Comprehensive Assessment of Immuno-oncology Biomarkers inAdenocarcinoma, Urothelial Carcinoma, and Squamous-cellCarcinoma of the Bladder
Andrea Necchi a[50_TD$DIFF],*, Russell Madison b, Daniele Raggi a, Joseph M. Jacob c, Gennady Bratslavsky c,Oleg Shapiro c, Julia A. Elvin b, Jo-Anne Vergilio b, Jonathan K. Killian b, Nhu Ngo b,Shakti Ramkissoon b, Eric Severson b, Amanda C. Hemmerich b, Richard Huang b, Siraj M. Ali b,Jon H. Chung b, Prasanth Reddy b, Vincent A. Miller b, Alexa B. Schrock b, Laurie M. Gay b,Brian M. Alexander b, Petros Grivas d, Jeffrey S. Ross b,c
a Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; b Foundation Medicine, Cambridge, MA, USA; cUpstate Medical University, Syracuse, NY, USA;dUniversity of Washington, Seattle Cancer Care Alliance, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
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Background: In patients with rare histologies of bladder cancer, including adenocarci-noma of the bladder (ACB) and squamous-cell carcinoma (SCC), there are limitedstandard therapy options, defining an unmet medical need.Objective: In this comparative comprehensive genomic profiling (CGP) study, genomicalterations (GAs), and immuno-oncology (IO) biomarkers have been analyzed.Design, setting, and participants: Within the Foundation Medicine database, 143 caseswith centrally reviewed pure ACB, 2142 with pure urothelial carcinoma (UC), and83 with pure SCC were subjected to CGP. All patients developed advanced diseasefollowing a primary diagnosis of bladder cancer.Intervention: CGP using a hybrid capture–based assay and immunohistochemistry(IHC).Outcome measurements and statistical analysis: Tumor mutational burden (TMB) wasdetermined on 1.1 Mbp of sequenced DNA, and microsatellite instability (MSI) wasdetermined on 114 loci. Programmed cell-death ligand-1 (PD-L1) expression wasdetermined by IHC (Ventana SP-142 assay), with >1% tumor cells (TCs) or tumor-infiltrating lymphocytes (TILs) scoring positive.Results and limitations: Pure ACB patients were younger and more often female thanpure UC and pure SCC patients. UC and SCC had a significantly higher median TMB thanACB (p [62_TD$DIFF]< 0.001). Rare CD274 (PD-L1) amplification cases were more frequently seen inSCC than in UC (5% vs 1%), and were not seen in ACB. MSI high status was veryuncommon in all tumor types (0–1%). The frequencies of PD-L1 expression in bothTCs and TILs was higher in UC and SCC (both 30%) than in ACB (18%). The results arelimited by their retrospective nature and lack of clinical data annotation.Conclusions: Deep sequencing revealed significant differences in IO biomarkers amongthe threemajor subtypes of bladder carcinomas. UC and SCC revealed higher frequenciesof PD-L1 expression and higher TMB than ACB, and SCC has the highest frequency of
* Corresponding author. Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale deiTumori, Via G. Venezian 1, Milano 20133, Italy. Tel. +39 02 2390 2402, Fax: +39 02 2390 3150.E-mail address: [email protected] (A. Necchi).
Locally advanced and metastatic urothelial carcinoma (UC)is an aggressive disease with few survival possibilities atlong term with the use of conventional chemotherapyoptions. In the last 2 [63_TD$DIFF]yr; however, the therapeutic landscapeof these patients has been revolutionized by the advent ofnew immunotherapy options [1]. Among these options,pembrolizumab and atezolizumab are US Food and DrugAdministration (FDA)- and European Medicines Agency(EMA)-approved standard therapy options in patients whoare ineligible to receive cisplatin-based chemotherapy andhave a tumor with highly expressing programmed cell-death ligand-1 (PD-L1), as well as in thosewho progress on/after a platinum chemotherapy regimen (along with othercheckpoint inhibitors for the latter indication) [2–5]. Inaddition to the available standard therapy options, severalclinical trials are currently being offered to patients withadvanced UC. These trials include immunotherapy andchemoimmunotherapy combinations or combinations withother novel agents.
UC has a propensity for divergent differentiation: the2016 World Health Organization (WHO) classificationrefined criteria of pathological features defining severalvariant histologies of bladder cancer [6]. In addition to thesevariant histologies mixed with UC, there are tumorscharacterized by a pure non-UC histology that are rarephenotypes (accounting for <5% of all bladder tumors),usually displaying an aggressive clinical course and poorresponse to conventional chemotherapy [7]. Unfortunately,patients presenting with pure non-UC histologies are notallowed to be enrolled in the majority of the availableclinical trials of bladder cancer, with rare exceptions.
In this study, we analyzed the molecular characteristicsof pure rare histologies of bladder carcinoma, and selectedthe pure adenocarcinoma of the bladder (ACB) and puresquamous-cell carcinoma (SCC) subtypes, by focusingprimarily on putative immuno-oncology biomarkers.
2. Patients and methods
2.1. Study population and objective
In this retrospective study, conducted from the entiredatabase of Foundation Medicine Inc., from June to July2019, we identified 137 cases of pure nonurachal ACB, six
cases of urachal ACB (143 total ACB), and 83 cases of pureSCC, and compared them with 2142 cases of pure UC of thebladder. The histology of all cases was centrally reviewed bya group of experienced board-certified pathologists. Allthese cases had an advanced stage, had progression on/afterconventional chemotherapies, and had their tumor ana-lyzed, between June 2012 and July 2018, at the time ofdisease progression as part of routine practice outside ofclinical trials. The analyzed samples were taken fromtreatment-naïve primary tumor samples, chemotherapy-treated radical cystectomy samples, or biopsies of meta-static sites. Approval for this study, including a waiver ofinformed consent and a HIPAAwaiver of authorization, wasobtained from the Western Institutional Review Board(protocol no. 20152817). The study objectivewas to describethe rationale for testing novel therapy options, primarilyimmunotherapy, in patients harboring uncommon histo-logical features.
2.2. Tumor biomarker analyses
DNAwas extracted from formalin-fixed paraffin-embeddedtissue obtained from clinically advanced cases that hadprogressed to metastatic disease at the time of sequencing.Comprehensive genomic profiling (CGP)was performed in aClinical Laboratory Improvement Amendments (CLIA)-certified, College of American Pathologists (CAP)-accreditedlaboratory (Foundation Medicine, Cambridge, MA, USA).The pathological diagnosis of each case was confirmed onroutine hematoxylin and eosin–stained slides, and allsamples forwarded for DNA extraction contained a mini-mum of 20% tumor nuclear area, compared with benignnuclear area. In brief, �50 [64_TD$DIFF]ng DNAwas extracted from 40 [65_TD$DIFF]mm of tumor samples and assayed by CGP using adaptorligation and hybrid capture performed for all coding exonsof 315 cancer-related genes plus select introns of 31 genesfrequently rearranged in cancer (Supplementary Table 1).Sequencing of captured libraries was performed using theIllumina HiSeq technology to a mean exon coverage depthof >500�, and resultant sequences were analyzed for basesubstitutions, insertions, deletions, copy number altera-tions (focal amplifications and homozygous deletions), andselect gene fusions, as previously described [8,9]. Germlinevariants documented in the dbSNP database (dbSNP142;http://www.ncbi.nlm.nih.gov/SNP/), with two or morecounts in the ExAC database (http://exac.broadinstitute.
CD274 amplification. The presence of pure SCC features should not disqualify patients forinclusion in IO trials.Patient summary: Tumor samples from patients diagnosed with advanced pure adeno-carcinoma of the bladder (ACB) or pure squamous-cell carcinoma (SCC) have beenanalyzed in terms of frequency of putative immunotherapy biomarkers. The resultsindicated that pure SCC of the bladder was characterized by genomic features thatportend similar response possibilities to immunotherapy compared with the classicalpure urothelial carcinoma. Conversely, for pure ACB there might be different therapeuticopportunities, such as targeted therapies against peculiar genomic alterations in selectedpatients.
org/), or recurrent variants of unknown significance thatwere predicted by an internally developed algorithm to begermlinewere removed,with the exception of knowndrivergermline events (eg, documented hereditary BRCA1/2 anddeleterious TP53 mutations). Known confirmed somaticalterations deposited in the Catalog of Somatic Mutations inCancer (COSMIC v.62) were highlighted as biologicallysignificant. All inactivating events (ie, truncations anddeletions) in known tumor suppressor genes were alsocalled significant. To maximize mutation-detection accura-cy (sensitivity and specificity) in impure clinical specimens,the test was previously optimized and validated to detectbase substitutions at a �5% mutant allele frequency (MAF),indels with a �10% MAF with �99% accuracy, and fusionsoccurring within baited introns/exons with >99% sensitivi-ty. Tumor mutational burden (TMB) was determined on1.1 megabases (Mb) of sequenced DNA for each case basedon the number of somatic base substitution or indelalterations per Mb, after filtering to remove known somaticand deleteriousmutations as previously described [8,9]. As-sessment of microsatellite instability (MSI) was performedfrom DNA sequencing across the coding regions of >300genes. To determine MSI status, optimized homopolymerrepeat loci on the CGP sequencing panel were analyzed forlength variability and compiled into an overall MSI score viaprincipal components analysis [10]. Each microsatellitelocus had repeat length of 7–39 bp. The next-generationsequencing-based “MSI score” was translated into categor-ical MSI-high, MSI ambiguous, or microsatellite stable byunsupervised clustering of specimens for which MSI status
was previously assessed via gold standard methods(polymerase chain reaction and immunohistochemistry[IHC]). This method was 97% (65/67) concordant withcurrent standards [10]. In a subgroup of tumor samples, PD-L1 expression was determined on [66_TD$DIFF]5-mm tissue sectionsusing the Ventana SP142 anti-PD-L1 antibody, with scoresof �1% tumor cells or tumor-infiltrating lymphocytes (TILs)staining in the intra- or paratumoral regions considered tobe positive [2,11]. PD-L1 expression status was determinedbyan experienced board-certified pathologist. All the assayshave been performed blinded to the patient’s outcomes.
2.3. Statistical analyses
Descriptive statistics were used to present the studypopulation and biomarker findings. The primary endpointwas to present the frequency of biomarkers related toimmunotherapy benefit: elevated TMB and MSI-hightumors, PD-L1 IHC expression, and CD274 (PD-L1) geneamplification. The secondary endpoint was to present theproportion of other targeted therapy biomarkers. Compar-isons between the TMB values were made via t tests.
3. Results
3.1. Clinical characteristics and the landscape of genomic
alterations within the Foundation Medicine database
The main patient and disease characteristics are presentedin Table 1 [67_TD$DIFF]. Pure ACB patients were younger and more often
Table 1 – Patients and disease characteristics in the included histological cohorts.
Characteristic Statistics ACB UC SCC
Total patients N 143 2142 83Age (yr) Median (IQR) 58 (48–66) 67 (60–74) 62 (52–72)Female patients N (%) 86 (60) 545 (25.4) 38 (45.8)Source of the tumor sample analyzedPrimary tumorMetastases
% 5545
5248
6931
GA/tumor N 5.6 7.7 8.2Top 10 cancer-related GA (%) % TP53 79 TERT 68 TP53 70
Fig. 1 – Tile plots showing the frequency of genomic alterations in (A) pure adenocarcinoma and (B) pure squamous-cell carcinoma. Tumor mutationalburden values are indicated on top of each figure, and classified as low (<6 mut/Mb), intermediate (6–20 mut/Mb), or high (>20 mut/Mb). Mb =megabases; mut = mutations.
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females than the patients with pure SCC or pure UC. Ingeneral, UC displayed a higher rate of genomic alterationsper tumor compared with ACB and SCC. The landscape of“targetable” genomic alterations was different between thetumor types, as it is presented in the tile plots of Fig. 1, aswell in the longtail plots of Supplementary Figs. 1A–C,whereas themost frequent “targetable” genomic alterationsin each tumor subtype are shown in Table 1. The top-10kinase gene mutations within each tumor category areshown in Supplementary Table 2. When the primary tumorsamples and metastasis biopsy samples were compared, nosignificant differences in the overall genomic alterationlandscapes were seen. Fibroblast growth-factor receptor 3(FGFR3) mutations were significantly more frequently seenin UC when compared with ACB and SCC cases. ERBB2alterations were seen in all the three subtypes, with UCshowing the highest rate (16%) followed by ACB at 8% andSCC at 6% of cases. MET alterations were more frequentlyidentified in ACB than in UC or SCC (5% vs 1% vs 1%). Themost frequently altered “so far untargetable” genomic
alterations varied with TP53 (79%), with KRAS (30%) and APC(8%) being more frequent in ACB and TERT (68%), andCDKN2A/B (35/28%) and DNA-repair genes (ARID1A [23%]and KDM6A [23%]) being more frequently altered in UC.“Targetable”MTOR pathway genomic alterations (includingPI3KCA [21%], TSC1 [9%], and PTEN [5%]) were more frequentin UC, as were “targetable” kinase alterations, such as FGFR3(19%) and ERBB2 (16%). In particular, Table 2 and Supple-mentary Table 2 show the proportion of cases presentingwith FGFR3 mutations or fusions across the histologicalsubtypes: these genomic alterations do not seem to bereliable therapeutic targets in ACB and SCC. Fig. 2 shows thedifferences in genomic alteration profiles between SCC orACB and UC, as the reference group. The most significantdifferences in the frequency of genomic alterations betweenSCC and UC involved CDKN2A, FAT1, andNFE2L2, all of whichbeing more frequently altered in SCC. ACB cases wereenriched in TP53 and KRAS alterations in particular. Twocase presentations of patients with pure ACB histology arepresented in Fig. 3. Genomic alteration lists for ACB, SCC,and UC are provided in Supplementary Tables 3–5.
3.2. Immuno-oncology putative biomarkers across the
histological subtypes
Table 1 also displays the landscape of genomic alterationsassociated with immunotherapy sensitivity, across thetumor subtypes. UC and SCC presented with a significantlyhighermedianTMB comparedwith ACB (p [68_TD$DIFF]< 0.001), and theproportion of cases presenting with TMB �10 or �20 mut/Mb was also higher. Interestingly, SCC showed the samemedian TMB of UC (7 mut/Mb). MSI-high feature was veryuncommon in all the tumor types, being 2% in ACB and 1% inthe remaining cohorts. Of note, a higher proportion of cases
[(Fig._2)TD$FIG]
Fig. 2 – Bubble plots illustrating the distribution of genomic alteration frequencies in (A) pure squamous-cell carcinoma versus pure urothelialcarcinoma and (B) pure adenocarcinoma versus urothelial carcinoma.
Table 2 – Proportion of FGFR3 alterations across the histologicalsubtypes.
ACB UC SCC
Total patients 143 2142 83Type of FGFR3 alteration: N (%)Mutation 1 (0.7a[49_TD$DIFF]) 298 (14) 6 (7.2b)Amplification 0 11 (0.5) 0Fusion 0 73 (3.4) 0Multiple alterations/sample 0 3 (1) 0
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[(Fig._3)TD$FIG]
Fig. 3 – (A) Pure adenocarcinoma of the bladder in a 79-yr-old Caucasian female that progressed to stage IV disease. Comprehensive genomic profilingrevealed major ERBB2 amplification at 112 copies/cell. TOPO2A was also coamplified. Additional alterations included short variant mutations in ATM,APC, STAG2, TERT, and TP53. (B) Needle biopsy of the liver in a 78-yr-old Caucasian man with stage IV pure adenocarcinoma of the bladder.Comprehensive genomic profiling revealed amplification of CCND3, EGFR, MET (13 copies), VEGFA, CDK6 HGF, and MYC. Additional short variantmutations were found in FOXP1, TBX3 and TP53.
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with pure SCC histology, reaching 5%, featured CD274amplification, compared with 1% of pure UC and none inpure ACB subtype.
A total of 265 patients had PD-L1 IHC staining availablefor analyses: fewer cases presented with PD-L1–positivetumor cells or TILs in the ACB group, whereas there was nodifference between SCC and UC, both of which having one-third of the patients with PD-L1–positive staining tumor.
4. Discussion
We presented a comprehensive assessment of the genomicalteration profiles of patients with bladder cancer acrossthree different histological subtypes, including pure UC,ACB, and SCC. To our knowledge, this is the largest studyoutlining the genomic features of these pure rare histologysubtypes of bladder tumors.
The main finding that emerges from our analyses is thatthere are overlapping molecular alterations between pureUC and pure SCC histology that may correlate withresponsiveness to immunotherapy as in the pure orpredominant UC. We believe that the presence of either apure SCC or an SCC component within the bladder UCshould not disqualify patients for inclusion in clinical trialswith immunotherapy as the backbone therapy. However, itwill require new prospective clinical trial data to confirmthat these immunotherapy predictive biomarkers will bevalidated for bladder cancer patients with pure SCC andvariant SCC histologies. This observation is corroborated byfindings from both genomic and IHC analyses on tumortissue. Therefore, the maximum limit of 50% of non-UCcomponent within the tumor slide, that is arbitrarilyallowed in order to include patients in clinical trials, maynot be necessarily valid for patients with SCC. Thisassumption may also be particularly important wheneverthere is no standard-of-care therapy if histological variantsare found, such as the nonmetastatic, muscle-invasivedisease [12,13]. Indeed for patients with pure SCC, theactivity of standard cisplatin-based neoadjuvant chemo-therapy may be poor, as well as the prognosis regardless ofthe therapeutic strategy that is offered, but clinical trial datawith the use of immunotherapy are emerging. In particular,updated results from the PURE-01 study focused on theactivity of neoadjuvant pembrolizumab in patients withpredominant variant histologies and found that six of sevenpatients (86%) with predominant SCC tumors achieveddownstaging to pT � 1 disease [14]. Of note, the genomicdata reported in this prospective study are similar to thosereported in the present study from metastatic cases. Inpatients with metastatic disease and variant histologies,preliminary results have been reported from clinical trials.The use of single agent atezolizumab was evaluated in thephase IV “real-world” SAUL study, which enrolled997 patients after failure of platinum-based chemotherapy[15]. This study, aimed to address the unmet medical needsof special populations by broadening the inclusion criteriaof the IMvigor trials [2,11], thus enrolled 47 patients withmixed variant histologies. The objective response rate (ORR)in this heterogeneous population was 9%, but no indication
emerged regarding potentially different drug sensitivity inspecific histological subtypes.
Another phase II study preliminarily evaluated theactivity of an immunotherapy combination, consisting of3 [69_TD$DIFF]mg/kg nivolumab and 1 mg/kg ipilimumab, every 3 wk,followed by 480 [70_TD$DIFF]mg nivolumab alone, every 4 wk, inpatients with variant histologies (NCT03333616) [16]. In19 patients enrolled in this study, the ORRwas 37%,with oneout of four patients (25%) responding in the ACB subtypeand two out of six (33%) in the SCC subtype. As alreadyreported in other solid tumors, it is possible that thelimitations of immune-checkpoint inhibitor sensitivity,represented by underlying adverse biological features, suchas low PD-L1 expression or low TMB,may be overcomewithcombination therapies, although additional data on morepatients within each variant histology subtype are needed.Further trials are currently evaluating combination thera-pies in variant histologies, including a study of durvalumaband tremelimumab (NCT03430895), durvalumab and cabo-zantinib (ARCADIA, NCT03824691), and nivolumab, ipili-mumab, and cabozantinib combination (ALLIANCE A031702ICONIC study, NCT03866382), among possible others.
Our study also revealed different opportunities forclinical trials with the use of targeted therapies acrossthe histological subtypes of bladder tumors. FGFRmolecularalterations currently represent the most prominent re-search opportunity in UC, with the pan-FGFR tyrosine-kinase inhibitor erdafitinib having accelerated FDA approvalbased on the results of a single-arm phase II trial inmolecularly selected patients (BLC2001, NCT02365597)[17]. The patients in this trial had a spectrum of previoustreatments, and the ORR was 40.4%. In addition toerdafitinib, other FGFR inhibitors such as rogaratinib,pemigatinib, and infigratinib have been explored inseparate phase I and II trials, with an ORR of 25% [18–20]. Results of vofatamab (B-701) monotherapy or incombination with docetaxel have been recently reported,with a confirmed ORR of 10% and 33%, respectively[21]. Based on the robust association between the tumorbiomarker (eg, FGFR mutation or fusion) and the activity ofthis class of agents, it is clear from the present study thatthis therapeutic possibility is mostly confined to UC, withonly a very small proportion of SCC (<10%) and no cases ofACB that are potentially responsive. For the latter tumors,peculiar possibilities, restricted to 5–10% of cases, arerepresented by EGFR, ERBB2, and MET inhibitors. Amplifi-cation of ERBB2 has been reported in 7–9% of bladder UCcases [22–25]. ERBB2 amplification frequency in bladderACB found in this study and in the literature is similar to thatfor UC. In one study, HER2 overexpression was identified in19% of bladder cancer cases, with significant enrichment ingrade 3 and muscle-invasive tumors [26]. As widelydemonstrated, ERBB2 amplification may predict sensitivityto therapies targeting HER2, including antibodies, such astrastuzumab, pertuzumab, trastuzumab emtansine (T-DM1), and dual EGFR/HER2 kinase inhibitors, such aslapatinib, afatinib, neratinib, and dacomitinib [27,28]. Inpatients with bladder cancer, concurrent PIK3CA or PTENalterations that activate the PI3K pathway have been
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associated with resistance to therapies that target HER2[25]. Therefore, understanding the landscape of simulta-neously occurring genomic alterations is always importantwhen delineating the response probabilities to a targetedtherapy. MET activation associated with high-level geneamplification results in signaling partly mediated by theRAS-RAF-MAPK and PI3K pathways to promote prolifera-tion. High-level amplification of MET has been reported in0.5–2% of bladder carcinomas [22,29]. Overexpression ofMET has been reported in 5–45% of UC [30,31]. METoverexpression in bladder carcinoma has been shown to becorrelated with poor long-term survival, higher pathologi-cal stage, and tumor grade [30]. Strong evidence suggeststhat MET activation may predict sensitivity to targetedtherapies, such as crizotinib and cabozantinib[32,33]. Strong clinical data also suggest sensitivity ofMET-altered tumors to various other MET inhibitors inclinical development [34].
Limitations in this study should be acknowledged. Thecentral confirmation of “pure” ACB, SCC, and UC histologywas based only on the sample received for DNA sequencingand the accompanying pathology report. The primarytumor biopsy samples (typically from the transurethralresection of the bladder tumor) and metastasis biopsysamples may have represented only a portion of thepatient’s tumor burden, and the remainder of the patient’scarcinoma was not evaluated histologically. Similarly, forthe cystectomy samples, only one tissue block wassubmitted and central pathology reviewwas not performedon the tissue blocks from the resections that were notsubmitted for sequencing. In addition, the smoking status,prior bladder infection, and a number of other clinicaldetails were not available for this study. DNA sequencingwas performed with somatic DNA obtained from theextraction of either the primary tumor or a metastasisresection. No clinical germline samples were sequenced inthis study, and germline results have not been reported.Additional limitations were the relatively small numbers ofpatients whose tumor samples had undergone PD-L1 IHC,potential selection and confounding factors relevant to theretrospective study nature, unavailability of full clinicalannotation, and lack of comparison with other histologicalsubtypes. Of the other subtypes, neuroendocrine tumorsusually show good chemotherapy sensitivity, which makesthem eligible to receive standard cisplatin-based chemo-therapy in either the (neo)adjuvant or the first-linemetastatic setting [35].
5. Conclusions
In summary, when examined by CGP, the genomic land-scapes of classic UC and SCC of the bladder are similar inhaving high frequencies of genomic alterations per tumor.The type and frequencies of “untargetable” and “targetable”genomic alterations in ACB, SCC, and UC appear differentwith potential opportunities for matching small subsets ofpatients with a variety of targeted therapies, includingmultiple kinase inhibitors and MTOR inhibitors. Patients
with pure ACB histology appear potentially less responsiveto immunotherapy. For these patients, after failure of thefew standard chemotherapy options that are used in routinepractice, there may be intriguing possibilities for targetedtherapy, for example, against ERBB2, EGFR, and MET inselected patients. In addition, based on our findings, SCCand UC also share the potential benefit from immunother-apy-based treatments, which should be tested further inclinical trials.
This paperwas presented in a poster session, 2019 annualmeeting of the European Society for Medical Oncology(ESMO), September 26–30 2019, Barcelona, Spain.
Author contributions: Andrea Necchi had full access to all the data in thestudy and takes responsibility for the integrity of the data and theaccuracy of the data analysis.
Study concept and design: Necchi, Ross.Acquisition of data: All authors.Analysis and interpretation of data: Necchi, Ross, Madison.Drafting of the manuscript: Necchi.Critical revision of the manuscript for important intellectual content: Allauthors.Statistical analysis: Necchi, Ross.Obtaining funding: Ross.Administrative, technical, or material support: None.Supervision: None.Other: None.
Financial disclosures: Andrea Necchi certifies that all conflicts ofinterest, including specific financial interests and relationships andaffiliations relevant to the subject matter or materials discussed in themanuscript (eg, employment/affiliation, grants or funding, consultan-cies, honoraria, stock ownership or options, expert testimony, royalties,or patents filed, received, or pending), are the following: R. Madison, J.A.Elvin, J.A. Vergilio, J.K. Killian, N. Ngo, S. Ramkissoon, E. Severson, A.C.Hemmerich, R. Huang, S.M. Ali, J.H. Chung, P. Reddy, V.A. Miller, A.B.Schrock, L.M. Gay, B.M. Alexander, J.S. Ross: employees and stock ownersof Foundation Medicine Inc. A. Necchi: consultant for Merck, AstraZeneca, Janssen, Incyte, Roche, Rainier Therapeutics, Clovis Oncology,Bayer, and Astellas/Seattle Genetics; grant/research support fromMerck,Ipsen, and Astra Zeneca; travel expenses/honoraria from Roche, Merck,Astra Zeneca, and Janssen. P. Grivas: consulting for AstraZeneca, Bayer,Biocept, Bristol-Myers Squibb, Clovis Oncology, Dendreon, Driver, EMDSerono, Exelixis, Foundation Medicine, Genentech, Genzyme, HeronTherapeutics, Janssen, Merck & Co., Mirati Therapeutics, Pfizer, SeattleGenetics, and QED Therapeutics; educational program (not current, withdirect input in content): Bristol-Myers Squibb and Genentech; researchfunding to institution: AstraZeneca, Bayer, Genentech/Roche, Merck &Co., Mirati Therapeutics, Oncogenex, Pfizer, Clovis Oncology, BavarianNordic, Immunomedics, Debiopharm, and Bristol-Myers Squibb.
Funding/Support and role of the sponsor: This work was supported byFoundation Medicine Inc., Cambridge, MA, USA.
Appendix A. Supplementary data
Supplementary material related to this article can befound, in the online version, at doi:https://doi.org/10.1016/j.eururo.2020.01.003.
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