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number of 36 patients (justified on the basis of frequentist

criteria). Our results were PP = 0 and PP = 1 for PD-L1–

negative and PD-L1–positive patients, respectively. These

figures clearly indicate that no further trial expansion or

enrichment is needed and that treatment efficacy is restricted

to biomarker-positive patients. This is not necessarily true,

however, in other scenarios. For instance, by shifting the

efficacy criterion to ORR


30% (chosen to represent a 10%

minimum improvement over the 20% baseline rate) and

requiring PP


0.95 as the stopping criterion, enrichment

with eight patients for the PD-L1–positive stratum would be

required. In addition, other results for different compounds

did not show clear-cut differences between patient sub-




To conclude, we aimed to provide investigators with

easy-to-use statistical tools for the design and analysis of

biomarker-based, early-phase, noncomparative trials. Sce-

narios that are not clear-cut might arise in future trials on

immune checkpoint inhibitors for which application of

formal yet manageable and efficient methods might better

support the therapeutic development process.

Conflicts of interest:

Andrea Necchi has received consultancy and

advisory fees from Roche, Merck Sharp & Dohme, Astra Zeneca, Bayer,

and Janssen, and has received institutional research funding from

Millennium Takeda, Amgen, Merck Sharp & Dohme, Astra Zeneca,

GlaxoSmithKline. The remaining authors have nothing to disclose.



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Rosenberg JE, Hoffman-Censits J, Powles T, et al. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial. Lancet 2016;387:1909–20.


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Sharma P, Bono P, Kim JW, et al. Efficacy and safety of nivolumab monotherapy inmetastatic urothelial cancer (mUC): results from the phase I/II CheckMate 032 study. J Clin Oncol 2016;34(Suppl):4501


Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

*Corresponding author. Department of Medical Oncology, Fondazione

IRCCS Istituto Nazionale dei Tumori, Via G. Venezian 1, 20133 Milan,

Italy. Tel. +39 02 23902402; Fax. +39 02 23903150.

E-mail address:

(A. Necchi).

September 28, 2016

Mutagenic Factors and Complex Clonal





Multifocal Urothelial Cell Carcinoma

Yiqing Du

a ,


, Ruoyan Li




, Zhanghua Chen




, Xiaofeng Wang

a ,

Tao Xu

a , * ,

Fan Bai


, *

Synchronous and metachronous development of multifocal

tumors in the urinary tract is a well-known characteristic of

urothelial cell carcinoma (UCC). Several theories have been

proposed to interpret the formation of these genetically

related and unrelated tumors


. However, the underly-

ing mechanisms leading to multifocal UCC are unclear. To

shed light on this question, we


performed exome sequenc-

ing of 13 tumor specimens (including 4 bladder, 6 ureter,

and 3 renal pelvis tumors) from five patients with

multifocal UCC (

Fig. 1

A, Supplementary Fig. 1, and

Supplementary Table 1).

On average, we obtained 77 exonic coverage (Supple-

mentary Table 2) and identified 2168 (421–4170) somatic

mutations in each tumor (Supplementary Table 1 and

Supplementary Fig. 2). The hypermutational feature may be

related to certain mutagenic factors in the carcinogenesis of

multifocal UCC. To uncover potential mutagenic factors, we

examined the mutational signature of each tumor. Striking-

ly, in all the tumors we found predominant T



transversion, with significant transcriptional strand bias.

The proportion of this transversion was markedly high in the





motif (

Fig. 1

B, Supplementary Figs. 3 and 4).

This remarkable mutational signature was consistent with

the mutagenic features of aristolochic acid (AA), a natural

herb-extracted compound that exists widely in Chinese

traditional medicine




The typical AAmutational signature

was also found in a morphologically normal urothelium

specimen (P5NT) surrounding a tumor (Supplementary Figs.

3 and 4). Taken together, although the current study cohort

is relatively small, these findings imply that AA is a potential

mutagenic factor leading to multifocal UCC.

Intriguingly, the mutational signature of sample P5U2

was slightly different to that in a typical AA-affected

tumor such as sample P5U1 (

Fig. 1

C). By dissecting

mutations according to their clonal fractions (Supple-

mentary Fig. 5), we found a latent mutational signature

other than the AA signature. This C


T predominant

mutational signature was largely consistent with the

signature of defective DNA mismatch repair (

Fig. 1



. Coincidently, we found a frame shift deletion in the

E U R O P E A N U R O L O G Y 7 1 ( 2 0 1 7 ) 8 3 7 – 8 4 3