Biology of Human Tumors

TP53 Mutation and Its Prognostic Significancein Waldenstrom's MacroglobulinemiaSt�ephanie Poulain1,2,3, Christophe Roumier2,3, Elisabeth Bertrand3,Aline Renneville2,3, Aur�elie Caillault-Venet2, Emmanuelle Doye2,Sandrine Geffroy3, Sheherazade Sebda2, Olivier Nibourel2,3,Morgane Nudel4, Charles Herbaux4, Loic Renaud2, C�ecile Tomowiak5,6,St�ephanie Guidez5,6, Sabine Tricot1, Catherine Roche-Lestienne2,3,Bruno Quesnel3,4, Claude Preudhomme2,3, and Xavier Leleu5,6

Abstract

Purpose: TP53 is a tumor-suppressor gene that functions as aregulator influencing cellular responses to DNA damage, andTP53 alterations are associated with pejorative outcome in mostB-lymphoid disorders. Little is known regarding TP53 alterationin Waldenstrom's macroglobulinemia (WM).

Experimental Design:Here, we have explored the incidence ofTP53 alteration using Sanger sequencing and ultradeep-targetedsequencing in 125WM and 10 immunoglobulin M (IgM)mono-clonal gammopathy of undetermined significance (MGUS),along with the clinical features and the associated genomiclandscape using single-nucleotide polymorphism array andmutational landscape in an integrative study.

Results:Overall, we have identified alteration of TP53 locusincluding mutation, deletion, and copy-neutral LOH in 11.2%of WM. TP53 mutation was acquired in 7.3% of patients withWM at diagnosis, being absent in IgM MGUS, and was highly

correlated to deletion 17p. No correlation with CXCR4 muta-tions was observed. Patients with TP53 alteration had a greaternumber of genomic abnormalities. Importantly, WM withTP53 alteration had a significantly shorter overall survival,particularly in symptomatic WM, and independently of theinternational prognostic scoring system for Waldenstrom mac-roglobulinemia (IPSSWM) score. Specific treatment for WMwith TP53 may have to be studied. Nutlin-3a–targeted p53signaling induced cytotoxicity preclinically, along with newcompounds such as ibrutinib, PrimaMet, or CP31398 thatbypass p53 pathway in WM, paving the path for future treat-ment-tailored options.

Conclusions: Our results highlight the clinical significance ofdetection of TP53 alteration inWM to determine the prognosis ofWMand guide the treatment choice.ClinCancerRes; 23(20); 6325–35.�2017 AACR.

IntroductionTP53, that encodes for a tumor-suppressor protein, p53, is

mapped at the chromosome 17p13 locus and has proved toplay a key role in cancer physiopathology. p53 is a transcriptionfactor and acts as a critical regulator of cellular proliferation,cell-cycle checkpoint, DNA repair, and apoptosis (1, 2). Func-tional activation of p53 occurs through posttranslational mod-ification that prolongs its half-life and thereby allows its accu-

mulation in the nucleus, where it transactivates target genes,including p21 and MDM2 (1, 3). Specific inactivation of theTP53 tumor-suppressor gene in mature B cells was shown to besufficient to promote oncogenic transformation into IgMþ cells(4). TP53 is thus essential for both maintenance of cellulargenetic integrity and the cytotoxic effect of DNA-damagingchemotherapy drugs.

The deletion of the short armof chromosome 17 (Del17p) thatcontains the TP53 gene is often monoallelic, and mutations mayoccur on the remaining allele of TP53 (5–7). The prevalence ofTP53 alterations, eithermutation or deletion, differs considerablybetween tumor types and stages of lymphoid disorders; never-theless, p53 inactivation is often associated with cancer progres-sion and drug resistance (1, 6, 8, 9). The mutations of TP53,especially when located in the DNA-binding domain, are associ-ated with a greater risk of more aggressive disease, poorerresponses to conventional chemoimmunotherapy, and shortsurvival in numerous lymphoid disorders, including chroniclymphocytic leukemia (CLL) and marginal zone lymphoma(6, 10–12). BTK inhibitors were recently proposed to treat tumorcells and bypass the defective p53 pathway (3, 13). An alternativestrategy could be to target the overexpressed p53-mutant proteinby directly modifying its conformation to restore its proapoptotictranscriptional function (1–3).

Waldenstrom macroglobulinemia (WM) is a low-grade B-celllymphoplasmacytic lymphoma associated with monoclonal

1Service d'H�ematologie- Immunologie- Cytog�en�etique, Centre Hospitalier deValenciennes, Valenciennes, France. 2Laboratoire d'H�ematologie, Centre deBiologie Pathologie, Centre Hospitalier R�egional et Universitaire de Lille, Lille,France. 3INSERM UMR 1172, Team 4, Cancer Research Institute of Lille, Lille,France. 4Service des Maladies du Sang, Hopital Huriez, Centre HospitalierR�egional et Universitaire de Lille, Lille, France. 5Service d'H�ematologie etTh�erapie cellulaire, Centre d'Investigation Clinique, Hopital de la Mil�etrie, CentreHospitalier Universitaire de Poitiers, Poitiers, France. 6INSERM U1402, CentreHospitalier Universitaire de Poitiers, Poitiers, France.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Xavier Leleu, Service d'H�ematologie et de Th�erapieCellulaire, Hopital La Mil�etrie, CHU de Poitiers, France. Phone: 33-549-443-717;E-mail: xavier.leleu@chu-poitiers.fr

doi: 10.1158/1078-0432.CCR-17-0007

�2017 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 6325

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

immunoglobulinM (IgM) secretion andMYD88 L265Pmutation(14, 15). Deletion 17pwas initially reported at a low frequency inheterogeneous WM cohorts (16, 17). TP53 deletion by FISH maybe associated with significantly shorter time to progression (18).However, the exact relationship between TP53 mutation, p53activity, and the genomic landscape of WM remains to beinvestigated.

The goal of our study was to analyze the TP53 mutationincidence and genomic features, to investigate the functionalconsequences of TP53 mutation, along with understanding theclinical significance of this genetic alteration in WM.

Materials and MethodsPatients

A total of 125 (82 males, 43 females) patients diagnosed withWM and 10 patients with IgMMGUS were included in this study(14, 19). The median age was 67 years (range, 36–92). Patientswere untreated at time of bone marrow (BM) collection atdiagnosis, and 65 patients with WM (52%) have been treatedsince. Patients gave informed consent prior to research samplingin accordance with the declaration of Helsinki. Approval forthis study was obtained from the Institutional Review Board ofthe tumor bank of CHRU of Lille (agreement number ofthe project: CSTMT045). The characteristics of patients aredescribed in Supplementary Table S1. Front-line therapy includ-ed rituximab-based regimens in 76%of cases, alkylating agent in78% of cases, and fludarabine in 6% of cases.

Cell lines and drugsThe following cell lines were used in the study, BCWM1,

MWCL1, MEC-1, RL, and MM.1S. BCWM1 and MWCL1 are B-cell lines developed in patients withWM (20, 21). These cell lineswere chosen because they harbor various types of TP53 dysfunc-tion: (i) MM1S (myeloma cell line) and BCWM1 cell line lackboth TP53mutation (TP53Wild); (ii) MWCL1, RL, and MEC-1 celllines carry both 17p deletion (Del17p) and TP53 mutations(TP53Del/Mut). All TP53 statuses were confirmed in the presentstudy: MWCL1 TP53 V143A, RL TP53 A138P, and MEC-1 TP53

V147G. BCWM1 and MM1s harbored no TP53mutation. Nutlin-3a, Prima Met, and CP31398 were purchased from Tocris andSigma (France), respectively.

Viability, apoptosis assay, immunoblotting, andflowcytometryViability and cell growth of treated cells were determined

using the MTS assay according to the manufacturer's proto-col. Apoptosis was detected using study of mitochondrialmembrane potential (MitoTracker Red CMXRos-Special Pack-aging; Invitrogen) using flow cytometry (Navios, BeckmanCoulter).

For Western blot analysis, whole-cell lysates were subjected to4% to 12% SDS–PAGE and transferred to polyvinylidene fluoridemembrane (Invitrogen), followed by incubation with primaryantibodies. Immunoblotting was performed using the followingantibodies, anti-P53, anti-P21, anti-MDM2, anti-PUMA, anti-Bcl2, anti-Bax, anti-BclXl (Cell Signaling Technology), and anti-GAPDH. Signal quantification was performed using ImageQuantTL 8.1 (GE Healthcare). GAPDH value was arbitrary set at 1arbitrary unit. The expression of P53 and P21 was studied onCD19þ cells using flow cytometry (Navios, Beckman Coulter)with the following antibodies: P53–FITC, P21-PE, CD3-ECD,CD19-APC, CD45 (eBioscience). At least 10,000 events wererecorded for each experiment performed. The mean of fluores-cence intensity (MFI) ratio was calculated as the ratio of fluores-cence intensity of specific marker and isotypic control for eachexperimental condition.

Genomic studiesPatients' research sampling consisted of enriched tumoral cells

from BM using a B-cell isolation kit (Myltenyi Biotech) and Tlymphocytes from blood samples (22, 23). The purity of sampleswas confirmed by flow cytometry. Cytogenetic and FISH analysiswas performed onDSP30þ Il2-stimulated BM cells using a panelof probes including 17p12 (22, 23). Single-nucleotide polymor-phism array analysis (SNPa) studieswere performed as previouslypublished (24).

Gene mutations analysisAll studies were performed on DNA from B isolated cells.

Next-generation sequencing (NGS) was performed in all WMcases (n ¼ 125) using the Ion Torrent PGM platform (Lifebiotechnologies). Libraries covering the regions of interest weredesigned with an amplicon length ranging from 150 to 250 bp.Mutation calling was performed using the torrent suite variantcaller under the low stringency somatic settings (TS4.0). Wehave studied TP53 mutations (exons 2–11) along with MYD88L265P (exon 5), CXCR4, CD79A, and CD79B (ITAM domain).Complete sequence data of TP53 gene were generated at meandepth coverage of 1,900 per nucleotide, and at least 20 variantreads were needed to identify a mutation (24). NGS assay alsoallowed a better assessment of the percentage of TP53-mutantallele with a sensitivity at 1%. Exons 5 to 8 of TP53 weresequenced from genomic DNA by PCR as previously describedin all cases (22).

Statistical analysisThe relationships between the clinical, biological, and molec-

ular parameters were determined using a nonparametric test(Mann–Whitney), a t test, a c2, or Fisher exact test when appro-priate. Correlations were tested through Spearman correlation

Translational Relevance

TP53 that encodes for a tumor-suppressor protein, p53 ismapped at the chromosome 17p13 locus and has proved toplay a key role in cancer physiopathology in part due to itsrole as a mechanism of resistance to several agents. Little isknown inWaldenstrom's macroglobulinemia (WM).We havetherefore sought to unravel the different aspects of TP53mutations in WM and to characterize the genetic backgroundusing targeted next-generation sequencing and single-nucleo-tide polymorphism arrays. TP53 alteration is observed in11.2% of WM, of which 7.3% is mutated, highly correlatedto deletion 17p and complex genomic features, but indepen-dent of CXCR4 mutations. TP53 alterations, including TP53mutations, are highly associated with poor outcome in WM,particularly in symptomatic WM. New compound such asibrutinib or p53 reactivator may bypass TP53 mutation alsoidentified as a new potential genomic subgroup of WM withpoor prognosis.

Poulain et al.

Clin Cancer Res; 23(20) October 15, 2017 Clinical Cancer Research6326

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

coefficient. Survival was studied using the Kaplan–Meier test, andcomparisons weremadewith the log-rank test. Finally, we studiedsurvival in multivariate cox model analysis, and we have enteredall variables with at least P ¼ 0.1 significance on univariateanalysis. All statistical analysis was done with the SPSS 15.0software.

ResultsFrequency of TP53 mutation in WM at diagnosis

No mutation of TP53 was observed in the MGUS IgM group(n ¼ 10). TP53 mutation was observed in nine of 125 patientswith WM (7.3%) including three frameshifts and six missensemutations (Fig. 1A). Each of the nine patients had presence of onemutation type, different frompatient to patient. All themutationswere somatic.

The mutation load of TP53 varied from 13% to 98.9% (mean:62.0%) using VAF study by NGS (n ¼ 8).

We observed that TP53mutation was present in the dominantclone as defined by more than 40% of VAF, and subclonal in twocases in WM (Fig. 1B).

Mutation of TP53 alters the DNA-binding domain and thefunction of p53 in WM

We observed that all mutations were localized in the DNA-binding domain (exons 5–8). We then sought to verify that TP53mutation had a relevant negative impact on the function of p53 atthe protein level inWMcell lines.We have thereforemeasured theactivation of p53 and its transcriptional targets, p21, PUMA, andMDM2, at protein level in response to nutlin-3a, an mdm2inhibitor, knowing that p53-mutated cells are insensitive tonutlin-3a (2, 3, 28).

BCWM1had amarked induction of p53 targets upon treatmentwithnutlin-3a, in contrast toTP53Mut cell lines, includingMWCL1(Fig. 2A and B). Treatment with nutlin-3a also induced adecreased viability using MTS assay and increased apoptosis inBCWM1 in contrast to TP53Mut cell lines, including MWCL1(Fig. 2C–E). We next examined the effects of nutlin-3a on WMpatients' CD19þ cells genotyped for TP53 mutation. Nutlin-3aincreased the expression of p53 and p21 in TP53Wild WM (n ¼ 6)anddecreasedviability incontrastwithTP53MutWM(n¼3;Fig.3).Overall, these data tend to demonstrate that p53 is functional inTP53WildWMcell lines and patients' cells, the opposite of TP53Mut

WM cells.

TP53 mutation is highly correlated with deletion 17pWe then studied the minimally deleted region (MDR) on 17p

chromosomeusing SNParray.We found12patientswithdeletion17p, with two additional with subclonal 17p deletion detectedusing FISH. The MDR of 17p was mapped in WM and contained79 genes, among which was systematically observed the loss ofTP53 (Supplementary Fig. S1).

We then sought to study the relationship between TP53mutation and deletion 17p (TP53), using FISH and/or SNPa(n ¼ 115). Five patients had only deletion 17p withoutTP53 mutation. TP53 mutation and TP53 deletion were asso-ciated with 58.3% (7/12) of the cases. Consequently, weobserved a high correlation between TP53 mutation and dele-tion 17p (P < 0.001).

In two cases, TP53 mutation was observed without dele-tion17p, nonetheless including one case with copy numberwithout LOH (UPD) of TP53 locus demonstrated by SNPa (there-fore biallelic alteration of TP53; Supplementary Table S1).

0

20

40

60

80

100

120

WM L194P WM V272M WM G244S WM C176W WM P191sfs WM C242Y WM P301fs WM Y236C

D el17p D el17p D el17pD el17p UPD17pD el17pD el17p

VAF

of T

P53M

uta�

on (%

)

A

B

Num

ber o

f pa�

ents

0102030405060708090

100110120

WM Pa�ents MGUS Ig M Pa�ents

Presence of TP53 muta�on

Absence of TP53 muta�on

n = 10n = 125

0% of TP53 muta�on

7.3% of TP53 muta�on

Figure 1.

TP53 mutations in WM. A, Representationof TP53 mutation frequency in patientswith WM and MGUS IgM. The X axisindicates the group of patient, Y axisindicates the number of patients, andpercentage of TP53 mutation in eachgroup is indicated. B, Representation ofVAF of TP53 mutation in each patientusing NGS. In this histogram, each columnrepresents a patient with the TP53mutation (X axis). To quantify themutatedclone, we have analyzed the VAF definedas the number of reads that map eachstudied position, divided by all fragmentscovering the site. Percentage indicatesVAF of TP53mutation determined by NGScorrected by the percentage of tumoralcells in the B selected samples in eacheight patients (Y axis). Light gray columnsindicate subclonal TP53 mutation inpatients with WM. The presence of adeletion 17p is indicated by del17p, andUPD indicates the presence of copy-neutral LOH without copy-numbervariation of TP53 using the SNP array.

TP53 Mutation in Waldenstrom's Macroglobulinemia

www.aacrjournals.org Clin Cancer Res; 23(20) October 15, 2017 6327

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

In summary, TP53 mutation was highly associated with TP53alteration either by deletion 17p or by UPD in WM (P < 0.001).

Genomic alteration spectrum associated with TP53 alterationWe then sought to study whether a specific high-throughput

SNPa signature was associated with TP53 alteration in 62 patientswith WM, including 14 patients with TP53 alterations, nineTP53Mut and 5 TP53Del. For the first analysis, we have consideredthe presence of any one genomic abnormality, including gain,

loss, and/or UPD. We found a relationship between alteration ofTP53 locus (TP53Alt) by any cause (mutation, UPD, or deletion)and a greater frequency of genomic aberrations in WM comparedwith TP53Wild (P ¼ 0.01 and P ¼ 0.06, respectively; Fig. 4). Agreater frequency of UPD was observed in TP53Mut and TP53Alt

groups (respectively, 44% vs. 7.5%, P ¼ 0.0093 and 28.5% vs.8.3%, P ¼ 0.05).

We then evaluated the number of genomic abnormality as areflection of the genomic complexity. We found that TP53Mut

0

20

40

60

80

100

MWCL-1MM.1SBCWM.1 MEC-1 RL

%of

Livi

ng c

ells

rela

�ve

toco

ntro

l

DMSO

Nutlin-3a

EBCWM1 MWCL1 MEC1 RL

Bcl2

Bax

Bcl-xL

GAPDH

Nutlin-3a - + - + - + - +- +

MM1S

D

*

0

20

40

60

80

100

%Vi

abili

tyre

la�v

eto

cont

rol

DMSO

Nutlin-3a

GAPDH

p53

p21

Puma

MDM2

- +

BCWM1 MM1S

- +

MWCL1 MEC1 RL

- + - + - +Nutlin-3a

C

*

**

A

B P53

P53 P53

P53 P21

P21

BCW

M1

MW

CL1

Nutlin-3a Nutlin-3a

Nutlin-3a: 15.9DMSO :2.5

Nutlin-3a: 12.3DMSO :2.25

Nutlin-3a: 1.2DMSO: 1.1

Nutlin-3a:13.3DMSO:11.1

Figure 2.

Nutlin-3a effects onWM cells. A, Impact of nutlin-3a(10 mmol/L, 16 hours) on p53 targets (p53, p21, puma,andMDM2) in different cell lines. Figure shows resultsfromWestern blot assay on the series of the five celllines, in particular BCWM1 andMM1Swith awild-typepattern, MWCL1, MEC1, and RLwith a mutant patternassociatedwith deletion 17p. Induction of p53 targetswas observed in TP53Wild cell lines. Only TP53Mut celllines showed basal expression of p53. For MEC1, P53band was observed at 40 kDa as previouslydescribed (7). GAPDH, glyceraldehyde 3-phosphatedehydrogenase, is used as a control (Santa CruzBiotechnology). Integration of signal value ispresented in Supplementary Fig. S2-1. B, Functionaltest for p53/p21 proteins using flowcytometry inWMcell lines. Basal expression of p53 was showed (grayline: isotypecontrol, blackp53staining). TheMFI ratiowas indicated for each experimental condition inarbitrary units. Profiles of response after 16-hour invitro culture of the indicated cell lines in the presenceof nutlin-3a (10 mmol/L; black line) or DMSO (grayline) for p53 and p21. Induction of p53 and p 21 wasobserved in BCWM1 as previously showed usingWestern blot. Studies were performed in triplicate,and results from a representative study set areshown. C, Cell viability was measured by using MTSassay (10 mmol/L, 48 hours) assay (CellTiter 96AQueous One Solution Cell Proliferation Assay;Promega) in different cell lines, and all data pointswere normalized to the vehicle controls (DMSO),whichwere arbitrary set at 100% in each experiment.Cell viability was calculated as a percentage withrespect to DMSO-treated cells. Data represent meanof triplicate experiments plus or minus SD. � pointsout statistical significantdifferencewith thecontrol,P< 0.05. D and E, Targeting p53 by nutlin-3a inducedapoptosis on TP53 wild-type cell lines, BCWM1, andMM1s in contrast with TP53-mutated cell lines(MWCL1, MEC1, and RL). D, Cell lines were culturedwith nutlin-3a (10 mmol/L) or vehicle (DMSO) for 16hours. The percentage of cell undergoing apoptosiswas studied using mitochondrial membranepotential assay by flow cytometry. E, Whole celllysates were subjecting to Western blotting usingBax, Bcl-xL, Bcl2, and GAPDH antibodies. Total levelsof the indicated proteins were evaluated byWesternblot analysis indifferent cell linesafter treatmentwith10 mmol/L nutlin-3a or DMSO (vehicle). Upregulationof proapoptotic proteins such as Bax and Bcl-xL wasobserved in TP53Wild cell lines. GAPDH(glyceraldehyde 3- phosphate dehydrogenase) isused as a control. Integration of signal value ispresented in Supplementary Fig. S2-2.

Poulain et al.

Clin Cancer Res; 23(20) October 15, 2017 Clinical Cancer Research6328

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

WM had a greater mean number of abnormality (6.8 vs. 2.6per patient, P ¼ 0.002). A higher frequency of patients withWM with more than three copy-number alterations identifiedby SNPa was observed in TP53Mut (28% vs. 88%, P ¼ 0.03),TP53Alt (64.2 % vs. 33%, P ¼ 0.008), and del17p WM (P <0.001). No association between TP53 alteration and chromo-thripsis was observed. Furthermore, we have observed that WMwith TP53Muthad a greater incidence rate of deletion 7q anddeletion 6q (P ¼ 0.001 and P ¼ 0.055, respectively).

In addition, karyotype was available in 83 cases in our cohort.Interestingly, translocations are found in 14 of 83 cases (16.9%),including five balanced translocations. Supplementary Table S1showed karyotype in WM with TP53 alteration. No recurrenttranslocation was described, and only one 14q32 rearrangementin a t(6;14)(p24;q32) was observed. In two cases, a desequili-

brated translocation led to a deletion of TP53 locus. In our series,we observed a greater frequency of translocations in the groupswith TP53Mut and del17p (28.5% vs. 5.8%, P¼ 0.02 and 50% vs.7.2% P ¼ 0.004, respectively).

Mutational landscape of TP53 alteration in WMWe then sought to study the relationship between mutations

described in WM, including MYD88L265P, BCR mutations(CD79A/CD79B), and CXCR4 and TP53 alteration (25–27). Noassociation was observed between TP53Mut and MYD88 muta-tions, and CD79A/CD79B or CXCR4 mutations (n ¼ 125). Wethen sought to study the incidence of ATM mutations associatedwith chemoresistance and or alteration in DNA damage responsein CLL (5, 6). No mutation of ATM was found in our cohortof WM.

0

20

40

60

80

100

WM 1 TP53wild

WM2 TP53wild

WM3 TP53wild

WM4 TP53wild

WM5 TP53wild

WM6 TP53mut

WM7 TP53mut

WM 8 TP53mut

DMSO

Nutlin 3a

CD3+ cellsCD19+ cells

P53P53

P53 P21

P21 P21

P21P53

B

WM

TP53

mut

WM

TP5

3 w

ild-t

ype

CD3+ cells :1.4

CD19+ cells:4.8

P53 P53P53 P53A

(2) TP53 Wild-type WM(1) TP53-Mutated WM

CD3+ cells:1.1

CD19+ cells:0.9

C

%Vi

abili

tyre

la�v

eto

cont

rol

Nutlin-3a: 4.9DMSO: 4.4

Nutlin-3a : 4.38DMSO: 0.88

Nutlin-3a : 3.55DMSO: 1.12

Nutlin-3a: 1.9DMSO: 1.8

Nutlin-3a : 2.26DMSO : 1.5

Nutlin-3a: 2.14DMSO: 1.2

Nutlin-3a: 1.5DMSO: 1.2

Nutlin-3a: 1.49DMSO: 1.13

WM

TP53

mut

WM

TP5

3 w

ild-t

ype

Figure 3.

Impact of nutlin-3a on primary WM cells. A, Functional test for p53/p21 proteins using flow cytometry in primary WM samples. Basal expression of p53 was shown(gray line: isotype control, black: p53 staining) in CD19þ and CD3þ cells on one patient with WM with TP53 mutation (1) and a wild TP53 sample (2). Histogramswere representative of six different patients with WM. The MFI ratio was indicated for each patient and for each condition in arbitrary units. B, Profiles ofresponse after 24-hour in vitro culture of the indicated cell lines in the presence of nutlin-3a (10 mmol/L; black line) or DMSO (gray line) for p53 and p21 in one patientwith TP53 mutation and one TP53 wild status. Histograms were representative of six different functional assays. The MFI ratio was indicated for each patientand for each condition in arbitrary units. C, Cell viability was measured by using MTS assay (nutlin-3a, 10 mmol/L; 48 hours) in WM CD19þ cells genotyped for TP53mutation samples. TP53 mutation is indicated by TP53Mut, wild-type pattern by TP53 wild, and all data points were normalized to the vehicle controls(DMSO, light gray column), which were arbitrary set at 100%. In each experiment, cell viability was calculated as a percentage with respect to DMSO-treated cells.Data represent mean of triplicate experiments plus or minus SD (�points out statistical significant difference with the control, P < 0.05).

TP53 Mutation in Waldenstrom's Macroglobulinemia

www.aacrjournals.org Clin Cancer Res; 23(20) October 15, 2017 6329

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

WM with TP53Alt has shorter survivalWM is often considered as a low-grade B-cell NHL associated

with prolonged survival, and on the other hand, TP53alteration,irrespective of TP53Mut or del17p, is often correlated to adverseprognosis in cancer. We sought to sort out the prognosis ofTP53alteration, TP53Mut, and del17p in WM.

With a median follow-up of 5 years, 28 (22%) patients haddied, the median (95% confidence interval) survival of thestudied cohort as a whole was 18 years (6;29), with 61% ofpatients surviving beyond 10 years. We then sought to dem-onstrate any relationship between survival prognosis to thepresence of TP53alteration, TP53Mut, and del17p in WM. Wehave first compared overall survival (OS) in the cohort as awhole (pool of indolent and symptomatic WM, n ¼ 115patients with available data), and the median OS was signif-icantly shorter in WM with TP53Alt (Fig. 5A), respectively 9(6;11) and 18 (6;29) years, P ¼ 0.003, irrespective of TP53Mut

(P ¼ 0.003) and del17p (P ¼ 0.002).We then looked separately at OS in the two groups and

discovered that either indolent or symptomatic WM had ashorter median OS by TP53Alt presentation, irrespective ofTP53Mut and del17p. Interestingly, in the group of TP53Alt WM,the median OS of symptomatic WMwas at 4 years (1.5;6.5) and

of indolent WM at 9 years (6.5;11.5), and always significantlygreater than WM with no TP53 alterations, respectively P ¼0.002 (Fig. 5B).

We then thought to identify whether the symptomatic WMwith TP53alteration displayed specific clinical and biologicalcharacteristics. These patients, regardless of TP53Mut or del17p,more often had b2m >3 mg/L (89% vs. 40%, P ¼ 0.012), and agreater IPSSWM score 2 and 3 (50% vs. 30%, P ¼ 0.041, respec-tively). No association was found with regard to other markerstaken independently: age, anemia, thrombocytopenia, lympho-cytosis,monoclonal component, adenopathy, and splenomegaly.TP53 alterationprognostic value appeared independent ofCXCR4or MYD88 L265P mutations.

Shorter time to treatment of indolent WM with TP53Alt

We have then tried to understand the reason for the shortersurvival observedwith the presence of TP53alteration, irrespectiveof TP53Mut or del17p. We first concentrated on patients that wereindolent (WM without treatment criteria at diagnosis and thosedid not need any treatment for at least 1 year following thediagnosis) at diagnosis [65 WM (52%) of 125], whom risk wastobecome symptomatic andneed to start therapy forWM,definedby the time to treatment (TTT). TheWMwith TP53Alt (n¼ 9) had a

MYD88L265Pmuta�on

CD79A/CD79B muta�on

TP53 Altera�on

Dele�on 17p

TP53 muta�on

4/GainTrisomy 4q

Dele�on 6q

CXCR4 muta�on

Dele�on 13q/Mir15aMir16

ofPresence UPDacquired

> 3 Abnormali�es

Dele�on 1p36/ARID1a

Gain 3p/MYD88

Dele�on 11q/ATM

Gain 18

Gain Xq

12Trisomy

Dele�on 8p

17q21/Altera�on NIK

IAltera�on KKalpha/1q24

7qDele�on

Figure 4.

Overview of copy-number alterations (CNAs) and copy-number neutral LOH (CN-LOH) detected in patientswithWM. Association network of genomic alterations inWM. The heatmap represents co-occurrence of TP53 mutation or alteration with other genetic alterations analyzed in 62 patients with WM using SNPa,cytogenetic, and FISH. Each row corresponds to key locus or chromosome targeted by CNA, UPD, or mutation in WM. The columns represent individual patientscolor-coded on the base of gene status. Each patient is represented by a virtual column (black ¼ presence of mutation, CN LOH, or deletion or gain of gene orlocus; white ¼ wild-type). The presence of MYD88 L265P, CXCR4, TP53, and CD79A/B mutations was reported by the black box in the corresponding column.

Poulain et al.

Clin Cancer Res; 23(20) October 15, 2017 Clinical Cancer Research6330

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

shortermedian TTT, 2 (1;3) years versus 5 (4;6) years forWMwiththe absence of TP53Alt (P¼ 0.001), similar to TP53 alteration anddel17p (Fig. 5C).

Shorter time to progression with TP53Alt in WMWe then studied the remaining WMwith symptomatic presen-

tation that had required therapywhomriskwas to relapse, definedby TTP. In this analysis, we only considered the 87 (70%)WMthatreceived a treatment for WM, of whom 47 (54%) had relapsed.The median TTP was 48 (IC95 38;57) and 18 (15;21) months inindolent WM according to the absence or presence of TP53Alt,respectively (P < 0.0001), similar to TP53 alteration and del17p(Fig. 5D).

Ibrutinib induces apoptosis independent of presence of TP53mutation

Ibrutinib is a BTK inhibitor that showed activity in WM (26)and in CLL with deletion17p (13). The effect of ibrutinib wasassessed using primary WM cells (n ¼ 8 including 3 TP53Mut),along with cell lines. Ibrutinib significantly reduced WM survival(P ¼ 0.001) using cell viability assay, but independently of theTP53Wild or TP53Mut inWM cell lines and patients withWM (23%vs. 46%, P < 0.05; Fig. 6A and B). Ibrutinib was not able to induce

p53 and downstream targets such as p21, MDM2, and Pumaexpression (Fig. 6C–E). The sensitivity to induction of cell deathwas similar, independently of the TP53 mutational status in thecell lines, including WM-derived cell lines, upon ibrutinib treat-ment (Fig. 6F and G).

PrimaMet and CP31398 induce cytotoxicity in WMCompounds that bypass loss of p53 function in relation to

TP53 mutation were described, including PrimaMet (APR-246), asmall molecule that reactivates mutant p53 by restoring its wild-type conformation and transcriptional functions. The compoundCP-31398 was identified as a substance that protects p53 fromthermal denaturation (2). A significant decrease of viability wasobserved inWM-derived cells lines and in primaryWM cells uponCP-31398 and PrimaMet treatment independently of TP53 muta-tional status (Supplementary Fig. S2A and S2B). Interestingly, wehave observed no induction of p53 in the different cell lines uponCP-31398 and PrimaMet exposure using Western blot and flowcytometry, including WM-derived cells lines, along with no sig-nificant variation of expression levels of p21 and the otherdownstream targets of p53 (Supplementary Fig. S2C and S2D).These results suggested that CP-31398 and PrimaMet-inducedWMcell death and cytotoxicity occurred independently of p53.

0 5

P = 0.003

P = 0.0001 P < 0.0001

P = 0.002

10

No

TP53alt

Yes

Yes/no OSMedian, 95% CI

15/94

8/14

18 (6-29)

9 (6-11)

No

TP53alt

Yes

Yes/no TTTMedian, 95% CI

34/56

3/9

5 (4-6)

2 (1-3)

No

TP53alt

Yes

Yes/no TTPMedian, 95% CI

50/60

5/5

48 (38-57)

18 (15-21)

No

No

TP53altWM

Yes

Yes

Indolent

Indolent

Symptom

Symptom

Yes/no OSMedian, 95% CI

6/49

9/45

5/9

3/5

18 (–)

-

9 (6.5–11.5)

4 (1.5–6.5)

Years15 0 5 10

Years1520

0 5 10Years

15 20

0.2

0.0

0.4

0.6

Cu

mu

lati

ve s

urv

ival

0.8

1.0A B

C D

0.2

0.0

0.4

0.6

Cu

mu

lati

ve s

urv

ival

0.8

1.0

0.2

0.0

0 50 100Months

150 200 250

0.4

0.6C

um

ula

tive

su

rviv

al

0.8

1.0

0.2

0.0

0.4

0.6

Cu

mu

lati

ve s

urv

ival

0.8

1.0

Figure 5.

Clinical prognosis of TP53Mut in WM. A,OS according to TP53Alt, P¼ 0.003. B,OS according to TP53Alt and either indolent or symptomatic status ofWM, P¼ 0.002.C, Time-to-treatment start according to TP53Alt, P ¼ 0.0001. D, Time to progression according to TP53Alt, P ¼ 0.0001.

TP53 Mutation in Waldenstrom's Macroglobulinemia

www.aacrjournals.org Clin Cancer Res; 23(20) October 15, 2017 6331

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

0

20

40

60

80

100

WM 1TP53wild

WM2TP53wild

WM3TP53wild

WM4TP53wild

WM5TP53wild

WM6TP53mut

WM7TP53mut

WM 8TP53mut

DMSO

Ibru�nib

B

C D

0

20

40

60

80

100

BCWM1 MM1S MWCL1 MEC1 RL

% V

iabi

lity

rela

�ve

to c

ontr

ol

DMSO

Ibru�nib

A

% V

iabi

lity

rela

�ve

to co

ntro

l

WM

cel

lsTP

53 m

utW

M c

ells

TP53

wild

P53

P53

P21

P21

MW

CL1

BCW

M1

P53

P53

P21

P21

* * ** * * * * * ** *

Ibru�nib : 2.7DMSO : 4.4

Ibru�nib : 1.2DMSO: 1.8

Ibru�nib : 0.8DMSO : 1.1

Ibru�nib : 1.3DMSO : 0.9

Ibru�nib : 1.47 DMSO : 1.12

Ibru�nib: 5.7 DMSO :7.8

Ibru�nib : 1.5 DMSO : 1.8

Ibru�nib : 1.8 DMSO : 0.73

MDM2

GAPDH

Puma

P53

P21

- + -- - +

BCWM1 MWCL1 MEC1

- + -- - +

- + -- - +

MM1S

- + -- - +

RL

- + -- - +

Nutlin-3a

Ibru�nib

GAPDH

Bcl-2

Bcl-xL

- - +

BCWM1 MWCL1 MEC1

Bax

- + --+ -- + --

RL

- - +

MM1S

+ --- - + - - + - - +

Nutlin-3a

Ibru�nib

E F

- +

1 1

G

BCWM.10.0

20.0

40.0

60.0

% o

f Liv

ing

cells

rela

�ve

to c

ontr

ol

80.0

100.0

MM.1S MWCL-1 MEC-1

DMSONutlin-3aIbru�nib

Figure 6.

Ibrutinib did not increase expression of the p53 target. A, Cell lines were cultured with ibrutinib (10 mmol/L) or vehicle (DMSO) for 24 or 48 hours (date notshown). Cell viability was measured by using MTS assay in different cell lines, and all data points were normalized to the vehicle controls (DMSO), which werearbitrary set at 100% in each experiment. Cell viability was calculated as percentage with respect to DMSO-treated cells. Data represent mean of triplicateexperiments plus or minus SD. (� points out significant difference with the control, P < 0.05). (Continued on the following page.)

Poulain et al.

Clin Cancer Res; 23(20) October 15, 2017 Clinical Cancer Research6332

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

DiscussionTo the best of our knowledge, our study recruited the largest

cohort of patients with WM for analysis of TP53 mutation,combining Sanger sequencing and ultradeep-targeted NGS. Inthis study, we described TP53mutation in less than 10% of WM,in a series of 125WMat diagnosis. Interestingly, our study showedseveral patterns of TP53 alteration including monoallelic loss,mutations, and/or UPD. We observed that TP53 mutation washighly associated with deletion 17p. TP53 mutations weredescribed in the DNA-binding domain and associated with acomplex genomic spectrum using SNPa. In addition, TP53Altwasassociated with a pejorative prognostic value in patients withWM independently of IPSSWM (30).

Frequency of deletion 17 was first described in small cohorts ofpatients with untreated or relapsed WM (16, 17). 17P/TP53deletion was previously observed in 8% of patients with WMusing FISH in a large cohort of 140 symptomatic patients withMW treated by fludarabine or chloraminophen (18). Patientswith TP53 deletion had short progression-free survival and shortdisease-free survivalwith amedian follow-upof nearly 39months(18). In this study, mutations of TP53 were not studied exten-sively, and there were some limitations, including that TP53mutation was studied using Sanger sequencing with low sensi-bility to detect subclones in unselected tumoral cells. Few studieson TP53 mutations were reported in WM so far. TP53 mutationwas observed in 6%of patientswithWM in a cohort of 30 patientsusing whole-genome sequencing (31). In our study, we haveaddressed the question using a deeper sequencing in order toallow a higher sensitivity in comparison with Sanger sequencingand the detection of potential subclonal mutation of TP53 (18).Nonetheless, the TP53mutation incidence inWM is lower than insplenic marginal zone lymphoma or refractory CLL (5, 10, 12).

The selective evolutionary process in cancer can lead todifferential development of subclones, each with their owncharacteristic profile of mutated genes. Nonetheless, an impor-tant dataset obtained in our series came from the quantificationof the allelic frequency of the variants (VAF) of TP53 mutationinto tumoral cells using NGS in our series. VAF allows todifferentiate between clonal (dominant clonal) and subclonalmutations by combining copy-number alteration using bothSNPa and mutation screening. The presence of TP53 mutationin subclones may be observed at diagnosis in WM. Develop-ment of minor clones of TP53 mutation overtime upon treat-ment may also be associated with pejorative outcome (11). We

also found that patients with TP53Mut as well as TP53Alt had ahigher degree of genomic complexity than those with TP53Wild.A higher incidence of copy-number alteration in patients withsymptomatic WM in comparison with indolent WM was pre-viously described (22, 32). Longitudinal analysis studies areneeded to explore the dynamics of clonal architecture and toidentify driver or additional mutations in WM that couldcontribute to clinical progression or chemoresistance.

In our study, all the mutations of TP53 were located in DNA-binding domain impairing DNA binding and target genetransactivation. However, the classical hotspot codons of TP53were not frequently targeted (3). To test the functional assess-ment of p53 response in TP53Mut cells, we used an in vitrofunctional assay based on TP53 activation by the nongenotoxicdrug, nutlin-3a, which is a potent and selective inhibitor ofTP53/MDM2 interaction, specifically active in TP53 Wild cases(7, 12, 28). As few MYD88 L265P WM cell lines are available,we limited the use of cell lines derived from WM, with BCWM1(TP53wild type) andMWCL1 (TP53 V143A; refs. 20, 21, 29). Inaddition, WM primary cells including three patients with muta-tions were used to confirm our results. Our results showed p53/p21 response, loss of viability, and apoptosis in TP53Wild WMcells in contrast with WM with TP53 mutation. These testsconsequently confirmed the functional impact of TP53 muta-tion in WM (2, 28).

In contrast with others lymphoid disorders, the prognosticvalue of TP53 alterations, including mutations, has never beenshowed in WM to that extent (1, 5, 8, 10, 18). Our study showedthat TP53alteration, irrespective of TP53Mut or del17p, defines ahigh-risk population in WM characterized with significantlyshorter TTP for symptomatic WM or TTT for indolent WM,although in a lesser extent for the latter, and subsequently OS.To date, few genomic alterations were associated with prognosisin WM. Recent studies have highlighted the potential adverseprognosis of MYD88 mutations in WM (3, 33), but most impor-tantly adverse prognosis was associated with CXCR4 mutations,along with ibrutinib resistance (24–26). Our study thus led us topropose that it might be primetime for a systematic evaluation ofTP53 alteration through mutation and/or deletion as part of theprognostic pretreatment package of WM at diagnosis, in order toidentify patients with TP53Alt that might benefit more from BTKinhibitors (13). Interestingly, we observed the absence of muta-tions of ATM in our cohort suggesting different genomic featuresbetween CLL andWM (5, 6). Similarly, we found no relationship

(Continued.) B, Study of viability in five patients with TP53Wild and three patients with TP53Mut using MTS assay. Experiments were performed on B selectedlymphocytes and data represent mean of triplicate experiments. Ibrutinib (10 mmol/L, 48 hours) induced cytotoxicity independently of TP53mutation status in eightpatients with WM (mean, 31%; range, 6–51; � points out significant difference with the control, P < 0.05). C and D, Flow cytometry analysis of p53 and p21expression after ibrutinib exposure (24 hours, 10 mmol/L). (C) in WM cell lines (studies were performed in triplicate, and results from a representative study set areshown, the MFI ratio was indicated for each experimental condition in arbitrary units.) and (D) flow cytometry analysis of p53 and p21 expression afteribrutinib exposure in primaryWM samples. The histograms show the levels for p53 and p21. Expression of p53 and p21was analyzed onCD19þCD45þ cells. Black line,ibrutinib exposure; gray line, DMSO condition. Absence of increased expression of p53 and p21 was observed in TP53Wild and TP53Mut patients (n ¼ 5), andrepresentative histograms of two different WM samples were showed. The MFI ratio was indicated for each experimental condition in arbitrary units. E,Immunoblotting depicting impact of ibrutinib exposure (48 hours, 10 mmol/L) in different cell lines. Nutlin-3a (10 mmol/L) was shown as reference of p53 targetinduction pattern. Modulation of p53 expression along with downstream targets was used as a surrogate marker for the activation of the p53 pathway. Absence ofinduction of p53 target (p53, p21, puma, andMDM2)was observed in ibrutinib-sensitive cell lines independently of TP53mutational status. Integration of signal valuesis presented in Supplementary Fig. S3-1. F andG, Ibrutinib induces apoptosis inWM cells CD19þ cells genotyped for TP53mutation and B-cell lines (BCWM1, MWCL1,RL, and MEC1). MM1S was resistant to ibrutinib. F, Western blot analysis of several apoptosis proteins. Variation of apoptosis proteins levels was observedindependently of TP53 mutational status. Integration of signal value is presented in Supplementary Fig. S3-2. G, The percentage of cell undergoing apoptosis wasstudied using mitochondrial membrane potential assay by flow cytometry. All data points were normalized to the vehicle controls (DMSO), which werearbitrary set at 100%.Data representmean of triplicate experiments plus orminus SD. (� points out significant differencewith the control,P<0.05.) Cellswere treatedwith ibrutinib or nutlin-3a (48 hours, 10 mmol/L).

www.aacrjournals.org Clin Cancer Res; 23(20) October 15, 2017 6333

TP53 Mutation in Waldenstrom's Macroglobulinemia

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

between TP53 alteration and CXCR4 and CD79ABmutations, thelatter associated with BCR inhibitor resistance in diffuse large B-cell lymphoma (25, 27). The question then for WM remains tounderstand how future drug development may improve survivalof the WM with TP53 alteration.

Number of new agents are becoming available that may actindependently of the DNA damage pathway and/or p53 (2,34). We thus investigated the effect of either of three com-pounds that showed activity in TP53Mut condition, indepen-dently of the mechanism of loss of function of p53. Preliminarydata showed the great interest for BTK inhibitors in relapsedWM and in CLL with TP53 alteration (6, 13, 26, 35). We haveevaluated the in vitro responses of WM to ibrutinib according totheir TP53 status and showed that ibrutinib may induce apo-ptosis and loss of viability in WM, independently of p53alteration. Our results may suggest that ibrutinib should bean interesting therapeutic option to WM in TP53AltWM. Furtherclinical trial may address this question in particular in the era oftargeted therapy or chemofree options.

As impaired TP53 function is one of the most characterizedfactors associated with chemoresistance, designing drug strategiesto target mutant p53 tumors is therefore challenging. Manycompounds that either bypass the defective p53 pathway orreactivate the p53 protein in cells expressing a mutant proteinwere tested with variable translation into useful therapeuticstrategies (2, 3). Our study showed preliminary results suggestingof the interest of PrimaMet and CP31398, described as "reactivator"of p53alteration, to induce apoptosis and loss of viability inWM.These compounds bind to mutant p53 proteins, interacting withthe DNA-binding domain, thereby promoting proper folding ofthe mutant protein and restoration of p53 function (2, 3, 28, 36).Taking together, these results support the framework for preclin-ical evaluation of new strategies with p53 "reactivator" in a "p53druggable" era.

In conclusion, this study identified TP53 alteration, mainlythrough mutation or deletion, and consequently loss of p53function as a key genomic alteration in WM, although presentin approximately 10% of WM. We thus propose that TP53alteration may be considered as a potential prognostic factor inWM, worth identifying prior to start treatment of WM. We also

suggested that the recently approvedBTK inhibitor, ibrutinib,maybe potentially proposed to rescue patients with WM with TP53alteration.

Disclosure of Potential Conflicts of InterestL. Renaud is an employee of Hospira. No potential conflicts of interest were

disclosed by the other authors.

Authors' ContributionsConception and design: S. Poulain, S. Geffroy, X. LeleuDevelopment of methodology: S. Poulain, C. Roumier, S. Geffroy, C. Roche-Lestienne, X. LeleuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Poulain, C. Roumier, E. Bertrand, A. Renneville,S. Sebda, M. Nudel, C. Herbaux, L. Renaud, C. Tomowiak, S. Guidez, S. Tricot,X. LeleuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Poulain, C. Roumier, E. Bertrand, A. Caillault-Venet,S. Geffroy, O. Nibourel, B. Quesnel, C. Preudhomme, X. LeleuWriting, review, and/or revision of the manuscript: S. Poulain, X. LeleuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Poulain, C. Roumier, A. Renneville, X. LeleuStudy supervision: S. Poulain, X. LeleuOther (sampling preparation and performed functional experiments):E. Doye

AcknowledgmentsWe thank Claudine Delsaut, Axelle S�eghir, and Pauline Vandycke for their

excellent technical assistance. The authors wish to thank the Unit�e de RechercheClinique of the Centre Hospitalier de Valenciennes for their financial andsupport assistance.

Grant SupportThis work was supported by the Comit�e du Septentrion de la Ligue

contre le Cancer, the Canc�eropole Nord Ouest, and the FondationFrancaise pour la Recherche contre le My�elome et les Gammapathies(FFRMG).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 2, 2017; revised June 3, 2017; accepted July 18, 2017;published OnlineFirst July 28, 2017.

References1. XuMonette Z,Medeiros J, Li Y, Orlowski RZ, AndreeffM, Bueso-RamosCE,

et al. Dysfunction of the TP53 tumor suppressor gene in lymphoidmalignancies. Blood 2012;119:3668–83.

2. Muller P, Vousden K. Mutant p53 in cancer: new functions and therapeuticopportunities. Cancer Cell 2014;25:304–17.

3. Soussi T, Wiman K. TP53: an oncogene in disguise. Cell Death Differ2015;22:1239–49.

4. Gostissa M, Bianco J, Malkin D, Kutok JL, Rodig SJ, Morse HC 3rd, et al.Conditional inactivation of p53 in mature b cells promotes generation ofnongerminal center-derived B-cell lymphomas. Proc Natl Acad Sci U S A2013;110:2934–9.

5. Rossi D, GaidanoG. The clinical implications of genemutations in chroniclymphocytic leukemia. Br J Cancer 2016;114: 849–54.

6. Zent C, Burack R. Mutations in chronic lymphocytic leukemia and howthey affect therapy choice: focus onNotch1, SF3B1, and TP53.HematologyAm Soc Hematol Educ Program 2014;2014:119–24.

7. Pozzo F, Dal Bo M, Peragine N, Bomben R, Zucchetto A, Rossi F, et al.Detection of TP53 dysfunction in chronic lymphocytic leukemia by an invitro functional assay based on TP53 activation by the non genotoxic drugNutlin-3: a proposal for clinical application. J Hematol Oncol 2013;6:83.

8. Hebraud B, Magrangeas F, Cleynen A, Lauwers-Cances V, Chretien ML,Hulin C, et al. Role of additional chromosomal changes in the prognosticvalue of t(4;14) and del(17p) in multiple myeloma: the IFM experience.Blood 2015;125:2095–100.

9. Lode L, Eveillard M, Trichet V, Soussi T, Wuill�eme S, Richebourg S, et al.Mutations in TP53 are exclusively associated with del(17p) in multiplemyeloma. Haematologica 2010;95:1973–76.

10. Parry M, Rose-Zerilli M, Ljungstrom V, Gibson J, Wang J, Walewska R,et al. Genetics and prognostication in splenic marginal zone lympho-ma: revelations from deep sequencing. Clin Cancer Res 2015;21:4174–83.

11. Rossi D, Khiabanian H, Spina V, Ciardullo C, Bruscaggin A, Fam�a R, et al.Clinical impact of small TP53 mutated subclones in chronic lymphocyticleukemia. Blood 2014;123:2139–47.

12. Marinelli M, Peragine N, Di Maio V, Chiaretti S, De Propris MS, Raponi S,et al. Identification of molecular and functional patterns of p53 alterationsin chronic lymphocytic leukemia patients in different phases of the disease.Haematologica 2013;98:371–5.

13. FarooquiM,Valdez J,Martyr S, AueG, SabaN,NiemannCU, et al. Ibrutinibfor previously untreated and relapsed or refractory chronic lymphocytic

Poulain et al.

Clin Cancer Res; 23(20) October 15, 2017 Clinical Cancer Research6334

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

leukemia with TP53 aberrations: a phase 2, single arm. Lancet Oncol 2015;16:169–76.

14. Owen RG, Pratt G, Auer RL, Flatley R, Kyriakou C, Lunn MP, et al. Guide-lines on the diagnosis and management of Waldenstrom macroglobuli-naemia. Br J Haematol 2014;165:316–33.

15. Treon SP, Xu L, Yang G, Zhou Y, Liu X, Cao Y, et al. MYD88 L265P somaticmutation in Waldenstrom's macroglobulinemia. N Engl J Med 2012;367:826–33.

16. Chang H, Samiee S, Li D, Patterson B, Chen C, Stewart A. Analysis of IgHtranslocations, chromosome 13q14 and 17p13.1(p53) deletions by fluo-rescence in situ hydridisation in Waldenstrom's macroglobulinemia: asingle center study of 22 cases. Leukemia 2004;18:1160–2.

17. Schop RF, Jalal SM, Van Wier SA, Ahmann GJ, Bailey RJ, Kyle RA, et al.Deletions of 17p13.1 and 13q14 are uncommon in Waldenstrom mac-roglobulinemia clonal cells and mostly seen at the time of disease pro-gression. Cancer Genet Cytogenet 2002;132:55–60.

18. Nguyen-Khac F, Lambert J, Chapiro E, Grelier A, Mould S, Barin C, et al.Chromosomal aberrations and their prognostic value in a series of 174untreated patients with Waldenstrom's macroglobulinemia. Haematolo-gica 2013;98:649–54.

19. DimopoulosMA,Kastritis E,OwenRG,KyleRA, LandgrenO,Morra E, et al.Treatment recommendations for patients with Waldenstrom macroglob-ulinemia (WM) and related disorders: IWWM-7 consensus. Blood 2014;124:1404–11.

20. Ditzel Santos D, Ho Aw, Tournilhac O, Hatjiharissi E, Leleu X, Xu L, et al.Establishment of BCWM.1 cell line forWaldenstr€om'smacroglobulinemiawith productive in vivo engraftment in SCID-hu mice. Exp Hematol2007;35:1366–75.

21. Hodge L, Novak A, Grote D, Braggio E, Ketterling RP, Manske MK, et al.Establishment and characterization of a novel Waldenstrom macroglob-ulinemia cell line, MWCL1. Blood 2011;117:e190–7.

22. Poulain S, Roumier C, Galiegue-Zouitina S, Daudignon A, Herbaux C,Aiijou R, et al. Genome wide SNP array identified multiple mechanisms ofgenetic changes inWaldenstrommacroglobulinemia. Am JHematol 2013;88:948–54.

23. Poulain S, Roumier C, Decambron A, Renneville A, Herbaux C, Bertrand E,et al. MYD88L265Pmutation inWaldenstrommacroglobulinemia. Blood2013;121:4504–11.

24. Poulain S, Roumier C, Caillault-Venet A, Figeac M, Herbaux C, Marot G,et al. Genomic landscape of CXCR4 mutations in Waldenstrom macro-globulinemia.Clin Cancer Res 2016;22:1480–8.

25. Roccaro AM, Sacco A, Jimenez C, Maiso P, Moschetta M, Mishima Y, et al.C1013G/CXCR4 acts as a driver mutation of tumor progression andmodulator of drug resistance in lymphoplasmacytic lymphoma. Blood2014;123:4120–31.

26. Treon S, Tripsas C, Meid K, Warren D, Varma G, Green R, et al. Ibrutinib inpreviously treated Waldenstrom's macroglobulinemia. New Eng J Med2015;372:1430–40.

27. Wilson W, Young R, Schmitz R, Yang Y, Pittaluga S, Wright G, et al.Targeting B cell receptor signalling with ibrutinib in diffuse large B celllymphoma. Nat Med 2015;21:922–6.

28. De Viron E, Michaux L, Put M, Bontemps F, Van den Neste E. Present statusand perspectives in functional analysis of P53 in chronic lymphocyticleukemia. Leuk Lymphoma 2012;53:1445–51.

29. DrexlerH, Chen S,MacLeodR.Would the realWaldenstrom cell line pleasestand up?. Leuk Lymphoma 2013;54:224–6.

30. Morel P, Duhamel A, Gobbi P, DimopoulosMA, DhodapkarMV,McCoy J,et al. International prognostic scoring system for Waldenstrom macro-globulinemia. Blood 2009;113:4163–70.

31. Hunter ZR, Xu L, Yang G, Zhou Y, Liu X, Cao Y, et al. The genomiclandscape of Waldenstrom macroglobulinemia is characterized byhighly recurring MYD88 and WHIM-like CXCR4 mutations, and smallsomatic deletions associated with B-cell lymphomagenesis. Blood2014;123:1637–46.

32. Paiva B, Corchete L, Vidriales MB, García-Sanz R, Perez JJ, Aires-Mejia I,et al. The cellular origin and malignant transformation of Waldenstrom'smacroglobulinemia. Blood 2015;125:2370–80.

33. Treon S, Cao Y, Xu L, Yang G, Liu X, Hunter Z. Somatic mutations inMYD88 and CXCR4 are determinants of clinical presentation andoverall survival in Waldenstrom macroglobulinemia. Blood 2014;123:2791–6.

34. Dimopoulos MA, Kastritis E, Ghobrial IM. Waldenstrom's macroglobuli-nemia: a clinical perspective in the era of novel therapeutics. Ann Oncol2016;27:233–40.

35. Dimopoulos M, Trotman J, Tedeschi A, Matous J, MacDonald D, Tam C,et al. Ibrutinib for patients with rituximab-refractory Waldenstr€om'smacroglobulinaemia (iNNOVATE): an open-label substudy of aninternational, multicentre, phase 3 trial. Lancet Oncol 2017;18:241–50.

36. Tessoulin B,DescampsG,Moreau P,Ma€�ga S, Lod�e L, GodonC, et al. PrimaMet induces myeloma cell death independent of p53 by impairing theGSH-ROS balance. Blood 2014;124:1626–36.

www.aacrjournals.org Clin Cancer Res; 23(20) October 15, 2017 6335

TP53 Mutation in Waldenstrom's Macroglobulinemia

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007

2017;23:6325-6335. Published OnlineFirst July 28, 2017.Clin Cancer Res   Stéphanie Poulain, Christophe Roumier, Elisabeth Bertrand, et al.   Macroglobulinemia

Mutation and Its Prognostic Significance in Waldenstrom'sTP53

  Updated version

  10.1158/1078-0432.CCR-17-0007doi:

Access the most recent version of this article at:

  Material

Supplementary

  http://clincancerres.aacrjournals.org/content/suppl/2017/07/28/1078-0432.CCR-17-0007.DC1

Access the most recent supplemental material at:

   

   

  Cited articles

  http://clincancerres.aacrjournals.org/content/23/20/6325.full#ref-list-1

This article cites 36 articles, 19 of which you can access for free at:

  Citing articles

  http://clincancerres.aacrjournals.org/content/23/20/6325.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.org

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/23/20/6325To request permission to re-use all or part of this article, use this link

on October 30, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0007