Co esponding au ho : Andoh Sheikh A a-ullah
Copy igh © 2025 Au ho (s) e ain he copy igh o his a icle. This a icle is published unde he e ms o he C ea i e Commons A ibu ion Liscense 4.0.
Role o mu a ional signa u es and clonali y assessmen s in ailo ing a ge ed
he apies o lymphoma
Andoh Sheikh A a-ullah 1, *, Uche Philip 2 and Sampson Janice Candy 3
1 Depa men o Hema ology, Fujian P o incial Cance Hospi al a ilia ed o Fujian Medical Uni e si y, Fujian Medical
Uni e si y, China.
2 Depa men o Hema ology and Oncology, Union Hospi al a ilia ed o Fujian Medical Uni e si y, China.
3 Depa men o In e nal Medicine, Fi s A ilia ed Hospi al o Fujian Medical Uni e si y, Fujian Medical Uni e si y, China.
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 26(02), 2847-2864
Publica ion his o y: Recei ed on 30 Ma ch 2025; e ised on 14 May 2025; accep ed on 17 May 2025
A icle DOI: h ps://doi.o g/10.30574/wja .2025.26.2.1936
Abs ac
Lymphomas a e highly he e ogeneous malignancies cha ac e ized by di e se gene ic, epigene ic, and pheno ypic
p o iles, which complica e diagnosis, p ognosis, and ea men . T adi ional bulk sequencing me hods mask he
complexi y o in a umo al a ia ion, o en o e looking a e subclones ha may d i e disease p og ession and
he apeu ic esis ance. Single-cell genomics has eme ged as a ans o ma i e app oach o deciphe umo he e ogenei y
a unp eceden ed esolu ion. This echnology enables he dissec ion o indi idual cellula popula ions wi hin
lymphomas, o e ing insigh s in o clonal e olu ion, ansc ip ional di e si y, and mic oen i onmen al in e ac ions. By
applying single-cell RNA sequencing (scRNA-seq), ch oma in accessibili y assays (scATAC-seq), and single-cell DNA
sequencing (scDNA-seq), esea che s can un a el lineage ela ionships, iden i y esis an subpopula ions, and ack
dynamic changes in esponse o he apy. In lymphomas such as di use la ge B-cell lymphoma (DLBCL), ollicula
lymphoma (FL), and man le cell lymphoma (MCL), single-cell app oaches ha e e ealed dis inc malignan and non-
malignan cell s a es ha co ela e wi h ea men ou comes. Mo eo e , in eg a ing single-cell da a wi h spa ial
ansc ip omics and immune p o iling enhances he unde s anding o he umo mic oen i onmen , including immune
e asion mechanisms. These insigh s can in o m pe sonalized ea men s a egies, iden i y no el he apeu ic a ge s,
and enable ea ly de ec ion o elapse. Despi e echnical challenges such as da a complexi y, sample iabili y, and cos ,
he applica ion o single-cell genomics in lymphoma esea ch is apidly ad ancing. Fu u e di ec ions include mul i-
omics in eg a ion, eal- ime pa ien moni o ing, and clinical ansla ion o p edic i e bioma ke s. This e iew
unde sco es he pi o al ole o single-cell genomics in esol ing umo he e ogenei y and p edic ing he apeu ic
esis ance, posi ioning i as a co ne s one o nex -gene a ion p ecision oncology in lymphomas.
Keywo ds: Single-cell genomics; Tumo he e ogenei y; The apeu ic esis ance; Lymphoma; P ecision
oncology; Clonal e olu ion
1. In oduc ion
1.1. Backg ound on Lymphoma Sub ypes (Hodgkin s. Non-Hodgkin)
Lymphomas ep esen a di e se g oup o hema ologic malignancies o igina ing om lymphoid cells, p ima ily a ec ing
he lymph nodes and ela ed immune issues. These cance s a e b oadly ca ego ized in o Hodgkin lymphoma (HL) and
non-Hodgkin lymphoma (NHL), each demons a ing dis inc his opa hological and molecula cha ac e is ics. Hodgkin
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lymphoma, ma ked by he p esence o Reed-S e nbe g cells, accoun s o app oxima ely 10% o all lymphoma cases and
is mo e p e alen in younge adul s [1]. In con as , non-Hodgkin lymphoma includes a wide a ay o sub ypes such as
di use la ge B-cell lymphoma (DLBCL), ollicula lymphoma (FL), man le cell lymphoma (MCL), and Bu ki lymphoma
(BL), wi h DLBCL being he mos common [2].
NHLs a y g ea ly in e ms o clinical agg essi eness, ea men esponse, and p ognosis. Indolen o ms, such as FL,
end o ollow a slow cou se bu a e o en incu able, whe eas agg essi e sub ypes like BL may p og ess apidly ye a e
mo e amenable o cu a i e he apy [3]. The clinical he e ogenei y o NHL unde sco es he necessi y o nuanced
classi ica ion sys ems ha go beyond mo phology and immunopheno yping..
1.2. Limi a ions o Con en ional The apy
Despi e ad ances in immunochemo he apy egimens such as R-CHOP ( i uximab wi h cyclophosphamide, doxo ubicin,
inc is ine, and p ednisone), he long- e m ou comes o many pa ien s wi h agg essi e o elapsed lymphomas emain
subop imal [4]. Resis ance o i s -line he apy and disease elapse a e equen challenges, pa icula ly in cases
in ol ing high-g ade ans o ma ion o e ac o y disease biology. Fu he mo e, con en ional he apies a e associa ed
wi h conside able oxici y, and hei e ec i eness is o en comp omised by he molecula di e si y wi hin and be ween
umo s [5].
Ano he limi a ion lies in he lack o pe sonalized ea men app oaches. Mos clinical p o ocols ely on a s anda dized
app oach, ailing o conside he gene ic and clonal landscape o indi idual umo s. This one-size- i s-all s a egy
o e looks biologically dis inc disease subg oups ha may espond di e en ly o he apy, ul ima ely limi ing he
e icacy o sys emic ea men s [6].
1.3. Rise o P ecision Oncology in Hema ologic Malignancies
The ad en o p ecision oncology has e olu ionized he ea men landscape o solid umo s and is inc easingly
gaining momen um in hema ologic malignancies. P ecision oncology is cha ac e ized by he in eg a ion o genomic,
ansc ip omic, and p o eomic da a o guide indi idualized he apeu ic s a egies. In lymphomas, genomic p o iling has
enabled he iden i ica ion o ecu en mu a ions, ch omosomal abe a ions, and pa hway dys egula ions, pa ing he
way o mo e a ge ed and a ional ea men designs [7].
Recen de elopmen s in high- h oughpu sequencing echnologies ha e u he acili a ed he cha ac e iza ion o
lymphoid malignancies a unp eceden ed esolu ion. Fo ins ance, la ge-scale e o s such as he Lymphoma/Leukemia
Molecula P o iling P ojec (LLMPP) ha e unco e ed no el bioma ke s and s a i ica ion ools ha ha e al eady
in luenced clinical p ac ice [8]. These ini ia i es highligh he easibili y o using molecula signa u es o classi y disease
sub ypes mo e accu a ely and p edic he apy esponse.
Mo eo e , p ecision oncology o e s he po en ial o iden i y ac ionable mu a ions and assess disease dynamics o e
ime. Unlike s a ic diagnos ic ools, eal- ime genomic su eillance can guide adap i e he apeu ic in e en ions,
pa icula ly in diseases cha ac e ized by clonal e olu ion and esis ance mechanisms [9].
1.4. De ine Mu a ional Signa u es and Clonali y: Rele ance o Lymphoma
Mu a ional signa u es e e o cha ac e is ic pa e ns o soma ic mu a ions ha e lec speci ic DNA damage and epai
p ocesses. These pa e ns, iden i iable h ough s a is ical modeling o genomic da a, can e eal insigh s in o he
biological his o y o a umo . Signa u e analysis has been ex ensi ely applied in solid umo s and is now inc easingly
employed in lymphomas o un a el he mu a ional p ocesses ha d i e pa hogenesis [10].
Fo example, ac i a ion-induced cy idine deaminase (AID) and APOBEC enzyme ac i i y a e known con ibu o s o he
mu a ional bu den in B-cell lymphomas. These signa u es no only help di e en ia e sub ypes bu may also ca y
p ognos ic and he apeu ic implica ions [11]. Some signa u es ha e been associa ed wi h inc eased umo
agg essi eness o esis ance o s anda d ea men s, making hem aluable ools o isk s a i ica ion and he apy
selec ion.
Clonali y, on he o he hand, pe ains o he gene ic ela edness o umo cell popula ions. Clonal assessmen s aim o
de e mine whe he a umo is de i ed om a single p ogeni o cell o comp ises mul iple e ol ing subclones. In
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lymphomas, clonali y is especially ele an gi en he hie a chical s uc u e o B-cell de elopmen and he equen
occu ence o soma ic hype mu a ion and class-swi ch ecombina ion [12].
Assessing clonali y helps iden i y dominan clones, ack subclonal dynamics o e ime, and e alua e he e ec s o
he apy on umo composi ion. Impo an ly, unde s anding clonal e olu ion can elucida e mechanisms o d ug
esis ance and in o m he iming o ea men escala ion o de-escala ion [13]. Fo ins ance, he expansion o mino
subclones ha bo ing esis ance mu a ions du ing he apy o en p ecedes clinical elapse and could be a ge ed
p eemp i ely in a pe sonalized ea men plan.
1.5. Pu pose and Scope o he A icle
Gi en he limi a ions o con en ional app oaches and he p omise o p ecision medicine, his a icle aims o explo e he
ole o mu a ional signa u es and clonali y assessmen s in ailo ing a ge ed he apies o lymphoma. I seeks o
syn hesize cu en knowledge on he genomic unde pinnings o lymphoid malignancies and demons a e how hese
molecula ea u es can guide mo e e ec i e and pe sonalized in e en ions.
The subsequen sec ions will discuss he de ec ion and in e p e a ion o mu a ional signa u es, highligh hei
p e alence in a ious lymphoma sub ypes, and examine he ools used o assess clonal a chi ec u e. Addi ionally, he
a icle will explo e how in eg a ing hese da a poin s suppo s clinical decision-making and enhances ou comes h ough
mo e p ecise he apeu ic a ge ing [14].
We will also add ess p ac ical challenges, including da a in eg a ion, assay s anda diza ion, and clinical implemen a ion
ba ie s. Real-wo ld case s udies and eme ging echnologies will be used o illus a e he ansla ional impac o hese
app oaches in mode n lymphoma ca e. Ul ima ely, his a icle ad oca es o a pa adigm shi —mo ing om gene ic
p o ocols o genomically guided, clonali y-in o med he apy s a egies ha e lec he ue complexi y o lymphoid
cance s [15].
2. Unde s anding mu a ional signa u es in lymphoma
2.1. De ini ion and Biological Basis
Mu a ional signa u es a e cha ac e is ic pa e ns o soma ic mu a ions wi hin he cance genome, e lec ing he ac i i y
o unde lying mu a ional p ocesses. These p ocesses may be endogenous, such as spon aneous deamina ion, eplica ion
e o s, o he ac i i y o enzymes like APOBEC o AID, o exogenous, such as exposu e o ul a iole adia ion, obacco
smoke, o chemo he apeu ic agen s [16]. Each mu a ional p ocess lea es a dis inc i e “ inge p in ” in he DNA, which
can be ma hema ically decomposed and classi ied in o speci ic signa u es.
The mos commonly used amewo k o ca ego izing hese pa e ns is p o ided by he Ca alogue O Soma ic Mu a ions
In Cance (COSMIC), which g oups hem in o single base subs i u ions (SBS), double base subs i u ions (DBS), and
inse ions and dele ions (indels) [17]. SBS signa u es, he mos ex ensi ely s udied, desc ibe poin mu a ions occu ing
a single nucleo ide si es, ypically wi hin speci ic inucleo ide con ex s. Fo ins ance, SBS1 a ises om spon aneous
deamina ion o me hyla ed cy osine, while SBS2 and SBS13 a e a ibu ed o APOBEC ac i i y [18].
Double base subs i u ions a e ela i ely a e bu p o ide dis inc insigh s in o DNA damage caused by mu agens o
eac i e oxygen species. Indel signa u es, meanwhile, e lec DNA epai pa hway de ec s, including misma ch epai
(MMR) o homologous ecombina ion de iciencies. No ably, he in e p e a ion o hese signa u es equi es s a is ical
modeling using compu a ional echniques such as non-nega i e ma ix ac o iza ion (NMF), which decon ol es
mu a ion ca alogs in o dis inc , biologically ele an componen s [19].
In lymphomas, hese signa u es se e as bo h e iological ma ke s and p edic i e ools, enabling a deepe unde s anding
o umo de elopmen , p og ession, and he apeu ic ulne abili y [20]. As mu a ional signa u e da abases g ow, hei
applica ion in lymphoid malignancies is expec ed o become inc easingly obus and clinically ac ionable.
2.2. Me hods o De ec ion and Analysis
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Iden i ying mu a ional signa u es in lymphoma equi es high- esolu ion genomic sequencing da a. Whole-genome
sequencing (WGS) emains he gold s anda d o comp ehensi e signa u e analysis, as i cap u es bo h coding and non-
coding egions, allowing o he de ec ion o mu a ion pa e ns ac oss he en i e genome [21]. Howe e , he high cos
and compu a ional demand o WGS limi i s widesp ead clinical use. Whole-exome sequencing (WES), which ocuses on
p o ein-coding egions, is a mo e accessible al e na i e bu p o ides a na owe iew o mu a ional ac i i y, po en ially
o e looking c i ical non-coding signa u es [22].
Following sequencing, mu a ion calls a e anno a ed using bioin o ma ics pipelines such as Mu ec 2, Va Scan, o S elka.
These calls a e hen agg ega ed in o inucleo ide o o he con ex ual ma ices o quan i y he con ibu ion o a ious
mu a ional p ocesses. Non-nega i e ma ix ac o iza ion (NMF) and hie a chical Bayesian models a e ypically applied
o decon olu e hese da a in o disc e e signa u es ha can be ma ched agains he COSMIC e e ence da abase [23].
To s anda dize his p ocess, compu a ional ools such as SigP o ile , decons uc Sigs, and Mu a ionalPa e ns ha e been
de eloped, allowing esea che s and clinicians o inpu a ian da a and ecei e signa u e p o iles in e u n. These ools
acili a e ep oducibili y and imp o e he compa abili y o s udies ac oss di e en pla o ms and labo a o ies [24].
Ne e heless, he e a e se e al limi a ions o cu en signa u e de ec ion wo k lows. Fi s , sample pu i y and
sequencing dep h can signi ican ly a ec he accu acy o mu a ion calls and he de ec ion o low- equency signa u es.
Second, echnical a i ac s in oduced du ing lib a y p epa a ion o sequencing can con ound genuine biological signals.
Las ly, in e -sample a iabili y and umo he e ogenei y necessi a e la ge coho s and ca e ul s a is ical modeling o
a oid misclassi ica ion [25].
S anda diza ion e o s a e unde way o add ess hese challenges. The Pan-Cance Analysis o Whole Genomes
(PCAWG) conso ium, o example, has p oposed benchma ks and quali y con ol me ics o signa u e a ibu ion,
enhancing he obus ness o signa u e-based diagnos ics [26]. Mo eo e , mul i-ins i u ional e o s a e beginning o
inco po a e hese ools in o ansla ional and clinical wo k lows, especially in ials in es iga ing genomically guided
he apy selec ion.
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Figu e 1 Wo k low diag am illus a ing he p ocess o mu a ional signa u e analysis in lymphoma, om sample
p epa a ion and sequencing o compu a ional decon olu ion and clinical in e p e a ion
As he ield ad ances, in eg a ing mu a ional signa u e analysis wi h eal- ime sequencing and decision-suppo
sys ems may become ou ine in managing agg essi e lymphomas o acking he apy esponse, pa icula ly whe e
esis ance is media ed by speci ic mu agenic p ocesses.
2.3. Cha ac e ized Mu a ional Signa u es in Speci ic Lymphoma Sub ypes
While mos esea ch on mu a ional signa u es has ocused on solid umo s, a g owing body o wo k is now
cha ac e izing speci ic signa u es ac oss di e en lymphoma sub ypes. These include DLBCL, FL, MCL, and BL, each o
which exhibi s unique mu a ional p o iles ha can in o m diagnosis, p ognosis, and ea men s a egy [27].
In di use la ge B-cell lymphoma (DLBCL), one o he mos p e alen and agg essi e sub ypes, signa u es associa ed
wi h ac i a ion-induced cy idine deaminase (AID) and APOBEC enzyme ac i i y a e equen ly obse ed. AID, c i ical
o soma ic hype mu a ion and class-swi ch ecombina ion in ge minal cen e B cells, is implica ed in o - a ge
mu a ions leading o SBS9 and SBS84 signa u es [28]. These mu a ions ypically a ec oncogenes such as MYC, BCL6,
o PIM1 and may d i e ea ly lymphomagenesis.
APOBEC- ela ed signa u es (e.g., SBS2 and SBS13) a e also de ec ed in a subse o DLBCL and a e associa ed wi h a
hype mu a ed pheno ype ha may in luence esponse o immuno he apy o PI3K inhibi o s [29]. In ollicula
lymphoma (FL), AID- ela ed mu agenesis p edomina es, e lec ing i s ge minal cen e o igin. FLs also exhibi high
clonal di e si y and equen mu a ions in epigene ic egula o s such as EZH2, CREBBP, and KMT2D, many o which a e
shaped by ecu ing mu a ional p ocesses [30].
Man le cell lymphoma (MCL) o en demons a es less he e ogenei y in e ms o mu a ional signa u es bu may ha bo
indel pa e ns e lec i e o DNA epai de iciency, especially in blas oid a ian s. These indel signa u es can po end
poo p ognosis and esis ance o con en ional egimens [31].
In Bu ki lymphoma (BL), cha ac e ized by MYC ansloca ions and a highly p oli e a i e pheno ype, unique mu a ional
signa u es ha e been linked o misma ch epai de iciency (MMRd) and ul a iole ligh exposu e in ce ain endemic
cases. SBS6, SBS15, and SBS44, o en associa ed wi h MMRd, con ibu e o he ele a ed mu a ion bu den obse ed in a
subse o BL umo s, especially hose occu ing in immunocomp omised indi iduals [32].
F om a clinical s andpoin , he p esence o speci ic mu a ional signa u es can aid in di e en ial diagnosis, especially in
ambiguous his ologies. Mo eo e , signa u e p o iling may assis in p edic ing he apy esponse o iden i ying
he apeu ic ulne abili ies. Fo ins ance, umo s wi h misma ch epai -de icien signa u es may bene i om immune
checkpoin blockade, while APOBEC- ich lymphomas may equi e no el agen s ha mi iga e DNA hype mu a ion [33].
The clinical in eg a ion o mu a ional signa u es emains an e ol ing ield, bu ea ly e idence sugges s ha hese
pa e ns can s a i y pa ien s beyond con en ional genomic bioma ke s. As such, signa u e-guided classi ica ion could
become an indispensable componen o lymphoma p ecision medicine, pa icula ly when combined wi h clonal
p o iling and eal- ime moni o ing.
3. Clonali y and clonal e olu ion in lymphoma
3.1. Concep s o Clonali y and In a umo al He e ogenei y
He e ogenei y The concep o clonali y in cance desc ibes he gene ic lineage o umo cells ha o igina e om a
common ances al cell, known as he ounde clone. In lymphomas, he ounde clone ypically a ises om a single B-
o T-cell p ecu so ha has unde gone malignan ans o ma ion [34]. Howe e , due o ongoing gene ic ins abili y and
selec i e p essu es wi hin he umo mic oen i onmen , his ounde popula ion o en gi es ise o subclones —
gene ically dis inc o shoo s ha may a y in hei p oli e a i e capaci ies, esis ance p o iles, and me as a ic po en ial.
These subclonal popula ions o m he basis o in a umo al he e ogenei y, a hallma k o cance e olu ion ha
complica es bo h diagnosis and ea men . Subclones may ei he emain mino o expand h ough clonal sweeps, whe e
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selec i e ad an ages—such as esis ance o he apy o enhanced p oli e a ion—allow ce ain clones o domina e he
umo popula ion [35].
An impo an ela ed concep is clonal selec ion, whe eby ex e nal o ces like chemo he apy o immune su eillance
p e e en ially elimina e sensi i e clones while spa ing o en iching esis an ones. This Da winian p ocess d i es
disease p og ession and elapse in lymphoma pa ien s, especially in agg essi e sub ypes like di use la ge B-cell
lymphoma (DLBCL) o man le cell lymphoma (MCL) [36].
The deg ee o he e ogenei y and clonal a chi ec u e has been di ec ly associa ed wi h disease agg essi eness and
p ognosis. Tumo s wi h high clonal di e si y end o be mo e adap able and less esponsi e o mono he apy. Fo
ins ance, a high clonal bu den a diagnosis co ela es wi h sho e p og ession- ee su i al in ollicula lymphoma (FL)
and poo e ou comes in elapsed/ e ac o y DLBCL [37].
Unde s anding hese e olu iona y dynamics is c i ical o p ecision oncology. Accu a e assessmen o clonal s uc u es
enables oncologis s o ack disease p og ession, p edic ea men esponse, and ailo he apeu ic s a egies ha
add ess bo h dominan and eme ging esis an subclones [38].
3.2. Technological Ad ances in Clonali y Assessmen s
The las decade has seen signi ican ad ances in echnologies used o assess clonali y in hema ologic malignancies.
T adi ionally, clonali y was in e ed om bulk sequencing app oaches, which analyze DNA o RNA om a pool o cells
and p o ide an a e aged iew o mu a ional landscapes. While in o ma i e, bulk me hods canno dis inguish be ween
indi idual clones, especially when subclones a e p esen a low equencies [39].
To o e come his limi a ion, single-cell sequencing has eme ged as a ans o ma i e ool in lymphoma esea ch. I
enables he di ec analysis o indi idual umo cells, e ealing no only hei mu a ional p o iles bu also ansc ip omic
s a es, lineage ajec o ies, and epigene ic modi ica ions. This echnology has unco e ed ex ensi e clonal di e si y e en
wi hin mo phologically homogeneous umo s [40]. Howe e , i s clinical adop ion emains cons ained by cos , echnical
complexi y, and da a in e p e a ion challenges.
Ano he powe ul ool is digi al PCR (dPCR), which p o ides highly sensi i e quan i ica ion o a e clonal a ian s. dPCR
is pa icula ly use ul o de ec ing minimal esidual disease (MRD), whe e adi ional me hods may lack he sensi i i y
o iden i y esidual malignan clones pos - ea men . MRD moni o ing ia dPCR has become a alida ed p ognos ic ool
in mul iple lymphoma sub ypes, guiding he apy du a ion and in ensi ica ion decisions [41].
In B-cell lymphomas, clonali y is also assessed by examining B-cell ecep o (BCR) gene ea angemen s. Du ing no mal
lymphocy e de elopmen , unique ecombina ions o V(DJ) gene segmen s occu in he immunoglobulin hea y chain
(IGH), gene a ing a di e se BCR epe oi e. The de ec ion o a dominan , monoclonal IGH ea angemen indica es a
clonal expansion, while he p esence o mul iple ea angemen s may sugges biclonal disease o subclonal
di e si ica ion [42].
Simila ly, T-cell ecep o (TCR) ea angemen s a e used o e alua e clonali y in T-cell lymphomas. High- h oughpu
sequencing pla o ms such as Adap i e Bio echnologies' ImmunoSEQ o A che Dx's Va ian Plex enable deep
in e oga ion o immune ecep o epe oi es, suppo ing bo h diagnos ic and MRD applica ions [43].
Despi e hei u ili y, clonali y assessmen echniques a y in sensi i i y, esolu ion, and clinical applicabili y. The
ollowing able summa izes key compa a i e ea u es:
Table 1 Compa ison o Techniques o Clonali y De ec ion in Lymphoma
Me hod
Resolu ion
Sensi i i y
Clinical U ili y
Bulk DNA Sequencing
Low
Mode a e
S anda d o mu a ion calling
Single-Cell Sequencing
High
High
Resea ch-g ade; no ye ou ine clinically
Digi al PCR (dPCR)
Mode a e
Ve y High
MRD de ec ion and he apy moni o ing
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BCR/TCR Rea angemen PCR
High (clone-speci ic)
High
Diagnosis, MRD, and clonali y con i ma ion
S anda dizing hese ools o clinical p ac ice emains a p io i y, especially as nex -gene a ion sequencing (NGS)
becomes in eg a ed in o lymphoma diagnos ic algo i hms [44].
3.3. Clonal Dynamics in The apy Response and Relapse
The apy exe s powe ul selec i e p essu e on umo popula ions, o en al e ing hei clonal a chi ec u e. This
phenomenon, known as clonal e olu ion, plays a cen al ole in he de elopmen o esis ance and elapse in lymphoma.
A diagnosis, a umo may appea o be domina ed by a single clone, bu he apy can lead o he expansion o p e iously
mino subclones ha ha bo su i al ad an ages [45].
A classic example is he eme gence o chemo- esis an subclones ollowing R-CHOP he apy in DLBCL. While ini ial
esponse a es a e high, a signi ican p opo ion o pa ien s elapse wi h umo s ha possess new mu a ions in genes
associa ed wi h d ug esis ance, such as TP53 o BCL2. These gene ic al e a ions a e o en absen o subclonal a
baseline, highligh ing he dynamic na u e o clonal selec ion [46].
Longi udinal moni o ing o clonal composi ion can p o ide ea ly wa nings o he apeu ic ailu e. In ollicula lymphoma,
s udies ha e shown ha he appea ance o a dominan EZH2-mu an clone du ing he apy co ela es wi h poo
p ognosis and sugges s ea ly clonal di e gence [47]. In man le cell lymphoma, high clonal complexi y pos -induc ion
he apy has been associa ed wi h apid p og ession and limi ed bene i om main enance egimens.
Impo an ly, no all esis an clones a ise de no o. Some a e he apy-induced as a esul o ea men -associa ed
mu agenesis. Fo example, cy o oxic d ugs can in oduce new mu a ions ha con e i ness ad an ages o p e iously
quiescen clones, leading o he apy-d i en e olu ion a he han simple selec ion om p e-exis ing popula ions [48].
The in eg a ion o se ial sequencing, bo h a he bulk and single-cell le el, enables esea che s and clinicians o map he
ajec o y o clonal changes o e ime. This includes iden i ying when a esis an subclone i s eme ges, how apidly i
expands, and how i esponds o second-line he apy.
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2854
Figu e 2 Longi udinal clonal e olu ion in lymphoma showing clonal dynamics a diagnosis, du ing he apy, and pos -
elapse, wi h mu a ion acking ac oss ime poin s
These insigh s in o m adap i e he apeu ic s a egies. Ins ead o wai ing o elapse, oncologis s may in e ene
p oac i ely based on clonal ends—escala ing he apy, swi ching agen s, o adding no el ea men s ha a ge
esis an clones [49].
Mo eo e , unde s anding he clonal basis o esis ance opens he doo o a ional d ug combina ions designed o a ge
bo h dominan and eme gen clones simul aneously. This could educe he isk o elapse and imp o e long- e m
disease con ol. Fo example, combining BCL2 inhibi o s wi h immune checkpoin blockade has shown p omise in
a ge ing mul iple clonal compa men s in elapsed FL and DLBCL [50].
Ul ima ely, inco po a ing clonal assessmen s in o s anda d ca e can pe sonalize ea men ac oss all s ages o
lymphoma— om ini ial diagnosis o sal age he apy—ma king a key ad ancemen in p ecision hema ology.
4. In eg a ion o mu a ional signa u es and clonali y in p ecision oncology
4.1. Linking Genomic P o iles o Ta ge able Mu a ions
Genomic p o iling in lymphoma has enabled he iden i ica ion o ac ionable d i e mu a ions ha con ibu e o
oncogenesis and umo main enance. These include al e a ions in genes in ol ed in B-cell ecep o signaling (e.g.,
CARD11, CD79B), apop osis egula ion (BCL2, TP53), and ch oma in emodeling (EZH2, CREBBP, KMT2D) [51]. Such
d i e mu a ions o en eme ge om selec i e e olu iona y p essu es and ep esen a ac i e he apeu ic a ge s o
pa hway-di ec ed agen s.
In eg a ing mu a ional signa u es enhances he in e p e i e powe o genomic p o iling by connec ing mu a ion
pa e ns o speci ic biological mechanisms. Fo example, umo s exhibi ing AID- ela ed mu a ional signa u es o en
ha bo ea angemen s o mu a ions in MYC, BCL6, and o he genes ha d i e ge minal cen e -de i ed lymphomas
[52]. Simila ly, he p esence o APOBEC signa u es may poin o genomic ins abili y, ende ing cells mo e suscep ible
o syn he ic le hal s a egies a ge ing DNA epai pa hways.
The p inciple o syn he ic le hali y—whe e co-occu ing gene dis up ions lead o cell dea h—o e s a compelling
s a egy o a ge ing umo s wi h de ined mu a ional con ex s. Fo ins ance, lymphomas wi h mu a ions in DNA
damage esponse genes (e.g., ATM, CHEK2) may be ulne able o PARP inhibi o s [53]. The combina ion o signa u e
analysis and mu a ion p o iling can hus pinpoin c i ical ulne abili ies no e iden h ough adi ional sequencing
alone.
Fu he mo e, hese signa u es o en illumina e pa hway dependencies, allowing o a ional d ug de elopmen and
epu posing. Tumo s wi h MMR-de icien signa u es (e.g., SBS6, SBS15) exhibi heigh ened sensi i i y o immune
checkpoin blockade due o inc eased neoan igen bu den [54]. In eg a ing his in o ma ion wi h clonali y da a allows
clinicians o design he apies ha selec i ely dis up he e olu iona y co e o he disease, p e en ing ecu ence and
clonal escape.
Ul ima ely, linking genomic p o iles o ac ionable mu a ions ia mu a ional signa u e in e p e a ion ans o ms s a ic
sequencing da a in o dynamic, clinically ac ionable insigh s, enabling a ge ed in e en ions ailo ed o indi idual umo
biology [55].
4.2. P edic i e Value o Signa u es o T ea men Sensi i i y
Mu a ional signa u es se e no only as e iological clues bu also as p edic i e bioma ke s o ea men sensi i i y.
Ce ain signa u es co ela e s ongly wi h he apeu ic esponse, p o iding a amewo k o e ining pa ien
s a i ica ion and guiding he selec ion o p ecision he apies.
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2855
A p ominen example is misma ch epai (MMR) de iciency, o en e lec ed by mu a ional signa u es such as SBS6,
SBS15, and SBS26. These umo s exhibi high umo mu a ion bu den (TMB), a condi ion ha enhances he gene a ion
o neoan igens and subsequen ly ende s he malignancy mo e esponsi e o immune checkpoin inhibi o s (ICIs) like
an i-PD-1 o an i-CTLA-4 he apies [56]. Al hough p ima ily s udied in solid umo s, MMR de iciency has also been
obse ed in subse s o agg essi e lymphomas, including p ima y medias inal B-cell lymphoma and Rich e ’s
ans o ma ion [57].
Ano he signa u e o he apeu ic ele ance is APOBEC-associa ed hype mu a ion, ypically ep esen ed by SBS2 and
SBS13. These mu a ions gene a e localized clus e s o cy osine- o- hymine subs i u ions, con ibu ing o inc eased
mu a ional bu den and genomic ins abili y. In lymphoma, APOBEC ac i i y is equen ly associa ed wi h esis ance o
con en ional chemo he apy bu pa adoxically may con e sensi i i y o PI3K inhibi o s due o pa hway ewi ing [58].
Eme ging da a sugges ha APOBEC-en iched umo s a e mo e likely o ha bo PIK3CA, PTEN, and AKT1 mu a ions—
al e a ions ha make he PI3K/AKT/mTOR axis an ac ionable he apeu ic a ge . Thus, mu a ional signa u es p o ide
a laye o con ex ha enhances he p edic abili y o d ug esponse beyond simple mu a ion p esence o absence [59].
Impo an ly, signa u es also in o m esis ance p edic ion. In DLBCL, o ins ance, SBS17—a pa e n linked o oxida i e
s ess—has been associa ed wi h ea ly elapse a e R-CHOP he apy, sugges ing an agg essi e pheno ype and po en ial
esis ance o an h acyclines [60]. Iden i ying such pa e ns allows o ea ly he apeu ic escala ion o inclusion in clinical
ials explo ing no el agen s.
The u ili y o mu a ional signa u es in p edic ing he apeu ic e icacy is apidly gaining ac ion and is now being
inco po a ed in o bioma ke panels alongside adi ional genomic ma ke s, immune cell in il a ion me ics, and MRD
s a us [61]. These in eg a i e app oaches o e a holis ic iew o umo beha io , enhancing pe sonalized ea men
s a egies and imp o ing ou comes in pa ien s wi h e ac o y o high- isk lymphoma.
4.3. Clonali y-Guided Risk S a i ica ion and The apy Design
The e ol ing unde s anding o umo clonali y has shi ed he pa adigm o he apy design om a ge ing a uni o m
disease en i y o add essing a he e ogeneous popula ion o e ol ing clones. S a i ying pa ien s based on clonal
a chi ec u e allows oncologis s o an icipa e disease ajec o y and op imize ea men selec ion.
One o he mos signi ican ad an ages o clonali y analysis is i s abili y o di e en ia e dominan om eme ging
subclones. Dominan clones, o en esponsible o bulk disease a p esen a ion, may be e ec i ely a ge ed by i s -line
he apies. Howe e , mino subclones— equen ly o e looked in bulk analyses—can ha bo mu a ions con e ing
esis ance, such as TP53 loss o MYD88 mu a ions, and expand unde ea men p essu e [62].
The apies designed o elimina e dominan clones while supp essing he ou g ow h o mino subclones a e mo e likely
o yield du able emissions. Fo example, he use o ene oclax, a BCL2 inhibi o , has shown ac i i y in BCL2-dominan
ollicula lymphomas, while combina ion wi h agen s a ge ing EZH2-mu a ed subclones can u he delay esis ance
[63].
Clonal da a also guide combina ion he apy decisions. By cha ac e izing he subclonal a chi ec u e o a umo , clinicians
can choose d ug pai s o iple s ha simul aneously dis up mul iple e olu iona y pa hways. In elapsed/ e ac o y
DLBCL, dual inhibi ion o CD79B ( ia an ibody-d ug conjuga es) and BCL6 (a ansc ip ional ep esso ) has
demons a ed e icacy in umo s wi h bi u ca ed clonal d i e s [64].
Impo an ly, unde s anding clonali y enhances isk s a i ica ion. Pa ien s wi h high clonal di e si y, apid clonal
u no e , o he apy-induced clonal eshaping a e mo e likely o expe ience elapse and may bene i om ea ly
hema opoie ic s em cell ansplan a ion o en ollmen in adap i e clinical ials [65].
Table 2 Ta ge ed The apies Associa ed wi h Speci ic Mu a ional Signa u es and Clonal A chi ec u es
Mu a ional Signa u e / Clonal Fea u e
Associa ed Ta ge able Pa hway
The apeu ic Agen (s)
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