*Co esponding au ho : Za een Fa ima
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 License 4.0.
Technological Ad ances in Ci cula ing Tumo DNA (c DNA) De ec ion o Moni o ing
Minimal Residual Disease (MRD)
Za een Fa ima 1, *, Ayesha Nadeem 2, Ai zaz Sajid 1, Pakeeza Rehman 3 and Kinza Ta iq 2
1 Ai zaz Lab and Diagnos ic Cen e Mandi Bahauddin, Punjab, Pakis an.
2 Depa men o Biochemis y, Facul y o science, Uni e si y o Ag icul u e, Faisalabad, Punjab, Pakis an.
3 Depa men o Allied heal h sciences, acul y o science, uni e si y o Sa godha Punjab, Pakis an.
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 24(02), 068-080
Publica ion his o y: Recei ed on 23 Sep embe 2025; e ised on 01 No embe 2025; accep ed on 03 No embe 2025
A icle DOI: h ps://doi.o g/10.30574/wjbphs.2025.24.2.0939
Abs ac
The abili y o assess minimal esidual disease (MRD) h ough ci cula ing umo DNA (c DNA) is e olu iona y wi hin
oncology as i p o ides he mos p ecise modali y o iden i ying esidual cance pos cu a i e he apies. This e iew
examines he ad ancemen s in he echnologies o c DNA-based MRD de ec ion published om he yea 2020 o
p esen while b ie ly men ioning he echnologies de eloped p io as a e e ence o he ime line. This e iew explains
he ans o ma ion o he olde PCR-based echnologies o nex gene a ion sequencing echnologies which include
digi al PCR, umo -in o med assays like CAPP-seq, and no el echnologies ha analyze he umo epigenome and
agmen ome as well. All o hese echnologies demons a e he inc easing abili y o he p ac i ione o iden i y and
moni o cance du ing ea men o cus om he ea men and he apy o he pa ien . F om he ea men o a ious
di e en cance s he e is speci ic disease, ea men and he apy moni o ing and cus omizing a ailable o he
p ac i ione . Howe e , he e a e s ill nume ous issues such as s anda diza ion, analy ical alida ion and ou ine clinical
in eg a ion o he echnology emain. This e iew aims o desc ibe he a ailable echnologies, hei in eg a ion wi hin
he clinical pa adigm, assess hei alue and echnologies and o ecas he p ospec i e o c DNA MRD su eillance.
Keywo ds: Cell- ee DNA; Technologies; Cance ; digi al PCR; Nex gene a ion sequencing
1. In oduc ion
Ci cula ing umo DNA (c DNA) consis s o sho segmen s o cell- ee DNA ha a e eleased om apop o ic and nec o ic
umo cells, p o iding a unique pe spec i e on umo biology and e olu ion (1). This in eg a ion o he de ec ion and
quan i ica ion o c DNA in o liquid biopsy echnology ep esen s a g oundb eaking ad ancemen , pa icula ly in he
ield o moni o ing MRD a e cu a i e in en ea men (2). Minimal Residual Disease, o MRD, is de ined as small,
clinically unde ec able, esidual cance cells ha emain pos p ima y ea men (3). As a esul , cance cells ha emain
and a e de ec able h ough c DNA es ing a e o a signi ican clinical and ope a i e change o pa ien ca e, as clinicians
can p o ide imely and a ge ed ca e be o e he pa ien unde goes a clinical elapse.
Moni o ing MRD h ough c DNA is p edica ed by he no ion ha umo al cells, h ough nec osis, apop osis, and ac i e
sec e ion, incessan ly elease agmen s o shed DNA in o he bloods eam (4). Ad anced cell- ee DNA molecula
echniques allow o he iden i ica ion o umo -de i ed agmen s o c DNA as opposed o “no mal” ci cula ing cell- ee
DNA (c DNA) and hese agmen s o c DNA con ain umo -speci ic, gene ic, and epigene ic al e a ions (5). The clinical
applica ions o c DNA o MRD cance de ec i
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on has been documen ed o colo ec al cance , lung cance , b eas cance , and melanoma, and whe e pos - ea men
c DNA is p esen , he e is a no able isk o disease ecu ence (6).
Moni o ing MRD equi es di e en analy ical app oaches as he sensi i i y and speci ici y equi ed o MRD moni o ing
is dis inc om diagnos ic unc ionali ies. When diagnosing a cance , he umo ac ion is highe (0.1-10%) as
compa ed o MRD moni o ing whe e he ac ion o ci cula ing umo DNA is only a minu e 0.001-0.01% o he o al
c DNA. This p omp ed he de elopmen o mo e ad anced analy ical echniques om basic quan i a i e PCR o nex
gene a ion sequencing sys ems wi h molecula e o co ec ion and umo -in o med designed s a egies (7).
2. Con en ional me hods
In he ea ly days, me hods de eloped o c DNA de ec ion we e based on s anda d quan i a i e PCR and allele-speci ic
PCR echniques ha ocused on ho spo mu a ions(8). While hese app oaches we e uncomplica ed and inexpensi e,
hey lacked sensi i i y enough o analy ical pu poses whe e he de ec ion h esholds we e 0.1% - 1% o he a ian
allele ac ion (9). In he case o c DNA as used o MRD applica ions, whe e he concen a ion is se e al o de s o
magni ude lowe , he app oaches became e en mo e inadequa e, causing high alse nega i e a es and limi ing he
u ili y o he me hod clinically (10).
In he MRD se ing, s anda d app oaches o nex -gene a ion sequencing also do no employ molecula e o co ec ion
and ace he same challenges. Wi h sequencing and DNA polyme ase, he e o a es o e.g. 10-3 o 10-4 c ea es a noise
loo in low equency a ian s (11). Issues such as low me hod speci ici y a ising om alse-posi i e esul s gene a ed
om PCR ampli ica ion, sequencing e o s, and DNA damage a i ac s (12). Con en ional app oaches also end o
emain ocused on a ge ed gene panels o ho spo mu a ions. This ixa ion may no only o e look pa ien -speci ic
a ian s bu also umo he e ogenei y and clonal e olu ion conce ns (13). In addi ion, he misiden i ica ion o clonal
hema opoiesis o inde e mina e po en ial (CHIP) mu a ions in c DNA could esul in a alse posi i e. Age- ela ed
mu a ions may be misiden i ied as umo -de i ed a ian s (14). Such limi a ions uel he de elopmen o mo e obus
and inclusi e de ec ion app oaches.
3. Digi al PCR (DPCR) Technologies
Compa ed wi h qPCR, digi al PCR (dPCR) has ad anced he de ec ion o ci cula ing umo DNA (c DNA) by o e ing
absolu e quan i ica ion and g ea e sensi i i y o low- equency a ian s (15). This is due o he pa i ioning o he
sample in o housands o millions o indi idual eac ions which allows a bina y (yes/no) de ec ion o a ge molecules
in a eac ion wi hou s anda d cu es (16). The wo p ima y sys ems o pe o ming dPCR a e d ople digi al PCR and
chambe -based digi al PCR sys ems.
D ople digi al PCR (ddPCR) is he mos equen ly used dPCR echnology o c DNA o ddPCR. The Bio-Rad QX200 and
QX ONE sys ems p oduce 20,000 nanoli e -sized d ople s pe sample ha can be used o indi idual PCR eac ions (17).
Such pa i ioning allows he de ec ion o a e a ian s a 0.001% equency and g ea e (18). Recen imp o emen s o
ddPCR echnology ocused on d ople gene a ion, luo opho e chemis y, and da a analysis (19). Ex ensi e alida ion
o he ddPCR analy ical pe o mance o MRD used nume ous cance ypes. Fo de ec ion o KRAS, BRAF, and PIK3CA
mu a ions in colo ec al cance , ddPCR is he mos sensi i e op ion and app oaches de ec ion le els o 0.01% VAF (20).
Fo lung cance , ddPCR has ocused on EGFR mu a ions and p o ided s ong clinical ou come co ela ions, while ends
in c DNA clea ance p o ided p ognos ic in o ma ion (21).
The cons uc ion o mul iplexed ddPCR assay echnology is one o he ecen ad ancemen s. This new echnology has
he abili y o simul aneously iden i y se e al a ge s which is a conside able imp o emen o e he o iginal digi al PCR
which had ex ensi e limi a ions in his ega d (22). The design o p obes using Locked Nucleic Acids (LNA) and Pep ide
Nucleic Acids (PNA) has esul ed in inc eased speci ici y and dec eased backg ound noise (23). The in oduc ion o
au oma ed sample p epa a ion has dec eased he amoun o ime spen on p epa a ion and a ied he ep oducibili y
o ddPCR which b ings his new echnology e en close o ou ine clinical applica ion (24).
None heless, wi h ega d o he comp ehensi e moni o ing o MRD, ddPCR has some basic sho comings. The
echnology is designed o he p ecise de ec ion o a limi ed, known, a ge ed a ian , which lacks he b oad-spec um
co e age expec ed om sequencing. Thus, he limi ed h oughpu o ddPCR echnology also excludes i om popula ion
sc eening and o he esea ch applica ions whe e mul iple samples o se e al analyses equi e simul aneous p ocessing
(25).
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4. Compa ison o ep esen a i e MRD echnologies
Table 1 Compa ison o MRD echnologiesD awn om supplied s udies (5, 6, 8)
Technology
Typical LOD epo ed
Pe sonaliza ion
equi ed
Clinical alida ion examples
ddPCR ( a ge ed ho spo s)
p ac ical low-pe cen o
0.01% scale wi h op imized
inpu and wo k lows (5), (6)
No (pe -locus
assays)
HPV16 c DNA de ec ion wo k lows;
compa a i e ho spo s udies (5), (6)
Tumo -in o med NGS
(pe sonalized panels, UMI)
down o ~0.02% a ian
allele ac ion epo ed o
deep panels; de ec ion
imp o es wi h numbe o
acked a ian s (8)
Yes ( umo
issue in o ms
panel)
La ge e ospec i e/p ospec i e MRD
coho s and Signa e a-s yle wo k lows
showing MRD co ela es wi h
ecu ence (8), (9), (10)
Plasma-only
epigenomic/ agmen omic
assays
a iable; combining genomic
+ epigenomic inc eased
sensi i i y by ~25–36%
e sus genomic alone in CRC
coho s
No
( umo -naï e)
Plasma-only MRD in colo ec al cance
wi h high PPV and imp o ed
sensi i i y when in eg a ing
epigenomic signa u es
4.1. Tumo In o med Pe sonalized Sequencing
By moni o ing pa ien speci ic panels o umo issue ha include dozens o housands o soma ic al e a ions, he
acking o minimal esidual disease becomes inc easingly mo e sensi i e as he in o ma ion om mul iple independen
a ian s is consolida ed. E idence, bo h comme cial and academic, abou pos ope a i e c DNA posi i i y and
subsequen elapse is mul iple and illus a es he e ec i eness o hese me hods (8), (9), (10).
4.2. Me hyla ion Assays
In pilo alida ions ac oss mul iple umo ypes, he a ge ed me hyla ion qMSP panels (e.g., PcMM qMSP) demons a ed
low copy numbe limi s o de ec ion (app oxima ely 10 o 30 me hyla ed copies) wi h a compe i i e u na ound and
cos p o ile in compa ison o NGS (7).
4.3. E o Supp ession and Panel Design
S a egies o cu b alse posi i e esul s in he con empo a y MRD panel landscape in ol e unique molecula iden i ie s,
ma ched whi e blood cell sequencing o emo e clonal hema opoiesis and backg ound noise da abases (8, 11). The
a o emen ioned s a egies, alongside pa e ned panel design, sc a ch he su ace o he e o s being made o esol e
he challenges ha MRD p esen s.
5. Nex -Gene a ion Sequencing App oaches
Ad anced sequencing echnologies, coupled wi h molecula e o co ec ion, ha e enabled he analysis o mul iple
genomic egions o ocus on sensi i i y and speci ici y in de ec ing c DNA-based MRD (26). These echniques can be
ca ego ized in o umo -in o med and umo -agnos ic me hods, each p o iding dis inc ad an ages and applicable
scena ios.
Rega ding he a ious nex -gene a ion sequencing c DNA MRD me hods, in aligned echnical cons uc s, hese ha e
consolida ed in o h ee b oad class dis inc ions. These include umo -in o med pe sonalized sequencing whe e
mul iple pa ien -speci ic a ian s a e moni o ed, umo -nai e a ge ed panels wi h deep e o supp ession, and he
epigenomic o agmen omic s a egies ha de i e u ili y om me hyla ion and agmen a ion pa e ns (1, 3, 8). Key
s a egies ocused on MRD de ec ion p esc ibed limi s and heigh ened con idence o MRD decla a ions include e o
supp ession (unique molecula iden i ie s, s a is ical in eg a ion ac oss many loci) and he b oadening o a ian lis s
(1, 8).
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5.1. Cance Pe sonalized P o iling by Deep Sequencing (CAPP-Seq)
CAPP-Seq is a new and inno a i e echnique ha combines ul a-deep sequencing o plasma c DNA wi h pe sonalized
umo p o iling and was es ablished by Diehn and cowo ke s (27). CAPP-Seq makes use o umo issue sequencing o
de ec pa ien -speci ic a ian s, and hen cus omizing assays o ci cula ing umo DNA (c DNA) by a ge ing hese
iden i ied a ian s. The o iginal CAPP-Seq p o ocol in eg a ed sophis ica ed bioin o ma ics algo i hms on op o
molecula ba coding o supp ess sequencing e o s and achie ed a de ec ion limi o 0.02% Va ian Allele F equency
(VAF) (28). The mo e ecen e sions o CAPP-Seq use mo e ad anced echnology. The addi ion o duplex sequencing
echnology has has g ea ly diminished he e o a e, allowing he de ec ion o a ian s a equencies abo e 0.001%
(29). The implemen a ion o machine lea ning algo i hms on cap u e p obes has imp o ed on- a ge cap u e e iciency
and diminished o - a ge cap u e (30). The mos ecen ad ances in s anda dized p o ocol de elopmen and quali y
con ol me ics ha e made CAPP-Seq mo e widely adop ed in mo e cen e s (31).
CAPP-Seq has demons a ed obus clinical alida ion while expanding i s use o a ious cance s. Fo clinical easibili y
o CAPP-Seq-based MRD de ec ion in non-small cell lung cance , i su passed s anda d imaging, achie ing an imp essi e
median lead ime o 5.2 mon hs be o e imaging showed disease p og ession (2). In s udies in ol ing colo ec al cance ,
indings we e simila , wi h a pos -ope a i e c DNA posi i e diagnosis and a disease ecu ence isk se a 7.2 (32).
5.2. Ta ge ed E o Co ec ion Sequencing (TEC-Seq)
TEC-Seq is an al e na i e umo -in o med me hod ha eaches ul a-high sensi i i y h ough he implemen a ion o
unique molecula iden i ie s (UMIs) and sophis ica ed e o co ec ion echniques (33). Like CAPP-Seq, umo
sequencing esul s a e in eg a ed in o cus omized pa ien cap u e panels, bu TEC-Seq uses di e en molecula
ba coding and consensus calling echniques (34). TEC-Seq has be e h oughpu and lowe cos pe sample while
main aining CAPP-Seq compa able de ec ion limi s. The unique ea u e o TEC-Seq is i s highly sophis ica ed e o
co ec ion echniques ha combine molecula ba coding, s and-awa e consensus calling, and noise backg ound
modeling (35). This enables he highly con iden dis inguishing o ue low- equency a ian s om high- equency
a i ac s. Machine lea ning has been applied o imp o e he accu acy o a ian calling and he educ ion o alse-posi i e
a es (36).
5.3. Sa e-Sequencing Sys em (SAFE-Seq)
The i s Ea ly NGS based ul a-sensi i e a ian de ec ion sys em is SAFE-Seq, c ea ed by Vogels ein and o he s (26).
Unique molecula iden i ie s and p ime ampli ica ion a e inco po a ed in he echnology o make i e sa ile enough
o de ec a e a ian s (37). Al hough i was no ailo ed o MRD sys ems, he basic p inciples o SAFE-Seq ha e been
modi ied in o a numbe o comme cial and esea ch sys ems (38). The g ea es con ibu ion o SAFE-Seq is he
inco po a ion o unique iden i ie s o e e y DNA s and be o e ampli ica ion, allowing o he de ec ion and
ec i ica ion o ampli ica ion and sequencing e o s (39). This has been in aluable in he de ec ion o low equency
mu a ions in di icul o wo k wi h samples such as FFPE issues and deg aded c DNA (40).
6. Tumo -Agnos ic App oaches
Al hough umo -in o med s a egies ha e g ea e sensi i i y o known a ian s, umo -agnos ic me hods ha e mo e
comp ehensi e scope and do no necessi a e p io analysis o umo issue (41). These me hods end o use la ge gene
panels o pe o m whole-exome sequencing and use sophis ica ed bioin o ma ics o de ec umo -de i ed a ian s (42).
Gua dan 360 and Founda ionOne Liquid CDx a e examples o clinically-implemen ed, comme cially a ailable umo -
agnos ic app oaches o MRD moni o ing (43). These sys ems u ilize p op ie a y e o co ec ion algo i hms and
clinically alida ed algo i hms o a ious cance s o analyze 70-500+ genes (44). Howe e , and pa icula ly o he
MRD applica ions, he sensi i i y o hese sys ems is lowe han ha o umo -in o med me hods because umo -
agnos ic me hods mus analyze mo e genomic e i o y han can be sequenced wi h he a ailable sequencing dep h
(45).
7. Eme ging Technologies and No el App oaches
The de ec ion o ci cula ing umo DNA (c DNA) con inues o de elop, pa icula ly wi h new echnologies ha enhance
minimal esidual disease (MRD) moni o ing. These e o s emphasize inc easing he de ec ion sensi i i y, dec easing
cos s, inc easing sample p ocessing capabili ies, and b oadening he ypes o molecula da a ha can be ob ained om
cell- ee DNA (c DNA) samples.
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7.1. Epigenomic App oaches
Pa e ns o DNA me hyla ion in c DNA s ill need o be exploi ed o umo -speci ic in o ma ion (46). Because o hei
mo e signi ican popula ion co e age, unlike gene ic mu a ions which a e limi ed o ce ain subclonal umo
popula ions, epigene ic al e a ions a e mo e likely o be de ec ed in cases o MRD (47). The e a e se e al me hodologies
o examining me hyla ion in c DNA, including bisul i e sequencing, me hyla ion-speci ic PCR, and a ge ed me hyla ion
a ays (48). C is iano and colleagues de eloped he Cance SEEK pla o m, which in eg a es gene ic and epigene ic
in o ma ion o enhance he sensi i i y o cance de ec ion (49). Recen modi ica ions o his me hodology o MRD
moni o ing a e me hyla ion-based signa u es, which p o ed o be highly in o ma i e in combina ion wi h mu a ion-
based de ec ion (50). Fo ad anced cases o diseases, me hyla ion echniques help in he de ec ion o neoplasms ha
ha e low mu a ion bu dens, including speci ic pedia ic cance s and some adul cance s (51).
7.2. F agmen omic Analysis
Due o a ia ions in size, dis ibu ion, and agmen a ion o c DNA, i is possible o ace DNA back o he umo and
di e en ia e i om no mal c DNA (52). F agmen omic analysis s udies he dis ibu ion and leng h o agmen s and
he speci ic end mo i s (53). This app oach has acili a ed he design o amewo ks o he in eg a ion o gene ic and
epigene ic analyses, whe e bo h a e employed as mul i-modal de ec ion echniques (54). Recen s udies show con as s
in he leng h o umo -de i ed c DNA agmen s and no mal c DNA agmen s, wi h he o me being sho e and ha ing
dis inc i e pe iodici y (55). Machine lea ning models a e c ea ed o cap u e hese di e ences o use in c DNA de ec ion
sys ems (56). DELFI (DNA E alua ion o F agmen s o Ea ly In e cep ion) is one o he i s sys ems designed o
agmen omic analysis in cance sc eening and moni o ing (57).
7.3. P o ein-Based App oaches
Wi h espec o he in eg a ion o p o ein liquid biopsies wi h c DNA o c ea e p o ein mul i-analy e liquid biopsies, his
has jus begun o ga ne in e es in he ield (58). Fu he in o ma ion wi h espec o c DNA analysis can be ga he ed
om ci cula ing umo cells (CTCs), ex acellula esicles, as well as soluble p o ein bioma ke s and umo al
in e s i ium (59).
7.4. A i icial In elligence and Machine Lea ning
In eg a ion o AI and ML in o he echnology ad ancemen o c DNA analysis (60). Se e al c oss-sec ional aspec s o
c DNA analysis would esul in he imp o ed accu acy o a ian calling, he supp ession o backg ound noise, and
clinical in e p e a ion eedback (61). In deep lea ning especially, algo i hms can e alua e complex pa e ns in
sequencing ha a e missed by olde app oaches (62). Nume ous comme cial pla o ms apply AI and ML algo i hms in
hei analy ics. In he case o he Exac Sciences Cologua d es , i employs machine lea ning o analyze me hyla ion
pa e ns (63). Likewise, he GRAIL Galle i es uses deep lea ning o analyze me hyla ion pa e ns o mul iple cance s
(64). Such echnologies a e likely o ad ance he ole o ML and AI in c DNA-based MRD moni o ing.
8. Clinical Applica ions and Valida ion S udies
The clinical ansla ion o c DNA-based MRD moni o ing is one a ea ha has ad anced e y quickly. Nume ous s udies
ha e demons a ed i s applicabili y o se e al cance s and di e en clinical en i onmen s. This led o he cons uc ion
o clinical guidelines and ecei ed he necessa y egula o y endo semen s o mo e o wa d as p ospec i e la ge-scale
ials.
8.1. Colo ec al Cance
Colo ec al cance is among he mos equen ly diagnosed cance cases in he wo ld. In addi ion, i has been he mos
s udied cance ype in c DNA MRD moni o ing, suppo ing i s clinical u ili y in la ge-scale s udies such as CIRCULATE-
Japan. In his s udy, which included o e 1,000 pa ien s wi h s age II-III colo ec al cance , i was shown ha c DNA
posi i i y a e su ge y co ela ed wi h a ecu ence isk ha was se e al imes g ea e (haza d a io 3.8, 95% CI 2.4-
6.1) (65). CIRCULATE-Japan also demons a ed ha c DNA as a p ognos ic ac o s ood independen ly o o he
clinicopa hological ac o s (66). DYNAMIC is he i s andomized con olled ial ha s udies c DNA-guided ea men
in colo ec al cance (67). Pa ien s wi h pos -su gical c DNA posi i i y we e andomized o ecei e ei he adju an
chemo he apy o su eillance, while pa ien s wi h c DNA nega i i iy we e moni o ed wi hou any ea men (68).
DYNAMIC demons a ed ha ea men based on c DNA le els p o ided simila ou comes as he s anda d o ca e, hus
p o ing ha o e ea men in pa ien s wi h nega i e c DNA could be a oided (69).
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Mo e s udies ha e also been done in use o c DNA moni o ing du ing and pos adju an chemo he apy. In he COBRA
s udy, i was shown ha ecu ence isk inc eased when c DNA was pe sis en ly p esen , whils enewed posi i i y
a e c DNA clea ance was associa ed wi h wo se ou comes (70). These we e he indings ha mos likely in luenced
he inclusion o c DNA es ing in clinical guidelines and he cons uc ion o c DNA-guided ea men p o ocols (71).
8.2. Lung Cance
Non-small cell lung cance (NSCLC) is a majo applica ion a ea o c DNA-based MRD moni o ing. c DNA de ec ion
p eceded adiog aphic p og ession by a median o 70 days in he TRACERx s udy (a landma k longi udinal analysis o
umo e olu ion) (72). Using a umo -in o med app oach, he s udy also demons a ed ha , in he majo i y o pa ien s,
c DNA moni o ing coupled wi h con en ional imaging p o ided he ea lies diagnosis o disease ecu ence (13).
Mul iple in es iga ions ha e been conduc ed on c DNA moni o ing in he adju an ea men s age o ea ly-s age
NSCLC. The ADAURA s udy, which ocused mos ly on osime inib e icacy and conduc ed c DNA analysis, showed ha
disease- ee su i al was sho e when pa ien s p esen ed wi h c DNA posi i e NSCLC (73). Fu he in es iga ions
showed ha he e was an associa ion be ween imp o ed ou comes and c DNA clea ance du ing ea men wi h
osime inib (74).
Combining c DNA moni o ing wi h he use o immuno he apy has been pa icula ly p omising in lung cance . S udies
ha e shown ha du ing ea men wi h immune checkpoin inhibi o s, c DNA kine ics can p edic esponse and iden i y
pa ien s who a e mo e likely o expe ience disease p og ession (75). Moni o ing c DNA alongside immune bioma ke s
like umo mu a ion bu den could be use ul o p edic ion (76).
8.3. B eas Cance
Resea ch on ci cula ing umo DNA (c DNA) in b eas cance concen a es p ima ily on ea ly diagnosis and moni o ing
pa ien s du ing he adju an phase (i.e. pos -su gical ea men s aimed a minimizing he likelihood o cance
ecu ence). The c-TRAK TN s udy, o example, illus a ed he po en ial o c DNA as a moni o ing ool o agg essi e
b eas cance ( iple-nega i e b eas cance ). The s udy epo ed ha he p esence o c DNA pos -su ge y was
associa ed wi h conside ably sho e disease- ee in e als (77). The s udy demons a ed ha c DNA, using a umo -
in o med app oach, p o ided p ognos ic in o ma ion ha was conside ably g ea e han wha s anda d clinical me ics
(i.e. umo size/s age) p o ided (78).
Fo ho mone ecep o -posi i e b eas cance , c DNA esea ch has been p ima ily aimed a in o ming adju an
endoc ine he apy (i.e. ho mone-blocking he apy). The iden i ica ion o ESR1 mu a ions in c DNA has been desc ibed
as a de ini i e way o lagging esis ance o a oma ase inhibi o s, a key ea men o ad anced b eas cance (79).
Mo eo e , he in eg a ion o c DNA wi h o he liquid biopsy echniques, like ci cula ing umo cell analysis, is iewed
as an impo an s a egy o s eamline he assessmen and longi udinal managemen o he disease (60).
9. Challenges and Limi a ions
Signi ican ad ancemen s ha e been achie ed in c DNA-based MRD moni o ing. Howe e , o comp ehensi e clinical
applica ion, he e a e s ill gaps ha need o be illed.
9.1. Analy ical Challenges
De ec ing minimal esidual disease (MRD) is pa icula ly challenging because he concen a ions o ci cula ing umo
DNA (c DNA) a e so low ha e en he mos ad anced echnologies a e likely o miss e idence o clinically ele an
disease (80). The unp edic able pa e ns o umo shedding c DNA c ea e addi ional challenges, as nega i e esul s do
no always signi y ha no esidual disease is p esen (81). Mo eo e , p eanaly ical a iables, such as he collec ion,
p ocessing, and s o age o samples, may con ibu e o ins ances whe e c DNA is no p esen when i is expec ed o be,
leading o challenges a ound disease de ec ion. Con inued e o s a e needed o de ise mo e e ec i e s a egies o
mi iga e hese challenges (82). In addi ion, he non-uni o mi y o es ing amewo ks and labo a o y se ups is a majo
obs acle. The absence o cohe en p o ocols esul s in highly dispa a e ou comes ha physicians canno in e p e
consis en ly (83). To add ess hese p oblems, he de elopmen o e e ence ma e ials, calib a ion s anda ds, and
p o iciency es ing in quali y con ol will be essen ial in aligning he dispa a e labo a o ies in he wo ld (84).
9.2. Biological Challenges
Unde s anding he biology behind he shedding o ci cula ing umo DNA (c DNA) pe plexes esea che s. Tumo
cha ac e is ics such as size, loca ion, blood supply, and issue ype in luence he le els o ci cula ing umo DNA e en
when he umo bu den is simila in clinical cases (85). Ce ain cance s, pa icula ly hose wi h ewe mu a ions o
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 24(02), 068-080
74
speci ic his ological sub ypes, elease cogna e DNA a e en lowe le els and challenge he limi s o con empo a y
de ec ion me hods (1). Clonal hema opoiesis adds complexi y as well. Age- ela ed mu a ions in blood cells esul in cell-
ee DNA (c DNA) agmen a ion ha can be classi ied as umo - ela ed mu a ions (86). Al hough sequencing ma ched
leukocy es p o ides some cla i y, c DNA analysis becomes e en mo e con olu ed and cos ly (87).
9.3. Clinical Challenges
The e a e also challenges associa ed wi h in eg a ing c DNA in o ou ine clinical p ac ice. Fo example, in u gen clinical
scena ios, he ypical 7-14 day u na ound ime is la gely inadequa e (88). Mo eo e , he expensi e umo -in o med
es ing s a egy ha in ol es bo h umo issue and plasma sequencing is u ile and bu densome in decision-making
(89). Mo eo e , physicians equi e adequa e educa ion o in e p e c DNA esul s accu a ely. Familia i y wi h he es s’
cons ain s, hei analy ical capabili ies, and hei ole in he whole clinical con ex o he pa ien is essen ial (90).
Imp o ed clinical decision suppo sys ems along wi h mo e e ined ea men pa hways will acili a e clinicians' abili y
o employ hese es s op imally (91).
9.4. Regula o y and Reimbu semen Challenges
As he egula o y en i onmen a ound c DNA es ing con inues o de elop, an asymme y o compliance manda es o
lab-de eloped es s and FDA-app o ed ones will emain (80). The e idence needed o suppo egula o y app o al, and
o ob ain eimbu semen o clinical use, is conside able and, in some cases, will equi e ob aining e idence h ough
la ge andomized con olled ials (92). The lack o s anda d eimbu semen policies o c DNA es ing, and pa ial
eimbu semen o i s clinical uses, add o he challenges. To jus i y comple e eimbu semen , cos -e ec i eness
analyzes o ea men plans guided by c DNA mus be done (93). Es ablishing billing codes and eimbu semen policies
is c ucial o he clinical use o c DNA es ing (94).
10. Compa a i e Analysis o Technologies
A ange o challenges and oppo uni ies a ises om he a ious echnologies in c DNA de ec ion conce ning hei
clinical implemen a ion. Each me hod’s p os and cons help de ine i s clinical u ili y.
Table 2 Analysis o echnologies
Technology
Sensi i i y
(VAF)
Th oughpu
Tu na ound
Time
Cos
Mul iplexing
Capabili y
Key
Ad an ages
Key
Limi a ions
ddPCR
0.001-
0.01%
Low-
Medium
4-6 hou s
Low-
Medium
Limi ed (1-4
a ge s)
High p ecision,
absolu e
quan i ica ion
Limi ed
b ead h,
single/ ew
a ge s
CAPP-Seq
0.001-
0.02%
Medium
7-14 days
High
High (100+
a ge s)
Ul a-high
sensi i i y,
pe sonalized
Requi es
umo issue,
complex
wo k low
TEC-Seq
0.001-
0.01%
Medium
7-14 days
High
High (50-200
a ge s)
Excellen e o
co ec ion
Requi es
umo issue,
high cos
Tumo -
agnos ic NGS
0.01-0.1%
High
7-14 days
Medium-
High
Ve y High
(300+
a ge s)
No umo
issue equi ed
Lowe
sensi i i y,
highe
backg ound
Me hyla ion-
based
0.01-0.1%
Medium-
High
7-14 days
Medium
High
Applicable o
low-mu a ion
cance s
Less ma u e
echnology
F agmen omics
Va iable
High
2-7 days
Low-
Medium
N/A
Rapid, cos -
e ec i e
Requi es
combina ion
wi h o he
me hods
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11. Fu u e Pe spec i es and Technological De elopmen s
Pos -ma ke c DNA MRD moni o ing is g owing and new echnologies a e being de eloped o add ess some o he ield’s
limi a ions and expand c DNA MRD moni o ing’s a ious clinical applica ions.
11.1. Nex -Gene a ion Technologies
A a ie y o di e en echnologies a e being buil and e ined ha will enhance c DNA de ec ion. Single-molecule
sequencing echnologies, like he ones being de eloped by Ox o d Nanopo e and Paci ic Biosciences, will allow he di ec
analysis o c DNA which will elimina e he need o PCR ampli ica ion, cu down on some ampli ica ion a i ac s, and
enhance sensi i i y o c DNA (95). These echnologies will enable clinicians o pe o m eal- ime analyses and ecei e
es esul s in a ma e o minu es (96). Recen de elopmen s in mic o luidic echnology will op imize he c DNA
moni o ing p ocedu e o he poin whe e i can be conduc ed a he si e o clinical ca e and e u ned wi hin hou s.
Clinicians will be enabled o make clinical decisions in eal ime a he han depending on bu densome logis ical
a angemen s ypical o cen alized es ing (97).
11.2. A i icial In elligence and Machine Lea ning
The in eg a ion o AI, ML, and au oma ion echnologies wi h c DNA analysis is likely o be g oundb eaking.
Compu a ional ools will enhance he p ecision and accu acy o a ian calling, iden i y alse-posi i e a ian s, and
au onomously de ec complex analy ical pa e ns ha a e cu en ly unde ec ed by human analys s (98). Deep lea ning
o he i s ime will help au oma e he analysis o complex genomic pa e ns and he in eg a ion o a ious da a ypes
(99). AI-d i en clinical decision suppo sys ems migh analyze c DNA epo s and help in making ea men decisions.
These sys ems migh gene a e ecommenda ions conside ing unique pa ien a iables, his o ical ea men da a, and
con empo aneous ea men moni o ing sys ems (100). Fu u e liquid biopsies will mos p obably in ol e mo e han
one analy e and mo e han one de ec ion echnology. Disease moni o ing can be mo e comp ehensi e i analyses o
c DNA a e coupled wi h o he p o iling echniques o in eg a i e assessmen o disease ma ke s such as p o ein
a iables, ci cula ing umo cells, ex acellula esicles, and me abolomic p o iling (58).
11.3. Regula o y E olu ion
The e olu ion o he guidance documen s and pa hways o he egula ion o MRD moni o ing applica ions will shape
he u u e o egula ion o c DNA es ing. The FDA is expec ed o con inue app o ing c DNA es s as mo e clinical
e idence eme ges and se e al o hese es s ha e al eady been app o ed o di e en indica ions d. The app o al and
clinical implemen a ion o egula ed companion diagnos ic es s o speci ic he apies will p obably be o ob ain
egula o y app o al as e (80, 92).
11.4. Clinical In eg a ion
The implemen a ion o pa hways and algo i hms o clinical in eg a ion o c DNA moni o ing will be essen ial o
es ablishing comp ehensi e in eg a ion. The e is ea ly s age wo k being done o es ablish guidance documen s o
speci ic clinical applica ions o c DNA, bu he mo e impo an wo k is he de elopmen o guidance documen s o
b oade clinical applica ions and he consensus o hese o be adop ed (91). The clinical suppo amewo ks and
p o ide educa ion will be undamen al o he app op ia e u iliza ion o he es s (90).
12. Conclusion
The speci ic cance ca e app oaches ha inco po a e ci cula ing umo DNA (c DNA) opened new op ions o
pe sonalized and p ecise MRD moni o ing echnimolecula app oaches wi hin ea men . Mode n e o -co ec ed
sequencing iden i ies c DNA wi h high sensi i i y o aid nume ous cance s and o assess esponse, e alua e ecu ence,
and p edic he apy. The applica ion o c DNA con inues o expand wi h CAPP-Seq, de elopmen s wi hin epigene ics,
and agmen omics. P oblems wi hin s anda diza ion, cos , and clinical adop ion o hese echnologies s ill pose
challenges. Eme ging esou ces like AI and poin -o -ca e es ing a e expec ed o mee hese challenges. Rou ine clinical
in eg a ion o c DNA moni o ing will imp o e diagnosis and ou comes. This is dependen upon collabo a ion and a shi
o eal-wo ld implemen a ion om scien is s, clinicians, and policymake s.
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Compliance wi h e hical s anda ds
Disclosu e o con lic o in e es
No con lic o in e es o be disclosed.
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