Academic Edi o : Alexanda Tzanko
Recei ed: 3 Decembe 2024
Re ised: 10 Janua y 2025
Accep ed: 16 Janua y 2025
Published: 5 Feb ua y 2025
Ci a ion: Fe nández-Cas illejo, S.;
Badia, J.; de la C uz-Me ino, L.; Ma ín
Ga cia-Sáncho, A.; Ca nice o-
González, F.; Palazón-Ca ión, N.;
Ríos-He anz, E.; de la C uz-Vicen e,
F.; Rueda-Domínguez, A.; Ma ínez-
Banaclocha, N.; e al. Ke one Bodies
A e Po en ial P ognos ic Bioma ke s in
Relapsed/Re ac o y Di use La ge
B-Cell Lymphoma: Resul s om he
R2-GDP-GOTEL T ial. Cance s 2025,
17, 532. h ps://doi.o g/10.3390/
cance s17030532
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
Ke one Bodies A e Po en ial P ognos ic Bioma ke s in Relapsed/
Re ac o y Di use La ge B-Cell Lymphoma: Resul s om he
R2-GDP-GOTEL T ial
Sa a Fe nández-Cas illejo 1,2,† , Joan Badia 1,2,† , Luís de la C uz-Me ino 3,4 , Alejand o Ma ín Ga cia-Sáncho 5,6 ,
Fe nando Ca nice o-González
7
, Na alia Palazón-Ca ión
3,4
, Edua do Ríos-He anz
8
, Fá ima de la C uz-Vicen e
9
,
An onio Rueda-Domínguez 10 , Na i idad Ma ínez-Banaclocha 11 , José Gómez-Codina 12, Jo ge Lab ado 13 ,
F ancisca Ma ínez-Madueño 1,2,14, Nú ia Amigó 14,15 , An onio Sala -Sil es e 16, Del ys Rod íguez-Ab eu 17,
Lau a Gál ez-Ca ajal
10
, Ma ga i a Sánchez-Bea o
18
, Ma iano P o encio-Pulla
19
, Ma ia Gui ado-Risueño
20
,
Es eban Nogales 3,4, Víc o Sánchez-Ma gale 21 , Ca los Jiménez-Co egana 21 , Guille mo Rod íguez-Ga cía 9,
Raquel Cume as 1,22,* and Josep Gumà1,2,14 on behal o he Spanish Lymphoma Oncology G oup (GOTEL)
1
T ansla ional, Epidemiological and Clinical Oncological Resea ch G oup (GIOTEC), Depa men o Oncology,
Ins i u d’In es igació Sani à ia Pe e Vi gili (IISPV), 43204 Reus, Ta agona, Spain;
sa a. e nandez@iisp .ca (S.F.-C.); joan.badia@iisp .ca (J.B.); [email p o ec ed] (F.M.-M.);
[email p o ec ed] (J.G.)
2Ins i u d’Oncologia de la Ca alunya Sud (IOCS), Hospi al Uni e si a i San Joan de Reus,
43204 Reus, Ta agona, Spain
3Cance Immuno he apy G oup, Oncohema ology and Gene ics Depa men , Biomedicine Ins i u e o
Se ille (IBIS)/CSIC, 41013 Se ille, Spain; [email p o ec ed] (L.d.l.C.-M.);
[email p o ec ed] (N.P.-C.); [email p o ec ed] (E.N.)
4
Depa men o Clinical Oncology, Uni e si y Hospi al Vi gen Maca ena and School o Medicine, Uni e si y o
Se illa, 41013 Se illa, Spain
5Depa men o Hema ology, Hospi al Uni e si a io de Salamanca, Ins i u o de In es igación Biomédica de
Salamanca (IBSAL), Uni e sidad de Salamanca, 37007 Salamanca, Spain; [email p o ec ed]
6CIBER de Cánce (CIBERONC), Ins i u e o Heal h Ca los III, 28029 Mad id, Spain
7Depa men o Hema ology, San Ped o de Alcán a a Hospi al, 10003 Cáce es, Spain; ca nice [email p o ec ed]
8Depa men o Hema ology, Hospi al Uni e si a io Vi gen de Valme, 41014 Se illa, Spain; [email p o ec ed]
9Depa men o Hema ology, Hospi al Uni e si a io Vi gen del Rocío, 41013 Se illa, Spain;
[email p o ec ed] (F.d.l.C.-V.); [email p o ec ed] (G.R.-G.)
10 Depa men o Clinical Oncology. Hospi al Uni e si a io Vi gen de la Vic o ia, 29010 Málaga, Spain;
[email p o ec ed] (A.R.-D.); [email p o ec ed] (L.G.-C.)
11 Depa men o Oncology, D . Balmis Gene al Uni e si y Hospi al, Alican e Ins i u e o Heal h and
Biomedical Resea ch (ISABIAL), 03010 Alican e, Spain; [email p o ec ed]
12 Depa men o Clinical Oncology, Hospi al Uni e si a io y Poli écnico La Fe, 46026 Valencia, Spain;
[email p o ec ed]
13 Depa men o Hema ology, Hospi al Uni e si a io de Bu gos, 09006 Bu gos, Spain;
[email p o ec ed]
14 Facul y o Medicine and Heal h Sciences, Uni e si a Ro i a i Vi gili (URV), 43201 Reus, Ta agona, Spain;
[email p o ec ed]
15 Bios e Teslab, 43206 Reus, Ta agona, Spain
16 Depa men o Hema ology, Hospi al del Ma , 08003 Ba celona, Spain; asala @hospi aldelma .ca
17 Depa men o Clinical Oncology, Hospi al Uni e si a io Insula de G an Cana ia,
35016 Las Palmas de G an Cana ia, Las Palmas, Spain; [email p o ec ed]g
18 Lymphoma Resea ch G oup, Depa men o Medical Oncology, Hospi al Uni e si a io Pue a de
Hie o-Majadahonda, IDIPHISA, 28222 Majadahonda, Mad id, Spain; [email p o ec ed]g
19 Depa men o Clinical Oncology, Hospi al Uni e si a io Pue a De Hie o-Majadahonda, IDIPHISA,
28222 Majadahonda, Mad id, Spain; [email p o ec ed]
20 Depa men o Clinical Oncology, Hospi al Uni e si a io de Elche, 03203 Elche, Alican e, Spain;
[email p o ec ed]
21
Medical Biochemis y and Molecula Biology and Immunology, Hospi al Uni e si a io Vi gen de la Maca ena,
41009 Se illa, Spain; [email p o ec ed] (V.S.-M.); [email p o ec ed] (C.J.-C.)
22 Depa men o Elec ical and Au oma ic Elec onic Enginee ing, Uni e si a Ro i a i Vi gili (URV),
43002 Ta agona, Spain
*Co espondence: aquel.cume as@iisp .ca
†These au ho s con ibu ed equally o his wo k.
Cance s 2025,17, 532 h ps://doi.o g/10.3390/cance s17030532
Cance s 2025,17, 532 2 o 16
Simple Summa y: Pa ien s wi h elapsed o e ac o y di use la ge B-cell lymphoma
(DLBCL) ha e poo ou comes and limi ed ea men op ions. A phase II ial by GOTEL
e alua ed he R2-GDP egimen (combina ion o lenalidomide, i uximab, gemci abine,
dexame hasone, and cispla in), demons a ing easibili y and e ec i eness. Baseline se um
me abolomic analysis o 69 pa ien s en olled in he ial iden i ied wo independen me abo-
li es, 3-hyd oxybu y a e (3OHB) and ace one, as being signi ican ly associa ed wi h o e all
su i al and p og ession- ee su i al. Ele a ed 3OHB le els (>141
µ
M) we e speci ic o
he ABC sub ype o DLBCL, while ace one le els we e ele a ed in bo h ypes o DLCBL
bu mo e p onounced in ABC cases. These bioma ke s, i espec i e o sex, age, and BMI,
could help p edic ou comes and guide ea men s a egies in elapsed/ e ac o y DLBCL.
Abs ac : Backg ound: Pa ien s wi h elapsed o e ac o y (R/R) di use la ge B-cell
lymphoma (DLBCL) who a e ineligible o high-dose chemo he apy ha e limi ed ea -
men op ions and poo li e expec ancy. The pu pose o his s udy is o iden i y a se um
me abolomic p o ile ha may be p edic i e o ou come in pa ien s wi h R/R-DLBCL.
Me hods: This s udy included 69 R/R DLBCL pa ien s om he R2-GDP-GOTEL ial
(Eud aCT 2014-001620-299). Se um samples we e collec ed a baseline, and he mean
leng h o ollow-up was 41 mon hs. Se um me aboli es we e analyzed by nuclea magne ic
esonance (NMR). Me aboli es we e co ela ed wi h ea men esponse, p og ession- ee
su i al (PFS), and o e all su i al (OS). Resul s: Se um le els o 3-hyd oxybu y a e
(3OHB) and ace one we e signi ican ly (p< 0.001) associa ed wi h PFS (3OHB: haza d
a io [HR] 7.7, 95% con idence in e al [CI] 2.5–24.1; ace one: HR 9.32, 95% CI 2.75–31.6)
and OS (3OHB: HR 9.32, 95% CI 2.75–31.6; ace one: HR 1.92, 95% CI 1.36–2.69). Se um
alues o 141
µ
M o 3OHB and 40
µ
M o ace one we e he op imal cu o s associa ed
wi h he su i al ou comes. Ele a ed 3OHB le els (>141
µ
M) we e speci ic o he ABC
sub ype o DLBCL, while ace one le els we e ele a ed in bo h ypes o DLCBL bu mo e
p onounced in ABC cases. In a mul i a ia e su i al analysis, including he In e na ional
P ognos ic Index (IPI) sco e and e ac o iness s a us (R/R), 3OHB and ace one emained
signi ican . To aid oncologis s employing he R2-GDP egime, we cons uc ed PFS and
OS nomog ams o R/R-DLBCL isk s a i ica ion, inco po a ing 3OHB le els o ace one
le els, IPI sco e, and e ac o iness s a us. The nomog am wi h 3OHB and e ac o iness
s a us showed a ime-dependen AUC o 0.86 o 6-mon h PFS and 0.84 o 12-mon h OS.
These nomog ams p o ide a comp ehensi e ool o indi idualized isk assessmen and
ea men op imiza ion. Conclusions: The ke one bodies 3OHB and ace one a e po en ial
p ognos ic bioma ke s o poo ou come in R/R DLBCL pa ien s ea ed wi h he R2-GDP
egimen, independen ly o IPI sco e and chemo e ac o iness s a us.
Keywo ds: ke one bodies; di use la ge B-cell lymphoma (DLBCL); elapsed/ e ac o y
lymphoma; 3-hyd oxybu y a e; 3OHB; ace one; p ognos ic bioma ke s; me abolomics
1. In oduc ion
App oxima ely 60% o pa ien s wi h di use la ge B-cell lymphomas (DLBCL) a e cu ed
using up on he apy wi h he CHOP-R egimen o o he an h acycline and i uximab-
based chemo he apies, whe eas he emaining 40% a e e ac o y o elapsed (R/R) ol-
lowing i s -line chemo he apy. A he ime his clinical ial was conduc ed, he s anda d
ea men o R/R DLBCL pa ien s was second-line con en ional chemo he apy ollowed
by consolida ion wi h high-dose chemo he apy in chemosensi i e pa ien s. Pallia i e
chemo he apy is an op ion o pa ien s who a e unable o ecei e high-dose chemo he apy
Cance s 2025,17, 532 3 o 16
o CAR-T he apy; ne e heless, he bes he apeu ic op ion may in ol e en olling he
pa ien in in es iga ional clinical ials.
The iden i ica ion o bioma ke s capable o p edic ing he ou come o DLBCL pa ien s
has been he ocus o inc easing in e es due o he ma ked gene ic and molecula he e o-
genei y ha unde lies disease agg essi eness and umo p og ession. Me abolomics is a
powe ul ool ha can iden i y cance bioma ke s and d i e s o umo igenesis. In he
ield o lymphomas, di e en s udies ha e e alua ed un a ge ed me abolomics using gas
(GC) o liquid ch oma og aphy (LC) coupled wi h mass spec ome y (MS) in pa ien s
wi h lymphoid neoplasms and heal hy popula ions [
1
–
5
]. Al hough mos s udies showed
me abolomic di e ences be ween pa ien s and heal hy subjec s, he iden i ica ion o di -
e en ial me aboli es has been inconsis en . This is likely due o di e ences in labo a o y
echniques (GC-MS and LC-MS) and/o biological samples (blood, u ine, and eces). How-
e e , he me abolomic p o ile in pa ien s wi h DLBCL as a p ognos ic ac o o su i al
has been e alua ed in only wo s udies. S enson e al. [
6
] used nuclea magne ic esonance
(NMR) spec oscopy in 87 pa ien s wi h DLBCL p io o s a ing i s -line ea men wi h
chemoimmuno he apy. S a is ically signi ican di e ences we e ound in he me abolomic
p o ile be ween pa ien s who achie ed a comple e esponse and long su i al and hose
who we e e ac o y o ea men o elapsed. Pa ien s who had been cu ed showed highe
le els o aspa a e, aline, o ni hine, and py oglu ama e, whe eas R/R pa ien s had highe
concen a ions o lysine, a ginine, cada e ine, and 2-hyd oxybu y a e. In a second s udy,
Mi e al. [
7
] used GC-MS o assess p e- ea men se um samples om 80 DLBCL pa ien s
and epo ed ha highe le els o py oglu amic and hexadecenoic acids and lowe le els
o aline we e associa ed wi h signi ican ly highe o e all su i al.
The Spanish G oup o he T ea men and S udy o Lymphomas (GOTEL) conduc ed a
phase II clinical ial o e alua e he combina ion o lenalidomide wi h R-GDP ( i uximab,
gemci abine, dexame hasone, and cispla in) in pa ien s wi h R/R DLBCL who we e ei he
unsui able o high-dose chemo he apy o whose ea men had no wo ked. A o al o
78 pa ien s we e included in he R2-GDP-GOTEL s udy, and a e a median ollow-up o
37 mon hs, 7.9% o pa ien s we e s ill ali e wi hou p og ession a 24 mon hs [
8
]. Taking
ad an age o baseline da a om his clinical ial, he p esen s udy was designed o iden i y
a se um me abolomic p o ile ha may be p edic i e o ou comes in pa ien s wi h R/R
DLBCL ea ed wi h he R2-GDP combina ion.
2. Ma e ials and Me hods
2.1. S udy Design and Pa ien s
The R2-GDP-GOTEL clinical ial was a phase II, mul icen e , open-label, and single-
a m s udy ca ied ou in 78 R/R DLBCL pa ien s who we e ea ed in 18 Spanish hospi als
be ween Ap il 2015 and Sep embe 2018. B ie ly, he R2-GDP egimen included an induc ion
ea men wi h a combina ion o lenalidomide, i uximab, gemci abine, dexame hasone,
and cispla in (R2-GDP), o up o 6 cycles (e e y 3 weeks), ollowed by main enance wi h
lenalidomide o up o 24 mon hs, unless he e was a p og ession, unaccep able oxici y, o
olun a y wi hd awal [
8
]. Addi ional in o ma ion abou he d opou a e (a i ion a e) and
a powe analysis o he R2-GDP ial ha e been p e iously epo ed [
8
]. Since his ial has
a single-a m design, andomiza ion and blinding do no apply. Eligible pa icipan s we e
pa ien s wi h chemo e ac o y o elapsed DLBCL unsui able o high-dose chemo he apy,
wi h an Eas e n Coope a i e Oncology G oup (ECOG) pe o mance s a us o 0–1, and who
had p e iously ecei ed a leas one line o immunochemo he apy, including i uximab.
Pa ien s wi h lep omeningeal o cen al ne ous sys em (CNS) in ol emen , and hose wi h
hema ological, enal, o li e dys unc ion, we e excluded om he ini ial R2-GDP-GOTEL
clinical ial. Since all he lymphomas included in his s udy we e DLBCL, all he included
Cance s 2025,17, 532 4 o 16
pa ien s exp essed CD45 and he pan B-cell ma ke s CD19, CD20, CD22, and CD79a in he
ini ial diagnos ic immunohis ochemical s udy.
The objec i e o he p esen subs udy was o assess he baseline me abolomic p o ile
o R/R DLBCL pa ien s and o iden i y se um me aboli es p edic i e o ou come. Fo
ha pu pose, 69 pa ien s om he R2-GDP-GOTEL clinical ial o whom a su icien
blood sample was a ailable o me abolomic p o iling we e included in he me abolomic
p o iling subs udy.
2.2. Sample P epa a ion and Me abolomic P o iling
A comp ehensi e me hodological app oach o high- h oughpu sc eening by NMR
spec oscopy using a B uke A ance 600 MHz NMR spec ome e (B uke BioSpin,
E lingen, Ge many
) was used o analyze a b oad spec um o me aboli es in se um sam-
ples [
9
]. The analysis included he lipop o ein, glycop o ein, and me aboli e p o iles om
in ac se um, in addi ion o he lipid p o ile om lipid se um ex ac s ob ained by a biphasic
ex ac ion wi h me hyl e -bu yl e he (MTBE). Lipid ex ac s we e d ied and econs i u ed
in 0.01% e ame hylsilane (TMS) solu ion (0.067 mM) and deu e a ed sol en s. All analy-
ses we e ca ied ou a Bios e Teslab (Reus, Ta agona, Spain). In addi ion, 1D Nuclea
O e hause E ec Spec oscopy (NOESY) was used o cha ac e ize small molecules such as
amino acids and small ca bohyd a es, while la ge molecules like lipop o eins and glyco-
p o eins we e de ec ed using LED Di usion (Di ) expe imen s. All he sequences an a
37 ◦C
in quan i a i e condi ions. Samples we e coded o main ain he subjec s’ anonymi y.
The Liposcale
®
es (IVD-CE) was used o de e mine he lipid composi ion, pa icle
size, and concen a ion o majo lipop o ein classes as well as he pa icle concen a ion o
nine subclasses [
10
]. Ci cula ing glycop o eins we e ob ained by decon olu ing he speci ic
NMR spec al egions and quan i ying a eas co ela ing o he concen a ion o he ace yl
g oups o N-ace ylglucosamine and N-ace yl galac osamine (GlycA) and ace yl g oups
o N-ace ylneu aminic acid (GlycB). A Ca –Pu cell–Meiboom–Gill (CPMG) il e on he
1H-NMR spec a was used o p o ile and absolu ely quan i y he me abolomics p o ile. The
BUME p o ocol [
11
] was used o lipid quan i ica ion, based on he Lipspin so wa e [
12
].
Succinc ly, we used lineshape i ing analysis o spec al egions o quan i y he lipids.
2.3. Endpoin s
The p ima y endpoin s we e he cha ac e iza ion o he baseline me abolomic p o ile
in R/R DLBCL pa ien s acco ding o esponse o ea men and ou come. Key seconda y
endpoin s we e o de e mine he p edic i e pe o mance o nomog ams o R/R DLBCL
isk s a i ica ion based on he me aboli es iden i ied and hei op imal cu o s associa ed
wi h he ou come. Tumo esponse was e alua ed acco ding o he In e na ional Wo king
G oup C i e ia [
13
] using compu ed omog aphy (CT) a e he hi d induc ion cycle and
posi on emission omog aphy (PET) in he ollowing 4 weeks a e he las cycle o he
induc ion phase. Ou come included p og ession- ee su i al (PFS) and o e all su i al
(OS). PFS was de ined as he ime be ween he i s dose o he R2-GDP schedule o he
p og ession o disease o dea h, and OS was de ined as he pe iod om he i s dose o he
R2-GDP schedule o dea h om any cause.
2.4. S a is ical Analysis
S a is ical analyses we e pe o med wi h R so wa e using he R S a s Package ( .4.3.2.),
su i al ( .3.5-7), su mine ( .0.4.9), maxs a ( .0.7-25), ca e ( .6.0-94), ms ( .6.7-1), and
su i alROC ( .1.0.3.1). The analyses we e es ic ed o me aboli es iden i ied in >90% o
pa ien s. Me aboli e missing alues we e no impu ed, and me aboli e concen a ions we e
only scaled in mul i a ia e analyses. Fo ep oducibili y, he andom seed was se a 123 ia
he se .seed unc ion (R base). Uni a ia e s a is ics was pe o med using he non-pa ame ic
Cance s 2025,17, 532 5 o 16
Mann–Whi ney–Wilcoxon s a is ical es . Su i al analysis was ca ied ou using he non-
pa ame ic Kaplan–Meie me hod and he log- ank es o he compa ison o su i al
cu es. Cu o s o nume ical a iables we e calcula ed using he maximally selec ed
ank s a is ics, wi h a 10% minimum p opo ion o obse a ions, and missing alues we e
assigned o he lowes g oup. Cox eg ession analyses we e un wice: once o sea ch o
s a is ically signi ican indi idual p ognos ic me aboli es, and again o con i m whe he
hey emained s a is ically signi ican in a mul i a ia e analysis wi h o he s a is ically
signi ican clinical p ognos ic ac o s, such as he In e na ional P ognos ic Index (IPI) [
14
]
and e ac o iness s a us. The haza d a io (HR) and he 95% con idence in e al (CI) we e
calcula ed. S a is ical signi icance was se a p< 0.05, and a alse disco e y a e (FDR)
p alue
adjus men was applied when necessa y. Fold change (FC) analyses we e used o
compa e he absolu e alue o change in he means o each me aboli e be ween esponde s
and non- esponde s.
The nomog am o R/R DLBCL isk s a i ica ion inco po a ed he signi ican me abo-
li es iden i ied in he uni a ia e Cox analysis and he signi ican clinical isk ac o s wi hin
a Cox p opo ional haza ds amewo k. A 70% ain spli was used o model i ing ia
boo s ap calib a ion (B = 1000), while he emaining 30% es spli was used o p edic i e
pe o mance e alua ion wi h a ime-dependen ecei e ope a ing cha ac e is ic cu e
(ROC) and a ea unde he ROC (AUC). The ime poin s selec ed we e 6 mon hs o PFS
and 12 mon hs o OS. Pea son’s p oduc -momen co ela ion coe icien ( ) was used o
assess he ela ionship be ween he me aboli es iden i ied.
3. Resul s
3.1. Baseline Cha ac e is ics o Pa ien s and Su i al
Six y-nine pa icipan s in he R2-GDP-GOTEL clinical ial (35 men and 34 women)
wi h a median age o 70 yea s we e included in he me abolomic p o iling s udy. Key base-
line da a a e displayed in Table 1. Ac i a ed B-cell-like (ABC) lymphomas, elapsed DLBCL,
and IPI low/high-in e media e isk ca ego y (0–3) we e he mos common cha ac e is ics.
Table 1. Key cha ac e is ics o he s udy popula ion.
Va iables 1To al Pa ien s (n= 69)
Sex, n(%)
Men 35 (50.7%)
Women 34 (49.3%)
Age, yea s, median (IQR) 70.1 (61.7–74.5)
Body mass index (BMI), kg/m2, median (IQR) 27.0 (23.9–30.2)
Cell-o -o igin (CoO), n(%) [n= 65]
Ge minal cen e B-cell-like 27 (41%)
Ac i a ed B-cell-like 39 (59%)
Re ac o iness, n(%)
Relapsed 40 (58.0%)
Chemo e ac o y 29 (42.0%)
In e na ional P ognos ic Index (IPI) sco e, n(%)
Low isk (0–1) 12 (17.4%)
Low/High-in e media e isk (2–3) 37 (53.6%)
High isk (4–5) 20 (29.0%)
Response, n(%)
Responde s 41 (59.4%)
Non- esponde s 28 (40.6%)
1IQR: in e qua ile ange (25 h–75 h pe cen ile).
In his subse o pa ien s, he o e all esponse o R2-GPD ea men was 59.4% (
n= 41
)
(comple e esponse 37.7%, pa ial esponse 21.7%), simila o hose p e iously epo ed
in he whole R2-GDP popula ion [
8
]. The indi idual clinical e olu ion o he 69 pa ien s
Cance s 2025,17, 532 6 o 16
is p esen ed in Supplemen a y Figu e S1. A e a median ollow-up o 41 mon hs, he
median PFS was 5 mon hs (36%, 16%, and 7.9% a 6, 12, and 24 mon hs, espec i ely) and
he median OS was 12 mon hs (66%, 47%, and 36% a 6, 12, and 24 mon hs, espec i ely)
(Supplemen a y Figu e S2A,B). Su i al analyses s a i ied by cell-o -o igin (CoO) showed a
median PFS o 6.0 mon hs (95% CI 3.0–11) o he ge minal cen e B-cell-like (GBC) sub ype
and 5.0 mon hs (95% CI 2–6) o ABC (p= 0.099) (Figu e S2C), simila o hose p e iously
epo ed in he whole R2-GDP popula ion [
8
]. The median OS was 16 mon hs (95% CI
6.2—no eached) o GBC and 9.3 mon hs (95% CI 5.3–24) o ABC (p= 0.59) (Figu e S2D).
Conce ning DLBCL s a us, pa ien s wi h chemo e ac o y disease showed a median PFS o
3 mon hs (95% CI 2–6), whe eas hose wi h elapsed disease had a median PFS o 6 mon hs
(95% CI 4–9) (p= 0.083) (Figu e S2E). The OS was signi ican ly longe o he elapsed g oup
(median 24 mon hs, 95% CI 12—no eached) han o he chemo e ac o y g oup (median
6.2 mon hs, 95% CI 4.5–13) (p= 0.0034) (Figu e S2F). Pa ien s in he low/high-in e media e-
isk IPI ca ego y showed a median PFS o 6 mon hs (95% CI 4–9), whils pa ien s in he
high- isk ca ego y had a median PFS o 2 mon hs (95% CI 2–7) (p= 0.0044) (Figu e S2G).
The median OS was also longe o he low/high-in e media e- isk IPI ca ego y (median
15 mon hs, 95% CI 9.3–33) compa ed wi h he high- isk IPI ca ego y (median 3.5 mon hs,
95% CI 1.8—no eached) (p= 0.074) (Figu e S2H).
3.2. T ea men Response Me abolomic P o ile
A o al o 66 me aboli es we e iden i ied, 7 o which we e excluded as hey we e de-
ec ed in less han 90% o he pa ien s. In Supplemen a y Table S1, he mean alues o he
ea men esponse o he 59 included me aboli es a e p esen ed. Twel e me aboli es we e
signi ican ly di e en (p< 0.05) in esponde s s. non- esponde s, including c ea inine,
lac a e, glycop o ein A, ee choles e ol, es e i ied choles e ol, in e media e-densi y lipop o-
ein (IDL) choles e ol, IDL iglyce ides, high-densi y lipop o ein (HDL) iglyce ides,
medium and la ge low-densi y lipop o eins (LDLs), and medium and la ge HDLs. All
me aboli es excep o c ea inine showed signi ican ly lowe mean alues in esponde s o
R2-GPD ea men han in non- esponde s. By con as , c ea inine was signi ican ly highe
in esponde s han in non- esponde s (Table 2). The box plo s o signi ican me aboli e
among esponde s and non- esponde s a e shown in Supplemen a y Figu e S3.
Table 2. Signi ican se um me aboli es in esponde s and non- esponde s.
Me aboli es 1Responde s (n= 41) Non-Responde s (n= 28) pValue Fold Change 2
Low molecula weigh
C ea inine, µM, mean (SD) 73.0 (43.3) 48.7 (17.6) 0.030 1.50
Lac a e, µM, mean (SD) 1174.2 (989.7) 2023.5 (1655.6) 0.034 0.58
Glycop o eins
Glycop o ein A, µmol/L, mean (SD) 846.0 (165.3) 915.5 (161.0) 0.028 0.92
Glycop o ein A, H/W a io, mean (SD) 24.7 (4.3) 27.04 (4.7) 0.024 0.92
Choles e ol
F ee choles e ol, mmol/L, mean (SD) 2.36 (0.71) 2.84 (0.83) 0.016 0.83
Es e i ied choles e ol, mmol/L, mean (SD) 4.76 (1.20) 5.36 (1.40) 0.043 0.89
Lipop o eins
IDL choles e ol, mg/dL, mean (SD) 14.14 (5.84) 17.39 (5.61) 0.015 0.86
IDL iglyce ides, mg/dL, mean (SD) 13.38 (4.59) 15.64 (4.07) 0.018 0.81
LDL iglyce ides, mg/dL, mean (SD) 18.94 (6.40) 22.90 (6.08) 0.005 0.83
Medium LDL-P, nmol/L, mean (SD) 340.83 (134.98) 424.09 (157.02) 0.008 0.80
La ge LDL-P, nmol/L, mean (SD) 180.15 (44.78) 201.32 (51.39) 0.043 0.89
Medium HDL-P, µmol/L, mean (SD) 10.18 (1.68) 11.27 (1.72) 0.006 0.90
La ge HDL-P, µmol/L, mean (SD) 0.30 (0.05) 0.33 (0.05) 0.005 0.90
1
SD: s anda d de ia ion; H/W: heigh /wid h o he NMR peak; IDL: in e media e-densi y lipop o ein;
LDL: low-densi y lipop o ein; HDL: high-densi y lipop o ein; P: pa icle numbe .
2
Fold change
(FC) > 1 indica es
an inc ease in me aboli e concen a ion and hose wi h FC < 1 indica e a dec ease in esponde s s. non- esponde s.
Cance s 2025,17, 532 7 o 16
3.3. Su i al Ou come and Me abolomic P o ile
Two me aboli es, 3OHB and ace one, we e signi ican ly associa ed wi h su i al
ou comes in he Cox uni a ia e eg ession analysis. Highe se um concen a ions o 3OHB
o ace one we e s a is ically signi ican (p< 0.001) p ognos ic ac o s o a wo se PFS (3OHB:
haza d a io [HR] = 7.7; ace one: HR = 1.83) (Figu e 1A) and OS (3OHB: HR = 9.32; ace one:
HR = 1.92) (Figu e 1B). Bo h 3OHB and ace one we e independen p edic o s ha we e
signi ican ly associa ed wi h PFS and OS in he mul i a ia e Cox eg ession model, which
included IPI isk ca ego ies and e ac o iness s a us (R/R) as classical DLBCL clinical
p ognos ic ac o s (Figu e 1C–F). In he o e all s udy popula ion, he mean se um le els o
3OHB we e 120.6
µ
M ( ange 0–1455.9
µ
M) (n= 67) and he mean le els o ace one
40.48 µM
( ange 0–534.2 µM) (n= 68).
Cance s 2025, 17, x FOR PEER REVIEW 8 o 18
Figu e 1. 3-Hyd oxybu y a e and ace one as p ognos ic me aboli es in he Cox uni a ia e analysis
(A,B) and mul i a ia e eg essions models (C–F) o p og ession- ee su i al (PFS) (A,C,E) and
o e all su i al (OS) (B,D,F).
The op imal cu offs o se um 3OHB and ace one we e iden i ied wi h an ou come-
o ien ed me hod (see Me hods) and we e se a 141 µM and 40 µM, espec i ely. Follow-
ing he applica ion o he cu off, he PFS and OS Kaplan–Meie su i al cu es we e sig-
ni ican ly diffe en . Fo se um 3OHB, he median PFS was 5 mon hs (95% CI 3–8) s. 2
mon hs (95% CI 1.0—no eached) (p = 0.044) (Figu e 2A). The co esponding alues o
OS we e 13 mon hs (95% CI 8.9–33) s. 3.2 mon hs (95% 2.0—no eached) (p = 0.0035),
Figu e 1. 3-Hyd oxybu y a e and ace one as p ognos ic me aboli es in he Cox uni a ia e analysis
(A,B) and mul i a ia e eg essions models (C–F) o p og ession- ee su i al (PFS) (A,C,E) and
o e all su i al (OS) (B,D,F).
Cance s 2025,17, 532 8 o 16
The op imal cu o s o se um 3OHB and ace one we e iden i ied wi h an ou come-
o ien ed me hod (see Me hods) and we e se a 141
µ
M and 40
µ
M, espec i ely. Following
he applica ion o he cu o , he PFS and OS Kaplan–Meie su i al cu es we e signi i-
can ly di e en . Fo se um 3OHB, he median PFS was 5 mon hs (95% CI 3–8) s.
2 mon hs
(95% CI 1.0—no eached) (p= 0.044) (Figu e 2A). The co esponding alues o OS we e
13 mon hs
(95% CI 8.9–33) s. 3.2 mon hs (95% 2.0—no eached) (p= 0.0035), espec-
i ely (Figu e 2B). Di e ences in PFS o se um ace one we e 5 mon hs (95% CI 3–9) s.
2 mon hs
(95% CI 1.0—no eached) (p= 0.0054) (Figu e 2C) and 15 mon hs (95% CI 9–33)
s.
2.5 mon hs
(95% CI 1.8—no eached) (p= 0.00014) o OS, espec i ely (Figu e 2D).
Signi ican di e ences we e also obse ed in uni a ia e Cox eg essions ollowing he
applica ion o he cu o . Fo se um 3OHB, he HR o PFS was 2.21, while he HR o
OS was 3.21. Fo se um ace one, he HR o PFS was 2.76 and he HR o OS was 3.71
(Supplemen a y Figu e S4).
Cance s 2025, 17, x FOR PEER REVIEW 9 o 18
espec i ely (Figu e 2B). Diffe ences in PFS o se um ace one we e 5 mon hs (95% CI 3–
9) s. 2 mon hs (95% CI 1.0—no eached) (p = 0.0054) (Figu e 2C) and 15 mon hs (95% CI
9–33) s. 2.5 mon hs (95% CI 1.8—no eached) (p = 0.00014) o OS, espec i ely (Figu e
2D). Signi ican diffe ences we e also obse ed in uni a ia e Cox eg essions ollowing
he applica ion o he cu off. Fo se um 3OHB, he HR o PFS was 2.21, while he HR o
OS was 3.21. Fo se um ace one, he HR o PFS was 2.76 and he HR o OS was 3.71
(Supplemen a y Figu e S4).
Figu e 2. Kaplan–Meie su i al cu es. (A,B), p og ession- ee su i al (PFS) and o e -
all su i al (OS) o he cu off o 3-hyd oxybu y a e (3OHB); (C,D) PFS and OS o he
cu off o ace one.
3.4. Nomog am o R/R DLBCL Risk S a i ica ion
Nomog ams o isk s a i ica ion in pa ien s wi h R/R DLBCL based on se um me-
aboli es 3OHB and ace one showed a high p edic i e pe o mance, sligh ly mo e a o a-
ble o 3OHB, wi h an AUC o 0.856 o 6-mon h PFS (Figu e 3A,B) and 0.844 o 12-mon h
OS (Figu e 3C,D). Se um ace one showed a simila p edic i e pe o mance, wi h an AUC
o 0.840 o 6-mon h PFS (Figu e 3D,E) and 0.830 o 12-mon h OS (Figu e 3F,G). A nom-
og am ha included bo h 3OHB and ace one was also explo ed; howe e , i pe o med
wo se (da a no shown) since bo h me aboli es showed a s a is ically signi ican co ela-
ion ( = 0.76, p < 0.001) (Supplemen a y Figu e S5). Le us conside he OS nomog am o
3OHB o wo pa ien s: Pa ien 1 and Pa ien 2. Al hough bo h pa ien s ha e an IPI sco e
o 4–5 (3 poin s) and a e bo h e ac o y (18 poin s), Pa ien 1 has a 3OHB concen a ion
o 800 µM (50 poin s), whe eas Pa ien 2 has a 3OHB concen a ion o 50 µM (3 poin s).
Hence, o he 3OHB OS nomog am, Pa ien 1 sco ed 71 poin s (<10% su i al p obabili y
a 12 mon hs), while Pa ien 2 sco ed 24 poin s (30% su i al p obabili y a 12 mon hs).
Figu e 2. Kaplan–Meie su i al cu es. (A,B), p og ession- ee su i al (PFS) and o e all su i al
(OS) o he cu o o 3-hyd oxybu y a e (3OHB); (C,D) PFS and OS o he cu o o ace one.
3.4. Nomog am o R/R DLBCL Risk S a i ica ion
Nomog ams o isk s a i ica ion in pa ien s wi h R/R DLBCL based on se um me abo-
li es 3OHB and ace one showed a high p edic i e pe o mance, sligh ly mo e a o able
o 3OHB, wi h an AUC o 0.856 o 6-mon h PFS (Figu e 3A,B) and 0.844 o 12-mon h
OS (Figu e 3C,D). Se um ace one showed a simila p edic i e pe o mance, wi h an AUC
o 0.840 o 6-mon h PFS (Figu e 3D,E) and 0.830 o 12-mon h OS (Figu e 3F,G). A nomo-
g am ha included bo h 3OHB and ace one was also explo ed; howe e , i pe o med
wo se (da a no shown) since bo h me aboli es showed a s a is ically signi ican co ela ion
(
= 0.76
,
p< 0.001
) (Supplemen a y Figu e S5). Le us conside he OS nomog am o 3OHB
o
wo pa ien s
: Pa ien 1 and Pa ien 2. Al hough bo h pa ien s ha e an IPI sco e o 4–5
Cance s 2025,17, 532 9 o 16
(
3 poin s
) and a e bo h e ac o y (18 poin s), Pa ien 1 has a 3OHB concen a ion o
800 µM
(50 poin s), whe eas Pa ien 2 has a 3OHB concen a ion o 50
µ
M (3 poin s). Hence, o he
3OHB OS nomog am, Pa ien 1 sco ed 71 poin s (<10% su i al p obabili y a 12 mon hs),
while Pa ien 2 sco ed 24 poin s (30% su i al p obabili y a 12 mon hs).
Cance s 2025, 17, x FOR PEER REVIEW 10 o 18
Figu e 3. Nomog ams o R/R DLBCL isk s a i ica ion based on single me aboli es and
hei espec i e ime-dependen ROCs. (A,B) 3-Hyd oxybu y a e (3OHB) nomog am o
p og ession- ee su i al (PFS) and i s ROC; (C,D) 3OHB nomog am o o e all su i al
(OS) and i s ROC; (E,F) ace one nomog am o PFS and i s ROC; (G,H) ace one nomo-
g am o OS and i s ROC.
4. Discussion
The p esen da a con ibu e o de ining he me abolomic p o ile linked o R/R-
DLBCL pa ien s’ esponsi eness o ea men and su i al. To he bes o ou knowledge,
no p e ious me abolomics s udy has been epo ed in his se ing. T ea men esponden s
Figu e 3. Nomog ams o R/R DLBCL isk s a i ica ion based on single me aboli es and hei
espec i e ime-dependen ROCs. (A,B) 3-Hyd oxybu y a e (3OHB) nomog am o p og ession-
ee su i al (PFS) and i s ROC; (C,D) 3OHB nomog am o o e all su i al (OS) and i s ROC;
(E,F) ace one nomog am o PFS and i s ROC; (G,H) ace one nomog am o OS and i s ROC.
Cance s 2025,17, 532 16 o 16
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Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .
