signifi cantly

enriched in the responder cohort ( Fig. 2A and Supplementary

Tables S5 and S6). Indeed, all ERCC2 nonsynonymous somatic






mutations occurred in the cisplatin-sensitive tumors ( P < 0.001;

Fisher exact test). ERCC2 remained signifi cantly enriched in

responders following false discovery analysis performed on

genes in which the mutation frequency afforded adequate

power ( q = 0.007; Benjamini–Hochberg; Fig. 2B ). Moreover, the

enrichment for ERCC2 mutations in the responder group was

signifi cant when adjusted for differences in the overall muta-

tion rate between responders and nonresponders ( P = 0.04;

binomial test). Toward this end, the median background muta-

tion rate for ERCC2 -mutant tumors (15.5 mutations/Mb) was

signifi cantly elevated compared with ERCC2 wild-type (WT)

tumors (5.1 mutations/Mb; P = 0.01; Mann–Whitney test; Sup-

plementary Fig. S1B), consistent with a possible DNA-repair

defect and prior reports ( 22 ). The somatic ERCC2 mutation frequency in the responder

cohort was also compared with two unselected bladder

Figure 2. Three tests examining selective enrichment of ERCC2 mutations in cisplatin-responder tumors. A, plot of MutSigCV gene-level signifi cance [−log 10 (MutSigCV P value)] and responder enrichment signifi cance [−log 10 (Fisher exact test P value)]. The size of the point is proportional to the number of responder patients who harbor alterations in the gene. Genes with a responder enrichment P value of <0.01 are shown in red; others are in gray, and the dashed line denotes a P value of 0.01. Only ERCC2 reaches statistical signifi cance in the responder cohort ( P < 0.001; Fisher exact test). B, among genes with suffi cient number of alterations for cohort comparisons ( n = 9), only ERCC2 somatic mutations occur exclusively in the cisplatin responders, which is signifi cant when accounting for the elevated mutation rate in responders compared with nonresponders ( * , P < 0.05). Compared with the unselected TCGA and Guo et al. urothelial carcinoma cohorts, C shows that ERCC2 somatic mutations are signifi cantly enriched in the responder cohort ( * , P < 0.01).

A

B C

ERCC2

Responder

Alterations

Max.

Min.

RB1TP53

KDM6A

ARID1A

Responder

sig

nific

ance

[–lo

g10

(P

-valu

e)]

MutSigCV significance

[–log10

(P-value)]

Responders (pT0/Tis)

ERCC2

NF1

PIK3CA

ERBB4

RB1

FAT4

ARID1A

KDM6A

TP53

*

*

RespondersUnselected

Nonresponders (pT2+)

Number of patients

Patients

with s

om

atic E

RC

C2

muta

tions (

%)

pT0/Tis

(n = 25)

TCGA

(n = 130)

Guo et al.

(n = 99)Number of patients

01

23

4

0 5 10 15

10 5 0 0 5 10

15

01

02

03

04

0






cancer populations: 130 cases from The Cancer Genome Atlas

(TCGA; ref. 21 ) and 99 cases from a Chinese patient cohort

(ref. 22 ; Fig. 2C ). When compared with these unselected popu-

lations, ERCC2 mutations were signifi cantly enriched in the

cisplatin responder cohort (36% of cases; P < 0.001; binomial

test; Fig. 2C ). ERCC2 mutation status does not seem to be

prognostic, as it had no impact on overall survival in the TCGA

cohort ( P = 0.77; log-rank; Supplementary Fig. S3). To deter-

mine the relative abundance of somatic ERCC2 mutations in

other tumor types, TCGA data from 19 tumor types ( n = 4,429)

were queried ( 25 ). Somatic ERCC2 mutations were observed at

low frequencies (<4%) in 11 other tumor types ( Fig. 3A and B ).

All identifi ed somatic ERCC2 mutations occurred at highly

conserved amino acid positions within or immediately adja-

cent to the helicase domains ( Fig. 3A and C and Supple-

mentary Fig. S4). Similarly, germline ERCC2 mutations in

Figure 3. ERCC2 mutation mapping and distribution across tumor types. A, a stick plot of ERCC2 showing the locations of somatic mutations in the responders compared with ERCC2 mutations observed in two separate unselected bladder cancer exome cohorts. The ERCC2 mutations cluster within or near conserved helicase motifs. B, the somatic ERCC2 mutation frequency in multiple tumor types from TCGA. SCC, squamous cell carcinoma. C, structure of an archaebacterial ERCC2 (PDB code, 3CRV) with mutations identifi ed in the responder cohort mapped to their equivalent position. These locations are shown in the context of canonical germline ERCC2 mutations responsible for XP, XP/Cockayne syndrome (CS), and trichothiodystrophy (TTD).

TCGA patients with alterations in ERCC2 (%)0

Bladder

Gastric

Prostate

Colorectal

Lung

adenocarcinoma

Cutaneous

melanoma

Head and neck

SCC

Low-grade

glioma

Cervical

Ovarian

Renal

Breast

2 4 6 8 10 12 14

E606 V242

N238

P463

Y24

Responders XP

TTDXP/CS

D609

G665

B C

Conserved

helicase motif Missense (responders)

Missense (Guo et al.)

Missense (TCGA)

D609G

D609EY24C

N238S

1 I

Y72C S209C R286W

S246Y

N238S

E86QM42V

Y14C

S44L

P463S

T484A/M

G607A H659Y G675S Q758E

IA II III IV V V 760

V242F P463L E606G G665A

A






patients with XP (complementation group D) and XP with

combined Cockayne syndrome (XP/CS), two disorders char-

acterized by NER function, clustered within helicase domains

( Fig. 3C ). Conversely, mutations causing trichothiodystro-

phy, a disease resulting from the alteration of ERCC2’s role in

transcription, were distributed throughout the protein ( 26 ).

ERCC2 Mutations Confer Increased Cisplatin Sensitivity In Vitro

These observations raised the possibility that the identi-

fi ed mutations disrupt ERCC2 function and interfere with

NER. To test this hypothesis, the fi rst fi ve of the identifi ed

ERCC2 mutants were stably expressed in an immortalized

ERCC2 -defi cient cell line derived from an XP patient, and

the cisplatin sensitivity profi le of each cell line was measured

(Supplementary Methods; Fig. 4A ). Expression of WT ERCC2

rescued cisplatin sensitivity of the ERCC2 -defi cient cell line,

whereas none of the ERCC2 mutants rescued cisplatin sensi-

tivity ( Fig. 4B ). The IC 50 for the ERCC2 -WT–complemented

cell line was signifi cantly higher than that for the ERCC2-

defi cient parent cell line ( P < 0.0001; ANOVA), whereas the

IC 50 for each ERCC2 -mutant–complemented cell line was not

signifi cantly different from the parent ERCC2 -defi cient par-

ent cell line ( Fig. 4C ). Similarly, the IC 50 for the ERCC2-WT

Figure 4. ERCC2 mutants fail to rescue cisplatin sensitivity of ERCC2 -defi cient cells. A, immunoblot of ERCC2 expression in cell lines created by transfection of the ERCC2-defi cient parent cell line (GM08207; Coriell Institute), with pLX304 (Addgene) encoding GFP (negative control), WT ERCC2, or a mutant ERCC2. The negative control ERCC2 -defi cient cell line (lane 1) expresses endogenous levels of inactive ERCC2 from the parent cell genome, whereas WT (lane 2) and mutant (lanes 3–7) ERCC2 cell lines show increased levels of ERCC2 expressed from the transfected gene. β-Actin is shown as a loading control. B, cisplatin sensitivity profi les of cell lines expressing WT or mutant ERCC2. Expression of WT ERCC2 in an ERCC2 -defi cient background rescues cisplatin sensitivity, whereas expression of the ERCC2 mutants fails to rescue cisplatin sensitivity. An IC 50 was calculated from the survival data for each cell line, and these values are shown in C. The difference in IC 50 between the parent ( ERCC2 -defi cient) cell line and the cell line expressing WT ERCC2 was statistically signifi cant, as was the difference between the WT ERCC2 cell line and each of the mutant ERCC2 cell lines ( P < 0.0001; ANOVA). The difference between the ERCC2 -defi cient cell line and each of the mutant cell lines was not statistically signifi cant.

A

B

C

1 2 3 4 5 6 7 1. ERCC2 –/– mock

ERCC2 –/– mock

ERCC2 –/– WT

ERCC2 –/– V242F

ERCC2 –/– P463L

ERCC2 –/– E606G

ERCC2 –/– D609G

ERCC2 –/– G665A

2. ERCC2 –/– WT

3. ERCC2 –/– V242F

4. ERCC2 –/– P463L

5. ERCC2 –/– E606G

6. ERCC2 –/– D609G

7. ERCC2 –/– G665A

1

0.1

0.01

0 1 2 3 4Cisplatin concentration (μmol/L)

P = NS

800

600

400

IC50

(n

mo

l/L

cis

pla

tin

)

200

0

ERCC2–/

– moc

k

ERCC2–/

– WT

ERCC2–/

– V242F

ERCC2–/

– P463L

ERCC2–/

– E606G

ERCC2–/

– D60

9G

ERCC2–/

– G66

5A

P < 0.0001

Re

lative

ce

ll via

bili

ty

ERCC2

β-Actin






cell line was signifi cantly higher than that for the ERCC2 -

defi cient and mutant cell lines ( P < 0.0001; ANOVA).

The NER pathway repairs DNA lesions other than cis-

platin adducts, so we also tested the effect of the identifi ed

ERCC2 mutations on NER-mediated repair of UV-induced

DNA damage. WT and mutant ERCC2 –complemented cell

lines were exposed to increasing doses of UV irradiation, and

clonogenic survival was measured ( Fig. 5A–C ). Whereas the

ERCC2 -WT–complemented cell line rescued UV sensitivity,

the UV sensitivities of the ERCC2 -mutant–complemented cell

lines were not signifi cantly different from that of the ERCC2 -

defi cient parent cell line. Taken together, these experiments

Figure 5. ERCC2 mutants fail to rescue UV sensitivity of ERCC2 -defi cient cells. A, a representative colony formation assay for the ERCC2 -defi cient cell line (top) as well as the ERCC2 -defi cient line transfected with WT ERCC2 (middle), or one of the ERCC2 mutants (D609G, bottom) following increased doses of UV irradiation. B, clonogenic survival data for negative control, WT ERCC2, and mutant ERCC2. WT ERCC2 rescues UV sensitivity of the ERCC2 -defi cient cell line, whereas the mutant ERCC2s fail to rescue UV sensitivity. C, UV IC 50 values for cell lines. The difference between the ERCC2 -defi cient cell line and the WT ERCC2 cell line was signifi cant ( P < 0.0001; ANOVA), whereas the difference between the ERCC2 -defi cient cell line and each of the ERCC2 -mutant cell lines was not statistically signifi cant (NS).

A

B

C

UV dose (J/m2)

ERCC2–/– mock

0 2 4 6 8

ERCC2–/– WT

ERCC2–/– D609G

1

0.1

0.01

0.001

0

ERCC2–/

– moc

k

ERCC2–/

– WT

ERCC2–/

– V24

2F

ERCC2–/

– P46

3L

ERCC2–/

– E60

6G

ERCC2–/

– D60

9G

ERCC2–/

– G66

5A

2

4

6

P < 0.0001

P = NS

0 2 4 6 8

Surv

ival ra

tio

UV dose (J/m2)

ERCC2–/– mock

ERCC2–/– WT

ERCC2–/– V242F

ERCC2–/– P463L

ERCC2–/– E606G

ERCC2–/– D609G

ERCC2–/– G665A

IC5

0 (

J/m

2)






suggest that the observed ERCC2 mutations result in loss of

normal NER capacity.

Because the overall mutation rate was higher in ERCC2 -

mutated tumors, we hypothesized that ERCC2 mutations

may be broadly contributing to genomic instability. Thus,

we measured rates of chromosomal aberrations in WT and

mutant ERCC2 –complemented cell lines before and after cis-

platin treatment. In the absence of cisplatin, background

rates of chromosomal aberrations were slightly lower in the

ERCC2 -WT–complemented cell line than in the ERCC2 - defi cient

or ERCC2 -mutant–complemented cell lines, but this differ-

ence was not statistically signifi cant ( Fig. 6A–D ). However,

following cisplatin exposure, signifi cantly fewer chromosomal

aberrations were observed in the ERCC2 -WT–complemented

cell line than in the ERCC2 -defi cient parent cell line ( P = 0.03;

ANOVA), whereas expression of the ERCC2 mutants resulted

in no rescue of chromosomal stability ( P > 0.5; Fig. 6D ). These

data suggest that the responder-associated ERCC2 mutations

may contribute to overall genomic instability in these tumors.

Other DNA Repair Gene Alterations Finally, we sought to determine whether other DNA repair

genes might undergo recurrent mutations in cisplatin-

sensitive tumors. No significantly recurrent mutations

were observed in other NER or homologous repair genes

in responders compared with nonresponders. However, in

two responder tumors that did not have ERCC2 mutations,

somatic nonsense (truncating) mutations were detected in

the homologous recombination DNA repair genes BRCA1

and BRCA2 (Supplementary Table S2). There were no non-

synonymous BRCA1 or BRCA2 mutations in the nonrespond-

ers (Supplementary Table S2). These results are consistent

with the known sensitivity of BRCA1/2 -mutant tumors (e.g.,

BRCA1/2 -mutant breast or ovarian cancers) to platinum-

containing regimens ( 27 ). Whereas somatic ERCC2 muta-

tions were the only DNA repair gene mutations enriched in

the responders, singleton missense mutations of uncertain

signifi cance were observed in DNA damage response genes

in both responders and nonresponders, and are of unknown

functional relevance.

DISCUSSION In bladder cancer, the clinical benefi t of neoadjuvant chem-

otherapy is most apparent when pathologic downstaging

to pT0 or pTis is achieved at surgical resection following

Figure 6. ERCC2 mutants fail to rescue genomic instability following cisplatin exposure. Representative mitotic spreads from an ERCC2 -defi cient cell line (A), and the same ERCC2 -defi cient cell line transfected with WT ERCC2 (B), or one of the ERCC2 mutants (V242F; C) following cisplatin exposure. D, chromosomal aberration data from ERCC2 -defi cient, WT ERCC2, and mutant ERCC2 cell lines. Rates of chromosomal aberrations following cisplatin exposure were signifi cantly lower in the WT ERCC2 cell line than in the ERCC2 -defi cient line or the cell lines expressing mutant ERCC2 ( P = 0.03; ANOVA).

ERCC2 –/– mock

P = NS

P = 0.03

ERCC2 –/– mock

ERCC2 –/– WT

ERCC2 –/– V242F

ERCC2 –/– P463L

ERCC2 –/– E606G

ERCC2 –/– D609G

ERCC2 –/– G665A

3

2

Aberr

ations p

er

cell

1

0

Untreated + Cisplatin

ERCC2 –/– WTA

D

B ERCC2 –/– V242FC






cisplatin-based chemotherapy. However, the lack of a predic-

tive biomarker for clinical benefi t from neoadjuvant cispla-

tin-based chemotherapy has limited the use of this approach

in the clinical community due to toxicity concerns. Using

an extreme phenotype analysis, we have identifi ed an asso-

ciation between somatic ERCC2 mutations and pathologic

complete response to neoadjuvant cisplatin-based chemo-

therapy in muscle-invasive urothelial carcinoma. Although

ERCC2 mutations occur in approximately 12% of unselected

cases, 36% of cisplatin-based chemotherapy responders in our

cohort harbored somatic ERCC2 nonsynonymous mutations.

Moreover, all ERCC2 -mutant tumors responded to neoadju-

vant chemotherapy.

The NER pathway is a highly conserved DNA repair system

that identifi es and repairs bulky DNA adducts arising from

genotoxic agents such as cisplatin. The NER helicase ERCC2

unwinds duplex DNA near the damage site through the coor-

dinated action of two conserved helicase domains. All ERCC2

mutations identifi ed in this study occurred at conserved

positions within or adjacent to these helicase domains, and

the identifi ed mutants all failed to complement cisplatin or

UV sensitivity of an ERCC2 -defi cient cell line. Together, these

data suggest that the mutations result in loss of normal

ERCC2 function, leading to increased tumor cell sensitivity

to DNA-damaging agents such as cisplatin.

Interestingly, in seven (78%) of the ERCC2 -mutant cases,

the ERCC2 mutation allelic fraction was <0.5 (Supplemen-

tary Table S2), suggesting that WT ERCC2 remains present

at one allele. Therefore, the cisplatin sensitivity phenotype

may result from a haploinsuffi cient or dominant-negative

effect of a heterozygous ERCC2 mutation, rather than as

a result of biallelic inactivating mutations (as in the tradi-

tional “two-hit” tumor suppressor model). The driving force

for heterozygous mutation of ERCC2 is unknown; however,

the prevalence of ERCC2 mutations in this study and other

cohorts (such as the TCGA) suggests that loss of nor-

mal ERCC2 function may provide a selective advantage for

tumors. Partial loss of DNA repair fi delity could aid tumor

growth by decreasing repair-associated delays in cell-cycle

progression. In addition, decreased NER capacity may result

in higher rates of error-prone repair or large-scale genomic

changes that further drive tumor growth.

Despite providing a potential growth advantage, muta-

tion of one ERCC2 allele may render tumor cells susceptible

to a DNA-damaging agent such as cisplatin if inadequate

levels of WT ERCC2 are present to support NER (i.e., haplo-

insuffi ciency). Alternatively, the mutated version of ERCC2

may bind, but not effi ciently repair, damaged DNA, thereby

preventing repair by an alternative DNA-repair pathway

and leading to a dominant-negative phenotype, as has been

described for mutants of the yeast ERCC2 homolog Rad3

( 28 ). Further studies are necessary to explore the effects of

ERCC2 loss on tumor growth, and the mechanism by which

the identifi ed ERCC2 mutations confer changes in tumor

NER capacity.

One possible explanation for the fi ndings observed in this

study is that somatic ERCC2 mutation is associated with

good prognosis or smaller tumors . However, this is not sup-

ported by the survival data from the TCGA bladder cancer

cohort, which excluded patients who received neoadjuvant

chemotherapy. No difference in overall survival was observed

on the basis of somatic ERCC2 mutation status (Supplemen-

tary Fig. S3). In the extreme response cohort reported here,

patients were generally noted to have obvious disease left

behind after initial transurethral resection based on opera-

tive reports and imaging, making the fi ndings unlikely to be

related to complete transurethral resection.

Although these data should not yet be used to justify

avoiding cisplatin-based treatment in ERCC2 WT patients,

our fi ndings raise the possibility that somatic ERCC2 muta-

tion status may provide a genetic means to select patients

most likely to benefi t from cisplatin-based chemotherapy,

while directing other patients toward alternative therapeu-

tic approaches. Of note, our study focused specifi cally on

somatic mutations that are exclusively in the tumor, and

not germline single-nucleotide polymorphisms in ERCC2 or

other genes. Thus, this approach is distinct from genome-

wide association studies that have examined germline ERCC1

or ERCC2 polymorphisms and their mixed impact on cispla-

tin sensitivity ( 29 ). Broadly, these fi ndings will require inde-

pendent clinical validation in prospective trials to establish

the clinical predictive power of somatic ERCC2 mutation

status for cisplatin response.

It is possible that some nonresponding urothelial tumors

will harbor somatic ERCC2 mutations in larger cohorts; if

observed, examination of the post–chemotherapy-resistant

tumor would be critical to understanding whether tumor

heterogeneity played a role in resistance. The patients ana-

lyzed in this extreme phenotype analysis were treated with

combination cisplatin-based chemotherapy regimens con-

taining non-cisplatin agents; however, cisplatin was the only

agent common to all regimens. Because half of patients with

bladder cancer are not candidates for cisplatin-based chemo-

therapy due to preexisting comorbidities, less toxic carbo-

platin-based neoadjuvant therapies may warrant study for

non–cisplatin-eligible patients with ERCC2 -mutant tumors.

To date, exceptional response genomic studies have

informed genomic mechanisms of response to targeted

therapies, such as response to everolimus in multiple dis-

ease contexts ( 30, 31 ). However, published studies have been

limited to individual case reports due to the rarity of such

events with targeted therapies. This study represents a new

approach for studying extraordinary responses to commonly

used cytotoxic chemotherapies using a case–control design,

which may be applied to other therapeutic settings in which a

signifi cant minority of patients achieve exceptional response

(e.g., neoadjuvant chemotherapies in other clinical settings).

Toward that end, a majority of responder cases in our cohort

had no recurrent genomic determinant of cisplatin response.

It is possible that alterations in DNA repair genes not readily

detectable with WES (e.g., epigenetic, expression-based) may

mediate cisplatin sensitivity in these cases.

In conclusion, this work provides new insights into the

relationship between somatic genetic alterations and clini-

cal response to cisplatin-based chemotherapy in urothelial

carcinoma. If further validated, these results may inform

therapeutic decision-making, novel therapeutic development,

and clinical trial designs for urothelial carcinoma and possi-

bly other ERCC2 -mutated tumors. Finally, these results show

that somatic genomic alterations may reveal the mechanistic






underpinnings of antitumor response to conventional cyto-

toxic chemotherapy.

METHODS Patients and Samples

Patients with muscle-invasive or locally advanced urothelial car-

cinoma, extreme responses to chemotherapy (defi ned as no residual

invasive carcinoma at cystectomy or presence of persistent muscle-

invasive or extravesical disease at cystectomy), and available pre-

chemotherapy tumor tissue who were enrolled on Institutional

Review Board (IRB)–approved tissue acquisition and DNA-sequencing

protocols were identifi ed (Dana-Farber protocols 02-021 and 11-334;

Memorial Sloan Kettering Cancer Center protocols 89-076 and

09-025). All patients provided written informed consent for genomic

testing used for this study. Specimens were evaluated by genitouri-

nary pathologists (J.A. Barletta/S. Signoretti—DFCI cohort, and

H. Al-Ahmadie—MSKCC cohort) to identify tumor-bearing areas for

DNA extraction. The minimum percentage of neoplastic cellularity

for regions of tumor tissue was 60%. Study specimens of frozen or

formalin-fi xed, paraffi n-embedded (FFPE) tissue sections were identi-

fi ed at the Dana-Farber Cancer Institute (DFCI) and Memorial Sloan

Kettering Cancer Center (MSKCC). Germline DNA was extracted

from either peripheral blood mononuclear cells or histologically nor-

mal nonurothelial tissue. Information about the source of germline

DNA is available in Supplementary Table S1.

WES and Statistical Analysis DNA extraction and exome sequencing Slides were cut from FFPE

or frozen tissue blocks and examined by a board-certifi ed patholo-

gist to select high-density cancer foci and ensure high purity of

cancer DNA. Biopsy cores were taken from the corresponding tis-

sue block for DNA extraction. DNA was extracted using Qiagen’s

QIAamp DNA FFPE Tissue Kit Quantitation Reagent (Invitrogen).

DNA was stored at −20°C. Whole exome–capture libraries were

constructed from 100 ng of DNA from tumor and normal tissue

after sample shearing, end repair, and phosphorylation and ligation

to barcoded sequencing adaptors. Ligated DNA was size-selected

for lengths between 200 and 350 bp and subjected to exonic hybrid

capture using SureSelect v2 Exome bait (Agilent Technologies). The

sample was multiplexed and sequenced using Illumina HiSeq tech-

nology for a mean target exome coverage of 121× for the tumors

and 130× for germline samples. Four cases did not complete the

exome-sequencing process due to sequencing process failure. All

BAM fi les generated for this study will be deposited in the database

of Genotypes and Phenotypes (dbGaP; phs000771.v1.p1) .

Sequence data processing Exome sequence data processing and

analysis were performed using pipelines at the Broad Institute. A

BAM fi le aligned to the hg19 human genome build was produced

using Illumina sequencing reads for the tumor and normal sample

and the Picard pipeline. BAM fi les were uploaded into the Firehose

infrastructure ( 32 ), which managed intermediate analysis fi les exe-

cuted by analysis pipelines. All BAM fi les will be uploaded to dbGaP

(phs000771.v1.p1).

Sequencing quality control Sequencing data were incorporated

into quality-control modules in Firehose to compare the tumor and

normal genotypes and ensure concordance between samples. Cross-

contamination between samples from other individuals sequenced in

the same fl ow cell was monitored with the ContEst algorithm ( 33 ).

Samples with >4% contamination were excluded ( n = 4).

Alteration identifi cation and annotation The MuTect algorithm

( 34 ) was applied to identify somatic single-nucleotide variants in

targeted exons. Indelocator was applied to identify small inser-

tions or deletions ( 35 ). Gene-level coverage was determined with the

Depth OfCoverage in the Genome Analysis Tool Kit ( 36 ). Alterations

were annotated using Oncotator ( 37 ). Power calculations for cover-

age were determined using the MuTect coverage fi le, which requires a

minimum of 14× coverage in the tumor. Samples with median allelic

fractions less than 0.05 and insuffi cient DNA for orthogonal valida-

tion with Fluidigm Access Array were excluded ( n = 1).

Alteration signifi cance MutSigCV ( 23 ) was applied to the aggregate

cohort of 50 cases to determine statistically altered genes in the cohort.

Alterations from all nominated signifi cant genes from MutSigCV were

manually reviewed in the Integrated Genomics Viewer (IGV; refs. 38,

39 ). Alterations that were invalid based on IGV review (as a result of

misalignment artifacts viewable in IGV) were subsequently excluded

from the fi nal results, which resulted in the exclusion of TGFBR1 and

DEPDC4 from the fi nal result (Supplementary Table S3).

Selective gene enrichment analysis All somatic mutations and

short insertion/deletions were aggregated for the cohort and split

between responders and nonresponders. Alterations with cover-

age at the tumor site of ≥30× and an allelic fraction of ≥0.1 were

considered for further analyses. Missense, nonsense, and splice-site

mutations, along with short insertion/deletions, were then assigned

a damaging score (range, 0–1) following previously reported methods

( 40 ). Missense mutations were scored using the Polyphen2 score ( 41 )

for the amino acid substitution. Missense mutations without avail-

able Polyphen2 scores (due to mapping errors or dinucleotide status)

were listed as “unavailable” in Supplementary Table S5 and excluded.

Nonsense mutations, splice-site mutations, and short insertion/

deletions were assigned a damaging score of 1. Damaging scores are

listed in Supplementary Table S5. Alterations with a damaging score

of ≥0.5 were then tabulated for occurrence in responders and nonre-

sponders. An altered gene would be counted only once per patient.

The Fisher exact test was performed to compare between cohorts to

derive a P value for each gene. Because a minimum of six alterations

were required to observe a P value of ≤0.01, only genes with ≥6 altera-

tions in the cohort (thereby representing >10% of the cohort and of

highest potential clinical signifi cance) were considered for multiple

hypothesis testing. These results are summarized in Supplemen-

tary Table S6 and Fig. 2A . The ERCC2 mutation frequency in the

responders was compared with the unselected TCGA (21) and Guo

and colleagues ( 22 ) cohorts with a binomial test. Results from this

analysis are made available in Fig. 2C . Comparison of ERCC2 muta-

tion distribution between responders and nonresponders adjusted

for elevated mutation rate in the responders was performed with

the binomial test conditional on observing nine mutations and

using the estimated ratio of mutation rates between responders and

nonresponders as the expected frequency of ERCC2 mutations in

responders under the null hypothesis [e.g., for ERCC2 , binomial test

( x = 9; n = 9; P = Mutation_Rate Responders /Mutation_Rate Nonresponders ;

alternative = “greater”)].

All statistical calculations were performed using the R statistical

package.

Mutation Validation Orthogonal validation of selected mutations and short insertion/

deletions (those presented in this article, including ERCC2 ) was

performed using the Fluidigm Access Array. Of 50 cases, 35 had suf-

fi cient DNA to generate suffi cient read depth for analysis. A total

of 85 candidate targets were submitted to Fluidigm for single-plex

PCR primer assay design. This resulted in the design of 65 assays

covering all 85 targets. Assay amplicons ranged from 163 to 199 bp

in size, with an average of 183 bp. All available samples were run on

the Access Array system (Fluidigm) using three 48.48 Access Array

IFC chips following the manufacturer’s recommendations using the






“4-Primer Amplicon Tagging protocol” for Access Array (Fluidigm;

P/N 100-3770, Rev. C1), with the exception that Access Array IFC

chips were loaded and harvested using a Bravo Automated Liquid

Handling Platform (Agilent Technologies), following the manufac-

turer’s recommendations. Resulting amplicons containing sample-

specifi c barcodes and Illumina adapter sequencers were pooled and

sequenced on a MiSeq sequencer (Illumina) with two runs of 150

base paired-end reads (V2 sequencing chemistry), using custom

Fluidigm sequencing primers following the manufacturer’s recom-

mendations (Fluidigm). All sites were manually reviewed in IGV to

determine the presence or absence of nonreference reads. Details

about validation results for ERCC2 and additional variants are given

in Supplementary Table S2. Variants in which there was inadequate

sample for validation or insuffi cient sequencing reads in the valida-

tion data to interpret manually in IGV were listed as “unavailable.”

Cloning and Cell Lines A site-directed PCR mutagenesis/BP (attB-attP) recombination

method ( 42 ) was used to generate WT and mutant ERCC2 open

reading frames (ORF). For each mutant, PCR products were gener-

ated such that fragments overlap at the region of the desired muta-

tion. The fragments were then introduced into the pDONR vector

through the BP reaction. The BP reaction mixture was transformed

into Escherichia coli and recombined to generate a pENTR vector. The

pENTR vector was then used to perform the LR (attL-attR) reaction to

create an expression plasmid.

The expression plasmids harboring WT ERCC2, GFP (negative

control), or mutant ERCC2 constructs were expanded in E. coli

TOP10 cells (Invitrogen) and purifi ed using an anion exchange kit

(Qiagen). Lentiviruses were propagated in 293T cells by cotransfec-

tion of the expression plasmid with plasmids encoding viral packag-

ing and envelope proteins. Unless otherwise noted, all human cell

lines were cultured in DMEM (Invitrogen) supplemented with 10%

FBS (Sigma) and 1% L -glutamine and grown at 37°C and 5% CO 2 .

The 293T cell supernatants containing virus were collected after 48

hours, fi ltered twice (0.45-μm syringe fi lter; Millipore), then added

directly to growing cultures of an SV40-transformed pseudodiploid

ERCC2 -defi cient fi broblast cell line derived from an XP patient of

genetic complementation group D (GM08207; Coriell Institute).

The cell line is a compound heterozygote harboring ERCC2 R683W and

ERCC2 -DEL 36_61 mutations. Polybrene (Sigma) was added to a

fi nal concentration of 8 μg/mL to increase the effi ciency of infection.

Stable integrates were selected by incubation for 5 days in media

containing 10 μg/mL blastocidin. Physical and biologic containment

procedures for recombinant DNA followed institutional protocols

in accordance with the NIH guidelines for research involving recom-

binant DNA molecules. The cell line was obtained in March 2013

from Corriell Cell Repositories (Camden, NJ) and authenticated by

microsatellite genotyping.

Cisplatin Sensitivity Assays Cells were transferred to 96-well plates at a density of 500 cells

per well. Six hours later, cisplatin (Sigma) was serially diluted in

media and added to the wells. After 96 hours, CellTiterGlo reagent

(Promega) was added to the wells, and the plates were scanned using

a luminescence microplate reader (BioTek). Survival at each cisplatin

concentration was plotted as a percentage of the survival in cisplatin-

free media. Each data point on the graph represents the average of

three independent measurements, and the error bars represent the

standard deviation. IC 50 concentrations were calculated using a four-

parameter sigmoidal model, and plots were generated using Prism

(GraphPad). A one-way ANOVA test was used to compare the IC 50

of the negative control cell line with the IC 50 of the WT and mutant

ERCC2 cell lines, and to compare the IC 50 of the WT line with the IC 50

of the negative control and mutant cell lines.

UV Clonogenic Survival Assays Cells were seeded in 6-well plates (Nunc) at a density of 1,500 cells

per well. The following day, the cells were washed once with PBS and

then exposed to increasing UV doses using a UV-B irradiator (Strata-

gene). Medium was replaced, and the cells were allowed to grow for

9 days. On day 10, cells were fi xed using a 1:5 acetic acid:methanol

solution for 20 minutes at room temperature. Cells were then stained

for 45 minutes using 1% crystal violet in methanol solution. Plates

were rinsed vigorously with water, allowed to dry, and colonies were

then manually counted. The number of colonies present at each

UV dose was plotted as a ratio of the number of colonies present

in mock-irradiated wells. Each data point represents the average of

three independent measurements, and the error bars represent the

standard deviation.

Chromosomal Breakage Analysis Approximately 1 × 10 6 cells were seeded per 10-cm dish. After

24 hours, 400 nmol/L of cisplatin was added, and cells were allowed

to grow for an additional 48 hours. Cells were exposed to colcemid

for 2 hours, harvested using 0.075 mol/L KCl, and fi xed in 3:1

methanol:acetic acid. Slides were stained with Wright’s stain, and

25 to 50 metaphases were analyzed for chromosomal aberrations

(chromosome or chromatid breaks, rings, translocations, deletions,

fragments/double minute chromosomes, tri- or quadraradials, di- or

tricentrics, and premature chromatid separation).

Immunoblots Frozen cell pellets were thawed and resuspended in RIPA buffer

[50 mmol/L TRIS (pH 7.3), 150 mmol/L NaCl, 1 mmol/L EDTA, 1%

Triton X-100, 0.5% Na-deoxycholate, and 0.1% SDS] supplemented

with complete protease inhibitor (Roche), NaVO 4 , and NaF. The cell

suspensions were centrifuged, and total protein concentration of the

supernatant was determined by colorimetry (Bio-Rad). Samples were

boiled with loading buffer (Bio-Rad) and electrophoresed in a 3% to

8% gradient TRIS-acetate gel (Life Technologies). Resolved proteins

were transferred to a polyvinylidene difl uoride (PVDF) membrane

(Millipore) at 90 V for 2 hours at 4°C. The membrane was blocked

for 1 hour in blocking solution (5% milk in TRIS-buffered saline-T)

and incubated with primary antibody in blocking solution at 4°C

overnight (mouse ERCC2, Abcam; rabbit β-actin, Cell Signaling

Technology). The following day, the membrane was rinsed and incu-

bated for 1 hour with peroxidase-conjugated secondary antibody in

blocking solution (anti-mouse and anti-rabbit; Cell Signaling Tech-

nology) and rinsed. Enhanced chemiluminescent substrate solution

(PerkinElmer) was added and signal was detected by fi lm exposure

(GE Healthcare).

Disclosure of Potential Confl icts of Interest E.M. Van Allen has ownership interest in a patent (pending). N.

Wagle has ownership interest in and is a consultant/advisory board

member for Foundation Medicine. D.F. Bajorin is a consultant/advi-

sory board member for Bristol-Myers Squibb. L.A. Garraway reports

receiving a commercial research grant from Novartis, has ownership

interest in Foundation Medicine, and is a consultant/advisory board

member for Novartis, Foundation Medicine, and Boehringer Ingel-

heim. J.E. Rosenberg has ownership interest in a patent (pending).

No potential confl icts of interest were disclosed by the other authors.

Authors’ Contributions Conception and design: E.M. Van Allen, K.W. Mouw, P. Kim,

G. Iyer, N. Wagle, R. Bambury, D. Farlow, P. Kantoff, T.K. Choueiri,

L.A. Garraway, J.E. Rosenberg

Development of methodology: E.M. Van Allen, G. Iyer, M. Lawrence,

B.H. Bochner, A. D’Andrea, J.E. Rosenberg






Acquisition of data (provided animals, acquired and managed

patients, provided facilities, etc.): E.M. Van Allen, K.W. Mouw,

P. Kim, G. Iyer, N. Wagle, H. Al-Ahmadie, C. Zhu, K.W. O’Connor,

J. Sfakianos, I. Garcia-Grossman, S. Bahl, N. Gupta, D. Farlow,

A. Qu, J.A. Barletta,J. Boehm, B.H. Bochner, T.K. Choueiri, D.B. Solit,

S. Gabriel, L.A. Garraway, J.E. Rosenberg

Analysis and interpretation of data (e.g., statistical analysis,

biostatistics, computational analysis): E.M. Van Allen, K.W. Mouw,

P. Kim, G. Iyer, H. Al-Ahmadie, I. Ostrovnaya, G.V. Kryukov, J. Kim,

R. Bambury, S. Bahl, N. Gupta, S. Signoretti, M. Lawrence, G. Getz,

T.K. Choueiri, D.F. Bajorin, A. D’Andrea, L.A. Garraway, J.E. Rosenberg

Writing, review, and/or revision of the manuscript: E.M. Van Allen,

K.W. Mouw, P. Kim, G. Iyer, H. Al-Ahmadie, C. Zhu, I. Ostrovnaya,

I. Garcia-Grossman, E.A. Guancial, R. Bambury, D. Farlow, A. Qu,

V. Reuter, P. Kantoff, B.H. Bochner, T.K. Choueiri, D.F. Bajorin,

D.B. Solit, L.A. Garraway, J.E. Rosenberg

Administrative, technical, or material support (i.e., reporting or

organizing data, constructing databases): E.M. Van Allen, P. Kim,

J. Sfakianos, I. Garcia-Grossman, R. Bambury, S. Bahl, D. Farlow,

A. Qu, T.K. Choueiri, J.E. Rosenberg

Study supervision: E.M. Van Allen, N. Gupta, D. Farlow, T.K. Choueiri,

A. D’Andrea, J.E. Rosenberg

Other (project management): N. Gupta

Acknowledgments The authors thank the patients for contributing tissue for research.

In addition, the authors thank Dr. Kenna Shaw, Dr. Jean C. Zenklusen,

and The Cancer Genome Atlas for allowing pertinent ERCC2 results

to be incorporated into their analyses. The authors also thank

Dr. Joaquim Bellmunt for thoughtful feedback and clinical review of

cases, the Broad Genomics Platform for sequencing and validation

activities, and Lisa Moreau for help with chromosomal studies.

Grant Support This study was supported by the Dana-Farber Leadership Council (to

E.M. Van Allen), the American Cancer Society (to E.M. Van Allen), the

Conquer Cancer Foundation (to E.M. Van Allen and K.W. Mouw), the

NIH/National Human Genome Research Institute (U54HG003067, to

L.A. Garraway and S. Gabriel), Cycle for Survival (to G. Iyer, I. Ostrov-

naya, and B.H. Bochner), the Geoffrey Beene Cancer Research Center

(to J.E. Rosenberg, D.B. Solit, H. Al-Ahmadie, and I. Ostrovnaya),

and the Starr Cancer Consortium (to J.E. Rosenberg, E.M. Van Allen,

G. Iyer, L.A. Garraway, I. Ostrovnaya, and H. Al-Ahmadie).

The costs of publication of this article were defrayed in part by

the payment of page charges. This article must therefore be hereby

marked advertisement in accordance with 18 U.S.C. Section 1734

solely to indicate this fact.

Received June 13, 2014; revised July 24, 2014; accepted July 28,

2014; published OnlineFirst August 5, 2014.

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2014;4:1140-1153. Published OnlineFirst August 5, 2014.Cancer Discovery Eliezer M. Van Allen, Kent W. Mouw, Philip Kim, et al. Muscle-Invasive Urothelial Carcinoma

Mutations Correlate with Cisplatin Sensitivity inERCC2Somatic

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